I mprovi ng t he Preservat i on Qual i t y of  Hi ghMoi st ure By- Product  Feedst uf f s by Ensi l age andUse of  Addi t i ves
著者（英）Oki ne Abdul  Razak Addy
学位名博士（農学）
学位授与機関岩手大学
学位授与年度2006
学位授与番号11201甲第367号
URLht t p: //i d. ni i . ac. j p/1588/00001405/
IMPROVING THE PRESERVATION QUALITY OF 
HIGH MOISTURE BY-PRODUCT FEEDSTUFFS 
BY ENSILAGE AND USE OF ADDITIVES 
          by   OKINE Abdul Razak Addy        
A dissertation submitted in partial fulfillment of  the requirements for 
the degree of  Doctor of  Philosophy in Agricultural Sciences with Major 
Chair of  Plant Production  
        Iwate University  March 2007   
Declaration 
I hereby declare that this dissertation, submitted in partial fulfillment of the 
requirements for the degree of Doctor of Philosophy in the Agricultural Sciences with 
Major Chair of Plant Production and entitled “Improving the preservation quality of 
high moisture by-product feedstuffs by ensilage and use of additives”, represents 
my own work and has not been previously submitted to this or any other institution for 
any degree, diploma or other qualification. 
------------------------------                               ----------------------------- OKINE Abdul Razak Addy                                  Date (Author) 
This dissertation is hereby approved by members of the Final Examination Committee: 
        
---------------------------------------                         ----------------------------- Professor Dr. Meiji OKAMOTO                              Date   (Major Advisory Professor)   
  ---------------------------------------                       ----------------------------- Associate Professor Dr. Masaaki HANADA                     Date    (Advisory Committee Member)  
---------------------------------------                        ------------------------------- Professor Dr. Akio HONGO                                 Date     (Advisory Committee Member)   ---------------------------------------                        ------------------------------- Professor Dr. Toshiyoshi TAKAHASHI                        Date     (Advisory Committee Member)    ---------------------------------------                        ------------------------------- Professor Dr. Suguru SAIGA                                  Date   (Dean of the United Graduate School of   Agricultural Sciences, Iwate University)   
(Signed) 
(Signed) 
(Signed) 
(Signed) 
(Signed) 
(Signed) 29/1/2007 
29/1/2007 
29/1/2007 
29/1/2007 
29/1/2007 
29/1/2007 
TABLE OF CONTENTS 
Page No.
Table of Contents…...……………………………………….………... iii 
List of Figures………………………………………………………… vii 
List of Tables………………………………………………………….. x 
List of Abbreviations…..……………………………………………... xiii 
Chapter 1.  General Introduction………………………………….. 1 
1.1. Importance of agricultural by-products………….…………. 1 
1.2. Definition of HMBF………………………………………... 2 
1.3. Relevance of HMBF in current production systems …… 3 
1.4. Preservation techniques for HMBF……………………..….. 3 
1.5. Environmental concerns on disposal of HMBF…...……….. 4 
1.6. Objectives of the study…………………………………….. 6 
Chapter 2. Pre-ensiling characteristics of a representative of 
HMBF……….…………………………………………. 
     7 
2.1. Introduction…………………………………………………  
2.2. Materials and methods……………………………………... 8 
2.3. Results………………………………………………………  10 
2.4. Discussion………………………………………………….. 17 
2.5. Summary…………………………………………………… 20 
Chapter 3. Potato pulp.......................................................................... 
3.1. Introduction……………………………….………………... 
3.2. Ensiling potato pulp with or without bacterial inoculants…. 
 3.2.1. Materials and methods….…..……………….………...... 
 3.2.2. Results………….………….…………….………….......  26 
 3.2.3. Discussion………………………………………………. 32 
 3.2.4. Conclusion……………………..………………………. 
3.3. Effect of temperature and storage duration on potato pulp    
    ensiled with fungal inoculants…………………………….. 
 3.3.1. Materials and methods……..…………………………… 
 3.3.2. Results……………………………………..……………. 
 3.3.3. Discussion……………..………………………………... 
 3.3.4. Conclusion………………..…………………………….. 
3.4. Effluent production and losses in potato pulp silage………. 
 3.4.1. Materials and methods…….…………………………… 
 3.4.2. Results……………….…………………………………. 
 3.4.3. Discussion………….…………………………………... 
 3.4.4. Conclusion……….…………………………………….. 
3.5. Summary…………………………………………………… 
 37   40 
Chapter 4. Daikon (Oriental radish)……………………………. 57 
4.1. Introduction………………………………………………… 57 
4.2. Ensiling of daikon by-product with wheat straw…............... 
 4.2.1. Materials and methods……………………………......... 
 58   4.2.2. Results………………………………………...………… 62 
4.3. Ensiling daikon with other absorbents……......…………….. 71 
 4.3.1. Materials and methods...................................................... 
 4.3.2. Results............................................................................... 
 4.3.3. Discussion......................................................................... 
    4.4. Summary................................................................................. 
Chapter 5.   Ensiling of brewer’s grain and apple pomace............. 
5.1. Introduction………………...………………………………. 
5.2. Brewer’s grain……………………………………………… 
 5.2.1. Materials and methods………………...…….………….. 
 5.2.2. Results…………………….………..…….……………... 82 
 5.2.3. Discussion………...…………………………………….. 85 
5.3. Apple pomace………………………………………………. 
 5.3.1. Materials and methods………………………………….. 
 5.3.2. Results………………………………………………….. 
 5.3.3. Discussion……………………………………………… 
5.4. Summary…………………………………………………… 
Chapter 6.  Nutritive value of HMBF…..……………..……………  
6.1. Introduction…...……………………………………………. 94 
6.2. Nutritive value of potato pulp silage….……………………. 95 
 6.2.1. Materials and methods…………......…….……………... 95 
 6.2.2. Results……..………………...………………………….. 
 6.2.3. Discussion......................................................................... 
 6.2.4. Conclusion........................................................................  
6.3. Intake and preference of daikon by-product silage................ 
 6.3.1. Materials and methods...................................................... 
 6.3.2. Results............................................................................... 
    6.3.3. Discussion.................................................................... 
    6.3.4. Conclusion................................................................... 
       6.4. Summary...........................................................................  
Chapter 7.  Factors affecting the ensiling of HMBF and their   
         relationships………………………………………………. 
        7.1. Introduction…………………………………………… 
       7.2. Materials and methods…………......…….………........ 
       7.3. Results………………………………………………… 
       7.4. Discussion……………………………………….……. 
       7.5. Summary…………………………………….………... 
 118  123 
Chapter 8.  General Discussion……………………………….... 
         8.1. Pre-ensiling characteristics of HMBF........................ 
           8.2. Ensiling of HMBF...................................................... 
        8.3. Nutritive value of HMBF........................................... 
        8.4. Factors affecting the ensiling of HMBF..................... 
        8.5. General Conclusions...................................................  
        8.6. Further Outlook.......................................................... 
Chapter 9.  General Summary ...……............................................ 
            Acknowledgements……………………………............. 
            References…………………………...…......................... 
 131  131 
List of Figures 
Figure No. Title                         Page No.
Figure 3-1. Change in pH, lactic and acetic acid concentrations of potato pulp 
during 50 days of ensilage (Experiment 1). (◆) PP, potato pulp silage 
without additive; (■) PL, lactobacillus alone inoculated potato pulp 
silage; (▲) PR, rhizopus alone inoculated potato pulp silage; (●) RL, 
rhizopus + lactobacillus inoculated potato pulp silage. 
Figure 3-2. Change in sugar, starch and pectin during 50 days of ensilage 
(Experiment 1). (◆) PP = potato pulp silage without additive; (■) PL, 
lactobacillus alone inoculated potato pulp silage; (▲) PR,  rhizopus 
alone inoculated potato pulp silage; (●) RL, rhizopus + lactobacillus 
inoculated potato pulp silage. 
Figure 3-3. Change of pH (a) and lactic acid concentration (b) during of storage 
under different temperatures in potato pulp silage. (◆) 4°C; (■) 12°C; 
(▲) 25°C.  
Figure 4-1. Changes in pH and lactic acid concentration in daikon by-product 
silages days of ensilage in Experiment 1. (◆) daikon control; (■) 
daikon inoculated with Lactobacillus plantarum, (LP); (▲), daikon 
adjusted moisture with wheat straw only; (●) daikon adjusted moisture 
with wheat straw and inoculated with LP. 
Figure 4-2. Development of lactic acid bacteria (LAB) and yeast populations in 
daikon by-product silages days of ensilage in Experiment 1. (◆) 
daikon silage control; (■) daikon inoculated with Lactobacillus 
plantarum, (LP); (▲), daikon adjusted moisture with wheat straw 
only; (●) daikon adjusted moisture with wheat straw and with LP.  
Figure 4-3. 
Effluent volume in daikon by-product silages during 30 days of 
ensilage. (◆), daikon silage control; (■), daikon silage inoculated with 
Lactobacillus plantarum, (LP); (▲), daikon adjusted moisture with 
wheat straw only; (●), daikon adjusted moisture with wheat straw and 
inoculated with LP. 
Figure 4-4. Changes in pH and lactic acid concentration in daikon by-product 
silages during 6 days of aerobic exposure in Experiment 1. (◆), 
daikon silage control; (■), daikon inoculated with Lactobacillus 
plantarum, (LP); (▲), daikon adjusted moisture with wheat straw 
only; (●), daikon adjusted moisture with wheat straw and inoculated 
with LP.  
Figure 4-5. Changes in volatile fatty acid concentrations in daikon by-product 
silages during 6 days of aerobic exposure in Experiment 1.(◆), daikon 
silage control; (■), daikon inoculated with Lactobacillus plantarum, 
(LP); (▲), daikon adjusted moisture with wheat straw only; (●), 
daikon adjusted moisture with wheat straw and inoculated with LP. 
Figure 4-6. 
Changes in pH (a) and lactic acid concentrations (b) in daikon 
by-product silages during 6 days of aerobic exposure in Experiment 2. 
(◆), daikon + dried beet pulp silage (DBP); (■), daikon + wheat straw 
silage (DWS); (▲), daikon + dried bean stalks and husks silage 
(DBH); (●), daikon + wheat bran silage (DWB). 
Figure 5-1. 
Figure 7-1. 
Figure 7-2.     Figure 7-3.        
Changes in pH and lactic acid concentrations in apple pomace silages 
during 7 days of aerobic exposure. (◆), apple pomace without additive, 
(AP); (■), apple pomace + dried beet pulp, ABP; (▲), apple pomace + 
rice bran, ARB; (●), apple pomace + wheat bran, AWB.            91 
Relationships between moisture (a), NDF (b), PWRC (c), and WRC (d) of 
some HMBF and effluent output from their silages.               122  
Scheme of water retention capacity (WRC) according to Robertson et al., 
2000.                                                   125  
PWRC of some HMBF materials and silages with error bars (a) and 
regression equation showing the relationship between HMBF and their 
silages (b).                                               128 
List of Tables 
Table No.  Title                        Page No.
Table 1-1. Biological oxygen demand (B.O.D.) of some agricultural wastes 
and domestic sewage 
Table 2-1. Moisture content of selected HMBF from vegetable, fruit and 
agro-industrial sources 
Table 2-2. pH values of selected HMBF from vegetable, fruit and 
agro-industrial sources 
Table 2-3. Buffering capacity (BC) of selected HMBF from vegetable, fruit 
and agro-industrial sources 
Table 2-4. 
Lactic acid bacteria (LAB) population of selected HMBF from 
vegetable, fruit and agro-industrial sources 
Table 2-5. 
Yeast population of selected HMBF from vegetable, fruit and agro- 
industrial sources 
Table 2-6. 
Water soluble carbohydrates (WSC) of selected HMBF from 
vegetable, fruit and agro-industrial sources 
Table 2-7. 
Potential water retention capacity (PWRC) of selected HMBF from 
vegetable, fruit and agro-industrial sources 
Table 3-1. 
Chemical composition and microbiological characteristics of potato 
pulp  
Table 3-2. 
Pre-ensiling quality of potato pulp and fermentation quality in 
potato pulp silages without additive (PP), inoculated with 
Lactobacillus rhamnosus alone (PL), Rhizopus oryzae alone (PR) 
and a combination of the two inoculants (RL) after 50 days in two 
separate experiments  
Table 3-3. 
Chemical characteristics of potato pulp material, main effects and 
interaction between inoculants, storage temperatures and duration 
of storage in fermentation characteristics of potato pulp silage after 
40 days of ensilage 
Table 3-4. 
Contrasts between control and inoculated silages and effects 
between pre-silage material and silages with respect to storage 
temperatures and duration of storage in potato pulp fermentation 
characteristics 
Table 3-5. 
Chemical and silage characteristics of potato pulp after 30 days of 
ensiling  
Table 4-1. Chemical and microbiological characteristics of daikon  62 
Table 4-2. Chemical composition of daikon by-product material and silages 
after 30 days of storage in pipe silos  
Table 4-3. Total losses of dry matter, gross energy and nitrogen, effluent 
volume, weight losses as effluent and gas in daikon by-product 
after 30 days of ensilage  
Table 4-4. 
Pre-silage and silage chemical characteristics of daikon by-product 
ensiled with different absorbents  
Table 5-1. 
Table 5-2. 
Table 6-1. 
Table 6-2. 
Table 6-3. 
Table 6-4.  
Table 6-5. 
Table 7-1. 
Table 7-2. 
Table 7-3. 
Effect of absorbents and pressure levels on chemical characteristics 
of brewer’s grain after 30 days of ensilage 
Chemical characteristics of apple pomace before and after ensiling 
for 30 days 
Chemical compositions of potato pulp, hay, soybean meal (SBM) 
and potato pulp silages without additive (PP), inoculated with 
Lactobacillus rhamnosus alone (PL), Rhizopus oryzae alone (PR) 
and a combination of the two inoculants (RL)  
Chemical composition of diets used in the digestibility trial  
Dry matter intake, apparent nutrient digestibility and nutritive value 
of diets and potato pulp silages  
Chemical composition of alfalfa hay and daikon by-product silages 
used for feed palatability/preference test 
Feed intake and preference by sheep of daikon by-product silages 
alone, as indicated by intakes over three test periods and feeding 
positions 
Chemical parameters of some HMBF and effluent output from their 
silages 
Mean fermentation characteristics of the silages used in the study 
Water retention capacity (WRC) of some HMBF and dry 
absorbents 
List of Abbreviations 
ADF, acid detergent fiber 
B.O.D., biological oxygen demand 
BC, buffering capacity  
BW, body weight 
CFU, colony forming unit 
cm, centimeter 
CP, crude protein 
Cu, copper 
DCP, digestible crude protein  
DE, digestible energy 
DM, dry matter 
EE, ether extract 
Fe, iron  
FM, fresh matter 
g, gram 
GE, gross energy 
h, hour 
HCl, hydrochloric acid 
HClO4, perchloric acid 
HMBF, high moisture by-product 
feedstuff 
kg, kilogram 
L, liter  
LAB, lactic acid bacteria  
LDH, lactate dehydrogenase 
m, meter 
meq, milliequivalent  
Mg, magnesium  
mg, milligram 
MJ, mega joule  
ml, milliliter 
N, nitrogen 
Na, sodium 
NDF, neutral detergent fiber 
oC, degrees Celsius  
OM, organic matter 
PVC, poly vinyl chloride 
PWRC, potential water retention   
capacity 
SD, standard deviation 
Se, selenium 
TDN, total digestible nutrients 
TN, total nitrogen 
VBN, volatile basic nitrogen 
VFA, volatile fatty acid  
vs., versus 
WRC, water retention capacity 
WSC, water soluble carbohydrate 
wt, weight  
w:w, weight per weight 
Zn, zinc 
μ m, micrometer 
Chapter 1 
1. General Introduction 
  1.1. Importance of agricultural by-products  
   Agricultural by-product feedstuffs have gained global socio-economic importance in 
recent years due to their increasing relevance to livestock feeding systems in most 
developing countries. A by-product feedstuff by definition is ‘a secondary product 
obtained during harvesting or processing of a principal commodity and has value as an 
animal feed’ (Grasser et al., 1995) or ‘a product that has value as animal feed and is 
obtained during the harvesting or processing of a commodity in which human feed or 
fiber is derived’ (Fadel, 1999). These could be either from plant or animal origin. Crop 
residues derived from the production of maize, rice, wheat, barley, beet pulp, bagasse, 
etc.; cakes remaining after the extraction of oil from plant sources such as soybean, 
palm kernel, copra, corn gluten and groundnut; and agro-industrial sources such as 
potato pulp, brewer’s grain, apple pomace, etc. constitute about 65% of the one billion 
metric tons of world dry matter tonnage of by-product feedstuffs of crop origin (Fadel, 
1999). Future projections suggest that by-product feedstuff production will increase in 
developed countries and will remain the same or increase slightly in developing 
countries relative to population growth.  
   Although the use of cereal grains as supplements for livestock feed is a common 
practice in developed countries, this is hardly possible in developing countries, 
especially for the small holder producer, due to the use of such grains in human diets. 
Alternatively, there is growing emphasis on the recovery, recycling and upgrading of 
wastes in today’s society driven by rising costs and often decreasing availability of raw 
materials together with the growing concern about environmental pollution. This is 
particularly true for the food and food processing industry in which wastes, residues and 
by-products can be recovered and often recycled to higher value and useful products. 
However, transport costs and sales problems due to the low quality of these residues in 
developed countries have led to alternative utilization concepts such as their use as 
building materials or conversion concepts like composting and biogas production 
(Laufenberg et al., 2003). Nevertheless, there is patronage of low moisture crop residues 
in most developing countries largely in their use as supplementary feed for ruminants 
mainly in pasture-based livestock feeding systems, and the most extensively used in this 
respect is straw, attracting a lot of reviews and evaluations. On the contrary, high 
moisture agricultural by-product feedstuff mostly from fruit and vegetable sources like 
citrus pulp, pineapple waste and reject bananas, most of which cannot be quantified, are 
often available but underutilized in ruminant nutrition or scarcely fed in dried form.        
       
1.2. Definition of HMBF  
       
  The term high moisture by-product feedstuff (HMBF) is used in this dissertation to 
denote ‘any product or commodity that is obtained during the harvesting, production, or 
processing of feed or fiber containing a moisture content of more than 750 g/kg in fresh 
matter, and has value as an animal feed’. For the sake and scope of this investigation, 
this has been scaled down to include largely by-products from fruit and vegetable 
sources (either as by-product rejects or culls of no commercial value), canneries, or 
breweries. 
1.3. Relevance of HMBF in current production systems 
   Although there is no data in the literature that quantify the amount of HMBF 
produced on a global level, reports (Grasser et al., 1995; van Horn and Hall, 1997; Fadel, 
1999) have indicated that HMBF constitute a sizable portion of by-product feedstuffs 
produced worldwide. The obvious advantages of feeding livestock with by-products are 
the low cost, the reduction in reliance on grains that could be used as feed, the 
elimination of costly waste management programs (Grasser et al., 1995), and the high 
energy value of some by-products.  The utilization of HMBF in livestock feeding 
systems in particular is however, constrained and limited in scope due to their bulky 
nature, short shelf life, and most often distance between production centers and 
livestock farms. Moreover, Laufenberg et al. (2003), in enumerating the current systems 
in place for the utilization of by-products from vegetable sources (those containing high 
moisture, emphasis mine) and the various concepts in use indicated that possibilities for 
the conversion and utilization or recycling of most of these wastes as animal feed are 
few. Since global production of by-products is, and would continue to preponderate in 
the near future (Fadel, 1999), the importance of animals as consumers of these 
by-products cannot be over-emphasized.  
1.4. Preservation techniques for HMBF   
  Techniques for the conservation of feed are numerous and include, among others, 
drying, salting, sweetening, freezing, radiation, or changing of the pH (as in pickling or 
ensiling). However, preservation of fresh feed in particular involves the application of 
two basic measures: the prevention of enzyme activity in the product, and the protection 
of the product from external deterioration factors such as bacteria, molds, yeasts, insects, 
rodents, etc. (Ashbell, 2003). 
   Conservation by ensilage has been practiced for many centuries. Ensilage is defined 
as ‘the material produced by the controlled fermentation of a crop of high moisture’ or 
‘the preservation of a crop by natural fermentation under anaerobic conditions’ 
(McDonald et al., 1991a). This definition, although restricting the material to ‘crop’, 
could be employed to encompass wet by-products such as HMBF. The advantages of 
preservation or conservation by ensilage are numerous and well established, and it is not 
the intention of this thesis to review them again. However, what is new and, in my 
opinion, an emerging necessity is the technology to preserve through ensiling, of 
agricultural by-products that are rich sources of nutrients and potential feed for livestock, 
yet contain very high moisture contents. Although developments in wet by-product 
preservation by ensilage have been reported (Ashbell and Donahaye, 1984; Ashbell et 
al., 1987; Weinberg et al., 1988; Bampidis and Robinson, 2006), literature on this aspect 
of study is thin. 
1.5. Environmental concerns on disposal of HMBF 
   The disposal of by-products, especially those containing high moisture including 
HMBF, if not done properly, imposes a huge burden on the environment; if ensiling of 
HMBF is not conducted properly, this may add to the burden through effluent 
production. Effluent is the single most important factor affecting ensilage of wet crops, 
not only due to losses encountered in the silo (the fermentation vessel), but also due to 
the considerable loss of nutrients in effluent and drainage through silage, with the 
attendant environmental consequences. Silage effluent is considered one of the most 
common agricultural pollutants of water courses (Woolford, 1978). In a comparative 
study of various farm wastes with that of domestic sewage (Spillane and O’Shea, 1973), 
it was concluded that the biological oxygen demand (B.O.D.) or the water-polluting 
potential of organic matter of silage effluent far exceeded that of other pollutants as 
given in the table below. 
Table 1-1. Biological oxygen demand (B.O.D.) of some agricultural wastes and 
domestic sewage (adapted from Spillane and O’Shea, 1973) 
B.O.D. (mg O2/L) 
Silage effluent 
90,000 
Pig slurry 35,000 
Cow urine 19,000 
Cow slurry 5,000 
Domestic sewage 500 
The obvious challenges militating against the interest in research into ensiling of 
particularly wet materials are: 
i. the risk of high effluent production with attendant environmental consequences 
ii. loss of nutrients through ensiling  
iii. difficulties in producing good quality silages from the wet materials 
iv. the aerobic instability or low shelf life of the silages 
1.6. Objectives of the study 
   Methodologies involved in addressing problems related to preservation by ensilage 
of wet materials are varied but often subjective and product specific, or largely refer to 
grasses and crops, and thus highlight the need for strategies in addressing problems 
specific to HMBF.  
The broad objectives of this study were therefore: 
a. to investigate the ensiling potential of some selected HMBF 
b. to improve preservation and extend shelf life of HMBF 
c. to control effluent and nutrient losses during the course of ensiling HMBF 
d. to evaluate the nutritive value of HMBF 
e. to identify the main factors affecting the ensiling of HMBF, to determine their 
relationships, and to establish a method suitable for ensiling HMBF 
To realize these objectives, four major experiments were conducted; the first involved a 
study of pre-ensiling characteristics of a selection of HMBF, followed by three others 
involving the ensiling of  representatives of HMBF (potato pulp, daikon or oriental 
radish by-product, brewer’s grain and apple pomace), determination of the aerobic 
stability, and nutritive value in vivo using sheep.  
Important factors affecting the ensiling of HMBF and their relationships are discussed. 
           
Chapter 2 
Pre-ensiling characteristics of a representative of HMBF 
2.1. Introduction  
   To fully comprehend the suitability for ensilage of any crop, and for that matter, 
HMBF, it is imperative that a study of the pre-ensiling characteristics of the material is 
conducted to provide an insight into the potential for ensilage. This would provide a 
basic and a preliminary assessment of its suitability for the ensiling process and an 
important point of reference. Of critical importance are the chemical and 
microbiological characteristics of the material, factors that intrinsically dictate the 
outcome of the ensiling process. 
  Considerable attempts have been made to score some vegetable wastes and fruits 
according to their nutritional potential (Teli et al., 1983; Gupta et al., 1993; Haque et al., 
1997) and some by-products according to their ensiling characteristics (Ishigoro, 1994). 
However, no comprehensive approach has been made to study the specific ensiling 
characteristics of HMBF. 
  The objective of this study was to investigate the ensiling potential of a representative 
of HMBF from vegetable and fruit sources through chemical and microbiological 
characterization as a prelude to their use as silage materials. 
2.2. Materials and methods 
2.2.1. Pre-ensiling characteristics   
  The HMBF investigated were those from vegetable sources (cabbage, cucumber, 
carrot pulp, and daikon [Oriental radish] Raphanus sativus L.), fruit sources (banana 
peel, apple pomace, orange peel, and pineapple skin) and one from an agro-industrial 
source (potato pulp).  
   A total of at least nine samples with three replications for each of the 
above-mentioned commodities except potato pulp were purchased from local 
supermarkets in Hokkaido, northern Japan, at different times during the harvesting 
season in 2004 and taken to the laboratory for the analyses of the ensiling characteristics. 
To ensure variability, each type of commodity was selected based on area of production 
in Japan.  Vegetables were cut into pieces to about 2 cm long and 2 cm thick with a 
kitchen knife, while in the case of fruits, their juices were extracted to provide a 
semblance of their by-product similar to that in the cannery or extraction factories. 
Potato pulp, a by-product from the extraction of starch from raw potatoes, was obtained 
from three different factories in Hokkaido, northern Japan at different times during the 
production season in 2004.  These were mixed, and representative samples were taken 
for dry matter determination. Part of the mixture was blended with an electric blender 
and sampled for the following determinations:  70 g was macerated with 140 ml of 
de-ionized water for measuring pH, 10 g for determination of the buffering capacity, 
and 10 g for the determination of microflora (lactic acid bacteria, LAB; yeast numbers). 
Water retention capacity (WRC) and water-soluble carbohydrates (WSC) were 
determined from dried samples. 
2.2.2. Chemical analyses 
   The dry matter content of the fresh materials was determined by freeze-drying for at 
least 24 h.  The pH of aqueous extracts of the material was measured using a pH meter 
(model HM-30G, TOA Electronics Ltd, Tokyo, Japan). WSC were determined by the 
anthrone method as described by Morimoto (1971). WRC, employed as a novelty to 
determine the moisture absorptive capacity of silage / absorbent material, was 
determined according to the method of Robertson et al. (2000), with slight 
modifications. One gram of air-dry weight of material was put into a 50 ml centrifuge 
tube and hydrated with 30 ml distilled water containing 0.02 g sodium azide per 100 ml 
as a bacteriostat. Samples were equilibrated for 18 h at room temperature, transferred to 
a glass filter with a pore size of 100-160 μm, (1G P160, Sibata Company, Tokyo, Japan) 
and drained under a pressure of 2 g/cm2 with a pressure pump (Compact air pump, 
NUP-1, AS-ONE Company, Tokyo, Japan). The glass filter was weighed, oven-dried at 
135oC for two hours, and weighed again. The WRC of the material was calculated as the 
amount of water retained by the pellet (g/g dry weight) after transfer to the glass filter. 
The potential WRC (PWRC), a novel terminology used in this study, is defined as ‘the 
amount of moisture in grams that a HMBF can retain per 100 grams of its own fresh 
matter weight’ and is calculated as WRC x DM of the material and given in g/100g FM. 
Buffering capacity (BC) was determined according to the method of Playne and 
McDonald (1966). This included the maceration of 10 g fresh weight of sample with 
250 ml of distilled water, titration of the macerate with 0.1 N hydrochloric acid to pH 3, 
then titration to pH 6 with 0.1 N sodium hydroxide. The BC was expressed as 
milliequivalent of alkali required to change the pH from 4 to 6 per kg of dry matter, 
after correction for the titration value of 250 ml water blank. Microbial analyses (LAB 
and yeast counts) were carried out on representative samples of the materials as follows: 
10 grams of sample were weighed into a sterile zippered bag, 90 ml of sterile water was 
added and homogenized by shaking for 3 min. The sample was serially diluted, and 1 
ml and 0.2 ml of dilutions were taken for LAB and yeast determinations, respectively. 
Lactic acid bacteria were enumerated on pour plate Difco YMS agar (Difco 
Laboratories, Becton Dickson and Company, Sparks, Maryland, USA) supplemented 
with 1 mg/ml of both cycloheximide and sodium azide, respectively, to inhibit yeast 
growth. The plates were incubated at 32oC for 72 h and LAB colonies counted directly 
on the plates. Yeasts were enumerated on spread-plate malt extract agar (Difco 
Laboratories, Becton, Dickson and Company, Sparks, USA) and incubated at 28oC for 4 
days, and colonies were counted directly on the agar plates.  
2.2.3. Statistical analyses 
  Means from the data of each material and the standard deviations were calculated 
using Excelstats for Windows 2004 (Microsoft Corporation, USA).  
2.3. Results  
   The chemical and microbiological (LAB and yeast populations) compositions 
relevant to ensilage are given in Tables 2-1 to 2-8. 
   The selected products had high moisture contents, with those from vegetable 
sources a level over those from fruit sources (Table 2-1). Potato pulp was lowest in this 
respect with a high standard deviation, an indication of the variability of the sources 
from where the materials were procured. 
Table 2-1. Moisture content of selected HMBF from vegetable, fruit and agro-industrial 
sources 
                           
