the present inventors have studied the utilization of viable microbial mixtures which show cooperative behavior for some time , and have investigated and prepared mixtures of three kinds of bacteria consisting of lb , sb and bb , and also have succeeded in utilizing such mixture as effective components of a fish feed additive . the mixture of three kinds of bacteria induces a symbiotic phenomenon in the intestinal tract of fishes and then increases useful bacteria . the resultant normal bowel movement due to the modification and maintenance of normal intestinal flora enhances the ability of the intestinal tract to absorb nutritional components . the facts suggesting that the mixture of three kinds of bacteria shows a tendency to enhance the growth of bacteria in the living body , as a result of cooperative action , are proved by the following experiments . when lb grown in a single culture or in the same medium but supplemented with the filtrate of saccarificating microbial culture solution were compared , the count was shown to be about 100 times more in the latter method than that in the former . this cooperative action is considered to be based on the following : proteinase and amylase yielded by sb in the course of cultivation decompose the surrounding proteins and sugars to produce amino acids ( e . g . glutamic acid and aspartic acid ) and glucose , respectively , which are essential for the nutritional resources of lb , and thus promote the growth of lb . in the comparison of a single culture of respective bateria with a mixed culture of both bacteria , the bb counts were observed to be about ten - fold higher in the latter method than in the former , and the latter method also increased lb counts several times over that of the former . a possible explanation of this cooperative action is that respective bacteria produce a growth promoting factor useful for each other during their cultivation . proper samples of respective bacteria composing the mixture of three kinds of bacteria will be listed below . three kinds of viable bacteria are prepared as the active principle containing 10 6 - 10 10 bacteria / g each and then mixed to obtain a mixture of three kinds of bacteria . the mixing ratio is preferably 1 to 8 parts of sb or bb against 1 part of lb . incidentally , when sb and bb are used after forming spores , their properties such as heat resistance , dry resistance , and drug resistance are enhanced . for the embodiment of the present invention , both proteinase and lipase should be selected according to the sort of constituent of an assorted feed to which the present preparation is added . examples of proteinase used are bacterial proteinases , which are distinguished by the sort of bacteria , actinomyces proteinases and filamentous fungus proteinases . especially , filamentous fungus proteinases , yielded by bacteria belonging to the genus aspergillus and genus rhizopus , have been used frequently . furthermore , according to optimal ph , an acid proteinase , neutral proteinase , or alkaline proteinase is chosen . as for a lipase , the enzymes yielded by bacteria belonging to the genus rhizopus and genus candida are used frequently . other than the aforesaid effective components , nutrition supplement materials such as a yeast , gluten meal , wheat flour , rice - bran oil cake , corn starch , potato starch , lactose , soybean cake and glucose are also effective and added at need . especially , since a yeast serves as a growth promoting factor to viable bacteria , if added to the effective components , it makes it possible to maintain the viabl microbial count constant . fish cultivated in accordance with the present invention include the yound yellowtail ( seriola quinqueradiata ), porgy , salmon , eel , rainbow trout , carp and ayu . during cultivation , the additive of the present invention is combined with the raw food , moist pellet and assorted feeds . ordinarily , the feed according to the present invention is added at a ratio of 0 . 1 to 2 . 5 %, the ratio being adjusted depending on the conditions , for example , the variety of fish , age , type of feed and environment in the water area of cultivation , such as water temperatures and the number of fish cultured . in fish cultured with the feed supplemented with the feed additive according to the present invention , beneficial intestinal bacteria increase due to the cooperative action of three kinds of bacteria , a normal bowel movement results , due to the modification , and improved maintenance of intestinal flora and digestive action occurs as a result of digestive enzymes which work synergistically to promote the digestion and absorption of nutrients , all of which serve to improve the fish under cultivation . the beneficial results of ingestion of food supplemented with the additive of the invention include : elongation of the body , increment of fish ratio , quick ingestion of feeds , increase in secretion of slime at the surface of the body , improvement in color of the body , inclusion of proper fat in fish and improvement in taste , decrease in both the morbidity and mortality , reduction of the smell of feces and , coincidently , solidification of feces into a small grain , and resulting reduction of pollution in the water area of fish cultivation due to the easy elimination of feces . the following examples describe the present invention in detail , but the present invention is not limited to these examples . ______________________________________composition of feed additive per 1000 g : ______________________________________active principle of lb ( note 1 ) 20 g ( 5 × 10 . sup . 