Values of pH of the selected products were variable, with those from vegetable sources 
higher in comparison with those from fruit sources (Table 2-2).  
     The BC, or the ability of a material to resist changes in pH, was higher in the 
vegetable sources (Table 2-3). Cabbage and daikon were particularly high, with the 
latter having a high standard deviation, indicative of the variation in the samples 
  Moisture (g/kg) SD 
Daikon 951 10.7 
Cabbage  933 7.5 
Cucumber 967 2.0 
Carrot pulp 912 3.6 
Banana peel 908 9.1 
Apple pomace 845 71.6 
Orange peel 811 5.1 
Pineapple skin 806 50.7 
Potato pulp 789 57.2 
g/kg, gram per kilogram; SD, standard deviation. 
analyzed. 
Table 2-2.  pH values of selected HMBF from vegetable, fruit and agro-industrial 
sources 
                               
This result pre-supposed difficulty in the ensilage of daikon from a theoretical point of 
view. The opposite is true for potato pulp, which was conspicuously low in this 
characteristic.  
  pH SD 
Daikon 5.57 0.97 
Cabbage  6.18 0.25 
Cucumber 6.23 0.17 
Carrot pulp 5.84 0.04 
Banana peel 5.52 0.12 
Apple pomace 4.26 0.25 
Orange peel 4.41 0.03 
Pineapple skin 3.75 0.09 
Potato pulp 5.31 0.37 
Table 2-3.  Buffering capacity (BC) of selected HMBF from vegetable, fruit and agro- 
industrial sources 
                                        
     LAB and yeast populations in the materials investigated were particularly low, with 
no clear consistency between vegetable and fruit materials. Potato pulp was highest and 
lowest in LAB and yeast, respectively (Tables 2, 4-5). 
  BC ( meq/kg DM)§    SD 
Daikon 703.4 255.6 
Cabbage  433.3 190.3 
Cucumber 1276.3 135.0 
Carrot pulp 562.3 94.7 
Banana peel 618.2 73.9 
Apple pomace 114.8 34.4 
Orange peel 199.3 57.6 
Pineapple skin 364.4 204.1 
Potato pulp 21.5 9.1 
§ meq/kg DM, milliequivalent per kilogram dry matter. 
Table 2-4.  Lactic acid bacteria (LAB) population of selected HMBF from vegetable,   
      fruit and agro-industrial sources 
                                                   
  LAB (log 10 CFU/g FM)      SD 
Daikon 4.82 2.09 
Cabbage  3.24 1.03 
Cucumber 4.91 1.42 
Carrot pulp 4.56 0.49 
Banana peel 5.31 0.60 
Apple pomace 3.40 0.37 
Orange peel 2.59 0.16 
Pineapple skin 5.32 0.08 
Potato pulp 6.4§ 1.1 
CFU/g FM, colony forming units per gram fresh matter. 
§ Based on Saito et al., 2006. 
Table 2-5.  Yeast population of selected HMBF from vegetable, fruit and  agro-industrial sources                                                              
  All materials except potato pulp were rich in WSC (Table 2-6). This indicated that 
sugar was the main available substrate and would be sufficient to maintain and direct 
the pattern of lactic acid fermentation during silage.  
  Yeast (log 10 CFU/g FM)      SD 
Daikon 5.45 1.00 
Cabbage  4.42 0.52 
Cucumber 5.30 0.85 
Carrot pulp 5.10 0.61 
Banana peel 4.57 0.54 
Apple pomace 3.21 0.71 
Orange peel 2.58 0.70 
Pineapple skin 4.57 0.31 
Potato pulp 2.4§ 1.2 
CFU/g FM, colony forming units per gram fresh matter. 
§ Based on Saito et al., 2006. 
Table 2-6.  Water soluble carbohydrates (WSC) of selected HMBF from vegetable, 
fruit and agro-industrial sources  
                                     
   The PWRC of the materials was low in cucumber and daikon (the lowest in 
moisture contents) but high in orange peel and potato pulp (Table 2-7). There was no 
clear consistency among other materials in this respect. 
           
  WSC (g/kg DM) SD 
Daikon 382 90.4 
Cabbage  377 20.2 
Cucumber 198 30.9 
Carrot pulp 547 110.3 
Banana peel 278 45.3 
Apple pomace 445 71.0 
Orange peel 417 57.1 
Pineapple skin 322 36.6 
Potato pulp 64 16.8 
Table 2-7.  Potential water retention capacity (PWRC) of selected HMBF from vegetable, fruit and agro-industrial sources                                                                                    2.4. Discussion          
   The chemical and microbiological characteristics relevant to ensilage of the 
materials studied showed some variations in terms of availability or not, versus the 
suitability to the ensiling process. The moisture content of the materials from vegetable 
sources were higher due to the mode of determination, ( i.e., without juice extraction) 
and  this provided a realistic approach of their state prior to  use as silage materials, in 
  PWRC (g/100g FM)   SD 
Daikon 16.9 1.24 
Cabbage  44.2 0.47 
Cucumber 16.0 0.80 
Carrot pulp 55.8 0.94 
Banana peel 39.9 0.43 
Apple pomace 40.5 1.46 
Orange peel 83.0 1.02 
Pineapple skin 57.4 0.28 
Potato pulp 72.8 0.75 
contrast with those from fruit and agro-industrial sources which principally undergo 
some sort of an extraction process.  Potato pulp was highly variable in terms of its 
moisture content, and this was partly dependent on the mode of starch extraction and the 
product line of the extraction factory. On use as silage materials, highly absorbent 
materials need to be employed to soak or retain excess moisture to forestall high silage 
effluent production. The pH, BC, and, to a large extent, the WSC of materials from the 
vegetable sources were higher than those from fruit sources, providing a rather 
unpredictable and contrasting indication of their role in an ensiling process. In a typical 
silage fermentation process, a quick reduction of pH during the initial stages of the 
fermentation process is necessary to neutralize and prevent the proliferation of 
undesirable microorganisms with a gradual increase in lactic acid production from the 
available substrate (WSC) which is mainly sugar. Materials with high buffering 
capacities may delay the reduction in pH even in the presence of sufficient WSC. 
Addition of an inoculum of LAB, prior to ensiling of material from vegetable sources 
may be a viable proposition. On the contrary, fruit by-products and potato pulp, from a  
theoretical point of view, provided strong indications of being ideal silage materials 
judging from their chemical analysis, as evidenced by their low pH and buffering 
capacities and relatively high WSC contents (except potato pulp). Usually a material 
containing 150 g/kg in dry matter available WSC would be enough for a good 
fermentation process. Potato pulp would need an ‘alternate’ source of substrate to 
sustain effective silage fermentation. The LAB populations of the materials investigated 
(excluding potato pulp) were generally low and did not exceed the minimum threshold 
of 106 organisms/g dry matter required to quick-start a highly effective lactic acid 
fermentation process. However, low initial population of LAB may not be a critical 
criterion for the success of the fermentation process. What is crucial is the efficiency of 
the indigenous LAB to initiate a rapid fermentation and sustain a rapid fall in pH 
(Woolford, 1984a). Yeast load was quite low in the investigated materials, especially in 
potato pulp. This augurs well for good silage fermentation. Again, what is critical is the 
yeast population at the end of the ensiling process (i.e., after opening of the silage). The 
rule is that if the number of yeasts exceeds the level of 105   organisms/g dry matter, 
rapid fungal deterioration would occur after opening of the silage (Jonsson and Pahlow, 
1984). From a microbiological point of view, potato pulp may be an ideal silage 
material. 
   The potential water retention capacity (PWRC), a terminology employed as a 
novelty in this study is, to a large extent, a function of moisture content (See Chapter 7). 
An important feature in the ensiling of particularly wet crops is the ability of the 
material to retain its moisture during the course of ensilage. Most wet crop silage 
materials are unable to retain moisture when ensiled, and consequently the system of 
drying prior to ensiling has been widespread. The PWRC describes the capacity of a 
material to retain or release its moisture under different silo conditions during the course 
of ensilage. This is an important factor vis-à-vis effluent production. The higher the 
PWRC, the stronger the ability of the ensiled material to retain effluent, and vice versa.  
   Judging by the PWRC of the materials investigated and barring any external factors, 
orange peel and potato pulp have the potential to produce less effluent than the other 
materials investigated during ensilage. The validity of this assertion is, however, subject 
to ipso facto ensiling of the two materials.  
2.5. Summary 
   High moisture by-product feedstuffs (HMBF) contain ensilable characteristics such 
as water soluble carbohydrates sufficient for lactic acid fermentation. Vegetable 
by-products are high in buffering capacity and pH compared with those from fruit 
sources. Due to the high moisture contents, means of controlling effluent production 
during their use as silage materials are imperative. 
                                       
Chapter 3  Potato pulp    
3.1. Introduction    
          Potatoes are the most important agricultural product in Japan in terms of annual 
production, after rice and sugar beets. Each year, over two and a half million metric tons 
of potatoes are harvested (Statistical Handbook of Japan 2006), leading to a 
simultaneous production of potato pulp, a by-product of the potato starch industry. The 
pulp, a remnant of the cell wall of potato tuber and fruit liquid, contains other 
components such as starch, cellulose, hemicelluloses, pectin, proteins, and mineral salts 
(Mayer and Hillebrandt, 1997). Even after separation of the fruit liquid from the 
particulate fraction (pulp), the pulp contains up to 90% water. Thus it is a bulky 
by-product of considerable environmental concern. Several strategies for the reduction 
of the huge amount of this by-product are available including its use as fertilizer, 
substrate for cultivation of fungi, syrup for use in the cosmetic industry (Mayer and 
Hillebrandt, 1997), or as ruminant feed (Hanada et al., 2004; Pen et al., 2005; Okine et 
al., 2005). Still, several tons of potato pulps are returned to the soil with adverse effects 
not only to the topsoil but also to the environment, due to the high content of mineral 
salts. Ensiling potato pulp would be a more economical alternative to drying for 
preservation and consequent use as livestock feed especially for ruminants.  
   In the experiments to be described, a series of strategies involving the use of 
bacterial and fungal inoculants were employed to preserve fresh potato pulp by ensilage. 
Following that description is a discussion of the role of these inoculants in the 
enhancement of the preservation quality of potato pulp silage.  
3.2. Ensiling potato pulp with or without microbial inoculants            
  The use of microbial inoculants to improve the fermentation quality of crops during 
ensilage has undergone a tremendous evolution in recent times.  Several commercial 
products aimed at increasing lactic acid production in crop silages are on the market 
today, including Lactobacillus rhamnosus, which is widely used in Japan. The use of 
fungi, an aerobic inoculant in an anaerobic process such as ensiling was, until recently 
(Oda et al., 2002; Okine et al., 2005; Okine et al., 2007), a novelty.  
   Studies have been conducted on the use of the fungus Rhizopus oryzae in lactic acid 
production (Skory, 2000) and in the pharmaceutical industry for its therapeutic qualities 
(Peimin et al., 1997). Although the use of starch, which is a major chemical component 
in potato pulp, as a substrate for the production of lactic acid is considered to be rare 
(Woolford, 1984c), it has been shown that Rhizopus oryzae can produce lactic acid 
using cornstarch as a substrate (Peimin et al., 1997), and that production of lactic acid 
by Rhizopus cultures is often preferred to bacterial fermentations because of the ease 
and ability of the fungus to utilize both complex carbohydrates and sugars (Skory, 2000). 
This is due to the fact that it contains the enzymes pectinase and lactate dehydrogenase 
(LDH), which can hydrolyze carbohydrates into glucose resulting in the production of 
lactic acid (Skory, 2000; Erdogan and Mahinur, 2000). However, information on the use 
of Rhizopus oryzae as a fermentation stimulant in potato pulp silage is scanty. Oda et al. 
(2002) isolated the Rhizopus oryzae strain IFO 4707 as the best among thirty-eight 
others for its ability in rapid reduction in hardiness and pH of potato pulp, followed by 
gradual synthesis of lactic acid. There are no direct indications that Rhizopus oryzae 
organisms are hazardous (Coenen et al., 1997) and their use as inoculants could enhance 
the fermentation quality of potato pulp silage (PPS). 
   The objective of this study was to investigate the effect of the two inoculants, 
Lactobacillus rhamnosus and Rhizopus oryzae, separately and in combined application 
on changes in fermentation quality and nutrient composition with duration of storage of 
potato pulp silage. 
3.2.1. Materials and methods 
3.2.1.1. Silage preparation 
  The study involved two experiments: In Experiment 1, fresh potato pulp was 
inoculated with Lactobacillus rhamnosus (L) and Rhizopus oryzae IFO 4707 (R). The 
two inoculants, both freeze-dried cultures, were added to potato pulp in the ratio of 0.5 
and 1.0 g/kg in fresh matter, respectively, enough to produce at least 1x106 colony 
forming units (CFU) g –1 as per manufacturer’s statement. A combined application of the 
two inoculants (R + L, included at 0.5 and 1.0 g/kg in fresh matter, respectively) was 
also prepared and set against potato pulp without additive (control). Four treatments viz; 
control (PP), L alone treated silage (PL), R alone treated silage (PR) and R+L treated 
silage (RL) were thus prepared. These were ensiled in 400 ml laboratory glass bottles 
with 12 bottles for each treatment. The average fresh weight of potato pulp in each 
bottle was 450 g, thus the average packing density obtained was 1,125 kg FM m-3. 
These were pressed to ensure compaction, closed with cap and sealed with vinyl tape to 
avoid entry of air then kept in a room (ambient temperature 15 ± 5oC).  Potato pulp 
material (day 0) was sampled for subsequent analysis and three bottles per treatment 
were opened on the 2nd , 8th , 32nd  and 50th  day post-ensiling to investigate the 
changes in the fermentation characteristics and starch, sugar and pectin content of the 
resultant silages. 
  In Experiment 2, four treatments (PP, PL, PR and RL), containing the same 
inoculants and ratios of preparations as in Experiment 1, were ensiled in polyethylene 
bags. Four hundred kilograms fresh weight of potato pulp was prepared for each 
treatment. For the sake of packing convenience, each treatment was separated into four 
polyethylene bags, 100 kg apiece. Each polyethylene bag was inserted into a plastic 
container (120 L capacity) prior to filling. After each bag was filled, air was evacuated 
the container and carefully sealed. The packing density was about 30% less than that of 
Experiment 1. These were kept in a large open container with an ambient temperature of 
12 ± 5oC. The silos were opened 50 days after filling and sampled for chemical 
analyses.  
3.2.1.2 Chemical analyses 
  The dry matter (DM) content of the potato pulp materials and silages was determined 
as described in Chapter 1. Organic matter (OM) content was obtained by difference 
following ashing at 550oC for 3 h. The crude protein (CP) and ether extract (EE) were 
determined using a Kjeldahl and a Soxhlet apparatus, respectively (AOAC, 1991). 
Neutral detergent fiber (NDF) assayed with a heat stable amylase and expressed 
inclusive of residual ash and acid detergent fiber (ADF) expressed inclusive of residual 
ash and without the use of sodium sulfite, were determined according to Van Soest et al. 
(1991). Sugar (net sugar, principally monosaccharides) was determined from dried 
samples by extraction with 800ml/L ethanol at a constant temperature of 80oC and acid 
hydrolysis with anthrone solution (containing anthrone and thiourea in a solution of 
concentrated sulfuric acid).  Hemicellulose was calculated as the difference between 
NDF and ADF. Starch was determined from the dried residue following sugar 
extraction with 600ml/L perchloric acid (HClO4) and continuous boiling for 2 h. The 
starch concentration was then determined by its color reaction with glucose at room 
temperature using a test kit (Glucose B Test Wako, Wako Pure Chemical Industries Ltd, 
Tokyo, Japan). The sugar and starch concentrations were measured using a 
spectrophotometer with a standard solution from the test kit at peak transmissions of 
650 mμ and 420 mμ, respectively. These methods are fully described by Abe (1988). 
The pH of aqueous extracts of pre-ensiling material and silages were measured using a 
pH meter (model HM-30G, TOA Electronics Ltd, Tokyo, Japan). Lactic acid was 
determined from aqueous extracts of materials and silages by the colorimetric method of 
Baker and Summerson (1961), in which lactic acid in the extracts was converted into 
acetaldehyde by treatment with concentrated sulfuric acid, and then determined by its 
color reaction with p-hydroxybiphenyl. The color was read in a spectrophotometer at a 
peak transmission of 560 mμ. Pectin was determined by the HCl extraction method of 
Phatak et al. (1988). The method included removal of protein from a defatted dried 
material using protease and dispersion in a solution of sodium phosphate buffer at pH 
7.5 and incubation overnight. This was followed by filtration to isolate the residue and 
subsequent dispersion in water adjusted to a low pH (1.5) with HCl and continuous 
shaking at 80oC for 4 h. This was followed by filtration and precipitation of the filtrate 
with 4 volumes of 950ml/L ethanol, centrifugation, and lyophilization to isolate pectin. 
The concentrations of acetic acid, a volatile fatty acid (VFA), were analyzed by 
gas-liquid chromatography (Shimadzu GC-14 A, Kyoto, Japan) equipped with a 
flame-ionization detector and a capillary column (ULBON HR-52, 0.53mm i.d. x 30m x 
3.0 μm) by using 2-ethyl-n-butyric acid as the internal standard. The gross energy 
content of potato pulp, after drying at 60oC in a forced-draught oven for 48 h, was 
determined by an adiabatic bomb calorimeter (CA-4P, Shimadzu, Tokyo, Japan).  
3.2.1.3. Statistical analyses 
   Silage fermentation data were analyzed using ANOVA in a randomized block design, 
using the General Linear Model Procedure of SAS (1996) computer package. Contrasts 
between the pre-ensiling quality and treated silages were performed, and means 
differences were compared for all variables and P<0.05 considered as statistically 
significant. 
   3.2.2. Results   
3.2.2.1. Chemical characteristics of potato pulp 
  The main chemical characteristics of potato pulp are presented in Table 3-1. A 
discussion on some of these characteristics has been made previously in Chapter 2. An 
attempt is however made here to highlight in much broader terms the composition of the 
by-product.  
Table 3-1. Chemical composition and microbiological characteristics of potato pulp  
(Figures are means of at least three separate determinations and are given in g/kg dry 
matter, unless otherwise stated) 
 Range  Mean  SD 
Moisture  730-845  789  57.2 
Organic matter 974-983  980  4.9 
pH 4.89-5.59  5.31  0.4 
Buffering capacity (meq/kg DM) 13.6-23.1  21.54  9.1 
Lactic acid (Log10 CFU g/FM)§ 4.6-8.4  6.4  1.1 
Yeast (Log10 CFU g/FM) § 1.6-6.3  2.4  1.2 
Sugar 5.0-23.2  11.4  10.2 
Crude protein 47.0-49.2  48.4  1.2 
Neutral detergent fiber 348-353  350  2.5 
Acid detergent fiber 336-348  336  6.0 
Hemicellulose 11.0-18.0  14.1  3.5 
Pectin 207-181  177  5.5 
PWRC (g/100g) 70.1-73.6  72.8  0.75 
§Based on Saito et al, 2006. 
3.2.2.2. Silage fermentation quality 
  The fermentation qualities of potato pulp in both experiments after 50 days of 
ensilage are presented in Table 3-2. There were no significant treatment effects on the 
moisture content of the silages. In Experiment 1, the pH of PPS decreased sharply with 
or without the inoculants in the first few days of ensilage, but declined gradually after a 
week and continued to decrease until the 50th day of ensilage (Fig. 3-1a). Inoculation 
with R and L significantly (P<0.05) decreased the pH of PPS compared with the control. 
Lactic acid production increased in all treatments with days in ensilage (Fig. 3-1b), but 
was lower (P<0.05) with inoculation with R. Lactic acid production was higher 
(P<0.05) in PL than in PR after 50 days of ensilage, but not higher than the untreated 
silage. However, the combined application of the two inoculants did not have any 
significant effect on lactic acid production over the control. The acetic acid 
concentration, the only volatile fatty acid detected in PPS, increased with days in 
ensilage (Fig. 3-1c) and was higher (P<0.05) in PR and RL compared to the control. In 
Experiment 2, the pH decrease after 50 days of ensiling was similar to that of 
Experiment 1, although the values were slightly higher in Experiment 2. Lactic acid 
increased in all the silages to about six folds that of pre-ensiling content, but effect 
among treatments was not significant (P>0.05). The acetic acid concentration increased 
(P<0.05) in both PR and RL, but did not differ in PL, compared to the control, 
confirming the result in Experiment 1. The pH of pre-silage material in both 
experiments decreased (P<0.001) while lactic and acetic acid concentrations increased 
(P<0.001) in comparison with the silages. 
Table 3-2. Pre-ensiling quality of potato pulp and fermentation quality in potato pulp silages without additive (PP), inoculated with Lactobacillus rhamnosus 
alone (PL), Rhizopus oryzae alone (PR) and a combination of the two inoculants (RL) after 50 days in two separate experiments (1 and 2). Values are 
given in g/kg dry matter, unless otherwise stated 
  Silages    SignificanceContrast§ 
Fermentation quality Potato pulp PP PL PR RL SEM  level 1 2 3 4 
Experiment 1             
    Moisture(g/kg FM†) 830 839 835 835 831 3.84  NS *** NS NSNS 
    pH 5.51 3.36 a 3.32 b 3.21 c 3.21 c 0.004  *** *** NS * * 
    Lactic acid  3.0 63.2 ab 67.3a 55.31 c 61.9 b 1.03  ** *** ** ***** 
    Acetic acid 0.0 6.5 b 3.8 b 11.9 a 11.8 a 1.42  * *** NS *** 
   Sugar 4.9 14.5 10.1 13.2 28.3 0.03  *** *** *** ****** 
   Starch 177 198 119 100 120 0.29  *** *** *** ****** 
    Pectin 213 203 192 197 133 3.26  *** *** ** **NS 
Experiment 2             
    Moisture(g/kg FM†) 830 842 839 840 840 1.75  NS *** NS NSNS 
    pH 5.51 3.57 a 3.55 ab3.46 bc 3.43 c 0.034  * *** NS * * 
    Lactic acid 5.0 28.5 ab 32.7 ab 26.7 b 35.8 a 2.07  NS *** NS NSNS 
    Acetic acid  0.8 7.0 b 8.1 bc 10.7 ab 12.9 a 1.09  * *** NS *** 
    Sugar 5.0 15.8 20.2 13.2 18.9 1.19  *** *** NS **NS 
    Starch  206 195 196 208 214 6.45  NS NS NS NSNS 
   Pectin 213 213 215 207 199 8.26  NS NS NS NSNS 
Except potato pulp, means with different letters in a row differ significantly (P <0.05); NS, not significant. 
†Fresh matter;* P <0.05; ** P<0.01; *** P<0.001. 
§1= Potato pulp vs. silages; 2 = PP vs. PL; 3 = PP vs. PR; 4 = PL vs. PR. 
Fig 3-1.                                        
                                             (c)     
Fig. 3-1. a-c. Change in pH, lactic and acetic acid concentrations of potato pulp during 50 days of 
ensilage (Experiment 1). ( ◆) PP, potato pulp silage without additive; (■) PL, lactobacillus alone 
inoculated potato pulp silage; (▲) PR, rhizopus alone inoculated potato pulp silage; (●) RL, rhizopus 
+ lactobacillus inoculated potato pulp silage. 
Fig. 3-2.                                                                    
                      Fig. 3-2 a-c. Change in sugar, starch and pectin during 50 days of (Experiment 1). ( ◆) PP = potato 
pulp silage without additive; (■) PL, lactobacillus alone inoculated potato pulp silage; (▲) PR,  
rhizopus alone inoculated potato pulp silage; (●) RL, rhizopus + lactobacillus inoculated potato pulp 
silage. 
3.2.2.3. Change in nutrient composition 
   The change in the predominant carbohydrate fraction of potato pulp, starch, sugar and 
pectin in Experiment 1 is shown in Fig. 3-2 (a-c).  Pectin, and to a large extent starch, 
decreased, while sugar increased, with length of ensilage. The initial sugar content in 
potato pulp (about 5 g/kgDM) increased to about four-fold in the control, PL and PR but 
five-fold in RL with significant difference (P<0.05) among the treatments after 50 days of 
ensilage (Table 3-2). Starch content showed a level of inconsistency with days in ensilage, 
and PP was the least degraded at the end of the ensiling period. Pectin content decreased  
consistently in RL (Fig. 3-2c) to about a half of its original content after 50 days of 
ensilage, with significant differences (P<0.05) among all treatments compared to the 
control (Table 3-2). In Experiment 2, however, there were no treatment effects on the 
predominant carbohydrate fraction of potato pulp silage (Table 3-2). 
3.2.3. Discussion  
  The pH of all silages, irrespective of the type of inoculants added or silo used, was 
below 3.60, indicating a good fermentation process.  However, R when used alone or in 
combination with L, significantly (P<0.05) decreased the pH of PPS in both 
investigations.  The decrease with inoculation with R is in consent with the findings of 
Oda et al. (2002), who reported a rapid decrease in pH within one day of inoculation with 
Rhizopus oryzae in potato pulp. The defect in lactic acid production with R (in 
Experiment 1) may be based on two hypotheses: The first is probably due to the 
malfunction of LDH, the active enzyme for conversion of sugars to lactic acid in R in 
agreement with Skory (2000), who stated that the enzyme requires high concentrations of 
sugars for conversion into lactic acid. The low sugar content in potato pulp may have 
therefore hindered the activity of the enzyme. Secondly, since most fungi thrive well 
under aerobic conditions, the activity of R in the initial aerobic phase may have played a 
role in the early phase of ensiling, although Oda et al. (2002), observed lactic production 
with Rhizopus oryzae under airtight conditions in potato pulp. Their supposition that the 
narrow spaces present in potato pulp could permit aerobic fungal growth and 
consequently boost the activity of the fungus, however, runs contrary to the principles of 
ensilage that require anaerobic conditions in the silo. 
  Further, the ambient temperature during the ensiling period could have contributed in 
part.  Hachmeister and Fung (1993), in their review of the Rhizopus strain (among them 
Rhizopus oryzae) used for the fermentation of tempeh (a traditional food in Indonesia), 
stated that temperature, relative humidity and length of fermentation are three crucial 
factors that dictate the outcome of tempeh fermentation and that optimum temperature 
requirements are in the range of 25 to 37oC.  Since there is no available literature on the 
use of R fermentation in PPS, it is our subjective conclusion that the low temperature 
range in the current experiments may have restricted effective fermentation and for that 
matter, lactic acid production in PPS.  The higher fermentation quality in Experiment 1 
compared to Experiment 2, as evidenced by lower pH values and higher lactic acid 
concentrations does reflect the ideal conditions in the former than the latter and could be 
attributed to the differences in the densities of the silages, the type of silos used, and 
differences in storage temperature, all of which may have affected the rate of 
fermentation. On visual appraisal, the quality of silages appeared good, and no 
differences in smell were observed.  
   The significant increase in lactic acid production in PL over PR in Experiment 1 
(though not in Experiment 2 due to differences in ensiling conditions, as earlier 
suggested) does seem to confirm the efficiency of L, a lactobacillus strain over R, in 
agreement with Hang (1989), who, in a study with corn, reported that the production 
yield of Rhizopus oryzae in lactic acid (based on the total carbohydrate consumed) is 
relatively low in comparison with that produced by lactic acid bacteria. The effect of the 
combined application of the two inoculants on lactic acid production over the control 
silage was not observed, probably due to the counter effect of Rhizopus oryzae, which, as 
observed earlier, had shown defective lactic acid production. Future investigations should 
therefore focus on the optimum conditions necessary for increased lactic fermentation 
with Rhizopus oryzae alone in PPS.  
  The acetic acid contents in the silages were generally low compared to the lactic acid 
contents, indicative of the good preservation quality of the silages. Other volatile fatty 
acids that are usually produced during fermentation in silages were not detected in PPS. 
Oda et al. (2002) made similar observations. The increases in acetic acid concentration in 
PR and RL over the control and PL after 50 days of ensilage, though indicative of the 
good quality of the former over the latter, is of little significance since other VFA 
especially butyric acid, which are usually associated with badly preserved silages, were 
not present. The changes in the carbohydrate fraction of potato pulp seemed to assume a 
consistent pattern with advance in the period of ensilage. The most obvious of this pattern 
was observed in RL on the degradation of pectin, which led to a concomitant increase in 
sugar concentrations after 50 days (in Experiment 1) and could be due to the combined 
effect of the two inoculants.  The increases in the concentrations of sugar with days in 
ensilage were probably due to slight increases in concentrations of unspecified 
monosaccharides during fermentation, due to the attack by enzymes on starch and pectin 
and possibly due to chemical interactions between other sugars. Hence, sugars, or 
specifically monosaccharides, which are the end products of enzymatic degradation, 
increase with fermentation, whereas starch concentrations are reduced by fermentation 
(Van de Riet et al., 1987). Others had supported this assertion earlier, including De Man 
(1957), who indicated that lactic acid fermentation took place in potato pulp when stored 
in pits using carbohydrate substrates, mainly galactan and also from pectin. Increases in 
sugar contents in Experiment 2 without a corresponding change in starch and pectin 
contents could, therefore, have originated from other sources such as cellulose or 
hemicellulose, which are present in potato pulp (Mayer and Hillebrandt, 1997). The 
degradation of starch in the current study showed some level of inconsistency among 
treatments, and this could be due to the slow degradability of potato starch in general 
(Monteils et al., 2002). It could be noted that the decrease in pectin and the resultant 
increase in sugar with RL after 50 days of ensiling in Experiment 1 (Fig 3-1 a, c) did not 
have any significant effect on lactic acid concentration (Table 3-2). This could be 
explained by the counter effect of R, which had shown defective lactic acid production in 
the current study as observed earlier. The breakdown of starch and pectin with the use of 
inoculants as observed in the current experiment could, and in agreement with 
Dongowski and Stoof (1993), lead to reduction of the water holding capacity of PPS and 
consequently effluent production; therefore, ensiling of potato pulp on a large scale 
would have to take this into account.  
3.2.4. Conclusion 
   Ensiling potato pulp with Lactobacillus rhamnosus and Rhizopus oryzae and their 
combined application reduced the pH and improved the fermentation quality of the silage. 
However, potato pulp can ensile well with or without the bacterial inoculants. Lactic acid 
production was low with inoculation with Rhizopus oryzae alone, and further studies are 
required to ascertain the optimum conditions for the lactic acid production potential in 
potato pulp silage.  
3.3. Effect of temperature and storage duration on potato pulp ensiled 
with fungal inoculants  
      
   Ensiling of potato pulp is considered a viable method for its preservation and 
subsequent use as feed for ruminants due to its high nutritional value (Okine et al., 2005; 
Aibibula et al., 2004).  Although the use of bacterial inoculants in the enhancement of 
fermentation quality of silages is well established (Guan et al., 2002; Meeske et al., 1999; 
Gordon, 1989a), few studies (Okine et al., 2006a; Oda et al., 2002) have, however, 
investigated the use of fungal or aerobic inoculants in an anaerobic process such as 
ensiling.  
  Although the use of the fungus Rhizopus oryzae in aerobic fermentations is well 
documented (Skory et al., 1998; Skory, 2000; Erdogan and Mahinur, 2001), our (Okine et 
al., 2005) previous investigation on the use of R. oryzae in PPS fermentation did not 
confer any advantages over the silage without the inoculant in lactic acid production. Of 
the reasons considered for the lack of effect was the ambient temperature during the 
ensiling period. The success of microbial inoculants used to enhance ensiling 
fermentation is partly dependent on the temperature, since different inoculants have 
various temperature optima (McDonald et al., 1991b). Hachmeister and Fung (1993) 
reported that temperature (between 27 and 35oC) and length of fermentation were crucial 
factors in Rhizopus fermentation of tempeh, a mold-modified indigenous fermented food 
made from soybeans or cereal grains in southeast Asian countries. In temperate regions 
like Hokkaido, in northern Japan, where potato pulp is produced abundantly annually and 
where preservation by ensiling is an option, temperature may be important in determining 
the efficacy of the fungus in PPS fermentation. The fungus Amylomyces rouxii is 
taxonomically identical to R. oryzae in characteristics and ability to produce lactic acid 
(Abe et al., 2004). Both were used in this study because of their ability to hydrolyze 
starch into glucose and their use as starters for potato pulp fermentation (Abe et al., 2003; 
Abe et al., 2004).  
  This study investigated the effect of various temperature regimes and duration of 
storage on the fermentation quality of potato pulp silage inoculated with the two fungi.  
3.3.1. Materials and methods 
  Two fungi inoculants, Rhizopus oryzae IFO 4707 (IR) and Amylomyces rouxii CBS 
438-76 (IA), originally from freeze-dried cultures, were each grown on media containing 
malt extract agar for fungi and incubated at 25oC in Petri dishes 72 h prior to use. The 
fungi, including the media, were added separately to fresh potato pulp at the rate of 1.0 % 
(w/w) in fresh matter, enough to produce at least 1x106 colony forming units (CFU) g –1 
on use as per manufacturer’s statement. Three treatments made up of potato pulp without 
additive (control, C), IR treated silage (IPR) and IA treated silage (IPA) were prepared, 
and ensiling was done in polyethylene silos (300 mm x 200 mm x 0.15 mm). Eighty-one 
silos were made, 27 for each of the three treatments. Each silo was filled with 500 grams 
of potato pulp, and 5 grams fresh weight of the medium containing either IR or IA were 
added to 54 silos and mixed thoroughly before filling. To ensure compaction, the silos 
were pressed gently and a vacuum air deflator was used to remove any air from the silos 
followed immediately by heat-sealing and further strengthening with adhesive tape. 
Twenty-seven silos from each of the three treatments were kept under constant 
temperature regimes of 4, 12 and 25 ± 1oC, respectively. Pre-silage material was sampled 
on day 0 for immediate determination of pH and dry matter (DM) content. Three bags per 
treatment from every temperature regime were randomly selected and opened on days 7, 
24 and 40 post-ensiling and sampled to investigate treatment effects on the fermentation 
quality through measurement of DM, pH, lactic acid, volatile fatty acids (VFA), sugar 
and starch contents of the resultant silages.  
3.3.1.1. Chemical analyses 
   Potato pulp material and silages were dried for at least 24 h using a freeze-dryer, and 
sub-samples were ground to pass through a 1mm screen for subsequent analyses. Dry 
matter was determined from sub-samples by drying in a forced-draught oven at a constant 
temperature of 135oC for 2 h. The pH, lactic acid, sugar and starch contents of pre-silage 
material and silages were determined as described earlier. The concentrations of acetic 
acid were analyzed by gas-liquid chromatography (Shimadzu GC-14 A, Kyoto, Japan) 
equipped with a flame-ionization detector and a capillary column (ULBON HR-52, 
0.53mm i.d. x 30m x 3.0 μm) and using 2-ethyl-n-butyric acid as the internal standard.  
3.3.1.2. Statistical analyses 
      Silage fermentation data were analyzed in a 3 x 3 factorial design using ANOVA in 
the general linear model procedure of SAS (1996). Main effects and interaction between 
treatments and contrasts were performed for responses of inoculation to temperatures and 
durations of storage. Significant mean differences in effects were separated using the 
least significant difference test and P-values less than 0.05 were considered statistically 
significant. In the regression equations, the measured parameters (pH and lactic acid) 
served as the dependent variables (Y), and storage temperature (oC) and duration of 
storage (days) were the independent variables. 
3.3.2. Results 
3.3.2.1. Fermentation quality  
  The effects of inoculation, storage temperatures, and duration of storage and their 
interactions are given in Table 3-3. The pre-silage material contained 227.0 g/kg DM, 
1.90, 1.31, 322.0 and 6.0 g/kg DM as lactic acid, acetic acid (the only VFA detected in 
PPS), starch and sugar, respectively, while values for the final (day 40) control silage at 
25oC were 229.0 g/kg DM and, respectively, 73.1, 10.7, 308.0 and 3.1 g/kg DM for the 
same previous parameters. The pH was 5.62 and 3.32 for pre-silage material and silage, 
respectively, indicating a good fermentation process. The changes in pH, lactic acid, and 
acetic acid contents of the silages, as affected by the fungal inoculants under different 
storage temperatures and durations of storage, are given in Table 3-3.  
 3.3.2.2. Effect of inoculants 
 There were no treatment effects on the DM of silages which were between 210.0 
and 227.0 g/kg. The inoculants had no significant effect on the fermentation quality of 
PPS relative to the control silage, although numerically they reduced the pH and 
increased the lactic acid content. Acetic acid production was also not affected by 
inoculation.  
3.3.2.3. Effect of storage temperature  
The effect of storage temperature on the fermentation quality of PPS was obvious. 
Generally, the pH decreased (P<0.01) while lactic and acetic acid contents increased  
(P<0.01) with increase in storage temperatures.  There were no significant effects on 
lactic and acetic acid contents of the silages at 4 and 12oC. However, at 25oC both lactic 
and acetic acid contents increased (P<0.01). 
3.3.2.4. Effect of duration of storage 
The effect of duration of storage on the fermentation in PPS was similar to that of the 
storage temperature, decreasing (P<0.01) the pH and increasing (P<0.01) lactic acid 
production with progressive duration of storage. Acetic acid content was low at day 7, but 
increased and then decreased at 24 and 40 days of ensilage, respectively.  
  3.3.2.5. Starch and sugar contents in silages 
  The changes in starch and sugar contents are given in Table 3-3. Starch levels 
fluctuated with no clear tendency in the effect of inoculation, storage temperature level, 
and duration of storage. The only obvious effect, though not statistically significant, was 
that of numerical decreases for IPR and IPA relative to the control, an indication that the 
inoculants somewhat enhanced starch degradation in PPS. Effects of temperature and 
duration of storage were significant (P<0.05) for sugar, but the inoculant effect was not. 
At 25oC the sugar level was low in comparison with that at lower temperatures of 4 and 
12oC, while it increased significantly (P<0.05) at days 24 and 40, in comparison with day 
Table 3-3. Chemical characteristics of potato pulp material, main effects and interaction between inoculants, storage temperatures and duration of storage in fermentation characteristics of potato pulp silage after 40 days of ensilage (Values are least square means of three determinations given in g/kg dry matter, unless otherwise stated) 
Inoculants1 Temperature2 Duration of storage3 Significance4 
  RM C IPR IPAT4T12T25D7D24D40SEM I T D I vs. T I vs. DT vs. D
DM (g/kg) 227.0 214.0 211.1 213.3212.0 212.1 213.2214.0 215.1 210.33.60 NSNSNSNS NS NS 
pH 5.62 3.97 3.94 3.954.41a 3.96b 3.49c4.30a 3.86b 3.69c0.05 NS******NS NS *** 
Lactic acid 1.90 24.6 26.8 24.811.1b 18.9b 46.2a13.4b 21.6b 41.3a3.63 NS******NS NS *** 
Acetic cid  1.31 10.6 12.6 10.008.3b 09.3b 15.5a08.0b 19.3a 05.8b1.51 NS****** ** NS 
Starch  322.0 283.0 270.3 264.1244.2 288.2 285.1261.2 277.0 279.113.60 NSNSNSNS NS NS 
Sugar  6.0 8.0 9.4 8.28.8a 11.6a5.3b5.7b 10.1a9.7a1.11 NS**** NS NS ** 
RM: pre-silage material; DM: dry matter; SEM: standard error of the means. 
1 C: silage without additive (control); IPR: Rhizopus oryzae inoculated potato pulp silage; IPA: Amylomyces rouxii inoculated potato pulp silage. 
2 T4, T12, T25, storage temperatures of 4, 12 and 25°C, respectively.  
3 D7, D24, D40, duration of storage of 7, 24 and 40 days, respectively. 
4 I: inoculation; T: storage temperature; L: duration of storage; I vs. T: I×T interaction; I vs. D: I×D interaction; T vs. D: T×D interaction. 
Except for RM, figures followed by different letters in a row for inoculants, temperature and duration of storage differ significantly (P<0.05). 
* P<0.05; ** P<0.01; *** P<0.001; NS: not significant. 
3.3.2.6. Interaction between inoculants, storage temperature and duration of 
storage 
  Interactions between inoculation and both temperature and duration of storage were 
absent for all measured parameters except for acetic acid (Table 3-3). The pH, lactic acid 
content and sugar concentration showed significant (P<0.01) interactions for both 
temperature and duration of storage.  
3.3.2.7. Contrasts between effects of inoculation, storage temperatures and 
duration of storage 
  Contrasts between control and inoculated silages showed no statistical differences 
between IR and IA or the average response of inoculation relative to uninoculated silages 
(Table 3-4). There were, however, clear significant (P<0.01) effects between pre-silage 
material and silages with respect to temperature and duration of storage in all the 
measured parameters except for acetic acid and sugar concentrations. Of particular 
significance was the linear relationship (P<0.001, Table 3-4) between storage temperature 
and duration of storage for pH and lactic acid concentration. These relationships could be 
summarized in the following multi-linear equations: 
        
   pH   =    - 0.046 T - 0.021 D + 5.10              R2 = 0.81   (P<0.01) 
   LA   =      0.16 T + 0.08 D – 1.40                 R2  = 0.66   (P<0.01) 
where T, ensiling temperature ( oC ); D, duration of storage (days); LA, lactic acid 
content (% DM) and R, correlation co-efficient.  
   Starch content in pre-silage material decreased (P<0.05) with storage temperature and 
duration of storage (Table 3-4). However, significant (P<0.05) but inconsistent 
relationships were observed for acetic acid and sugar concentrations.  
   3.3.3. Discussion             
   This study was conducted to investigate the possible conditions necessary for 
improving fermentation in PPS inoculated with fungi as a follow-up to our previous study 
(Okine et al., 2005). The silo chosen for the study has been shown to be acceptable for 
ensiling without affecting the preservation quality (Ashbell et al., 2001). The chemical 
analysis of the pre-silage material and the final control silage showed that the changes in 
DM, pH and lactic and acetic acids were comparable to our previous study.  
   In the present investigation, the inoculant effect on PPS fermentation, especially lactic 
acid production, was minor, in confirmation of our previous results. We hypothesized that 
application of the fungi to potato pulp in conditions sufficient to maintain their growth 
would yield better inoculant responses in fermentation end products as compared to the 
freeze-dried form employed in the previous study. This was built on the premise that the 
initial aerobic phase of the fungi was likely to influence their efficacy, since oxygen is 
indispensable for fungal growth. Evacuation of the bags after filling, though  
                
Table 3-4. Contrasts between control and inoculated silages and effects between pre-silage material and silages with respect to storage temperatures and duration of storage in potato pulp fermentation characteristics 
Contrast1 
Inoculants Temperature Relationship  Duration of storage Relationship  
  C/IPR C/IPA C/IRARM/T4RM/T12 RM/T25Lin.Quad.  RM/D7 RM/D24RM/D40Lin.Quad.
Dry matter  NS NS NS *** *** *** NS NS  *** *** *** NSNS 
pH NS NS NS *** *** *** *****  *** *** *** ***** 
Lactic acid   NS NS NS *** *** *** *****  *** *** *** ***** 
Acetic acid  NS NS NS * ** NS NS **  *** NS NS ***** 
Starch  NS NS NS * * *** * NS  ** ** ** NSNS 
Sugar  NS NS NS *** NS * * ***  *** *** NS **** 
1 C/IPR: control silage vs. Rhizopus oryzae silage; C/IPA: control silage vs. Amylomyces rouxii silage; C/IRA: control silage vs. R. oryzae and A. rouxii silage. 
RM/T4: RM/T12, RM/T25: pre-silage material vs. storage temperatures of 4, 12 and 25°C, respectively. 
RM/D7: RM/D24, RM/D40: pre-silage material vs. storage duration of 7, 24 and 40 days, respectively.  
Lin.: linear relationship; Quad.: quadratic relationship.  
* P<0.05; ** P<0.01; *** P<0.001; NS: not significant.  
 Figure 3-3. Change of pH (a) and lactic acid concentration (b) during storage under different 
temperatures in potato pulp silage. ( ◆) 4°C; (■) 12°C; (▲) 25°C. [Values represent least square 
means (n=81)].  
0 72440
Duration of storage (days)
0 72440 
Duration of storage (days)
Lactic acid (g/kg DM)
necessary for anaerobiosis, may have inhibited slow diffusion of oxygen into the surface 
and consequently fungal activity, thereby impairing the inoculants’ potential for lactic 
acid production. However, this assumption does not explain the high final 
concentrations of lactic acid at the end of the period of investigation (Fig. 3-3). Mayer 
and Hillebrandt (1997) and later Saito et al. (2006), in investigations of microbial 
characteristics of raw potato pulp sampled from different varieties over a period of time, 
observed that the dominant microbe present was lactic acid bacteria (up to 108 CFU/g 
FM). This large load of lactic acid bacteria under favorable silo conditions, such as 
sustained anaerobiosis and sufficient substrate availability, would be enough to maintain 
fermentation even in the absence of a lactic acid fermentation stimulant. This report 
seems to support our previous conclusion (Okine et al., 2005) that potato pulp does 
ensile well without bacterial inoculants.  
Ensiling is based on fermentation, which involves biological and enzymatic activity 
and as such is strongly influenced by temperature. There was a common trend in the 
fermentation pattern of PPS irrespective of the inoculants used. The pH decreased, 
while the lactic acid content increased, with rise in storage temperatures and progressive 
duration of storage. The multi-linear equation elucidates this relationship. On the basis 
of the equation, to attain a pH of 3.50, for example, it takes only 21.4 days at a 
temperature of 25oC, while it requires 49.9 and 67.0 days at 12 and 4oC, respectively.  
Similarly, to attain a lactic acid content of 35.0 g/kg DM it takes 11.3, 37.3 and 53.3 
days at 25, 12 and 4oC, respectively. The pH was lower at elevated storage temperatures 
and extended periods of storage, while lactic acid production accelerated at higher 
storage temperatures and extended periods of storage (Fig. 3-3). There was a higher 
correlation for pH decrease than for the rise in lactic acid production, evident from the 
correlation co-efficients (R2 = 0.81 vs. 0.66). The insignificant differences in lactic acid 
content between 4 and 12oC and also at days 7 and 24 (Table 3-3) elicited the relevance 
of a combined effect of both storage temperature and duration of storage on PPS 
fermentation. 
  Although the acetic acid content fluctuated inconsistently, the increase (P<0.01) at 
25oC followed an increase in lactic acid content (Table 3-3), reflecting the conversion of 
the latter to the former and/or the permeability of some oxygen into the bags, in 
agreement with Ashbell et al. (2001). These effects, however, did not adversely affect 
the quality of the silages and may have protected them from development of butyric 
acid and molds, which were not detected in any of the silages. The starch content was 
not affected by the inoculation, temperature, or duration of storage. The hypothesis that 
Rhizopus cultures enhance potato pulp starch degradation or conversion to sugar for use 
as a substrate for lactic acid fermentation could not be validated by the present results. 
Although inoculated silages had numerically (Table 3-3) higher values relative to the 
control, starch in potato pulp was degraded with/without the fungal inoculants. The 
slow degradation of potato starch (Monteils et al., 2002) may be responsible for this, or 
during fermentation enough sugar may have been produced from hydrolysis of other 
carbohydrate sources such as pectin (De Man, 1957; Okine et al., 2005) through the 
enzyme pectinase (produced by the fungi) in amounts sufficient to maintain 
fermentation and this obviated the need to hydrolyze starch. The significant decrease in 
sugar levels (from 12 to 25oC) indicated that high ensiling temperatures increased the 
rate of sugar consumption as a substrate for lactic acid production.  
3.3.4. Conclusion          
   These data suggest that the fermentation quality of potato pulp silage is influenced 
most by ensiling temperatures and the duration of storage and least by inoculation with 
fungal inoculants. The use of the Rhizopus oryzae and Amylomyces rouxii in potato pulp 
silage fermentation should be weighed against their cost and other nutritional benefits. 
3.4. Effluent production and losses in potato pulp silage 
         In the previous experiments, the role of microbial inoculants in enhancing 
fermentation in potato pulp has been acknowledged. However, the use of inoculants, 
especially fungi, is related to the breakdown of pectin and starch in potato pulp silage, 
which may decrease the water holding capacity of PPS with undesirable consequences 
like increased effluent production and nutrient losses.  
   The objective of the following experiment (Experiment 3) was to quantify effluent 
losses in potato pulp silage inoculated with or without fungi.  
3.4.1. Materials and methods 
    Fresh potato pulp with estimated moisture content (based on visual appraisal) of 
900 g/kg was dehydrated by bagging and suspension in semi-porous polyethylene bags 
overnight. Three microbial inoculants; Lactobacillus rhamnosus (L), Rhizopus oryzae 
IFO 4707 (R) and Amylomyces rouxii CBS 438-76 (A), whose characteristics have been 
previously (Experiments 1 and 2) mentioned, were used. The inoculants were added at 
the rate of 0.5, 1.0 and 10 g/kg in fresh weight of potato pulp, respectively, and enough 
to produce at least 1x106 CFU g –1 on application and according to manufacturer’s 
instructions. These were set against potato pulp without inoculant as control. The two 
previous inoculants were added to fresh potato pulp in their freeze-dried forms. The 
later, originally in freeze dried form was incubated in a medium containing agar at 25oC 
for 48 hours prior to addition to potato pulp. Treatments were thus made up of the 
control without inoculant (EC), Lactobacillus rhamnosus added to potato pulp (EL), 
Rhizopus oryzae added to potato pulp (ER) and Amylomyces rouxii added to potato pulp 
(EA). All the three inoculants were mixed with wheat bran (1.0 g/kg in fresh matter 
weight of potato pulp material) prior to addition to potato pulp for the sake of 
application and mixture convenience. About 10 kilograms fresh weight (with three 
replications from each treatment) were ensiled in silos (1 m in height, 0.10 m in 
diameter and 0.05 m thick) made of cylindrical polyvinyl chloride (PVC) plastic pipe 
with two ends. The end was open, and the base was closed. The base of each pipe 
comprised a sealed conical narrow end with a drain opening fitted with rubber tube and 
a cork which when relaxed, enabled the flow of effluent into a calibrated glass flask 
fitted with a rubber stopper. This prevented infusion of ambient air into the silo via the 
tube during effluent measurement. A perforated stainless steel plate, further covered 
with cheesecloth, was inserted into the base of each pipe, creating a spacer between 
silage material and effluent. Each silo was filled and pressed with weights to 
compaction and the upper part was capped with a polyethylene bag and enforced with 
vinyl tape to prevent the infiltration of air. The silos were placed horizontally on a stand 
to facilitate collection of effluent, then stored at room temperature and effluent was 
collected each day for the first 3 days of ensilage. Then, at 3-day intervals, pH was 
measured immediately upon opening, and samples were taken and stored at -20oC until 
analyzed. The silos were opened on the 30th day of ensilage, weighed and sampled for 
subsequent analyses. 
3.4.1.1. Chemical analyses       
   The fermentation characteristics (moisture, pH, lactic acid and VFA), WRC, organic 
matter, sugar, and gross energy were determined using procedures previously described.  
3.4.2.1. Statistical analyses 
    Same as in the previous experiment. 
  3.4.2. Results 
3.4.2.1. Fermentation quality 
    The fermentation quality of potato pulp material and silages are presented in Table 
3-5. Even after dehydration for 24 hours, the moisture content was still high (840.1 
g/kg). The moisture content of the silages, though not statistically significant, 
numerically decreased in ER and EA. The pH of silages was low in the inoculated 
treatments relative to the uninoculated control silages and was particularly lowest in ER 
and EA. However, values were generally below 3.60 and consistent with the two 
previous experiments. Lactic acid concentration increased (P<0.05) in the inoculated 
silages except with ER. Sugar concentration increased significantly (P<0.05) in ER and 
EA, respectively. Water retention capacity decreased after ensiling and was more 
pronounced with inoculation, especially in EA.  
3.4.2.2. Effluent production and nutrient losses 
   Effluent volume increased significantly (P<0.05) in ER and EA, ranging from 619 
to 1,816 grams at the end of 30 days of ensilage (data not shown), corresponding to 
66.1-174.8 g/kg fresh weight of ensiled material, and was highest in EA. This had 
consequent adverse effects on the silages in losses of DM, OM, and gross energy.   
Table 3-5. Chemical and silage characteristics of potato pulp after 30 days of ensiling. 
[(Experiment 3). Values are means of three determinations and given in g/kg DM, 
unless otherwise stated] 1  
   Silages  
  PP  EC  EL  ER  EA 
 Moisture (g/kg)  840.1  830.2  831.1  823.3  811.0 
 pH  5.53  3.35b  3.39a  3.28c  3.25d  Lactic acid   -  60.7c  77.7b  62.3c  114.3a  Acetic acid   -  4.91c  6.12b  4.63c  9.71a  Sugar   23.1  19.2c  20.3c  23.0b  25.5a  Water retention capacity (g/g)  2.41  1.94a  1.84b  1.81b  1.73c  Effluent (g/kg of material)  -  66.1bc  62.9c  83.2b  174.8a  Total losses during ensilage   
   DM - 119.1bc 138.0b114.2c 204.3a 
   OM - 119.3bc 137.2b113.1c 201.1a 
   Energy - 116.0b 120.2b93.2b 184.3a 
1 PP, potato pulp pre-silage material; EC, potato pulp silage without additive; EL, ER, EA, potato pulp 
inoculated with Lactobacillus rhamnosus, Rhizopus oryzae and Amylomyces rouxii, respectively.  
Except PP, means within a row with uncommon letters differ significantly (P<0.05). 
          