8 bacteria / g ) active principle of sb ( spore ) ( note 2 ) 20 g ( 5 × 10 . sup . 6 / g ) active principle of bb ( spore ) ( note 3 ) 20 g ( 5 × 10 . sup . 7 / g ) proteinase ( note 4 ) 100 , 000 unitslipase ( note 5 ) 400 , 000 unitsbeer yeast ( a nutrition supplement ) proper quantitynatural alminium silicate ( a vehicle ) proper quantity______________________________________ ( note 1 ) streptococcus faecalis t110 , deposition no . 8936 , was used ; it was deposited with the international deposit agency in japan , fermentatio research institute , agency of industrial science and technology , ministry of international trade and industry . ( note 2 ) bacillus mesentericus toa , deposition no . 8934 . ( note 3 ) clostridium butyricum toa , deposition no . 8935 . ( note 4 ) alkaline proteinase yielded from the bacteria belonging to genus rhizopus . ( note 5 ) alkaline lipase yielded from the bacteria belonging to genus rhizopus . purpose : to investigate the effect of a feed additive on growth promotion of fishes , field studies were made using rainbow trout . test method : an outdoor test pond ( made of concrete ) was divided along a stream into three parts by a board , and two net crawls ( 5 - mm meshes ), 1 m × 1 m × 0 . 8 m ( depth ), in each division were settled at intervals of about 0 . 5 meters . spring water ( 14 . 5 °- 15 . 0 ° c .) was flowed into this pond at the rate of 300 l / min , and the depth of water was controlled at about 0 . 6 meters . the rainbow trout of the same venter , at about 7 months after hatching , were divided at random into 6 groups ( 30 fishes per group ) and cultivated preliminarily with the same feed as used in the field studies for two weeks , then field studies were carried out ( for 8 weeks in july to september ). according to the feed supply rate table by leitritz et al ., a test feed was given in principle at 2 . 3 % of body weight a day for the average body weight of 23 - 40 g and 1 . 9 % for 40 - 60 g . the feed was given twice a day , at 10 and 15 o &# 39 ; clock , for 6 days ( not given on sunday ). as a basic feed for the test feed , the assorted feed for cultivating the rainbow trout ( oriental mark , solid type no . 3 ), which meets standard requirements , was used . three sorts of test feeds , a control feed not containing additives , feeds containing additives in an amount of 0 . 5 % and 2 %, were prepared and used for field studies . prior to and after the studies , all fish were weighed , the length of the fish was measured , and the corpulence rate was estimated . total body weight in each group was measured every 2 weeks , and feed efficiency and body - weight increase rate were estimated . in a comparison control group , the feed not containing the additives ( group a ), that containing the additives in an amount of 0 . 5 % ( group b ), and that containing the additives in an amount of 2 % ( group c ) were supplied . the studies were carried out in duplicate and called a - 1 , a - 2 , b - 1 , b - 2 , c - 1 and c - 2 . groups belonging to 1 were located on the upper stream of the pond and groups belonging to 2 on the downstream . test results : as for the feed efficiency ( note 6 ), the average of control group ( a ) was 59 . 4 % ( a - 1 : 64 . 4 %, a - 2 : 54 . 4 %), the average of control group containing 0 . 5 % of additives ( b ) 67 . 4 % ( b - 1 : 67 . 5 %, b - 2 : 67 . 3 %), and the average of control group containing 2 % ( c ) 67 . 1 % ( c - 1 : 74 . 6 %, c - 2 : 59 . 5 %), and when compared with the average value , group b was higher than group a by 8 . 0 %, group c was higher than a by 7 . 7 %, and groups b and c are almost the same ( difference 0 . 3 %). comparisons among the groups located on the upper stream show that : group c was higher than group a by 10 . 2 % and higher than group b by 7 . 1 %, and on the downstream group b was higher than group a by 12 . 9 % and higher than group c by 7 . 8 %. that is to say , the difference in feed efficiency between the control group and addition group , fed food containing additive of the invention ; was shown to be 10 - 13 % in both groups located on the upper stream and downstream , and it was clear that the additives do take part in improvement of the feed efficiency . as for the increase rate of body weight ( note 7 ), group a was 72 . 7 % on the average ( a - 1 : 79 . 9 %, a - 2 : 65 . 4 %), group b 84 . 7 % ( b - 1 : 84 . 9 %, b - 2 : 84 . 5 %), and group c 84 . 2 % ( c - 1 : 94 . 5 %, c - 2 : 73 . 