3.4.3. Discussion         
   Although the moisture content decreased in the silages compared to the original 
material, it was more evident in ER and EA. This was due to the effect of R and A 
which may have broken the cohesion of the cell wall structure of potato pulp 
(Dongowski and Stoof, 1993), enabling the release of water and effluent and 
consequently reducing the moisture content of the resultant silages. Reduction in pH of 
potato pulp in the silage fermentation without or with bacterial inoculants has already 
been acknowledged and discussed in detail in the previous experiments. The results of 
the present study (Experiment 3) lend further support to that view. Although increase in 
lactic acid with inoculation with L was expected and in tandem with Experiment 1, the 
high increase with inoculation with A, relative to the control uninoculated silage was a 
novelty in this study. Several reasons could be attributed to this, among them, the rate, 
state and mode of application (incubation with a medium prior to inoculation as against 
direct application in freeze-dried form) and pre-mixture with wheat bran. Addition of 
wheat bran has been reported to increase lactic acid content of raw comminuted 
potatoes (Weise, 1964). Conspicuously low in lactic acid production was ER. This 
result validated earlier conclusions (Okine et al, 2005; Okine et al, 2007) on the defect 
of Rhizopus oryzae in lactic acid production in potato pulp silage. Increases in sugar 
with inoculation with ER and EA reflected the rate of the fermentation process and to a 
large extent, end products of hydrolysis of carbohydrates in potato pulp such as starch 
and pectin (Okine et al., 2005). 
   The significant increase in effluent production with inoculation with A could be 
attributed to the breakdown of starch and pectin as theorized in previous studies 
(Experiments 1 and 2) and a direct consequence of reduction of the WRC of the silages.  
The increase in effluent production in PPS with A was so consistent that had the period 
of investigation been extended, more effluent would have been produced. ER also 
produced a substantial amount of effluent compared with EC, a result that could be 
explained by the action of the enzymes pectinase and LDH on the residual starch in 
potato pulp, which, according to Dongowski and Stoof (1993), decreases the water 
binding capacity of potato pulp after the action of cell wall degrading enzymes. The 
results of this study suggested that A was more effective in breaking the water holding 
capacity of PPS, compared with the other inoculants. As a consequence, losses were 
higher in EA, a result that could adversely affect the nutritive value of the silages. The 
combination of high sugar concentration and effluent production with inoculation with 
R and A would not only have negative impacts on the environment but also on the 
aerobic stability of the silages, since the residual sugars would serve as substrate for the 
proliferation of spoilage organisms such as molds, further impairing the nutritive value 
of potato pulp silage.  
3.4.4. Conclusion 
   Ensiling potato pulp with Rhizopus oryzae and Amylomyces rouxii increased 
effluent volume and silage losses in potato pulp silage. Until evidence to the contrary 
arises, inoculation with fungal additives would seem to yield no benefits in potato pulp 
silage, and where their application is necessary, the use of in-silo absorbents prior to 
ensiling, is imperative.  
3.5. Summary  
     Ensiling potato pulp with Lactobacillus rhamnosus and Rhizopus oryzae and their 
combined application reduced the pH and improved the fermentation quality of the 
silage. However, potato pulp can ensile well with or without the bacterial inoculants. 
Lactic acid production was low with inoculation with Rhizopus oryzae alone.  A 
subsequent study suggested that the fermentation quality of potato pulp silage was 
influenced most by ensiling temperatures and the duration of storage and least by 
inoculation with fungal inoculants such as Rhizopus oryzae and Amylomyces rouxii in 
potato pulp silage. Moreover, ensiling potato pulp with Rhizopus oryzae and 
Amylomyces rouxii increased effluent volume and silage losses in potato pulp silage. 
Therefore, inoculation with fungal additives would yield little benefits in potato pulp 
silage and where their application is necessary, the use of in-silo absorbents prior to 
ensiling, is warranted.  
                      
Chapter 4  Daikon (Oriental radish) 
4.1. Introduction   
   Daikon, or Oriental radish (Raphanus sativus L.), is a popular root vegetable in 
Japan and East Asia. It goes by different names depending on the country of origin; lor 
bark (Cantonese Chinese), Mu (Korean), labanos (Filipino) or cu-cai trang 
(Vietnamese), and is grown all year round in areas having an oriental population. 
Daikon is consumed in different forms be it fresh, soaked, brined or dried.  In Japan, 
more daikon is produced than any other vegetable after potatoes and yearly production 
runs into nearly two million tons with an increasing tendency during the past five years 
(Statistical Handbook of Japan, 2005). Out of this about 20 g/kg representing the 
crowns, leaves, damaged or infested root parts and culls that are of no commercial value 
are disposed off during processing as waste. A similar situation may exist in other 
regions where daikon is produced. These unwholesome parts are returned to the soil 
with detrimental consequences to the topsoil due to their high content of mineral salts. 
Daikon has high crude protein, digestible energy and low ether extract contents (154, 15 
and 15 g/kg in dry matter, respectively) and contains easily digestible nutrients 
(National Agricultural Research Organization, 2001) thus, a potential animal feed 
resource. An intrinsic characteristic of daikon is its very high moisture content (about 
940 g/kg in fresh matter), which is a major limitation to its use as livestock feed. Drying 
of daikon by-product for use in ruminant diets is an option but the high cost involved 
makes this uneconomical. Ensiling with drier materials for subsequent use as ruminant 
feed is a viable alternative from nutritional, preservation and environmental 
perspectives. However, the taste of, the taste of fresh daikon involves some irritation 
and a burning sensation on the tongue (Lindsay, 1985) and has a characteristically 
pungent smell and aroma (Friis and Kjaer, 1966) that may limit its use as livestock feed. 
To date and to the best of our knowledge, the only quantitative data available on the 
ensiling of daikon or its by-product, its fermentation pattern or its use as feed are our 
reports (Okine et al., 2006a, Okine et al., 2006b).  
    The objective of this investigation was to explore the possibility of using a material 
very low in dry matter content (such as daikon by-products) as animal feed with 
emphasis on ensiling as a means of preservation and, to assess the role of absorbents on 
effluent retention in the silages.  
   The study involved a series of experiments conducted separately over two years. 
First an investigation of the chemical composition and pre-ensiling characteristics of 
daikon, followed by ensiling of daikon by-products initially with wheat straw as a sole 
in-silo absorbent, and then with other absorbents: dried beet pulp, dried bean stalks and 
husks and wheat bran. A study of the aerobic stability or shelf life of daikon silages was 
investigated in both experiments.  
4.2. Ensiling of daikon by-product with wheat straw (Experiment 4-1) 
 4.2.1. Materials and methods  
4.2.1.1. Chemical characteristics of daikon  
    A total of twelve samples of fresh daikon (roots) were purchased from local 
supermarkets at different times during the harvesting season between July and October 
2004 for the analyses of the chemical composition and ensiling characteristics. 
Procedures used in the determination of the ensiling characteristics have already been 
described in Chapter 2. The chemical composition (organic matter, OM; crude protein, 
CP; neutral detergent fiber, NDF; ether extract, EE; gross energy, GE; water-soluble 
carbohydrates, WSC) was determined from dried samples. 
4.2.1.2. Ensiling of daikon by-product  
   Fresh daikon by-product (roots and leaves) were chopped to fit into two types of 
silos used for this purpose namely pipe and bucket type silos.  
4.2.1.2a. Pipe silos  
Twelve silos, (1 m in height, 0.10 m in diameter and 0.05 m thick) made up of 
cylindrical polyvinyl chloride (PVC) previously described in Chapter 3, were used. 
Daikon was chopped to about 2 cm length and 2 cm thickness (together with the leaves 
using a forage cutter and mixed). This size was determined through a short experiment 
as the most appropriate size for filling in terms of convenience, type of the silos 
employed, and effect on minimizing effluent output. The moisture content of daikon 
was adjusted to 850 g/kg in fresh matter by addition with wheat straw which had been 
chopped to about 2 cm prior to mixture with the daikon. The mixing ratio of daikon to 
wheat straw was 4:1 (w:w). A commercial lactic acid bacteria inoculant, Lactobacillus 
plantarum (LP, Ecosyl Products Ltd, Tokyo, Japan) was added at the rate of 10 g/kg, 
calculated to contain at least 1x106 CFU per gram in fresh matter as per manufacturer’s 
instruction. The contents were thoroughly mixed prior to filling. Four treatments were 
prepared with three replications for each treatment consisting of control (DN-N), with 
LP inoculation (DN-L), adjusted moisture level without LP (DA-N), and with LP 
(DA-L). Each silo was filled and pressed with weights to compaction, with an average 8 
or 6 kg in fresh matter of pre-silage material for non-moisture and adjusted moisture 
levels, respectively. After filling, the upper part of each pipe was capped with a 
polyethylene bag and enforced with vinyl tape to prevent the infiltration of air. The silos 
were rested horizontally on a stand to facilitate collection of effluent, stored at room 
temperature and effluent volume was measured after 1, 2, 3 4, 5, 6, 8, 10, 12, 14, 16, 19, 
22, 25, 28 and 30 days. On each effluent measurement, the cork fitted on the drainage 
tube was relaxed, enabling effluent flow, then tightened, flask removed and replaced 
immediately with a fresh one. The silos were opened and weighed on the 30th day of 
ensilage, and representative samples were taken for immediate determination of pH and 
juice extraction for subsequent chemical analyses. Composite samples of the silages (a 
total of 5 kg fresh matter) of each of the four treatments were transferred into separate 
open containers, kept at room temperature and representative samples taken from each 
container daily for 6 days for the determination of the aerobic stability using changes in 
pH, lactic acid and VFA as indices.  
  4.2.1.2. Bucket silos  
Thirty-six 20-liter polyvinyl chloride (PVC) buckets, each equipped with a 
septum-enforced lid and nozzle enabling gas escape only, were used as silos and 
prepared simultaneously with the pipe silos. Treatments, including rate of addition of LP 
and moisture adjustment level, were the same as that of the pipe silos. Filling was done 
by uniform mixture of straw and daikon and compacted by pressing with weights. The 
average weight was 20 and 17 kg in fresh matter for non-moisture and adjusted 
moisture levels, respectively. The silos were stored at room temperature and three 
buckets from each treatment were opened after 7, 14 and 30 days post-ensiling and 
sampled to investigate proceeding changes in fermentation quality, development of 
LAB and yeast flora.   
4.2. 1.3. Chemical analyses 
    The DM, pH, lactic acid and VFA concentrations, WSC, ether extract, NDF, ADF, 
and gross energy contents of material and silages were determined by methods 
previously described (Chapters 2 and 3). Volatile basic nitrogen (VBN) was determined 
by the complete distillation method as described by Morimoto (1971). Organic matter 
content was obtained by difference following ashing at 600oC for 3 h. Total nitrogen 
(TN) was measured through Kjeldahl method and crude protein (CP) was calculated as 
TN x 6.25, all according to AOAC (1990).  
4.2.1.4. Statistical analyses 
   Silage fermentation data were analyzed using ANOVA in a randomized block design 
using General Linear Model Procedure of SAS (1996). Contrasts between fermentation 
quality of silages (inoculated with/without LP, and non/adjusted moisture, and the 
average response of the two factors to ensilage) were performed and means differences 
compared for all variables and P<0.05 considered as statistically significant. 
4.2.2. Results  
4.2.2.1. Chemical characteristics of daikon 
    The chemical composition and characteristics relevant to ensilage are given in 
Table 4-1. Daikon was characteristically low in DM, had high OM, CP and gross energy 
contents and low NDF, ADF and ether extract thus, a potential animal feed resource. 
The buffering capacity ranged 467-1001 meq/kg DM with a high standard deviation, 
which made it theoretically difficult to ensile in comparison with grass materials in 
general.  
  Table 4-1. Chemical and microbiological characteristics of daikon (n = 12). (Figures are given in g/kg DM, unless otherwise stated)§ 
 Range Mean SD 
Dry matter (g/kg ) 42.7- 63.5 49.0 10.7 
Organic matter  836 - 884 863.0 20.3 
pH 5.85 - 6.20 6.05 0.18 
Water soluble carbohydrates 262 - 480 382 90.4 
Buffering capacity (meq/kg DM) 467-1001 703 255 
Crude protein  131-210 180 35.0 
Neutral detergent fiber 151-194 184 21.5 
Acid detergent fiber  144 - 180 169 16.8 
Hemicellulose  7.5 - 21.0 14.9 5.6 
Ether extract 7.7 - 16.4 11.9 3.5 
Gross energy (MJ/kg DM) 17.0 - 17.7 17.4 0.28 
Lactic acid bacteria (Log10 CFU/g FM) 3.3 - 7.8 4.8 2.09 
Yeast (Log10 CFU/g FM) 4.7 - 6.9 5.5 1.00 
§DM, dry matter; meq/kg DM, milliequivalent per kilogram DM; SD, standard deviation; MJ, mega joule;  CFU, colony forming units; FM, fresh matter. 
4.2.2.2 Ensiling of daikon by-products 
Pre-ensiling characteristics indicated that daikon by-product had high pH, contained 
varying levels of LAB and yeast numbers per gram of fresh matter and contained 
enough WSC to maintain lactic acid fermentation.   
The chemical compositions of the pre-silage materials and silages in Experiment 4-1 
are presented in Table 4-2. The DM of daikon by-product was elevated from 64.0 to 
175.1 g/kg, and this had subsequent effects on the chemical composition of both 
pre-silage material and silages. Dry matter-adjusted material was low in sugar, buffering 
capacity and total nitrogen contents but had higher pH, WRC and gross energy values.  
The fermentation characteristics of the silages were good as indicated by low pH values 
and high lactic acid contents relative to the pre-silage materials. The silages contained 
some amounts of ethanol, fair amounts of acetic and propionic acids and traces of 
valeric acid but no butyric acid. Volatile basic nitrogen was low in all treatments. Within 
adjusted and non-adjusted moisture, inoculation with LP had no effect on all measured 
silage parameters except pH and lactic acid contents (main fermentation end products). 
Contrarily, contrasts between inoculation with/without LP, and non/adjusted moisture, 
and the average response of inoculation with LP and moisture adjustment on the silages 
were highly significant (P<0.01) except for pH.  
             
Table 4-2. Chemical composition of daikon by-product material and silages after 30 days of storage in pipe silos (Experiment 4-1) 1    Pre-silage material Silages  Pooled  Contrasts# 
 DN DA DN-N DN-L DA-N DA-L SEM P value1 2 3 
Dry matter (g/kg) 64.0 175.1 48.1b 47.3b 132.0a 131.3a 2.33 0.0001 ** ** ** 
pH 4.14 4.17 3.58a 3.50b 3.65a 3.51b 0.02 0.0020 NS NS NS 
Lactic acid (g/kg DM) 21.4 9.2 132.3b 179.8a 63.5c 68.7c 3.06 0.0001 ** ** ** 
Volatile fatty acids (g/kg DM)            
Acetic - - 12.7a 10.2b 5.4c 4.1c 0.62 0.0001 ** ** ** 
Propionic - - 9.7a 7.8a 1.9b 1.4b 1.10 0.0014 ** ** ** 
Butyric - - NF NF NF NF - - - - - 
Valeric - - 2.5a 2.4a 1.0b 1.1b 0.28 0.0070 ** ** ** 
Ethanol (g/kg DM) - - 3.3a 2.9a 0.90b 0.85b 0.16 0.0001 ** ** ** 
Sugar (g/kg DM) 274.0 53.0 11.8a 11.5a 2.6b 1.9b 0.47 0.0001 ** ** ** 
Water retention capacity (g/g) 2.59 3.20 2.38ab2.02b 2.72a 2.54a 0.13 0.0299 ** ** ** 
Buffering capacity (meq/kg DM) 830.7 341.2 - - - - - - - - - 
Total nitrogen  (g/kg DM) 31.6 13.4 31.9a 32.6a 12.7b 11.3b 0.16 0.0001 ** ** ** 
Volatile basic nitrogen (g/kg TN) - - 34.8 27.7 34.7 33.0 2.55 0.2394NS NS NS 
Density (kg/m3 fresh matter) 942 683 - - - - - - - - - 
Gross energy (MJ/kg DM) 17.5 18.3 17.3b 16.9b 18.2a 18.0a 0.22 0.0112 ** ** ** 
1 DN, daikon without moisture adjustment; DA, daikon adjusted moisture content with wheat straw; DN-N, DN-L, daikon silage with and without Lactobacillus plantarum (LP), respectively; DA-N, DA-L, daikon silage adjusted moisture content with wheat straw with and without LP, respectively. Except DN and DA, figures are means of three values. Means followed by different letters in a row differ significantly (P<0.05). DM, dry matter; meq/kg DM, milliequivalent per kilogram DM; SEM, standard error of the means; MJ, mega joule; TN, total nitrogen.  # 1, DN-N vs. DA-N; 2, DN-L vs. DA-L; 3, average response of inoculation and moisture adjustment to ensilage; NS, not significant; *, P<0.05; **, P<0.01. 
4.2.2.3. Change in fermentation characteristics, LAB and yeast population 
with days in ensilage 
    Changes in pH, lactic acid concentrations and development of LAB and yeast 
numbers during the course of ensilage in the bucket silos are shown in Fig. 4-1 and 4-2.  
The pH decreased sharply, followed by increases in lactic acid for all treatments during 
the first 7 days of ensilage, but continued steadily in the decrease and increase, 
respectively, till the end of the 30 th day.  Moisture adjusted silages had lower lactic 
acid contents while inoculation with LP increased concentrations of lactic acid relative 
to the uninoculated control silages.  
071430
Days of ensilage
Lactic acid (g/kg DM)
Fig. 4-1. Changes in pH (a) and lactic acid concentration (b) in daikon by-product 
silages days of ensilage in Experiment 4-1. (◆) daikon control; (■) daikon inoculated 
with Lactobacillus plantarum, (LP); (▲), daikon adjusted moisture with wheat straw 
only; (●) daikon adjusted moisture with wheat straw and inoculated with LP.  
071430
Days of ensilage
(a) (b) 
Lactic acid bacteria population increased in non-adjusted moisture in contrast with 
adjusted moisture during the first 7 days of storage, but both treatments decreased 
gradually till the end of the storage period. Yeast population followed a similar pattern 
and at the end of the duration of storage, had reduced considerably to about 30%. 
Inoculation with LP had lower LAB and yeast populations relative to the uninoculated 
silages. 
071430Days of ensilage 
Log 10 of yeast (CFUg/FM
Fig. 4-2. Development of lactic acid bacteria (LAB) (a) and yeast (b) populations in 
daikon by-product silages days of ensilage in Experiment 4-1.  
(◆) daikon silage control; (■) daikon inoculated with Lactobacillus plantarum, (LP); 
(▲), daikon adjusted moisture with wheat straw only; (●) daikon adjusted moisture with 
wheat straw and inoculated with LP.  
4.2.2.4. Effluent production and nutrient losses during ensilage 
There was high effluent production from the non moisture-adjusted silages (with or 
without LP inoculation) with progression of days of ensilage especially during the  first 
6 days (Fig. 4-3), and at the end of 30 days had reached a peak of 378.4 and 507.7 g/kg 
071430
Days of ensilage
Log 10 of LAB (CFUg/FM
(a) (b) 
fresh weight of ensiled material, respectively (Table 4-3). High dry matter, energy and 
nitrogen losses followed as a consequence. Adjustment with wheat straw minimized 
(P<0.01) these losses considerably. Total weight losses as effluent (calculated as the 
difference between initial and final total weight of silage minus effluent) were 
significantly (P<0.01) higher for non-adjusted moisture silages.  
123456810 12 14 16 19 22 25 28 30
Days of ensilage
Effluent  (g/kg)
Fig. 4-3. Effluent volume in daikon by-product silages during 30 days of ensilage. (◆), 
daikon silage control; (■), daikon silage inoculated with Lactobacillus plantarum, (LP); (▲), 
daikon adjusted moisture with wheat straw only; (●), daikon adjusted moisture with wheat 
straw and inoculated with LP.  
 The opposite was true for gas losses. Significant increases (P<0.01) in effluent 
production were observed only for LP inoculated relative to the uninoculated silage; 
otherwise, inoculation had no effect on losses within moisture adjusted and 
non-adjusted silages. 
Table 4-3. Total losses of dry matter, gross energy and nitrogen, effluent volume, weight 
losses as effluent and gas (g/kg of total weight losses) in daikon by-product 
after 30 days of ensilage (Experiment 4-1) 
     4.2.2.5. Aerobic stability of silages 
   Aerobic stability of the silages in Experiment 4-1 was determined over 6 days with 
changes in fermentation end products as indices (Ashbell et al., 1991) and is presented 
in Figures 4-4 and 4-5. The pH of non-adjusted moisture silages was somewhat stable 
till the 5th day of aerobic exposure in contrast with that of the moisture adjusted silages 
which increased rapidly. There was a similar rate of decrease in lactic acid content in 
both moisture adjusted and non-adjusted silages. However, a common pattern was 
observed for LP inoculated silages (adjusted or non-adjusted moisture) in sharp 
increases and decreases in pH and lactic acid contents, respectively, after the 5th day of  
 Silages§  
  DN-N DN-L    DA-N DA-L P value 
Dry matter (g/kg) 554.4a 532.4a 58.9b 120.7b 0.0001 
Gross energy (g/kg) 560.5a 535.8a 61.5b 122.3b 0.0002 
Nitrogen (g/kg) 500.1a 518.4a 141.1b 248.5b 0.0026 
Effluent (g/kg ensiled material) 378.4b 507.7a 21.3b 62.9b 0.0001 
Weight losses      
  Effluent (g/kg total weight loss) 519.0a 530.7a 25.0b 43.0b 0.0001 
  Gas (g/kg total weight loss ) 481.0b 469.3b 975.0a 957.0a 0.0001 
 § Same as in Table 4-2. Figures are means of three values. Means followed by different letters in a row differ significantly (P<0.05). 
aerobic exposure. There were gradual decreases in residual VFA (acetic acid) and 
123456Days after opening of silage
Lactic acid (g/kg DM)
Fig. 4-4. Changes in pH (a) and lactic acid concentration (b) in daikon by-product silages 
during 6 days of aerobic exposure in Experiment 4-1. (◆), daikon silage control; (■), daikon 
inoculated with Lactobacillus plantarum, (LP); (▲), daikon adjusted moisture with wheat 
straw only; (●), daikon adjusted moisture with wheat straw and inoculated with LP.  
increases (propionic acid) in all silages with progression of days of aerobic exposure of 
silages. Butyric acid, however, increased sharply after the 5 th day, indicative of the 
increase in putrefactory microorganisms in the silages. 
123456
Days after opening of silage 
123456
Acetic acid (g/kg DM)
123456
Propionic acid (g/kg DM)
123456
Days after opening of silages
Butyric acid (g/kg DM)
  Fig. 4-5 (a-c). Changes in volatile fatty acid concentrations in daikon by-product silages during 
6 days of aerobic exposure in Experiment 4-1. (◆), daikon silage control; (■), daikon inoculated 
with Lactobacillus plantarum, (LP); (▲), daikon adjusted moisture with wheat straw only; (●), 
daikon adjusted moisture with wheat straw and inoculated with LP. 
4.3. Ensiling daikon with other absorbents (Experiment 4-2). 
   The objective was to investigate the effect of other in-silo absorbents on the 
fermentation quality, effluent retention and aerobic stability of daikon by-product silage. 
4.3.1. Materials and methods  
    Daikon by-product was adjusted to lower moisture (four treatments with three 
replications each) with dried beet pulp (DBP), dried bean stalks and husks (DBH), 
wheat straw (DWS) and wheat bran (DWB) as in-silo absorbents(Table 4-4). The 
daikon: absorbent mixing ratio was 4:1 (w:w) for DBP, DBH and DWS. The ratio for 
DWB was 7:3 (w:w) due to its perceived low water retention capacity. Ensiling was 
done in 18-liter capacity PVC cylindrical bucket silos (Takagi Company, Tokyo, Japan). 
Each bucket is equipped with a perforated stand at the base, which served as a separator 
between silage material and effluent, and a drain tap which enabled the flow and 
measurement of effluent. Each treated material was put into a polythene bag, pressed 
and inserted upside- down into the bucket silo and a weight (calculated to exert a 
pressure of 40 g/cm3 per fresh weight of material) was placed on the silo. The purpose 
was to simulate conditions in a commercial silo and to investigate effluent retention of 
the treatments under pressure. The packing weights and densities are presented in Table 
4-4. The silos were kept at room temperature (18-24oC) and opened after 30 days of 
storage. Representative samples were taken from each treatment for the determination 
of the aerobic stability 3, 5 and 7 days after opening the silages using procedures similar 
to those in the previous experiment. 
4.3. 1.1. Chemical analyses  
   Analyses of chemical components in pre-silage materials and silages were done as 
previous described in this chapter.  
4.3.1.2. Statistical analyses 
   Silage fermentation data were analyzed using General Linear Model Procedure of 
SAS (1996). Statistical differences, where applicable, were declared at P<0.05.   
4.3.2. Results 
4.3.2.1. Fermentation quality, effluent and losses during ensilage  
   In Experiment 4-2, treatments were adjusted to lower dry matter contents based on 
observations of effluent production in silages from Experiment 4-1. The adjustments 
augmented the WRC of all the pre-silage materials except DWB (Table 4-4). Silage 
fermentation quality was relatively depressed compared with that of Experiment 4-1. 
There were significant (P<0.05) treatment effects on pH and lactic acid but none on 
VFA concentrations. Silage losses were mostly as effluent, and were significant 
(P<0.05) among treatments, with DWS producing zero effluent. Other treatments had 
fair amounts of effluent, with DWB producing the most.  
Table 4-4. Pre-silage and silage chemical characteristics of daikon by-product ensiled 
with different absorbents (Experiment 4-2) § 
             
  DBP DWS DBH DWB SEMP value
Pre-silage characteristics       
  Dry matter (g/kg) 18.4 18.9 17.6 28.7 - - 
  Water retention capacity (g/g )  4.25 4.08 3.95 2.49 - - 
  Packing weight (kg/silo) 12.1a 5.1c 6.4b 12.0a 0.060.0001
  Packing density (kg/m3) 671.1a 282.3c 356.1b 664.4a 3.360.0001
Silage characteristics       
  Dry matter (g/kg) 196.2c 229.1b 206.3c 253.1a 4.060.0010 
  pH 3.80b3.97a4.00a3.78b 0.03 0.0020 
  Lactic acid (g/kg DM) 44.5a 17.4c 34.6b 36.4b 2.29 0.0020 
Volatile fatty acids (g/kg DM)       
  Ethanol 0.01 0.20 0.04 0.07 0.06 0.2135 
  Acetic  0.2  0.2  0.4  0.2 0.45 0.5473 
  Propionic 0.0  0.1  0.1  0.1 0.10 0.4851 
  Valeric 0.0 0.1  0.1  0.1  0.64 0.2543 
Effluent (g/kg ) 152.0b 0.0d 103.1c 201.1a 12.550.0002
Losses       
    Effluent (g/kg total losses) 935.9a 0.0c 889.6a 714.4b 28.030.0001
    Gas (g/kg total losses) 64.1c 1000.0a110.4c 285.6b 28.030.0001§DBP, daikon + dried beet pulp; DWS, daikon + wheat straw; DBH, daikon + dried bean stalks and husks; DWB, daikon + wheat bran.  SEM, standard error of the mean. Except pre-silage dry matter and water retention capacity, values are means of three determinations; means followed by different letters in a row differ significantly (P<0.05). 
4.3.2.2. Aerobic stability of the silages 
   The changes in pH and lactic acid contents of the silages after exposure for 7 days 
are given in Fig. 4-6. The pH rose sharply from 3.80 to about 5.00 after 3 days of 
opening of the silages, and continued to rise steadily thereafter. In 7 days, the silages 
were all above 6.0 with DBH recording the lowest. The opposite pattern was observed 
for lactic contents in quick declinations in all silages after 3 days. At the end of the 
period of investigation, values were below 10.0 g/kg DM, irrespective of the type of 
silage. Of the different treatments, DWS and DBH were the most unstable during the 7 
day period. 
Days of aerobic exposure
Lactic acid ( g/kg DM )
Fig. 4-6. Changes in pH (a) and lactic acid concentrations (b) in daikon by-product silages 
during 6 days of aerobic exposure in Experiment 4-2. (◆), daikon + dried beet pulp silage 
(DBP); (■), daikon + wheat straw silage (DWS); (▲), daikon + dried bean stalks and husks 
silage (DBH); (●), daikon + wheat bran silage (DWB). 
      
Days of aerobic exposure
4.3.3. Discussion 
  Despite the high contents of OM, CP, WSC, GE and the low content of EE that 
underline its prospect as animal feed resource, pre-ensiling characteristics, especially 
the high moisture content, pH, buffering capacity and yeast population of daikon 
pre-supposed difficulties in its preservation by ensilage. Adjustment of the moisture 
contents and inoculation with LP were measures to address these presumed deficiencies 
and to improve the suitability for ensiling. Dry matter adjustment of pre-silage material 
in Experiment 4-1 led to reduced  concentrations of sugar, buffering capacity and total 
nitrogen due to low content of the absorbent (wheat straw) in these chemical 
components (not shown in data). Adjustment of DM content increased the WRC of 
daikon pre-silage material. The term WRC, defined as the amount of water retained by a 
known weight of fiber under the condition used (Robertson et al., 2000) or in other 
words, g moisture/g dry matter is employed as a novelty in this study. A major feature in 
the ensiling of particularly high moisture materials is the DM/WRC relationship 
vis-à-vis effluent production. From studies in our laboratory using low dry matter 
agriculture by-products, the WRC of silage materials is highly influenced by, and 
positively correlated to, the dry matter content (unpublished observation),.  In the 
present study, an upward adjustment of the DM of pre-silage material in Experiment 4-1 
from 64.0 to 175.1 g/kg (370 g/kg increase) led to about 810 g/kg increase in the WRC 
and an obvious effect on effluent retention in the silages (Fig. 4-3). Inoculation with LP 
reduced (P<0.01) the pH and final concentrations of lactic and acetic acids of daikon 
non-adjusted silages, obviously due to the initially high sugar concentration in them as 
typical of a lactic acid culture. This led to concomitantly, high effluent outputs with 
days in ensilage (Fig. 4-3) and consequently high nutrient losses (Table 4-3) relative to 
the moisture-adjusted silages. The amount of effluent produced from non-adjusted 
moisture silages with/without LP showed the non-feasibility of ensiling daikon 
by-product alone and underscored the utmost necessity of incorporation with absorbents 
prior to ensiling. The concentrations of VFA, apart from acetic acid, did not reveal 
differences between LP inoculations or otherwise, and were indicative of the relatively 
good preservation of the silages. This was particularly true with the absence of butyric 
acid, which is usually associated with badly preserved silages. Contents of silage VBN 
were low in all treatments and below the 80 g/kg of total N threshold suggested by 
Henderson (1993) of well preserved silage, a further indication of effective fermentation 
in the silages. The transitional behavior of the fermentation acids in the silages (bucket 
silos in Experiment 4-1) caused sharp decreases in pH and corresponding increases in 
lactic acid concentrations after 7 days of ensilage were typical of a good fermentation 
process (Woolford, 1984b). Between 14 and 30 days, a steady reduction in pH indicated 
stability, while gradual progression of lactic acid concentrations followed, reflective of 
substrate depletion with duration of storage. Growth of LAB peaked in the first 7 days 
in corroboration with the rise in lactic acid concentration in all silages (except 
moisture-adjusted silages), followed by decreases in LAB numbers. This phenomenon 
has been reported by several workers (Ashbell et al., 1987; Jonsson and Pahlow, 1984; 
Woolford, 1984b). The qualitative changes in lactic acid bacteria are generally attributed 
to their acid tolerance and acidifying potential (Woolford, 1984b).  The behavior of 
lactic acid bacteria during the later stages of ensilage revealed little information on the 
state of preservation and, as concurred by (Woolford, 1984b), pH is a more reliable 
indicator of whether or not further microbial development will occur. Moreover, lactic 
acid bacteria can produce acid after cessation of growth or when the population is 
decreasing (Woolford, 1984b). The attainment of a stable pH of below 3.80 after the 
first 7 days to a large extent suppressed LAB growth. Yeast population decreased with 
time due to sustained anaerobic conditions in the silage, and with inoculation, due to the 
elevated concentrations of lactic acid, in consent with Jonsson and Pahlow (1984), who 
reported that anaerobic ensiling results in continuous reduction in the number of yeast, 
especially lactate-utilizing yeasts. In the present study, high lactic acid concentrations 
especially during the first 7 days of ensilage may have inhibited certain metabolic yeast 
groups resulting in the decline in yeast numbers and not simply low pH. The low yeast 
numbers, however, augurs well for daikon silage fermentation, since their presence 
would otherwise in high numbers indicate the infiltration of air during the course of 
ensilage. The behavior of LAB and yeast in moisture-adjusted silages during the first 
week of storage cannot not be explained.  
In the first five days of aerobic exposure, the moisture adjusted silages in Experiment 
4-1 deteriorated faster due to higher dry matter content and to a lesser degree, to lower 
packing density of the pre-silage material which enabled easy infiltration of air causing 
rapid spoilage. After the fifth day, LP inoculated silages had faster rates of spoilage as 
indicated by high pH and butyric acid concentrations, probably due to increased activity 
of lactate assimilating yeasts (Filya et al., 2000; Jonsson and Pahlow, 1984). Care is 
therefore needed after unloading of daikon silage to forestall such losses. 
In Experiment 4-2, moisture levels were adjusted based on observations from the 
previous experiment. The hypothesis was that increasing the DM content to a level 
above that in Experiment 4-1 would lead to an increase in the WRC of the mixtures and 
consequently, to effluent retention in the silos. Although DM and WRC values were 
elevated, effluent outputs in DBP and DWB were more than the projected, suggesting 
that packing weight, density, and pressure exerted on the silages, or other factors such as 
fiber content (Offer and Al-Rwidah, 1989; O’Keily, 1991) and/or physical 
characteristics of ensiled material (Jones and Jones 1996) may have played a role in 
their ability to retain effluent. Daikon/wheat straw stemmed effluent production 
completely, while DWB, in spite of the high pre-silage DM, produced the most effluent. 
Straws are effective absorbents, but wheat bran and cereals in general have poor water 
absorptive capacities (Jones and Jones, 1996). It was obvious from the two experiments, 
however, that silage materials (singularly ensiled or with an absorbent material) with 
high water retention capacities tend to control effluent more effectively than those with 
lower water retention capacities. Although DBP had significantly lower pH and higher 
lactic acid contents compared with other treatments, silage fermentation in Experiment 
4-2 was generally restricted due to effect of the absorbents.    
  Aerobic stability of daikon silages with different absorbent in Experiment 4-2 was 
similar in character to the daikon wheat straw adjusted moisture of Experiment 4-1 as 
evidenced by sharp increases and decreases in pH and lactic acid contents, respectively, 
after 3 days of aerobic exposure of the silages. The rate of rise in pH and decline in 
lactic acid contents indicated a phenomenon that is common to HMBF; a possible 
proliferation of yeast numbers. Yeast, especially lactate assimilating types are mostly 
responsible for rapid decline in silage quality at feed out stage (Filya et al., 2000; 
Jonsson and Pahlow, 1984). DBW and DBH deteriorated faster, compared to DWB and 
DBP on aerobic exposure due to the state of the added absorbents (wheat straw and bean 
stalks) prior to addition to daikon by-product. The two absorbents are usually gathered 
from the field after harvesting of wheat and beans and thus normally come with some 
soil which may serve as a contaminant, in contrast with wheat bran and dried beet pulp, 
which are ‘cleaner’ products from factories. Another factor responsible for the quick 
deterioration of the two silages was their low packing densities which create air pockets 
that, with the least available substrate, provide fertile grounds for yeast growth and 
metabolism and consequently, enhance spoilage. 
4.4. Summary  
     These data have indicated that ensiling of daikon by-product is a viable venture 
and that the high moisture and buffering capacity not withstanding, daikon can be 
ensiled successfully using appropriate technology. Due to the high moisture content of 
daikon by-product, the use of bacteria or microbial inoculants is strongly discouraged 
since they increase effluent production. The use of in-silo absorbents, especially those 
capable of stemming effluent flow prior to ensiling of daikon by-product is sine qua non 
to the ensilage process.  The use of appropriate in-silo dry materials with the dual 
responsibility of stemming effluent flow and minimizing the effect of silage spoilage 
during feed out stage would be a great contribution to extending the shelf life of daikon 
by-product silage.  
   A study of the nutritive value of daikon by-product silage in vitro and in vivo is 
highly warranted. 
          
Chapter 5  Ensiling of brewer’s grain and apple pomace    5.1. Introduction      In the previous chapters, the ensilability of two representatives of HMBF from 
agro-industrial root and vegetable sources has been discussed. One of the fallouts of 
ensiling HMBF is effluent production. Effluent is produced from HMBF, whether 
ensiled alone (as in the potato pulp experiments) or with absorbents (as in daikon 
by-product), and the rate at which they are produced largely depends on the physical 
and chemical characteristics of the material used, the dry matter, and degree of 
consolidation of the silage material.  
   The previous experiments investigated the effect of chemical factors such as 
microbial inoculation and the use of absorbents in the control of effluent in HMBF. In 
the following experiments, the role of two other factors, pressure and packing density 
and their role on effluent production and retention using apple pomace and brewer’s 
grain as representatives of HMBF is discussed.  
5.2. Brewer’s grain 
      
  The use of brewer’s grain as animal feed is not new. It has been recognized as animal 
feed and a lot of reports on its nutritive value have been reported (Alawa et al., 1988; 
McCarthy et al., 1990; Chiou et al., 1998). 
  The purpose of this investigation, though, was to investigate the role of physical 
factors such as pressure and packing density on effluent flow and retention in brewer’s 
grain as a representative of HMBF, ensiled without or with dried beet pulp and wheat 
bran as in-silo effluent absorbents. 
5.2.1. Materials and methods 
   Fresh brewer’s grain procured from a local beer distillery was used in this study. The 
grain used in the distillery factory was wheat, and as such, the spent grains bore a 
typical brown color. Three treatments were prepared with brewer’s grain without an 
additive (Control, C),   with dried beet pulp (BP) and with wheat bran (WB). The 
mixing ratios of brewer’s grain to dried beet pulp and wheat bran were 4: 1 for BP and 
WB (w: w basis), respectively. A freeze-dried commercial lactic acid bacteria inoculum, 
Lactobacillus plantarum (Ecosyl Products Ltd, Tokyo, Japan) was added to all the 
treatments including C at the rate of 0.75 g/100kg in fresh matter (as per manufacturer’s 
instruction) to hasten lactic acid fermentation.  These treatments were separately and 
uniformly mixed and ensiled in bucket silos with drain taps for effluent measurement as 
previously described in Experiment 2 of Chapter 4. Three pressure regimes (P0, P1 and 
P2) were allotted to each treatment by placing weights (0, 15 and 30 kg) on top of each 
silo of the three treatments (C, BP and WB).  A total of 27 silos were thus prepared 
consisting of three treatments under three pressure regimes with three replications.  
Effluent was measured during 30 days of ensiling period. Samples from all treatments 
were taken after opening of the silages for subsequent chemical analyses.  
5.2.1.1. Chemical analyses 
  General fermentation characteristics were analyzed with procedures described 
previously in earlier chapters.  
5.2.1.2. Calculations  
   Packing density was calculated as mass of fresh pre-silage material (kg)/volume of 
the silo (m3). Pressure at the bottom of the silages was calculated as force (weight of 
material)/area (cm2) of silo. 
5.2.1.3. Statistical analysis 
   Silage fermentation data were analyzed using ANOVA in a randomized block design 
using the General Linear Model Procedure of SAS (1996) computer package. Contrasts 
between treatment and pressure levels were performed and means differences were 
compared for all variables. Significant differences among treatments were accepted at 
P<0.05. 
5.2.2. Results  
    The chemical characteristics of pre-silage material and silages are presented in Table 
5-1. The material used in the current experiment was quite low in moisture (760.1 g/kg). 
Adjustments with dried beet pulp and wheat bran further reduced (P<0.05) the moisture 
content to about 700 g/kg. This adjustment in turn reduced (P<0.05) the packing density 
of each silo from 957 kg/m3 to an average of 850 kg/m3 and was significant (P<0.05) 
among adjusted and non-adjusted moisture treatments. PWRC was higher in WB but 
highest in BP, relative to C. The pH of all silages were generally low (below 3.80), 
indicating an acceptable fermentation process. Lactic acid concentrations were, however, 
low in all treatments despite addition of an inoculum of Lactobacillus plantarum as a 
fermentation booster. The effect of pressure exerted at the bottom of the silos was 
obvious as the weights on the silos were increased. This had remarkable increases 
(P<0.05) in effluent outputs in the silages especially in the control silages. Addition of 
dried beet pulp to the brewer’s grain stemmed effluent production completely, 
regardless of the pressure level.  
5.2.2.1. Effect of absorbents  
   Addition of the absorbent (treatments) materials led to clear decreases (P<0.05) in 
pH, lactic acid contents and effluent production in the silages. Effects on moisture, 
pressure and packing density were inconsistent, with significant differences (P<0.05) 
only between C and the absorbent added treatments (BP and WB). 
5.2.2.2. Effect of pressure  
    Effect of pressure levels on fermentation characteristics was inconsistent. The only 
obvious (P<0.05) effect was on lactic acid and effluent production at P2; otherwise 
moisture, pH, or packing density did not change significantly.   
Table 5-1. Effect of absorbents and pressure levels on chemical characteristics of brewer’s grain after 30 days of ensilage. 
 Treatments Pressure levels  Contrasts# 
 C BP WB  P0 P1 P2 SEM1 2 3 4 5 6 
Pre-silage characteristics              
Moisture (g/kg) 760.1 698.3 701.2 - - - - - - - - - - 
pH 5.03 5.17 6.00           
PWRC (g/100g) § 57.6 89.4 71.9 - - - - - - - - - - 
Silage characteristics              
  Moisture (g/kg) 736.5a 679.8b 683.5b 707.5a 702.1a 690.4b 2.40** ** NSNS****
  pH 3.41c3.75a3.65b3.59a3.60a 3.61a 0.14** ** **NSNSNS
  Lactic acid (g/kg DM) 12.6c 13.4b 15.8a 14.4a 13.9b 13.5b 0.14** ** ******NS
  Effluent (g/kg ensiled material) 20.6a 0.0c 5.6b 7.7b 8.9ab 9.5a 0.49** ** **NS* NS
              
Pressure (g/cm2)§ 45.7a 42.9b 42.9b  22.5c 43.9b 65.1a 0.11* * NS******
Packing density (kg/m3) § 957.0a 850.2b 849.6b 881.9a 889.5a 886.3a 4.32** ** NSNSNSNSC, brewer’s grain without additive; BP, WB, brewer’s grain added with dried beet pulp and wheat bran, respectively; P0, P1,P2, pressure levels exerted at the bottom of the 
silages; PWRC, potential water retention capacity. 
Values apart from pre-silage characteristics, are least square means (n=27). Different letters following figures within a row are statistically significant (P<0.05); NS, not 
significant P>0.5; *, P<0.05; P<0.01. 
# 1, C vs. BP; 2, C vs. WB; 3, BP vs. WB; 4, P0 vs. P1; 5, P0 vs. P2; 6, P1 vs. P2. 
§ At day 0 of ensiling period. 
5.2.3. Discussion  
    The low moisture content of the brewer’s grain used in the current experiment 
would not have been the most ideal representative material of HMBF for this study 
given the objective of the experiment, but appropriate in the context of variety, since 
different materials produce different results which enhance the validity of the results.  
  Although pH values were generally low, the fermentation quality of the silages was 
restricted as indicated in the general low lactic acid concentrations, a result that 
obliterated the rather significant differences among the treatments. The low lactic acid 
content in the current experiment is in consent with Nishino et al., (2001), on brewer’s 
grain sampled from different factories in Japan. Several factors could be attributed to 
this, among them the chemical characteristics of the material. Wheat grain, the source 
material from which the by-product was derived, generally contains mainly starch as the 
principal carbohydrate source and has very little sugar (data not shown). After 
extraction of starch during the distillery process, the main nutrients remaining are 
mostly proteins and fiber (about 270 and 630 g/kg DM, respectively (NARO, 2001; 
Nishino et al., 2001). These, especially the former, are antagonistic to lactic acid 
fermentation due to the high buffering capacities of proteins in general (McDonald and 
Henderson, 1962). Brewer’s grains undergo heating processes which generally 
neutralize or nullify the effect of enzymes that may aid the ensiling process. 
Additionally, yeast is added during the brewery process which may have been present in 
large numbers in the by-product thereby impeding lactic acid production during ensilage. 
The fact that even addition of an inoculum of Lactobacillus plantarum did not enhance 
lactic production is an indication of this fact.    
   On visual appraisal, however, the quality of the silages were not drastically different 
from the pre-silage material, a fact that strengthens a personal assumption that brewer’s 
grain is better utilized fresh. Packing density and pressure have positive relationships 
with effluent production in silages, especially those ensiled from low dry matter 
materials (McDonald et al., 1960; Zimmer, 1974; Peters and Weissbach, 1977), and the 
results of this study amply demonstrated that. However, a combination of the two 
factors and PWRC appears to have had the most potent effects. The relationship 
between these factors and effluent production is fully discussed in Chapter 7.  
5.3. Apple pomace 
       
   Apple pomace is a by-product resulting from the extraction of juice from fresh 
apples. It comprises the seeds, peel, core and stem along with insoluble material. The 
pomace is characteristically high in moisture, above 700 g/kg (Kennedy et al, 1999), but 
a rich source of sugar and pectin and it has been acknowledged as ruminant feed 
(Smock, 1950; Fontenot et al., 1977; Toyokawa et al., 1977; Alibes et al., 1984).  
    The purpose of this study was to investigate the effectiveness of in-silo absorbents 
at ensiling, on the fermentation, effluent control and aerobic stability of apple pomace 
silage.  
   5.3.1. Material and methods          
  Fresh apple pomace was procured from an apple juice factory using local apple 
varieties and located in the Tohoku (northern) region of Japan. Dried beet pulp, rice 
bran and wheat bran were used as in-silo absorbents to adjust the pomace to lower 
moisture levels at ratios of 87.5:12.5, 80:20 and 76: 24 (pomace: absorbent on w:w 
basis) for the three materials, respectively. Four treatments consisted of apple pomace 
without additive (AP), with dried beet pulp (ABP), with rice bran (ARB), and with 
wheat bran (AWB) with three replications for each treatment. These were filled into 
bucket silos with a drain tap and ensiled following procedures similar to those described 
in the previous experiment. A weight of 15 kg was placed on each silo to provide an 
average pressure of 43 g/cm2 at the bottom on all silos. The silos were kept at room 
temperature for a 30-day ensiling period with regular measurement of effluent. After 
opening, silages were sampled for chemical analyses of the fermentation quality. 
Additionally, composite samples were taken from each treatment and exposed but 
protected from contamination of flies and rodents, at room temperature and sampled on 
the 2 nd, 4 th, 6 th and the 7 th day of exposure for the determination of the aerobic 
stability of the silages.  With the exception of pre-silage moisture and pH, which were 
determined from bulk samples, all treatments were sampled from each silo for chemical 
analysis. 
5.3.1.2. Chemical analyses 
   Pre-silage and silage chemical analyses were determined using procedures described 
in the previous experiment. 
5.3.1.3. Statistical analyses  
   Silage fermentation data were analyzed using Excelstats for Windows 2004 
(Microsoft Corp, USA). Significant differences among means were accepted at P<0.05 
level. 
    5.3.2. Results        
   The chemical characteristics of apple pomace and silages are given in Table 5-2. It is 
evident that given the high moisture of the pomace material (918.7 g/kg), adjustments to 
lower levels were inevitable. ABP, ARB and AWB were adjusted to 821.2, 708.2 and 
733.7 g/kg, respectively. These adjustments, in turn, reduced the PWRC values as 
shown in Table 5-2. The original low pH of the pre-silage material (3.75) facilitated the 
ensiling process with even lower values (below 3.60) in all the silages. Lactic acid 
content in the silages indicated a satisfactory fermentation process and was highest in 
AP, followed by ARB, AWB and ABP in that order. VFA in the silages were generally 
low, with no differences (P>0.05) among treatments. This result, especially for acetic 
and butyric acids, was indicative of the low incidence of secondary fermentation in the 
silages. Bulk density at ensiling was lower in ABP and AWB, respectively, relative to 
AP and ARB.  
   Effluent output was highest in AP (391.1 g/kg FM of ensiled material), followed by 
AWB and ARB, respectively. ABP silage had the lowest effluent production. 
   Aerobic stability of the silages was determined with changes in pH and lactic acid 
contents during the 7-day exposure period (Fig. 5-1) as indices. There was a steady rise 
in pH of all silages until the 6th day of aerobic exposure. On the 7th day, the rise was 
sharp except for AP. Conversely, lactic acid decreased in a similar pattern. At the end of 
the period of investigation, AWB and ABP were the most unstable, judging from the pH 
and lactic acid contents. 
              
Table 5-2. Chemical characteristics of apple pomace before and after ensiling for 30 days. (Values are given in g/kg DM, unless otherwise stated)    Treatments§       
Characteristics AP ABP ARB AWB 
Pre-silage      
 Moisture  918.7 821.2 708.2 733.7 
 pH 3.75 3.99 4.74 4.20 
 PWRC (g/100g ) 50.9c 90.7a 76.0ab88.2b 
 Packing density (kg/m3) 886.9a 818.8b 914.1a 776.3b 
Silage     
 Moisture  892.0a 834.4b 697.9d 744.8c 
 pH 3.43b 3.53a 3.51a3.48ab 
 Lactic acid  116.6a 62.6d 70.9b 68.9bc 
Volatile fatty acids     
   Acetic 16.8ab 19.8a 14.4ab11.3b 
   Propionic 3.40 2.41 2.05 1.75 
   Valeric 3.01 4.49 3.71 5.59 
   Butyric 2.37 1.32 0.34 0.22 
 Effluent (g/kg FM) 391.1a 37.2d 73.2bc93.0b 
§AP, apple pomace without additive; ABP, ARB, AWB, apple pomace added with dried beet pulp, 
rice bran and wheat bran, respectively; PWRC, potential water retention capacity. 
Except pre-silage moisture and pH, values are means of three silos; letters following figures 
within a row indicate a statistical difference (P<0.05). 
                  
Days after aerobic exposure
Lactic acid (g/kg DM)
    Fig 5-1. Changes in pH and lactic acid concentrations in apple pomace silages during 7 days of 
aerobic exposure. (◆), apple pomace without additive, (AP); (■), apple pomace + dried beet 
pulp, ABP; (▲), apple pomace + rice bran, ARB; (●), apple pomace + wheat bran, AWB. 
       5.3.3. Discussion   
   In the current study, adjustments of the moisture content were made based on results 
from the previous one. Dried beet pulp had proven its capacity in completely stemming 
effluent flow in the brewer’s grain experiment, and the assumption was that applying 
similar mixing ratios between fresh material and absorbents would yield similar results. 
Results from effluent outputs in the current experiment were, however, contrary to the 
projected, indicating that calculations based solely on dry matter/moisture assumptions 
are insufficient.  The PWRC results, in confirmation of previous results, gave a strong 
indication that barring other major determining factors such as the moisture content, 
Days after aerobic exposure
effluent production from any given silage material is largely a function of its PWRC 
(See Chapter 7).  
  The good fermentation quality of the AP silage (especially the low pH content) is in 
consent with earlier reports (Kennedy et al., 1999; Rumsey and Lindahl, 1982; Osaka, 
2001; Pirmohammadi et al, 2006) on apple pomace silage. This, in addition to its low 
buffering capacity (Chapter 2) and high lactic acid contents, in agreement with Anrique 
and Viveros (2002), Alibes et al., (1984) and Wyss (2003) indicated that apple pomace 
is an easy-to-ensile material. The high lactic acid content in AP silage was primarily due 
to the high WSC (Chapter 2). Other treatments had fairly low pH and high lactic acid 
contents due to effect of the high percentage of apple pomace material of the mixtures. 
The generally low contents of acetic and butyric acids typified the good fermentation 
quality of the silages.  
  Packing density was positively related to effluent production in the silages, as 
previously discussed in the study of brewer’s grain silage. Despite the high pre-ensiling 
packing density, AP silage produced the most effluent. The spongy-like nature of apple 
pomace fiber, with its ‘easy-to-absorb’ and ‘easy-to-release’ moisture capacity, may be 
largely responsible for this. Moreover, fibers derived from fruits and vegetables have a 
high proportion of soluble dietary fibers (Herbafood, 2002) and possess functional 
properties such as swelling and binding capacities. These properties are related to the 
porous matrix structure formed by polysaccharide chains which can hold large amounts 
of water through hydrogen bonds (Dawkins et al., 2001; Kethireddipalli et al., 2002). 
From this study, however, the high effluent from AP silage may be related to the 
pressure exerted on the silo through the weight, rendering the binding capacity of apple 
pomace vulnerable. In contrast, AWB, with significantly lower density, produced more 
effluent than ABP. Cereals like wheat bran are known to have poor absorptive capacities 
(Jones and Jones, 1996). However, given the low moisture and packing density, the 
relatively high effluent produced from AWB provides a rather contrasting relationship 
which warrants further investigation.  
  The silages were relatively stable until the 6th day of aerobic exposure, probably due 
to the low pH and high lactic acid contents which may have protected them from 
spoilage microorganisms and consequently enhanced the shelf life.  On the 7th day, 
ABP and AWB were the most unstable, probably due to their lower packing densities 
which may have made them susceptible to more air penetration, infiltration and 
proliferation of aerobic bacteria causing deterioration of the quality (Chapter 4). The 
aerobic stability of the silages, if anything, has proven that apple pomace silage could 
have a longer shelf life compared with other HMBF studied so far.  
5.4. Summary  
   High packing densities and pressures exerted on fresh brewer’s grain and fresh apple 
pomace, ensiled alone or in combination with absorbents prior to ensiling, generally 
increased effluent production from the silages. This was, however, influenced by the 
moisture content, the potential water holding capacity and type of material and 
absorbent used.   
More experiments using different absorbents and pressure levels are needed to clarify 
this hypothesis. 
Chapter 6    Nutritive value of HMBF   6.1. Introduction       
   In the previous chapters, the possibility of extending the shelf life by ensilage of 
various HMBF has been discussed. The nutritive value of silage depends on three major 
factors: voluntary intake, its nutrient digestibility and feed efficiency (Woolford, 1984d). 
The ambit of the silage-animal interface is, however, intrinsically the nutritive value or 
the acceptability by livestock who are the final determinants of the quality of the silages. 
Animal responses are therefore vital in the final equation of the fermentation quality of 
the silages. Not all well fermented silages have increased intakes of silages; in fact, 
several other factors such as type of silage, length of storage, physiological state of the 
animal etc. all play a role. However, there is ample evidence that well preserved silages 
(grass or whole crop), including those treated with or without an inoculum of lactic acid 
bacteria, have higher digestibility (McDonald et al., 1991c) and voluntary dry matter 
intakes (Gordon, 1989a) and would be more palatable and preferred by animals as 
opposed to ‘badly’ preserved ones (Michalet-Doreau, 1975).  Agricultural by-product 
silages, in contrast to grass or whole crop silages, are most likely to differ widely in 
terms of voluntary intake and palatability due to their ‘reduced’ nutrient state prior to 
ensilage. This may have an effect on the nutritive value and, consequently, on dry matter 
intake.  
   In the following experiments, the nutritive value of two representatives of HMBF 
silages were evaluated in vivo using sheep. 
6.2. Nutritive value of potato pulp silage        
   The objective of this study was to evaluate the intake and nutrient digestibility of 
potato pulp ensiled without/with microbial inoculants.  
6.2.1. Materials and methods 
6.2.1.1. Silage preparation  
   Silage preparation including inoculants, type of silo, and duration of storage has 
been described in Experiment 2 of Chapter 3.  The four silage treatments, potato pulp 
silages without additive control (PP), inoculated with Lactobacillus rhamnosus (L) 
alone (PL), inoculated with Rhizopus oryzae (R) alone (PR) and inoculated with a 
combination of R and L (RL), were placed into small polyethylene bags as 4 kg each, 
kept at -10oC and thawed on use in a digestion trial. 
6.2.1.2. Digestibility trial: animals, feed and experimental design   
   Four mature wether sheep (Suffolk breed) with initial live weights of 46 ± 4 kg were 
used in a 4 x 4 Latin square design digestion trial using the silages, Italian ryegrass 
(Lolium multiflorum) hay and soybean meal. After being de-wormed of endoparasites 
and treated against ectoparasites, the animals were kept in metabolic cages facilitating 
the separate collection of feces and urine. The procedures used in this trial were 
approved by Obihiro University of Agriculture and Veterinary Medicine Committee for 
Animal Use and Care, where the trial was conducted. A standardization period of 10 
days was allotted to the animals during which all animals were fed control silage ad 
libitum to allow for 100 g/kg refusal with regular hay and soybean meal supplements. 
This was followed by adaptation and collection periods of 7 and 5 days, respectively, 
during which time experimental diets comprising the control and treated silages, hay 
and soybean meal, representing 600, 250 and 150 g/kg of daily ration, respectively, 
were offered at 20 g/kg BW twice daily at 08.00 and 17.00 h. The hay was chopped to 
about 2 cm lengths with a mechanical forage cutter and mixed with the silages and 
soybean meal prior to offer to the sheep. The hay and soybean meal supplementation 
rates (250 and 150 g/kg DM on as fed basis of daily intake, respectively) contained an 
average of 130 g/kg crude protein and 18 MJ/kg DM of gross energy respectively, 
enough to satisfy the DCP and energy requirements for sheep according to NRC feeding 
standard (NRC, 1985).  Mineral block (Nihonzenyaku Ltd., Tokyo, Japan) and 
drinking water were provided ad libitum. The mineral block contained: Fe 1232 mg/kg, 
Cu 150 mg/kg, Zn 500 mg/kg, Mn 500 mg/kg, Se 15 mg/kg and Na 382 mg/kg. During 
each of the 5-day collection periods, feed offered from each animal were sub sampled, 
bulked and stored at -10oC for subsequent chemical analyses. Total feces from each 
animal were weighed, and kept at -10oC and bulked at the end of each collection period. 
Portions for DM determinations were taken from composite samples and the rest frozen 
at -10oC until analyzed.  At the end of a collection period, the animals were released 
into an enclosed pen for exercise for 24 hrs and fed a mixture of the four treatment diets. 
The following collection period was preceded by 7 days of adaptation period where the 
experimental diets were fed. This was to rid the rumen of traces of the previous diets 
and to allow the rumen bacterial population to respond to the change in diet. The 
process was repeated until the end of the fourth period.  
6.2.1.3. Chemical analyses  
   Analyses of chemical components in feed has been previously described (Chapter 3). 
Analyses of chemical components in feces were done from dried samples following the 
same procedures as in the feed analyses.  
6.2.1.4. Calculations  
   Digestible energy (DE) was calculated as the difference between gross energy in 
feed offered and in feces voided. Total digestible nutrients (TDN) in feed were 
calculated from the DE using the regression equation of Harris et al. (1972): 
                   TDN (for sheep) =  .0 04409DE  
where TDN is the total digestible nutrients of feed in g/kg DM; DE is the digestible 
energy in MJ/kg DM.  
The TDN of each treatment diet was calculated as the sum of TDN of the treatment feed, 
hay and SBM. The TDN of silages (corrected TDN of silages) was calculated as the 
difference between the TDN of diets and the sum of TDN of Italian rye grass hay and 
SBM (values based on Standard Tables of Feed Composition in Japan, 2001) of the 
treatment diets. The DE of silages was calculated in a similar manner. 
The nutritive value (TDN and DE) of potato pulp silages were estimated by 
extrapolation from the TDN and DE values of the diets minus those of Italian rye grass 
hay and soybean meal in the diets based on NARO (2001) and dividing by the rate of 
inclusion of the individual silage. 
6.2.1.5. Statistical analyses  
   Silage fermentation data were analyzed using ANOVA in a randomized block design, 
while data from the digestibility trial were analyzed as a 4 x 4 Latin square design using 
the General Linear Model Procedure of SAS (1996) computer package. Means 
differences were compared for all variables and P<0.05 was considered as statistically 
significant. 
6.2.2. Results  
   The chemical composition of potato pulp, silages and hay and soybean meal are 
shown in (Table 6-1). Potato pulp had low DM, CP, fiber and fat contents but was fairly 
high in organic matter, starch, pectin and gross energy. The silages were not markedly 
different from the material with the exception of sugar that increased about 4-fold that 
of the pre-ensiling content. The CP and NDF contents of the silages were generally low 
(average of 50 and 350 g/kg DM, respectively). Supplementation with soybean meal 
and Italian rye grass hay increased the CP and NDF considerably to about 130 g/kg DM 
and 380 g/kg, respectively (Table 6-2). Dry matter intake was higher (P<0.05) in PL 
compared with the control (Table 6-3). There were no statistical differences among 
treatments in the digestibilities of DM, OM, NDF and energy in the diets which were on 
average 0.70, 0.78, 0.64 and 0.74, respectively. Crude protein digestibility was higher 
(P<0.05) in sheep fed L inoculated compared to the control silage, but there were no 
significant effects among the inoculant added silages.   
   The DE and TDN in the diets averaged 13 MJ/kg DM and 709 g/kg DM, 
respectively, while those of PPS were on average 13.3 MJ/kg DM and 724 g/kg DM, 
respectively. There was no effect of inoculation on the nutritive value of the diets and 
PPS. No orts were recorded during the entire experiment. 
                                     
Table 6-1. Chemical compositions of potato pulp, hay, soybean meal (SBM) and potato pulp silages without additive (PP), inoculated with 
Lactobacillus rhamnosus alone (PL), Rhizopus oryzae alone (PR) and a combination of the two inoculants (RL) in g/kg DM§, except 
where indicated. 
                          
    Silages1 
  Potato pulp Hay SBM PP PL PR RL 
 Dry matter (g/kg) 170 842 864 158 161 159 160 
 Organic matter 974 943 934 974 969 974 969 
 Crude protein   49.0 91.0 502 49.0 49.6 50.3 49.9 
 Neutral detergent fiber         353 617 119 353 350 365 340 
 Acid detergent fiber 342 334 78 337 334 334 327 
 Ether extract 6.2 13.0 17.0 6.1 6.5 6.1 6.3 
 Starch 206 - - 195 196 208 214 
 Pectin 213 - - 213 215 208 199 
 Sugar 5.0 - - 16.0 20.0 13.0 19.0 
Gross energy  (MJ/kg DM) 17.1 19.0 20.0 17.7 17.3 17.4 17.4  Except with potato pulp, hay (Italian rye grass hay) and (SBM) soybean meal, values means are of four silos.  
§DM = dry matter; MJ, mega joule.  
1 PP, potato pulp silage without additive; PL, PR, Lactobacillus rhamnosus (L) and Rhizopus oryzae (R) inoculated potato pulp silage, respectively; RL, R and L inoculated potato pulp silage. 
Table 6-2. Chemical composition of diets used in the digestibility trial. Values are 
given in g/kg dry matter unless otherwise stated. 
                                        
 Diets§ 
  PP PL PR RL 
    Dry matter (g/kg) 43.5 43.7 43.6 43.6 
    Organic matter  960 958 960 957 
    Crude protein  127 128 128 128 
    Neutral detergent fiber  384 382 391 376 
    Acid detergent fiber  297 296 295 291 
    Gross energy (MJ/kg DM) 18.3 18.1 18.2 18.2 
§Diets comprise 600 g/kg DM of potato pulp silages, 250 g/kg DM of hay and 150 g/kg DM of
soybean meal; figures represent the sum of values of single feeds; PP, potato pulp silage without 
additive; PL, PR, Lactobacillus rhamnosus (L) and Rhizopus oryzae (R) inoculated potato pulp
silage, respectively; RL, R and L inoculated potato pulp silage.  
 Table 6-3. Dry matter intake, apparent nutrient digestibility and nutritive value of diets and potato pulp silages (PPS) 
                            
 Diets1   
 PP PL PR RL S.E.M Significance
Dry matter intake (kg /d) 1.07a 1.16b 1.10ab 1.13ab0.02 * 
Apparent nutrient digestibility         
   Dry matter  0.758 0.783 0.778 0.767 0.09 NS 
   Organic matter 0.768 0.790 0.787 0.776 0.08 NS 
   Crude protein 0.616a 0.678b 0.652ab 0.645ab0.16 § 
   Neutral detergent fiber 0.617 0.651 0.650 0.645 0.17 NS 
   Energy 0.724 0.748 0.747 0.732 0.11 NS 
Nutritive value of diets       
   Digestible crude protein (g/kg) 80.0 94.0 85.0 84 4.99 NS 
   Digestible energy (MJ/kg DM) 12.9 13.2 13.2 12.9 0.24 NS 
   TDN (g/kg DM) 701 716 717 701 12.66 NS 
Nutritive value of silages       
   TDN of silages (g/kg DM)# 711 736 737 711 12.66 NS 
   Digestible energy of silages (MJ/kg DM)# 13.1 13.6 13.6 13.1 0.24 NS 
  Means in a row with different letters differ significantly (P<0.05); NS, not significant; *, P<0.05. 1Same as in Table 6-2.   # Calculation based on Standard Tables of Feed Composition in Japan (2001). § P = 0.1866, although means were separated using least square means with a level of 0.05 and P value was 0.1866, but difference between treatmentswere significantly different. 
6.2.3. Discussion  
   The fermentation qualities of the silages, whether treated with the microbial inocula 
or not, were satisfactory and has been discussed previously (Chapter 3) in detail.   
Addition of hay and soybean meal to the silages prior to feeding increased not only the 
DM of the diets but the texture, which was highly appreciated by the sheep. The low CP 
and NDF contents in the silages were due to their low content in the potato pulp 
pre-silage material. In order to make up for the low N and fiber contents, soybean meal 
and Italian rye grass hay were introduced into the diets. These augmented the diet CP 
and NDF contents to levels in tandem with NRC feeding standard (NRC, 1985). The 
pulp material used in the present trial contained fair amounts of easily digestible 
carbohydrates such as starch and pectin, though the content of the former was low 
compared to the conventional potato pulp of about 370 g/kg DM (Mayer and 
Hillebrandt, 1997), and could be due to the mode and rate of starch extraction, which 
differs among factories. The diets contained an average gross energy of 18 MJ/kg DM, 
fairly sufficient for the energy requirements and ages of the sheep used in the trial. With 
the exception of PL which was higher (P<0.05) compared with PP, DM intake among 
silages was not affected by addition of the inoculants. Similar improvements in DM 
intakes of silages treated with an inoculum of lactic acid bacteria, especially with 
Lactobacillus plantarum, have been reported in grass silages in particular (Gordon, 
1989a; Gordon, 1989b), especially where there were improvements in the quality of the 
silages, although others (Hooper et al., 1984; Steen et al., 1989) have reported increased 
DM intakes with commercial inoculants with no obvious improvement in fermentation, 
as was the case in this study. It is noteworthy that the quality of the silages used in the 
digestibility trial was good and was readily eaten by the animals.  
   Crude protein digestibility was higher (P<0.05) in sheep fed silage inoculated with L 
compared to those fed control, a result that could not be explained. Moreover, other 
treatments, though not statistically significant, had numerically higher CP digestibilities 
compared with the control. Evidence is scanty on the efficiency of nitrogen utilization 
between treated and untreated silages. However, the phenomenon observed in the 
current trial could be related to the general improvement in nitrogen retention with 
feeding of PPS that may have improved microbial nitrogen synthesis within the rumen 
and/or reduced silage nitrogen degradability within the rumen. In a later study, and in 
agreement with these results, Aibibula et al. (2004) found no differences in nitrogen 
retention in sheep fed Lactobacillus rhamnosus and Rhizopus oryzae inoculated PPS 
based diets. However, it was observed that the concentration of rumen ammonium 
nitrogen and serum urea nitrogen decreased in PPS based diets compared with alfalfa 
hay, which led to consequent decreases in nitrogen excretion into the urine of sheep. 
Rooke et al. (1989), however, observed a marked difference in both nitrogen 
digestibility and nitrogen retention in sheep between treated and untreated grass silages. 
Studies are therefore warranted to investigate the influence of bacterial inoculants, 
especially Lactobacillus rhamnosus, in N digestibility with PPS.  
   Woolford (1984d) observed that starch-based materials when available in large 
proportions and used as material for ensilage would influence the nutritive value if they 
survive the ensiling process, since they would be available to amylotic organisms in the 
rumen. In the present study, though, the high DM contents of NDF and pectin, in 
addition to the starch contents of potato pulp, generally contributed to the high TDN 
values of the diets.  Dry matter digestibility and nutritive value were not affected by 
the inoculants (Table 6-3), a result that was confirmed in another study with sheep 
(Aibibula et al., 2004). The DM digestibility in the present study is in consent with the 
findings of Aibibula et al. (2004), who reported a value of 0.75 in sheep fed potato pulp 
silage based diets. The DE in this study is comparable to the values of the National 
Agricultural Research Organization (NARO, 2001) of 12.7 MJ/kg DM, but slightly 
higher than those reported by Aibibula et al. (2004) and Hanada et al. (2004), which 
were 12.1 and 10.3 MJ/kg DM, respectively.  The TDN values of PPS in the present 
trial are in good agreement with those of NARO (2001). The digestibility and nutritive 
value of PPS is comparable to other by products like beet pulp and citrus pulp (NARO, 
2001; O’Mara et al., 1999; Deaville et al., 1994). 
6.2.4. Conclusion 
    The nutritive value of potato pulp silage was not influenced by inoculation with 
Lactobacillus rhamnosus and Rhizopus oryzae or their combined application. The high 
nutritive value of potato pulp silage compares to other agricultural by-products such as 
citrus pulp and beet pulp and as such could be a useful feed ingredient in ruminant diets.  
                
6.3. Intake and preference of daikon by-product silage      In earlier chapters (2 and 4), the feasibility of ensiling daikon (Oriental radish), 
Raphanus sativus L. has been discussed in detail. Its nutritive value until now (Okine et 
al, 2006b), was unknown. Daikon, apart from its high moisture content, which may be a  
limitation on its use as a feed for ruminants, contains a characteristically pungent smell 
and aroma (Friis and Kjaer, 1966) and a burning sensation on the tongue when eaten 
fresh (Lindsay, 1985). These characteristics may have an effect on voluntary intake of 
daikon by-product silage.   
   The objective of the current study was to explore the possibility of using daikon 
by-product silage as ruminant feed through intake and preference test using sheep.  
 6.3.1. Materials and methods   6.3.1.1. Ensiling of daikon by product    Daikon was adjusted to lower moisture into with three replications each with dried 
beet pulp (DBP), dried bean stalks and husks (DBH) and wheat straw (DWS) as in-silo 
effluent absorbents (see Table 4-4, Chapter 4). A fourth treatment, daikon/wheat bran 
silage (DWB), was omitted from the group meant for the animal preference test. The 
daikon: absorbent mixing ratio was 4:1 (w:w) for all treatments and the same protocol 
for mixture and the packing density. The only differences were the type of silo used and 
packing density. In this case, 70 kilograms of the three treatments, DBP, DBH and DWS 
were filled into separate polythene bags and inserted into large plastic container silos 
and pressed with weights similar to that of the bucket silos in Experiment 2 of Chapter 4. 
The silages were kept at room temperature and opened on the 30th day of ensilage.  
6.3.1.2. Silage intake and preference test 
Three mature wether sheep weighing 55±5 kg were used in a 3 x 3 Latin square 
design (3 sheep x 3 feeds x 3 feeding positions) to study feed intake and preference  of 
the silages. The sheep were kept in metabolic cages for 19 days with an adjustment 
period of 10 days during which animals were fed alfalfa hay (in pellet form) as a basal 
diet (24.0 g/kg body weight (BW) in dry matter per day) with gradual introduction of a 
mixture of the experimental silages to allow for a 100 g/kg feed refusal. This was 
followed by three experimental periods of 3 days each as follows: each day 1.20 kg of 
each of the three types of silages (this amount was determined based on intake of silages 
during the adjustment period) was separated into three trays and offered simultaneously 
to a sheep at 08.00 h. A basal diet of alfalfa hay as in the adjustment period was offered 
at 16.00 h every day. Water and mineral block were provided ad libitum. The mineral 
block contained: Fe 1232 mg/kg, Cu 150 mg/kg, Zn 500 mg/kg, Mn 500 mg/kg, Se 15 
mg/kg and Na 382 mg/kg. Voluntary intake of silages was recorded after every 1, 2, 8 
and 24 h post feeding on all the test days.  Silage preference was determined as the 
highest feed intake time after silage allocation.  
6.3.1.3. Chemical analysis 
    Analyses of chemical components in feed have been previously described (Chapter 
6.3.1.4. Statistical analyses 
   Silage fermentation data were analyzed using ANOVA in a randomized block design 
using General Linear Model Procedure of SAS (1996) as previously described in 
Chapter 4. Data from the intake and preference test were analyzed as a 3 x 3 Latin 
square design with type of silage, period and feed position as factors. Means differences 
were compared for all variables and P<0.05 was considered statistically significant. 
6.3.2. Results 
6.3.2.1. Silage intake and preference test 
   The chemical compositions of feed used in the preference test are presented in Table 
6-4. Alfalfa hay was offered at 24.0 g/kg BW in DM/day/sheep, while the silages were 
offered at 1.20 kg in fresh matter per animal. Thus, the diets contained on average 877 
g/kg in dry matter, 887, 202, 464, 329 and 29 g/kg DM in OM, CP, NDF, ADF and EE, 
respectively, and 19.3 MJ/kg in gross energy (calculated from the chemical components 
of alfalfa hay and those of the single silages). These figures meet or exceed the daily 
maintenance requirements for sheep (NRC, 1985). Daikon/wheat straw and DBH 
silages had higher NDF and ADF but lower CP and EE contents compared with DBP 
silage. 
Silage preference was, to a large extent, influenced by its chemical composition 
(Table 6-5). Silage intake was significantly higher (P<0.01) in DBP, followed by DBH 
and DWS in that order for the first 2 hours after feed allocation; however, at 8 and 24 h, 
there were no statistical differences in intakes of DBP and DBH silages, respectively. 
Daikon/wheat straw silage was least in terms of intake and preference. Intake of silages 
between periods (except after 1 h of feed allocation) and effect of feed positions were 
not significant (P>0.05). Alfalfa hay was readily consumed immediately when offered 
to the sheep and as such did not feature in the preference test. 
  Table 6-4. Chemical composition of alfalfa hay and daikon by-product silages used for 
feed palatability/preference test 
   Silages§ 
  Alfalfa hay DBP DWS DBH 
Dry matter (g/kg) 881 179 140 145 
Organic matter (g/kg DM) 890 903 905 904 
Crude protein (g/kg DM) 211 143 69 92 
Neutral detergent fiber (g/kg DM) 428 414 725 595 
Acid detergent fiber (g/kg DM) 301 265 492 528 
Ether extract (g/kg DM) 27.0  16.5 52.5  39.3 
Gross energy (MJ/kg DM) 18.6 19.4 18.7 18.6 
§ DBP, daikon + dried beet pulp; DWS, daikon + wheat straw; DBH, daikon + dried bean stalks and husks.                        
Table 6-5. Feed intake and preference by sheep of daikon by-product silages alone, as indicated by intakes§ (kg FM/hour) over three test periods and feeding positions.  Values represent least square means of three sheep  
Silage 1 
Period2    
Position3    
P value 
Time after feed offer (h)  DBP DWSDBH 1 2 3  A B C 
SEM Silages Period 
Position  
1  1.08a 0.51c0.80b 0.69b0.83ab0.89a 0.79 0.81 0.80 0.462 0.00010.032 0.973 
2  1.13a 0.63c0.94b 0.83 0.95 0.95  0.89 0.92 0.91 0.481 0.00010.699 0.899 
8  1.19a 0.85b1.13a 1.02 1.08 1.07  1.05 1.06 1.05 0.329 0.00010.328 0.928 
24  1.20a 1.00b1.16a 1.09 1.12 1.15  1.11 1.12 1.13 0.20 0.00010.656 0.856 
§Alfalfa hay was offered at 24.0 g/kg BW in DM/day/sheep as basal diet and was readily consumed by sheep. All silages were offered simultaneously at 1.20 kg FM /silage/sheep/day. 1 Same as in Table 6-4. 2 Periods of three experimental days. 3 A, arrangement of silages according to the following order DBP, DWS , DBH; B, arrangement order of DWS, DBH, DBP; C, arrangement order of DBH, DBP, DWS.  SEM, standard error of the means. Means followed by different letters in a row differ significantly (P<0.05). 
6.3.3. Discussion  
Even after addition of the absorbents prior to ensiling, daikon by-product silages 
were still low in DM. Some amounts of effluent had settled at the base of the silos 
indicating that the absorbents had not effectively soaked up the moisture as expected. 
Higher daikon by-product:absorbent mixture ratios need to be considered in future 
experiments. Addition of alfalfa hay to the diet augmented the DM, nitrogen and energy 
contents of the diets and generally improved the texture of the diets in a way preferred 
by the animals. Daikon/dried beet pulp silage had the best feed intake and preference in 
comparison with the other treatments probably due to its fermentation quality, low fiber 
and physical characteristics (dry matter content, particle size and resistance to fracture), 
factors that have profound effects on feed intake (Inoue et al., 1994; Baumont et al., 
2000). Feeds that can be ingested fast and are rapidly digested are very palatable, 
provided they do not contain toxic compounds. Moreover, intake of feed, when supplied 
indoors, depends mainly on its nutritive value, fill effect and its sensory properties, 
provided it does not contain toxic compounds (Baumont et al., 2000), characteristics 
that were obviously higher in DBP than the other treatments.  Intake is a key factor in 
assessing feed preference (Moseley and Antuna Manendez, 1989); thus, the high rate of 
intake in DBP silage at the first two hours was a key factor in assessing its preference 
over the other treatments. At 8 and 24 h, intake did not differ between DBP and DBH 
silages, times when intake of the silages decreased continuously until satiety and 
preference became less obvious.  
This study indicated that dried bean stalks and hulls (a by-product produced 
abundantly but rarely utilized as a feed in Japan) can double as an absorbent and a 
worthy feed mixture in daikon silage due to the high intake of DBH by the sheep. 
Daikon/wheat straw silage had the lowest intake due to its high fiber and low CP 
contents, factors that affect nutritive value and intake. It is noteworthy that daikon 
silages were readily eaten by sheep and orts recorded were only after 24 h and were the 
hard, stemmy and undevourable portions of DWS and DBH.  Further, this study has 
somewhat proven that the pungent smell in fresh daikon could not be a limiting factor 
when offered as silage to sheep. The significant increments in intake between periods 1 
and 3 during the first hour, and insignificant but numeric increases with hours that 
followed, indicated that sheep ate more of the silages after becoming used to them.  
6.3.4. Conclusion  
Daikon by-product silages adjusted with dried beet pulp, bean stalks and husks and 
wheat straw were readily eaten when offered to sheep.  More feeding trials with 
different absorbents are required to ascertain the full potential of daikon by-product 
silages. 
6.4. Summary  
    Ensiling of potato pulp without or with microbial inoculants does not affect its 
feeding value using sheep. Since potato pulp silage is deficient in crude protein, 
supplementation with feed sources rich in protein is necessary.  The high nutritive 
value compares to other by agricultural by-products such as citrus pulp and beet pulp 
and as such could be a useful feed ingredient in ruminant diets.  
  Daikon by-product silage has the potential to be ruminant feed ingredient. It is 
imperative, however, that the by-product is adjusted with absorbents prior to ensiling 
and supplemented with hay or dry feed due to the high moisture content of the 
by-product. Since the silage was readily eaten when offered to sheep, more feeding 
trials with different absorbents and bigger ruminants are required to ascertain its full 
potential. 
Chapter 7 
Factors affecting the ensiling of HMBF and their relationships 
  7.1. Introduction       
   In the previous chapters, pre-ensiling characteristics, followed by the ensiling of 
representatives of HMBF and the role of absorbents in the control of effluent have been 
discussed. It is evident from the results that ensiling HMBF is a viable venture from 
preservation and animal feed resource perspectives. However, this process also entailed 
peculiar challenges not only in its feasibility as animal feed resource, but also in its 
relationship with the environment. In an era where the latter is of considerable concern, 
it is prudent that any endeavor towards enhancement of science be ‘environmentally 
friendly’ as possible. A major factor in the ensiling of HMBF, as previously discussed, is 
effluent. However, several other factors directly or indirectly affect this process. In this 
chapter, the role of some important factors affecting ensiling of HMBF is discussed with 
particular emphasis on its relationship to effluent production.  
7.2. Material and methods 
   Data were pooled from a series of experiments conducted separately over a period 
of five years. They involved the ensiling of some HMBF (n=27) ensiled without or with 
absorbents. These have been divided into 5 groups (Table 7-1), each group consisting of 
the pre-silage material and moisture adjusted treatments using various absorbents as 
follows: 
    Group 1 (Potato pulp A): This included four treatments consisting of silage 
material without moisture adjustment (control, PP1) and material adjusted with wheat 
bran (absorbent) to three different moisture levels (PP2, PP3 and PP4).  
    Group 2 (Potato pulp B): This included 7 treatments consisting of potato pulp 
adjusted without or with three different absorbents as follows: 
Control, without moisture adjustment (PPC); adjusted with wheat bran to two moisture 
levels (PPWB 1 and PPWB 2); adjusted with dried beet pulp to two moisture levels 
(PPBP 1 and PPBP 2); and adjusted with wheat straw to two moisture levels (PPWS 1 
and PPWS 2).  
   Group 3: Brewer’s grain consisting of three treatments adjusted without absorbent 
(C), with dried beet pulp (BP), or wheat bran (BW) (Chapter 5). 
   Group 4: Apple pomace consisting of four treatments adjusted without an absorbent 
(AP), with dried beet pulp (ABP), rice bran (ARB), or wheat bran (AWB (Chapter 5). 
   Group 5: Daikon by-product without absorbents consisting of three treatments; DKS, 
DKM, DKL and DN; with wheat straw absorbents (DA and DWS); with dried beet pulp 
(DBP), with bean stalks and hulls (DBH) and with wheat bran (DWB). All treatments in 
this group have been described previously in Chapter 4. 
   All groups were ensiled at ambient temperatures of between 18-25oC. Data of 
chemical components of pre-silage materials including moisture, WRC, PWRC, NDF, 
and silage characteristics (pH, lactic acid content and effluent output of the silages) 
were the parameters considered. These parameters were chosen for the sake of 
consistency and availability of data. Factors such as those that have a direct bearing on 
the fermentation process (sugar, buffering capacity, microbial numbers and physical 
characteristics such as pressure or density) were excluded because they have either been 
previously discussed in detail, or are limited by number of available data.  
 7.2.1. Chemical analyses  
   Parameters were analyzed with procedures similar to those described previously in 
earlier chapters. Water retention capacity (WRC) was adapted from the method of 
Robertson et al., (2000) and modified for the sake of convenience, and is described (see 
Chapter 2) in more detail here. One gram of air-dry weight of each HMBF material 
(dried at 60oC for 48 h and milled to < 1 mm) was measured into a 50 ml centrifuge 
tube and hydrated with 30 ml distilled water containing 0.02 g sodium azide per 100 ml 
as a bacteriostat. The tube containing the sample was then equilibrated for 18 h at room 
temperature and its contents were transferred to a glass filter with a pore size of 100-160 
μm, (1G P160, Sibata Company, Tokyo, Japan) and drained under a pressure of 2 g/cm2  
with a pressure pump (Compact air pump, NUP-1, AS-ONE Company, Tokyo, Japan) 
for 2 minutes (The vacuum time of 2 minutes was chosen as the most convenient 
following a short experiment using three different materials and three vacuum duration 
times). The glass filter containing the sample was weighed, oven-dried at 135oC for two 
hours and weighed again. The WRC of the material was calculated as the amount of 
water retained by the pellet (g moisture /g dry weight) after transfer to the glass filter. 
Potential WRC (PWRC), defined as ‘the amount of moisture in grams that a HMBF can 
retain per 100 grams of its own fresh matter weight’, is calculated as WRC x DM of the 
material and is given in g/100g fresh matter.   
   To enable comparison between WRC and PWRC and in essence validate the PWRC 
concept, the moisture, WRC and PWRC of some HMBF are provided (Table 7-3). 
7.2.2. Statistical analysis 
    Data were analyzed using the statistical analyses software Excelstats for Windows 
2004 (Microsoft Corporation, USA). Means from the effluent data were calculated from 
each group and analyzed separately. Regressions were performed to study the 
relationships between the studied parameters. A multi-linear equation describing the 
relationship between effluent (dependent variable) and moisture, PWRC and NDF 
(independent variables) was established. 
                 
7.3. Results    
7.3.1. Moisture content of pre-silage material 
  The chemical parameters of the HMBF materials and silage effluent outputs are 
presented in Table 7-1. Each group consisted of the control followed by adjustments 
into different moisture levels. In Group 1, the moisture content of potato pulp was 
adjusted from 88.1 to 64.3 % in fresh matter with wheat bran as a sole in-silo absorbent, 
while in Group 2 it was adjusted from 78.1 to 64.8 % in fresh matter with wheat bran, 
dried beet pulp, or wheat straw at two levels per absorbent. In the brewer’s grain and 
apple pomace experiments (Groups 3 and 4), adjustments were made with dried beet 
pulp, wheat bran and rice bran, which reduced the moisture contents from 81.5 to 64.0 
and 91.9 to 73.4 % in fresh matter for brewer’s grain and apple pomace, respectively. 
   In the daikon by-product study (Group 5), data were pooled from three separate 
experiments involving the use of the by-product without/with adjustment with various 
absorbents. These reduced the high moisture of the material from 96.9 to 73.6 % in 
fresh matter. The pooled mean for moisture content in all the experiments was 78.3%.  
7.3.2. Water retention capacity (WRC) and potential WRC 
   The WRC varied with type of HMBF, absorbent used and moisture content of the 
material alone or the mixture, with a pooled mean of 3.43 g/g.  However, a pattern was 
observed in reductions of WRC with increment in moisture contents of Groups 1, 2, 3 
and 4. In Group 5, the opposite was true. The WRC values of the adjusted moistures 
(DA, DBP, DWS, DBH and DWB) were higher than that of DN, the source material. 
There  
Table 7-1. Chemical parameters of some HMBF materials and effluent output from their silages§   MoistureWRC  PWRC NDF  Effluent 
Grouping HMBF (% FM)  (g/g DM)(g/100gFM) (% DM)   (g/100gFM)Group 1 Potato pulp (A)1      
   PP1 88.1 2.98 35.5 32.8 11.3a    PP2 79.4 2.44 50.2 35.3 8.9ab    PP3 74.3 2.22 57.1 36.6 6.8b    PP4 64.3 2.00 71.4 40.8 0.2c Group 2 Potato pulp (B) 2          PPC 78.1 2.43 53.1 32.6 0.7a    PPWB 1 74.1 2.37 61.3 33.5 0.4a    PPWB 2 68.4 2.25 71.0 39.3 0.0b    PPBP 1 73.1 2.44 65.7 35.1 0.0b    PPBP 2 68.0 2.45 78.3 37.5 0.0b    PPWS 1 70.0 2.16 64.7 54.0 0.0b    PPWS 2 64.9 2.17 76.3 58.0 0.0b Group 3 Brewers grain (BG) 3         C  81.5 3.11 57.6 38.3 18.4a    BP 66.4 2.66 89.4 40.5 0.0c    WB 64.0 2.00 71.9 42.0 4.9b Group 4 Apple pomace (P) 4         AP 91.9 6.27 50.9 46.6 39.1a    ABP 82.1 5.08 90.7 50.8 3.7c    ARB 70.8 2.60 76.0 35.6 7.3b    AWB  73.4 3.31 88.2 49.0 9.3b Group 5 Daikon (D) 5         DKM 96.9 7.02 21.5 21.0 53.9a    DKS 96.8 7.02 22.5 21.0 54.5a    DKL 96.6 7.03 23.6 21.0 49.6a    D N 93.4 2.40 15.8  21.1 37.8b    DA  83.7 3.52 57.4  69.0 3.2e    DWS 81.6 4.08 75.1 72.5 0.0e    DBH 79.5 3.95 80.9 59.5 10.3c    DBP 79.3 4.25 87.9 41.4 15.2c    DWB 73.6 2.49 65.6 50.7 20.1d  Statistical parameter         Mean 78.3 3.43 61.5 41.3 -    Standard deviation 10.3 1.6 21.8 13.6  - 
  § Effluent means (of three samples) within a column for each group with uncommon letters indicate a significantly effect at 
P<0.05; WRC, water retention capacity;   PWRC, potential WRC; NDF, neutral detergent fiber; FM, fresh matter; DM, dry matter; 
g, gram. 
1 PP1, potato pulp control (A); PP2, PP3, PP4, (A) adjusted to various moisture levels with wheat bran.  
2 PPC, potato pulp control (B); PPWB1, PPWB2, (A) adjusted with wheat bran; PPBP1, PPBP, (B) adjusted with dried beet pulp; 
PPWS1, PPWS2, adjusted with  wheat straw to different moistures, respectively. 
 3 C, brewers grain control (BG); BP, WB, (BG) adjusted with dried beet pulp and wheat bran, respectively.  
4 AP, apple pomace control (P); AP, ABP, ARB, AWB, (P) adjusted with dried beet pulp, rice bran and wheat bran, respectively. 
 5 DKM, DKS, DKL, DN, daikon by-product control (D); DA and DWS, DBH, DBP, DWB, (D) adjusted to moisture levels with 
wheat straw, bean stalks, dried beet pulp and wheat bran, respectively. 
was however, a clear consistency in the pattern of PWRC, increasing with decrease in 
moisture content (Table 7-1).  
7.3.3. Fiber content 
     The fiber contents (NDF) were low in the materials but increased according to the 
fiber content of the absorbent introduced. As a result, absorbents high in fiber, such as 
wheat straw and bean stalks, tended to increase the NDF of daikon by-product (DA, 
DWS and DBH). 
 7.3.4. Effluent output and relationship with pre-silage material 
    The silages underwent acceptable fermentation processes, as indicated by the 
average pH and lactic contents in Table 7-2. Effluent outputs varied with type, moisture 
and PWRC of the material. Differences in effluent outputs were obvious (P<0.01) with 
an increase in moisture or addition of absorbent within each group.  Daikon by-product 
silages produced the most effluent relative to the other HMBF silages studied. 
  Table 7-2. Mean fermentation characteristics of the silages used in the study  
  Moisture range (% DM)  pH Lactic acid (% DM) 
Potato pulp A 89-67  3.75 ± 0.14 5.59 ± 1.48 
Potato pulp B 80-67  3.39 ± 0.15 2.17 ± 0.62 
Brewer’s grain 84-67  3.60 ± 0.12 1.44 ± 0.16 
Apple pomace 89-70  3.49 ± 0.05  7.97 ± 2.48 
Daikon by-product 94-75  3.80 ± 0.18  4.73 ± 3.10 Values are means ± standard deviation.   
Obvious relationships were observed between the pre-silage characteristics of the 
HMBF studied and effluent production in the resultant silages and are presented in Fig 
7-1. There was a positive relationship between effluent and moisture but inverse 
relationships with PWRC and NDF of the HMBF materials studied, as given in the 
following multi-regression equation: 
Y = 1.569M – 0.1059PWRC – 0.2159NDF – 62.246              
                       (R2 = 0.7185, n=81, (P<0.01) 
where Y, effluent (g/100g fresh matter of material); M, moisture content of material 
(%); PWRC, (potential water retention capacity of material, in g/100g FM); NDF, 
neutral detergent fiber of material (% DM). 
                        
                                     
Fig. 7-1. Relationships between moisture (a), NDF (b), PWRC (c),  and WRC (d) of 
some HMBF (n=27) and effluent output from their silages (n=81). 
          
(a) (b)
(c) (d)
7.4. Discussion       Ensiling of wet materials is fraught with difficulties, not only in losses encountered 
in the silo, but also in the considerable loss of nutrients in effluent and seepage through 
silage, with the attendant environmental consequences. Most silage materials, especially 
grasses and whole crop materials, are unable to retain their moisture when ensiled, and 
consequently the system of drying prior to ensiling has been widespread. However, in 
cases where drying is unreliable or difficult, incorporation with drier materials is 
inevitable. In the ensiling of HMBF, one statement is valid: high moisture material = 
more silage effluent, and their relationship is conspicuously clear, as validated by the 
present results (Table 7-1). From an environmental perspective, the process by which 
the excess moisture in HMBF is contained during ensilage is of critical importance.       
Several methods have been suggested to address and control effluent production during 
ensilage from particularly low dry matter materials without compromising the quality of 
the resultant silage. Prominent among them is the inclusion of dry materials as in-silo 
effluent absorbents (Reaves and Brubaker, 1956; Done and Appleton, 1989; O’Keily, 
1991; Ferris and Mayne, 1994, Okine et al., 2006b), which invariably control effluent 
flow and relationships between the dry matter of silage material and effluent have been 
established (Jones and Murdoch, 1954; Sutter, 1957; Castle and Watson, 1973). In the 
current study, a relationship between the moisture content of HMBF and effluent was 
established: 
Y = 1.4966x – 103.4           (P<0.01), 
where Y, effluent (g/100g of ensiled material) and x, the moisture content of pre-silage 
material (%), with a regression co-efficient (r)  of 0.85 (Fig. 7-1a). In the given 
equation, when Y = 0, x = 69.1, i.e., the moisture content of an ensiled HMBF at which 
it is predicted that no effluent will be produced is 69.1 % (or 31.9 % DM). This is in 
good agreement with Sutter (1957), Zimmer (1967) and Bastiman (1976) for ensiled 
grasses and crops, who proposed dry matter content at zero effluent production to be 
between 29.9 and 30.7 %. From the pooled mean values of the moisture contents of 
materials ensiled in this study, the results are particularly significant. It is therefore 
prudent that the moisture content during ensiling of HMBF does not go beyond 70 %.    
  Water retention capacity (WRC), or the amount of water held by a known weight of 
fiber (Robertson et al., 2000), is one of the methods used to determine the hydration 
properties of dietary fiber in relation to nutrient absorption from the intestine and stool 
properties. In silage terminology (used interchangeably with ‘water holding capacity’), 
it represents the amount of moisture in grams that a dry absorbent can hold or retain per 
its dry matter weight  and is often used  to describe the absorptive capacity of dry 
materials in silage effluent control (reviewed by Jones and Jones, 1996). This 
measurement, however, has some shortcomings. First, it accounts only for the amount 
of moisture that the absorbent material can ‘hold’ or ‘absorb’ without considering how 
much moisture the silage material can ‘release’ or ‘retain’ or is available for absorption. 
Fibrous materials are known to hold water differently depending on the chemical 
activity of the water, pore size distribution of the fiber matrix and trapped water within 
the cell wall lumen (Robertson and Eastwood, 1981a). Secondly, the ability to retain 
effluent under the influence of some chemical or physical conditions within the silo is 
more relevant than the ability of the absorbent material per se. The use of microbial 
additives prior to ensiling and/or the application of external forces, such as surface 
pressure on the silo as pertains during ensilage, affect effluent outputs from HMBF.  
To elucidate this point, it is important to consider the measurement of WRC as proposed 
by Robertson et al. (2000) and shown in the scheme below (Fig. 7-2): 
Hydrate dry sample in a centrifuge tube   Centrifuge (3000 x g; 20min) §   Remove excess supernatant/soluble material   Record residue hydrated wt   Record residue dry weight   WRC (g/g) =      (residue fresh weight – residue dry weight) / residue dry weight      
Fig. 7-2. Scheme of water retention capacity (WRC) according to Robertson et al., 
2000.                                                              
§Centrifugation was substituted with filtration for the sake of convenience as described in ‘Materials and 
methods’.  
The WRC values of HMBF in this study (Table 7-3) were quite low compared with 
other reports for vegetable and fruit by-products in general (Cho et al., 1997a, b; 
Thibault et al., 1992) but comparable to those of Auffret et al., (1994) for wheat bran 
and pea hulls, and (Dexter, 1961; Hillman and Thomas, 1974) for dried beet pulp, and 
they only reflect the source of fiber and the method used. Water retention in fiber has 
been determined using different methods such as centrifugation, filtration, or suction 
pressure (McConnell et al., 1974; Robertson and Eastwood, 1981b, c; Chen et al., 1984), 
with each method measuring the amount of water associated with a sample under the 
defined conditions. The calculation of WRC, although valid for the measurement of the 
absorptive element of an in-silo effluent absorbent, discounts the characteristics and fate 
of the moisture of silage material. 
Table 7-3. Water retention capacity (WRC) of some HMBF and dry absorbents§ 
  WRC (g/g DM) 
HMBF  
  Cucumber 4.86 ± 0.80 
  Water melon residue 4.54 ± 1.26 
  Daikon (root) 3.58 ± 1.31 
  Cabbage  6.55 ± 0.47 
  Carrot pulp 6.33 ± 0.94 
  Banana peel  4.50 ± 0.43 
  Apple pomace  3.12 ± 0.54 
  Peach pomace 2.17 ± 0.15 
  Pineapple  (skin & pomace) 2.96 ± 0.28 
  Potato pulp 3.55 ± 0.75 
Absorbents  
  Dried beet pulp 2.96 ± 0.58 
  Wheat straw 3.08 ± 0.40 
  Bean stalks and husks 2.58  ± 0.52 
  Wheat bran 1.32 ± 0.62 
  Rice bran 0.92 ± 0.23 
  Wood shavings 2.44 ± 0.46 
§ Values are means of three determinations ± standard deviation. 
To address this shortcoming, the terminology potential water retention capacity 
(PWRC) is proposed: 
PWRC of silage material = WRC x specific DM of the material 
If WRC =DMWW21+, and specific DM of material (in fresh matter) = FMDM , 
where W1, original moisture content of material and W2, absorbed moisture,   
then, PWRC = FMWW21+ 
This equation takes into account the ‘moisture’ element of the material and the 
absorptive capacity of the dry in-silo absorbent and it provides a more realistic approach 
in determining the retentive capacity of the mixture vis-a-vis effluent output. This is 
particularly valid during the addition of absorbents prior to ensilage and gives a reliable 
prediction of effluent outputs from materials particularly high in moisture such as 
HMBF, since different materials have various dry matter contents. This is true, since 
during ensilage of HMBF, effluent output is intrinsically a function of its PWRC, as 
indicated by the following figures using different HMBF (Fig. 7-3 a, b).  
y = 0.8604x + 14.612r = 0.92
020406080100
Material PWRC (g/100g material)
Silage PWRC (g/100g material)
  Fig. 7-3. PWRC of some HMBF materials and silages with error bars (a) and regression 
equation showing the relationship between HMBF and their silages (b) (n=81).  
It is evident from the figures that after ensiling, the PWRC of HMBF reduces as effluent 
is released. The strong relationship between material and silage PWRC (r = 0.92, 
P<0.01) validates this theory. The data in Fig. 7-1c, d of the HMBF silage experiments 
showed a stronger effluent relationship with PWRC than with WRC (r = 0.76 vs. 0.41), 
a further indication that the former may be a more relevant index for the measurement 
of effluent retention in HMBF silages. (See Chapters 4 and 5 on PWRC effect on 
effluent outputs in some HMBF silages.) The regression equation between the PWRC of 
the HMBF and effluent outputs from the silages (see Fig. 7-1c) is given below: 
Y = - 0.6164x - 51.1      (P<0.01) 
where Y, effluent (g/100g FM of ensiled material) and x, PWRC (g/100g FM of 
pre-silage material).  
From the given equation, the theoretical PWRC of a silage material at which effluent 
production would be zero is 82.9. To achieve this, a combination of factors such as the 
moisture content and the WRC of both silage material and the absorbent need to be 
DaikonPotatopulpApplepomaceBrewersgrain
By-product
PWRC (g/100g)
Material
Silage
considered. The use of absorbents high in WRC and low moisture materials favor an 
elevated PWRC of the mixture.  
   The fiber content of both material and absorbent also play an important role in both 
WRC and PWRC of materials in relation to effluent retention in silages. A typical 
illustration of this is given in Table 7-1. The WRC of the adjusted moistures DA, DBP, 
DWS, DBH and DWB were higher than that of DN, the source material, while the 
opposite was true for the other groups (PP1, PPC, C and AP (Table 7-1). This could be 
attributed to the high NDF of the absorbents used which increased the low original NDF 
of DN (daikon by-product) by ≥ twofold.  In the other groups, addition of absorbents, 
however, did not affect the NDF contents of the mixtures. The PWRC values, however, 
were consistent in increases with an increase in NDF contents, and both had an inverse 
relationship with effluent outputs, as shown in the multi-regression equation described 
earlier. Since fibrous materials retain effluent better than non-fibrous materials (Jones 
and Jones, 1996), the PWRC/NDF relationship with effluent further validates the 
PWRC theory and could be an important parameter in ensiling of wet materials.  
   7.5. Summary       A major factor affecting the ensiling of HMBF is effluent production, which if not 
properly disposed, may have adverse consequences on the environment. Effluent is 
influenced mainly by such factors as the moisture and fiber (NDF) contents and the 
potential water retention capacity (PWRC) of pre-silage material. While the relationship 
with moisture is an inverse one, the effluent relationship with NDF and PWRC were 
positive. 
  The terminology PWRC is an important novel terminology in the ensiling of HMBF 
and a theory that warrants further validation.  
      
                            
Chapter 8  General Discussion      
   Feed conservation or preservation by ensilage is a viable method by which the shelf 
life of crops meant for feeding of ruminants can be extended.  This practice has 
evolved tremendously in recent times due to, among others, the discovery of new 
preservation techniques, silage additives and advances in silo technology.   
Methodologies involved in addressing problems related to preservation by ensilage of 
wet materials are varied but mostly refer to grasses and crops. Preservation by ensilage 
of agricultural by-products, especially those that contain high moisture contents, is still 
in embryonic stage. The aims of the experiments were to investigate the ensiling 
potential of HMBF, improve the preservation by incorporation with additives, control 
effluent flow and minimize nutrient losses from their silages, extend their shelf lives, 
evaluate the nutritive value, and to identify the main factors that affect the ensiling of 
HMBF and their relationship and establish a suitable method for ensiling HMBF. 
         
8.1. Pre-ensiling characteristics of HMBF      One of the aims of this research was to identify the suitability of type of HMBF for 
ensilage, through chemical and microbiological characterization as a prelude to the 
actual ensiling of the by-product. The chemical and microbiological characteristics 
relevant to ensilage of the materials studied here showed some variations in terms of 
availability or not, versus the suitability to the ensiling process. The moisture content of 
the materials was variable within and among the samples studied but generally higher in 
by-products from vegetable sources, compared with those from fruit and agro-industrial 
sources. Potato pulp and apple pomace were typical examples of this, partly due to the 
mode of extraction employed in the processing factory. The pH, buffering capacity, and, 
to a large extent the WSC of materials from vegetable sources, were higher than those 
from fruit sources, providing a rather unpredictable and contrasting indication of their 
role in an ensiling process. In a typical silage fermentation process, a quick reduction of 
pH during the initial stages of the fermentation process is necessary to neutralize and 
prevent the proliferation of undesirable microorganisms with a gradual increase in lactic 
acid production from the available substrate (WSC), which is mainly sugar. Materials 
with high buffering capacities may delay the reduction in pH even in the presence of 
sufficient WSC. Addition of an inoculum of LAB, prior to ensiling of material from 
vegetable sources may be a viable proposition. Conversely, fruit by-products and potato 
pulp, from a theoretical point of view, provided strong indications of being ideal silage 
materials judging from their chemical analysis, as evidenced by their low pH and 
buffering capacities and relatively high WSC contents (except potato pulp). Usually a 
material containing 150 g/kg in dry matter available WSC would be enough for a good 
fermentation process. The LAB populations of the materials investigated except potato 
pulp were generally low and did not exceed the 106 minimum organisms/g dry matter 
threshold required to quick-start a highly effective lactic acid fermentation process. 
However, low initial population of LAB may not be a critical criterion for the success of 
the fermentation process. What is crucial is the efficiency of the indigenous LAB to 
initiate a rapid fermentation and sustain a rapid fall in pH (Woolford, 1984a). Yeast load 
was quite low in the investigated materials especially in potato pulp. This augurs well 
for good silage fermentation. Again, what is critical is the yeast population at the end of 
the ensiling process, i.e., after opening of the silage. The rule is that if the number of 
yeasts exceeds the level of 105 organisms/g dry matter, rapid fungal deterioration would 
occur after opening of the silage (Jonsson and Pahlow, 1984). From a microbiological 
point of view, potato pulp may be an ideal silage material. 
   The potential water retention capacity (PWRC), a terminology employed as a 
novelty in this study is, to a large extent, a function of its moisture content (See Chapter 
7). An important feature in the ensiling of particularly wet crops is the ability of the 
material to retain its moisture during the course of ensilage. The PWRC describes this 
capacity of materials ensiled either alone or with an absorbent, under different silo 
conditions during the course of ensilage, and it is an important index in ensiling of 
HMBF (Chapter 7). The higher the PWRC, the stronger the ability of the ensiled 
material to retain effluent, and vice versa.   
8.2. Ensiling of HMBF  
8.2.1. Potato pulp 
   In all the three investigations on potato pulp silages, the pH was below 3.60, 
irrespective of the type of inoculants added or silo used, indicating a good fermentation 
process. There were, however, variations in character and efficiency of pH reduction of 
the inoculants used. Particularly effective was the fungus Rhizopus oryzae which, when 
used alone or in combination with Lactobacillus rhamnosus, decreased (P<0.05) the pH 
in two separate experiments. A third experiment confirmed this when Amylomyces 
rouxii, a fungus taxonomically identical to Rhizopus oryzae was used. This decrease 
after inoculation with the fungal inoculants is in good agreement with other reports 
(Oda et al., 2002; Okine et al., 2005), but it was rather overshadowed by a defect in 
lactic acid production. Hypotheses built on the lack of effect included the probable 
malfunction of LDH (the active enzyme responsible for conversion of sugars to lactic 
acid in Rhizopus oryzae), the lack of aerobic conditions, the ambient temperature during 
ensiling and the duration of storage.  Subsequent experiments, however, proved that 
ambient ensiling temperatures and lengths of ensilage were more important in 
determining the rate of fermentation than inoculation with the fungal inoculants (Okine 
et al., 2007). Pre-ensiling characteristics of potato pulp material, such as the low 
buffering capacity (Chapter 2) and bacteria load (Saito et al., 2006) in the pulp, 
facilitated and enhanced its ensiling capacity.  
  In all three investigations, the low VFA in potato pulp silage, especially the low acetic 
acid content and non-appearance of butyric acid, the latter which is usually associated 
with badly preserved silages, further boosted the ensiling potential of potato pulp, in 
consent with Oda et al., (2002). The fate of nutrient composition during the ensiling 
process was to a large extent, affected by inoculation with the microbial inoculants. The 
carbohydrate fraction of potato pulp, starch, sugar and pectin seem to assume a 
consistent pattern with ensilage time. The most obvious of these patterns was observed 
in the effect of Rhizopus oryzae on the degradation of pectin, which led to a 
concomitant increase in sugar concentrations. The increases in the concentrations of 
sugar over time were probably due to slight increases in concentrations of unspecified 
monosaccharides during fermentation (Van de Riet et al., 1987) or galactan or pectin 
(De Man, 1957), or from other sources such as cellulose or hemicellulose, which are 
present in potato pulp (Mayer and Hillebrandt, 1997). The degradation of starch, 
however, was not affected by inoculation or duration of storage and could be due to the 
slow degradability of potato starch in general (Monteils et al., 2002).       
   Although ensiling of potato pulp was effective from a preservation standpoint, the 
process involved some difficulties with effluent and losses in nutrients as observed in 
the third experiment. This phenomenon was more pronounced with the use of the fungal 
inoculants. The increase in effluent production upon inoculation with Amylomyces 
rouxii could be attributed to the breakdown of starch and pectin as described in previous 
studies (Experiments 1 and 2 of Chapter 3) with consequent reductions of silage WRC. 
Rhizopus oryzae equally produced a substantial amount of effluent compared with the 
uinoculated control silage, a result that could be explained by the action of the enzymes 
pectinase and LDH on the residual starch in potato pulp, which, as supported by 
Dongowski and Stoof (1993), decreases the water binding capacity of potato pulp. The 
results of this study suggested that Amylomyces rouxii was more effective in breaking 
the water holding capacity of PPS, compared to the other inoculants, with consequent 
higher losses compared to other treatments, a result that could adversely affect the 
nutritive value of the silages.  
8.2.2. Daikon  
 The two separate experiments conducted on the ensiling potential of daikon 
by-product provided information on an otherwise novel by-product and was a test case 
for the validity of the hypotheses on ensiling particularly high moisture by-products.  
For various reasons, this was fraught with many challenges. Despite the high contents of 
OM, CP, WSC, GE and low EE that underline its prospect as animal feed resource, 
pre-ensiling characteristics, especially the high moisture content, pH, buffering capacity 
and yeast population of daikon pre-supposed difficulties in its preservation by ensilage. 
Adjustment of the moisture contents and addition of an inoculum of Lactobacillus 
plantarum prior to ensiling were measures to address these presumed deficiencies and 
to improve the suitability for ensiling. Dry matter adjustment in Experiment 4-1 
increased the water retention capacity of daikon pre-silage material. A major feature in 
the ensiling of particularly high moisture materials is the DM/WRC relationship 
vis-à-vis effluent production. This study indicated that the WRC of silage materials is 
highly influenced by, and positively correlated to, the dry matter content.  Upward 
adjustments of the DM of pre-silage material led to significant increases in the WRC 
and an obvious effect on effluent retention in the resultant silages. Inoculation with 
Lactobacillus plantarum though improved silage fermentation, led to high effluent 
outputs with consequent high nutrient losses and as such would not be recommended. 
Changes in the pH, organic acid content and microbial numbers (LAB and yeast 
populations) were all important indices in assessing the fermentation quality of daikon 
by-product silages and this is amply supported by other works (Jonsson and Pahlow, 
1984; Woolford, 1984b; Ashbell et al., 1987). 
Moisture adjusted daikon by-product silage deteriorated faster on aerobic exposure 
due to higher dry matter content and, to a lesser degree, lower packing density of the 
pre-silage material, and was further accelerated by inoculation with Lactobacillus 
plantarum. Application of the inoculum is therefore not recommended.  
In Experiment 4-2, moisture levels were adjusted based on observations from the 
previous experiment. The hypothesis was that increasing the DM content a level above 
that in Experiment 4-1 would lead to an increase in the WRC of the mixtures and 
consequently, to effluent retention in the silos as well. Although DM and WRC values 
were elevated, effluent outputs in DBP and DWB were more than the projected, 
suggesting that packing weight, density, and pressure exerted on the silages, or other 
factors such as fiber content (Offer and Al-Rwidah, 1989; O’Keily, 1991) and/or 
physical characteristics of ensiled material (Jones and Jones 1996), may have played a 
role in their ability to retain effluent. Daikon/wheat straw stemmed effluent completely, 
while DWB, in spite of the high pre-silage DM, produced the most effluent. Straws are 
effective absorbents, but wheat bran and cereals in general have poor water absorptive 
capacities (Jones and Jones 1996). It is obvious from the two experiments, however, that 
silage materials (singularly ensiled or with an absorbent material) with high water 
retention capacities tend to control effluent more effectively than those with lower water 
retention capacities. Although DBP had significantly lower pH and higher lactic acid 
contents compared with other treatments, silage fermentation was generally restricted in 
Experiment 4-2 due to effect of the absorbents.    
   Aerobic stability of daikon silages with different absorbents in Experiment 4-2 was 
similar in character to the daikon wheat straw adjusted moisture of Experiment 4-1 in 
sharp increases and decreases in pH and lactic acid contents, respectively, after 3 days 
of aerobic exposure of the silages. The rate of rise in pH and decline in lactic acid 
contents indicated a phenomenon that is common to HMBF: a possible proliferation of 
yeast numbers. Yeast, especially lactate assimilating types, are mostly responsible for a 
rapid decline in silage quality at feed out stage (Filya et al., 2000; Jonsson and Pahlow, 
1984). DBW and DBH deteriorated faster, compared to DWB and DBP on aerobic 
exposure due to the state of the added absorbents (wheat straw and bean stalks) prior to 
addition to daikon by-product. The two absorbents are usually gathered from the field 
after harvesting of wheat and beans and they normally come with some soil which may 
serve as a contaminant, in contrast with wheat bran and dried beet pulp, which are 
‘cleaner’ products from factories. Another factor responsible for the quick deterioration 
of the two silages was their low packing densities, which create air pockets that, with 
the least available substrate, provide fertile grounds for yeast growth and metabolism 
and consequently enhancement of spoilage. 
8.2.3. Brewer’s grain and apple pomace      
  The low lactic acid contents of the silages confirmed a phenomenon typical to the 
fermentation quality of brewer’s grain silages in general and in consent with Nishino et 
al., (2001). Several factors could be attributed to this, including the characteristic of the 
material, the low buffering capacity and the yeast load in the material. However, the 
main aim of the experiment was to investigate the effects of packing density and 
pressure and their relationship with effluent production. The results in this study 
confirmed other reports on silages, especially those ensiled from low dry matter 
materials (McDonald et al., 1960; Zimmer, 1974; Peters and Weissbach, 1977). Rather 
significant, however, was the effect of the two factors in conjunction with the PWRC, 
which appeared to have had the most potent effects. This result was confirmed in the 
apple pomace silage experiment. Apart from pressure that had a significant influence on 
effluent outputs, PWRC gave a strong indication that, barring other major determining 
factors such as the moisture content, effluent production from any given silage material 
is largely a function of its PWRC (See Chapter 7).  
    Aerobic stability in the apple pomace silages confirmed earlier  results (Chapter 
4) that pH and lactic acid (fermentation end products) are important barometers of 
aerobic stability in HMBF silages, and that lower pre-silage packing density makes 
silages more prone to quick deteriorations in quality during the feed out stage.  
8.3. Nutritive value of HMBF   
   The high moisture content of HMBF, as a matter of necessity, dictates that feeding 
with them be in conjunction with other dry feeds. In the digestion trial with potato pulp 
silages, addition of hay and soybean meal to the silages prior to feeding increased not 
only the DM of the diets but the texture, which was highly appreciated by the sheep. 
This also augmented the diet CP and NDF contents to levels in tandem with NRC 
feeding standards (NRC, 1985).The pulp material used in the present trial contained fair 
amounts of easily digestible carbohydrates such as starch and pectin, though the content 
of the former was low compared to the conventional potato pulp of about 370 g/kg DM 
(Mayer and Hillebrandt, 1997), and could be due to the mode and rate of starch 
extraction as described in (Chapter 6). The diets contained an average digestible energy 
of 13 MJ/kg DM, fairly sufficient for the energy requirements and ages of the sheep 
used in the trial. With the exception of Lactobacillus plantarum inoculated silage, DM 
intake among silages was not affected (P>0.05) by addition of the inoculants. Similar 
improvements in DM intakes of silages treated with an inoculum of lactic acid bacteria, 
especially with Lactobacillus plantarum, have been reported in grass silages in 
particular (Gordon, 1989a; Gordon, 1989b), especially where there were improvements 
in the quality of the silages, although others (Hooper et al., 1984; Steen et al., 1989) 
have reported increased DM intakes with commercial inoculants with no obvious 
improvement in fermentation, as was the case in this study. The silages were acceptable 
to the sheep, as they were readily eaten by the animals.  
   Crude protein digestibility was higher (P<0.05) in sheep fed silage inoculated with 
Lactobacillus plantarum,  compared to those fed control, a result that could not be 
explained. This could be related to the general improvement in nitrogen retention with 
feeding of PPS that may have improved microbial nitrogen synthesis within the rumen 
and/or reduced silage nitrogen degradability within the rumen as implied by  Aibibula 
et al., (2004), who found no differences in nitrogen retention in sheep fed Lactobacillus 
rhamnosus and Rhizopus oryzae inoculated PPS based diets.  
   The DM, NDF and CP contents of the diets, in addition to the starch and pectin 
contents of potato pulp generally contributed to the high TDN values of the diets.  Dry 
matter digestibility and nutritive value were not affected by the inoculants in consent 
with other reports (Aibibula et al., 2004; Hanada et al., 2004). The digestibility and 
nutritive value of potato pulp silage is comparable to other by-products like beet pulp 
and citrus pulp (NARO, 2001; O’Mara et al., 1999; Deaville et al., 1994). 
In the experiment with daikon by-product silages, addition of alfalfa hay to the diet 
augmented the DM, nitrogen and energy contents of the diets and generally improved 
the texture of the diets in a way preferred by the animals. No N supplements were 
necessary since the silages and hay contained CP levels that met or exceeded their 
requirement (NRC, 1985). Daikon/dried beet pulp silage had the best feed intake and 
preference in comparison with the other treatments, probably due to its fermentation 
quality, low fiber and physical characteristics (dry matter content, particle size and 
resistance to fracture), factors that have profound effects on feed intake (Baumont et al., 
2000; Inoue et al., 1994). Intake was a key factor in assessing feed preference (Moseley 
and Antuna Manendez, 1989); thus, the high rate of intake in DBP silage in the first two 
hours was a key factor in assessing its preference over the other treatments. At 8 and 24 
h, intake did not differ between DBP and DBH silages, times when intake of the silages 
decreased continuously until satiety and preference became less obvious. The use of 
cheap and available dry by-products as absorbents in the ensiling of daikon is 
noteworthy from an economic perspective.  
8.4. Factors affecting the ensiling of HMBF 
   Several factors may directly or indirectly affect the flow or release of effluent from 
HMBF. However, two revelations are worth acknowledging in this study: the theoretical 
optimum moisture needed for zero effluent in HMBF silages, and, more significantly, 
the theory of PWRC. The moisture level for zero effluent production in HMBF (69.1%) 
is close to those of Sutter, (1957); Zimmer, (1967) and Bastiman (1976), which 
predicted between 29.9 and 30.7 % for ensiled grasses and crops on the optimum 
moisture content necessary to stem effluent in silages in general. As such the value in 
this study could therefore be an important index for ensiling HMBF.  
   The terminology PWRC, attempts to address a rather ignored and important aspect 
of effluent retention in silages. Although the absorptive capacities of several dry 
materials have been reported (Reaves and Brubaker, 1956; Done and Appleton, 1989; 
O’Keily, 1991; Ferris and Mayne, 1994), the relationship between the ‘absorbent’ and 
‘absorbed’ has not been considered, until now. The contention here is that if dry 
absorbents are capable of holding water several times their weight in dry matter, how 
much moisture is released or ‘allowed to be held’ by the absorbed material?  The 
PWRC theory provides an answer to this question. High moisture materials have varied 
capacities to release their moisture and therefore a provision must be made for this 
capacity when they are used as silage materials. The strength of this hypothesis, 
however, rests on further experimentation using different materials and a broad range of 
experimental data. The relationship between effluent and PWRC, both in the single and 
multi-regression equations (Chapter 7), validated the assumption that PWRC of the 
material is the most important determinant of effluent after moisture.   
  8.5. General Conclusions      To sum up, high moisture by-product feedstuffs could be preserved by ensiling 
based on the intrinsic characteristic of the material. The more the material is endowed 
with characteristics favorable to the ensilage process, the better the chances of success 
of preservation, and vice versa. Effluent production is an important factor affecting the 
ensiling of high moisture by-products feedstuff and, as such, the use of appropriate 
absorbents prior to ensiling is highly warranted in controlling effluent outputs from 
HMBF silages. 
  The aerobic stability of HMBF silages is highly variable, but mostly influenced by 
the pre-silage bulk density, pH and organic acid content of the silage. The lower the 
bulk density, the more susceptible the silage is to fast deterioration in quality. Rapid use 
of HMBF silages during the feed out stage is highly recommended.  
    The potential for use as ruminant feed is high and very promising due to the high 
nutritive value of some HMBF. 
    The potential water retention capacity, or the ability of the silage material to retain 
effluent, is a novel terminology and could be an important index in the ensiling of 
HMBF. The theoretical moisture content and PWRC at which there would be no effluent 
in HMBF silages were 69.1 % in fresh matter and 82.9 g/100g, respectively. 
8.6. Further outlook  
    The theory of potential water retention capacity (PWRC) requires further 
substantiation with a wide range of absorbent materials and HMBF. Future experiments 
should also consider the possibility of extending the ‘shelf’ or ‘bunker’ life of HMBF.     
Solutions and strategies should include experimentation with materials with proven 
ability to control secondary fermentation and aerobic stability with a focus on biological 
rather than chemical means.  
  Studies on rates of inclusion in ruminant diets and animal responses are equally vital 
in ascertaining the nutritive value of HMBF. 
                   
Chapter 9  General Summary         
   Agricultural by-products, principally residues from farm harvests or processing 
factories, are an integral part of livestock feeding in most developing countries. The 
term high moisture by-product feedstuff (HMBF) is used in this thesis to describe ‘any 
product or commodity that is obtained during the harvesting, production, or processing 
of feed or fiber and contains moisture content of more than 750 g/kg in fresh matter, and 
has value as an animal feed. There are several means of preservation or extension of the 
shelf life of HMBF including drying and freezing. However, preservation of HMBF by 
ensilage is, comparatively, a more viable and cost effective method. However, this, 
involves challenges both in terms of quality of the resultant silage and effect of the 
process on the environment, since effluent production is a major characteristic of 
HMBF silages. Methodologies involved in addressing problems related to preservation 
by ensilage of wet materials are varied but often subjective, product-specific, or largely 
refer to grasses and crops and thus highlight the need for strategies in addressing 
problems specific to HMBF.  
   The objectives of this study were therefore to investigate the ensiling potential of 
HMBF, improve the preservation by incorporation with additives, control effluent flows 
and minimize nutrient losses from their silages, extend their shelf life, evaluate the 
nutritive value, and to identify the main factors that affect the ensiling of HMBF and 
their relationship and establish a suitable method for ensiling HMBF.  
To realize these objectives the following experiments were conducted: 
1. Study of the pre-ensiling characteristics of a selection of HMBF     The objective of this study was to investigate the ensiling potential of 
representatives of HMBF from vegetable and fruit sources through chemical and 
microbiological characterization as a prelude to their use as silage materials. Materials 
investigated were from vegetable sources (cabbage, cucumber, carrot pulp and daikon or 
Oriental radish (Raphanus sativus L.), fruit sources (banana peel, apple pomace, orange 
peel, and pineapple skin) and one agro-industrial source (potato pulp). Pre-ensiling 
parameters investigated indicated that they were high in moisture and variable in pH, 
buffering capacity and microbial populations (lactic acid bacteria and yeast), but had 
water soluble carbohydrates sufficient for lactic acid fermentation. Vegetable 
by-products were generally high in buffering capacity and pH compared with those 
from fruit sources.       
   Due to the high moisture contents, means of controlling effluent production during 
their use as silage materials are imperative. 
  2. Ensiling of representatives of HMBF  Potato pulp: A series of strategies were used to preserve fresh potato pulp by ensilage. 
These involved the use of microbial inoculants in three separate experiments with a 
lactic acid bacteria inoculum (Lactobacillus rhamnosus) and two fungal inocula 
(Rhizopus oryzae and Amylomyces rouxii), and investigations on their role in enhancing 
the fermentation quality in potato pulp silage.  
  Results indicated that although these inoculants improved (P<0.05) the fermentation 
quality, potato pulp can ensile well with or without microbial inoculants. A subsequent 
study suggested that the fermentation quality of potato pulp silage was influenced most 
by ensiling temperatures and the duration of storage (P<0.01) and least (P>0.05) by 
inoculation with fungal inoculants. Moreover, ensiling potato pulp with Rhizopus oryzae 
and Amylomyces rouxii, in particular, increased (P<0.05) effluent volume and silage 
losses in potato pulp silage. It was concluded that inoculation with fungal additives 
would yield little benefits in potato pulp silage and where their application is necessary, 
the use of in-silo absorbents prior to ensiling was required.  
Daikon (Oriental radish): The study involved two experiments. First, an investigation 
of the chemical composition and pre-ensiling characteristics of daikon, followed by 
ensiling of daikon by-product with wheat straw as a sole in-silo absorbent and an 
inoculum of lactic acid bacteria (Lactobacillus plantarum), while the second involved 
ensiling with other absorbents (dried beet pulp, dried bean stalks and husks and wheat 
bran). A study of the aerobic stability or shelf life of daikon silages was investigated in 
both experiments.  
   Results indicated that ensiling of daikon by-product was a viable venture and that 
the high moisture content (951 g/kg) and buffering capacity (703.4 meq/kg DM) not 
withstanding, the by-product could be ensiled successfully using appropriate technology. 
Due to the moisture content of the by-product, the use of microbial inoculants is 
strongly discouraged, since they increase (P<0.01) effluent production and nutrient 
losses. There were variations in the efficiency of the absorbents used; wheat straw was 
the most effective (P<0.05) but least in effect on aerobic stability of the silages. The use 
of appropriate in-silo absorbents, with the dual responsibility of stemming effluent flow 
and minimizing effect of silage spoilage during feed out stage (aerobic stability), is 
needed to extend the shelf life of daikon by-product silage.  
   Brewer’s grain and apple pomace: The aim was to investigate the role of two 
physical factors (surface pressure and packing density) on the capacity of the two 
by-products ensiled without absorbents (as in brewer’s grain) or with absorbents (as in 
apple pomace) on effluent production and retention using the two by-products as 
representatives of HMBF. 
      High packing density and pressures exerted on fresh brewer’s grain and apple 
pomace ensiled alone or in combination with absorbents prior to ensiling, generally 
increased (P<0.05) effluent production from the silages. However, this was influenced 
by the moisture content, type of material, absorbent used and potential water holding 
capacity of the ensiled material.  
3. Nutritive value of HMBF 
   The nutritive value of two representatives of HMBF silages, potato pulp and daikon 
by-product silages were evaluated in vivo using sheep. First, potato pulp, ensiled with or 
without microbial inoculants was used in a 4 x 4 Latin square design experiment to 
investigate the nutritive value of potato pulp silage. The silages were supplemented with 
Italian ryegrass hay (Lolium multiflorum) and soybean meal. Addition of the microbial 
inoculants did not affect (P>0.05) its feeding value for sheep. The high nutritive value 
(digestible energy, (DE) 13 MJ/kg DM; total digestible nutrients (TDN), 709 g/kg DM) 
of potato pulp silage compares to that of agricultural by-products such as citrus pulp and 
beet pulp and as such could be a useful feed ingredient in ruminant diets.  
  In the second study, a 3 x 3 Latin square design experiment was undertaken to 
investigate intake and preference of daikon by-product ensiled with dried beet pulp 
(DBP), wheat straw (DWS) and bean stalk/husks (DBH) as absorbents. Alfalfa hay was 
offered as a basal diet at 24.0 g/kg BW/sheep/day with the silages. Intake and 
preference was best in DBP, followed by DBH and DWS, in that order. Since the silages 
were readily eaten when offered to sheep, more feeding trials with different absorbents 
and bigger ruminants are required to ascertain the full potential of the by-product. 
 4. Factors affecting the ensiling of HMBF     A study of the relationship between effluent and its major determinants during the 
ensiling of HMBF was investigated. Data were pooled from a series of experiments 
involving the pre-silage materials of potato pulp, brewer’s grain, apple pomace and 
daikon silages either alone or with various absorbents materials (n=27) and their silages 
(n=81).  
   Results indicated a high correlation (P<0.01) between silage effluent and moisture/ 
potential water retention capacity (PWRC) of the pre-silage material. The theoretical 
moisture content and PWRC at which there would be no effluent in HMBF were 69.1% 
in fresh matter and 82.9 g/100 g, respectively. 
      
          
   農作物の収穫および加工過程で生じる農産副産物は、多くの発展途上国におい
て重要な飼料資源である。この論文では、高水分農産副産物(HMBF)という語句
を、食料や繊維製品の生産過程で得られる植物由来の副産物で、水分含量が
750g/kg 以上と高く、家畜の飼料として価値を有するものとして用いる。高水分
農産副産物の保存や貯蔵期間の延長には、乾燥や凍結などさまざまな方法があ
るが、高水分農産副産物のサイレージ化は他の方法に比べ現実的であり、費用
のかからない方法である。しかし、高水分農産副産物をサイレージ化する過程
では排汁が生成しやすく、サイレージ品質の低下や環境への悪影響が懸念され
る。水分含量の高い原料を用いたサイレージ調製に伴う問題点の解決方法は
様々あるが、適切な対処方法はそれぞれの原料で異なるため、個々の高水分農
産副産物に対応できるような問題解決の戦略が必要となる。 
 本研究の目的は、高水分農産副産物のサイレージ化の可能性の検討、添加剤
利用による保存性の向上、排汁生成ならびに栄養損失の抑制、貯蔵期間の延長
ならびに高水分農産副産物サイレージの栄養価を評価することである。さらに
高水分農産副産物のサイレージ化に影響を及ぼす主要因を明らかにするととも
に、高水分農産副産物の適切なサイレージ化の方法を確立することである。 
 この目的を達成するために、下記に示した一連の実験を実施した。 
１．高水分農産副産物のサイレージ化適性に関する研究 
 この研究では、高水分副産物のサイレージ化適性を検討するため、野菜や果
物由来の副産物の化学的・微生物学的特性を調査した。研究で供した材料は、
キャベツ、キュウリ、ニンジン粕、ダイコンの野菜由来の副産物とバナナの皮、
リンゴ粕、オレンジ皮、ナイナップル粕の果物由来の副産物、さらにデンプン
加工工程排出される残渣物であるジャガイモデンプン粕(ポテトパルプ)であっ
た。これら材料の特徴を調べた結果、いずれの材料とも水分含量は高く、pH、
pH 緩衝能および乳酸菌や酵母数などは材料によって大きく異なっていたが、い
ずれの副産物も乳酸発酵に必要な水溶性炭水化物は十分に含まれていた。果物
由来の副産物に比べ野菜由来の副産物は、pH 緩衝能が大きく pH が高かった。こ
れらの農産副産物は水分含量が高いため、排汁生成の抑制は重要な課題である。 
２．高水分農産副産物のサイレージ化 
 ポテトパルプ：ポテトパルプを貯蔵するため、いくつかの方法を用いてサイ
レージ調製した。乳酸菌(Lactobacillus rhamnosus)と２種類の乳酸生成糸状菌
(Rhizopus oryzae, Amylomyces rouxii)を添加剤として用いサイレージ調製し、
これらの微生物添加剤がポテトパルプサイレージの発酵品質に及ぼす影響につ
いて調査した。 
 ポテトパルプをサイレージ調製する際に、乳酸発酵を促進させる微生物添加
剤を使用することによりサイレージの発酵品質は向上したが、添加剤を使用し
なくても良好な発酵品質のサイレージが得られることが示された。ポテトパル
プサイレージの発酵品質は、微生物添加剤の使用の有無よりも貯蔵期間の温度
や長さの違いによる影響のほうが大きいことが示された。さらに Rhizopus 
oryzae や Amylomyces rouxii といった乳酸生成糸状菌の添加は排汁の生成量を
増加させ、サイレージ損失につながることが示された。これらの結果からポテ
トパルプサイレージ調製における乳酸生成糸状菌の添加効果は小さく、これら
の微生物添加剤を使用する際には、水分吸収剤を同時に添加して排汁生成を抑
制する必要があると判断された。 
ダイコン：野菜の選果場から排出されるダイコン(ダイコン選別残渣)のサイレ
ージ調製に関して 2 つの実験を実施した。最初の実験ではダイコン選別残渣の
サイレージ化適性を調べると共に、水分吸収剤および乳酸菌(Lactobacillus 
plantarum)添加がダイコン選別残渣サイレージの発酵品質に及ぼす影響につい
て検討した。2 番目の実験では、水分吸収剤として乾燥ビートパルプ、マメの殻
と茎およびフスマを用い水分吸収剤の違いがダイコン選別残渣サイレージの発
酵品質および排汁生成量に及ぼす影響について検討した。また、いずれの試験
においても好気的条件下におけるダイコン選別残渣サイレージの安定性につい
て検討した。 
 これらの試験の結果、ダイコン選別残渣は水分含量や pH 緩衝能が高いが、適
切な手法によりサイレージ化できる材料であることが示された。ダイコンは水
分含量の高い副産物であるため、ダイコン選別残渣のサイレージ調製時におけ
る微生物添加剤の利用により排汁生成量や栄養損失の増加が認められた
(P<0.01)。水分吸収剤の添加効果は吸収剤によって異なり、小麦ワラは最も水
分吸収能に優れていたが、小麦ワラを添加したダイコン選別残渣サイレージの
好気的条件下における安定性は、他の水分吸収剤を添加したサイレージよりも
劣っていた。これらのことからダイコン選別残渣サイレージの保存性を高める
ためには、排汁生成量の抑制とともにサイロ開封後における好気的発酵の抑制
といった２つの目的を達成できるような添加剤の利用が必要であることが示さ
ビール粕とリンゴ粕：高水分農産副産物を用いたサイレージ調製時における物
理的条件(サイレージ表面にかかる圧力と詰め込み密度)が排汁生成に及ぼす影
響について、水分吸収剤を添加したとき(リンゴ粕を用いてサイレージ調製)と
しないとき(ビール粕を用いてサイレージ調製)で検討した。 
 その結果、ビール粕、リンゴ粕のいずれにおいても詰め込み密度やサイレー
ジ表面の圧力が高いと、水分吸収剤の添加の有無にかかわらずサイレージから
の排汁生成量が増加した(P<0.05)。しかし、排汁生成量は材料の水分含量や種
類、潜在水保持能力(potential water holding capacity，PWRC)によって左右
されることが示された。 
３．高水分農産副産物の栄養価 
 デンプン粕とダイコン選別残渣を用いて調製したサイレージをめん羊に給与
し、栄養価を査定するため２つの実験を実施した。最初の実験では添加剤を用
いずに調製したポテトパルプサイレージと微生物添加剤を用いて調製したサイ
レージの栄養価を調べるため、４×４のラテン方格法に基づいて消化試験を実
施した。飼料はポテトパルプサイレージの他にイタリアンライグラス乾草
(Lolium multiflorum)と大豆粕を給与した。その結果、サイレージ調製時にお
ける微生物添加剤の利用はポテトパルプサイレージの栄養価に影響を及ぼさな
かった(P>0.05)。ポテトパルプサイレージの可消化エネルギー含量は 13MJ/kgDM、
可消化養分総量は 709g/kgDM であり、オレンジ粕やビートパルプのような副産
物と同等のエネルギー価であった。これらのことからポテトパルプサイレージ
は反芻家畜のエネルギー飼料として利用価値があると判断された。 
 もう一方の試験では、水分吸収剤として乾燥ビートパルプ(DBP)、小麦ワラ
(DWS)および豆の殻と茎(DBH)を用いてサイレージ調製したダイコン選別残渣サ
イレージの採食性を調べるため、めん羊を用いた３×３ラテン方格法による採
食試験を実施した。めん羊にはサイレージとともに乾草を 24.0g/kgＢＷ/頭/日
給与した。ダイコン選別残渣サイレージの摂取量は水分吸収剤として DBP を給
与したサイレージで最も多く、次いで DWS、DBH を用いたサイレージであった。
これらのサイレージはいずれも給与後、速やかに採食された。このようにダイ
コン選別残渣サイレージはめん羊による嗜好性が高いため、ダイコン選別残渣
のもつ潜在的な飼料価値をさらに引き出すためには、さまざま水分吸収剤との
組み合わせによるサイレージ調製試験や大型の反芻家畜による給与試験の実施
が必要であろう。 
４．高水分農産副産物のサイレージ化に影響を及ぼす要因 
 高水分農産副産物のサイレージ調製過程における排汁生成に影響を及ぼす要
因明らかにするため、これまでに実施した試験のデータを用いて解析を行った。
解析に用いたデータには、デンプン粕、ビール粕、リンゴ粕、ダイコンなどの
高水分農産副産物とそれらに水分吸収剤を添加したサイレージ原料(n=27)とそ
れらを用いて調製したサイレージ(n=81)であった。 
 その結果、排汁量とサイレージ原料の水分および PWRC との間には高い正の相
関が認められ、排汁抑制の理論値は原料の水分含量が 69.1%以下、PWRC は
82.9g/100g 以下となった。 
                              
Acknowledgements      I would like to acknowledge the support of the Ministry of Education, Science and 
Culture of Japan for the scholarship program without which this work would not have 
been possible.  
   With a deep sense of gratitude, I would like to express my sincere thanks to both of 
my supervisors, Professor Meiji Okamoto and Associate Professor Masaaki Hanada for 
their immense support and guidance during the course of my studies. The fatherly 
guidance of Professor Okamoto has been very inspirational and his constant supports in 
my studies a great source of motivation. I am deeply grateful. On the other hand, the 
profound knowledge, valuable suggestions, and insightful directions from Professor 
Hanada on my entire research work are greatly acknowledged. What I know today about 
research, I learned from him. I am sincerely thankful. 
    I am grateful to Professor Toshiyoshi Takahashi of Yamagata University for his 
valuable suggestions and useful advice on my graduate program and the final 
manuscript.  
    My sincere thanks to Professor Akio Hongo of the Department of 
Agro-environmental Science, Obihiro University of Agriculture and Veterinary Science 
for the use of sheep during the animal trials, and for his counsel and useful remarks on 
the manuscript.  
    I am also indebted to Dr. Aibibula Yimamu, Mie University, Japan, for his 
immense help and selfless contributions towards my research work. The tremendous 
assistance offered during his presence in our laboratory is greatly appreciated.  
    I would also like to acknowledge the assistance of all my past and current Japanese 
colleagues and staff from the Laboratory of Grassland Utilization, including Ikehata 
Keita, Murata Satoshi, Izumita Mai, Hamabe Kazutaka and others for their untiring 
assistance during the experiments. This also includes my foreigner colleagues, Huiquan 
Li, Maimaijiang Zunong (China) and Shin In Sun (Korea) for their company and 
contributions to my work. 
   I also want to thank my parents, Mohammed Okine and Aishetu Larley, and to share 
this moment with my brothers and sisters for their support during my studies.  
   Finally, I am grateful to my wife Hajara, for her untiring moral and loving support, 
encouragement and patience, and to my son Abdul Gafar who, even at his tender age, 
endured the loss of my company at times when I was needed most. 
                          
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