8 %). the rate of increase in the addition groups was higher than that of the control group by about 12 % on the average , showing a similar tendency to the feed efficiency . from these results , it was confirmed that the feed additives for fish cultivation according to the present invention played a part in digestion and absorption of feeds in the cultivation of rainbow trout and had some effects of improving the feed efficiency and increasing the rate of body weight gain . ( note 6 ) ## equ1 ## ( note 7 ) ## equ2 ## g 1 : total body weight after the test g 2 : total body weight before the test g 3 : total body weight in the dead fish __________________________________________________________________________test results ( summary ) control addition ( 0 . 5 %) addition ( 2 %) a - 1 a - 2 b - 1 b - 2 c - 1 c - 2 ( upper - ( down - ( upper - ( down - ( upper - ( down - stream ) stream ) stream ) stream ) stream ) stream ) __________________________________________________________________________total number 1 start 30 30 30 30 30 30of fish 2 14 days 30 30 30 30 30 30 3 28 days 30 30 30 30 30 30 4 42 days 30 30 30 30 30 30 5 56 days 30 30 30 30 30 30total 1 start 1052 . 1 1015 . 7 1041 . 9 1054 . 7 1042 . 6 1122 . 3weight ( g ) 2 14 days 1170 1120 1180 1190 1170 1250 3 28 days 1410 1310 1410 1430 1420 1530 4 42 days 1620 1450 1640 1660 1690 1700 5 56 days 1892 . 6 1679 . 7 1926 . 2 1945 . 9 2027 . 6 1950 . 4average 1 start 35 . 1 33 . 9 34 . 7 35 . 2 34 . 8 37 . 4body 2 14 days 39 . 0 37 . 3 39 . 3 39 . 7 39 . 0 41 . 7weight ( g ) 3 28 days 47 . 0 43 . 7 47 . 0 47 . 7 47 . 3 51 . 0 4 42 days 54 . 0 48 . 3 54 . 7 55 . 3 56 . 3 56 . 7 5 56 days 63 . 1 56 . 0 64 . 2 64 . 9 67 . 6 65 . 0number of i term 0 0 0 0 0 0dead fish ii term 0 0 0 0 0 0 iii term 0 0 0 0 0 0 iv term 0 0 0 0 0 0 mortality 0 0 0 0 0 0quantity of i term 290 . 4 280 . 8 288 . 0 291 . 6 288 . 0 309 . 6feed ( g ) ii term 322 . 8 309 . 6 325 . 2 328 . 8 322 . 8 345 . 6 iii term 321 . 6 298 . 8 321 . 6 326 . 4 324 . 0 349 . 2 iv term 369 . 6 331 . 2 374 . 4 378 . 0 385 . 2 387 . 6increase i term 11 . 2 10 . 3 13 . 3 12 . 8 12 . 2 11 . 4rate of body ii term 20 . 5 17 . 0 19 . 5 20 . 2 21 . 4 22 . 4weight (%) iii term 14 . 9 10 . 7 16 . 3 16 . 1 19 . 0 11 . 1 iv term 16 . 8 15 . 8 17 . 5 17 . 2 20 . 0 14 . 7feed i term 40 . 6 37 . 1 48 . 0 46 . 4 44 . 2 41 . 2efficiency ii term 74 . 3 61 . 4 70 . 7 73 . 0 77 . 4 81 . 0 (%) iii term 65 . 3 46 . 9 71 . 5 70 . 5 83 . 3 48 . 7 iv term 73 . 8 69 . 4 76 . 4 75 . 6 87 . 6 64 . 7quantity of i term 0 0 1 . 38 1 . 38 5 . 52 5 . 52sample per ii term 0 0 1 . 38 1 . 38 5 . 52 5 . 53kg of body iii term 0 0 1 . 14 1 . 14 4 . 56 4 . 56weight ( g ) iv term 0 0 1 . 14 1 . 14 4 . 56 4 . 56__________________________________________________________________________ ______________________________________active principle of lb ( note 1 ) 25 g ( 1 × 10 . sup . 9 bacteria / g ) active principle of sb ( spore ) ( note 2 ) 40 g ( 1 × 10 . sup . 7 / g ) active principle of bb ( spore ) ( note 3 ) 55 g ( 1 × 10 . sup . 8 / g ) proteinase ( note 4 ) 150 , 000 unitslipase ( note 5 ) 300 , 000 unitsdry yeast ( a nutrition supplement ) proper quantitylactose ( a nutrition supplement proper quantityand vehicle ) wheat flour ( a nutrition supplement proper quantityand vehicle ) ______________________________________ ( note 1 ) lactobacillus acidophillus was used . ( note 2 ) bacillus subtilis was used . ( note 3 ) clostridium acetobutyricum was used . ( note 4 ) a proteinase yielded from the bacteria belonging to genus aspergillus , showing a maximum enzymatic activity at about ph 8 . 0 . ( note 5 ) a neutral of alkaline lipase yielded from the bacteria belonging to genus rhizopus . in the example , a proteinase showing a maximum enzymatic activity at about ph 8 . 0 was used , which is a property close to a proteinase in the pyloric cecum and intestine of the fish , especially rainbow trout , and hence it is a suitable one for cultivating the fish . 1 . effect of the additive of the present invention on digestion and absorption in the rainbow trout ( test period : may to september ) the rainbow trout , at 6 months after hatching were divided into 3 groups , 30 fishes a group , and tests were made ( the average body weight was 166 - 188 g ). spring water , which temperature was 12 ° c ., was flowed in at a rate of 202 l / min . the quantity of feed supplied was 1 % of the body weight a day according to the feed - supply rate table by leitritz et al . and the feed was given twice , at 9 and 14 hours . in the control group , the feed did not contain the additive . test feed contained the additive in an amount of 0 . 5 % and 2 . 0 %. as a basic feed , the assorted feed for growing the rainbow trout , which met standard requirements , was employed . to each feed chrome oxide was added by 0 . 5 % as an indicator . the digestion absorption rate of crude protein was 84 . 9 % in the control group , and 85 . 0 - 87 . 4 % in the addition groups , showing that the rate in the addition groups was statistically significant compared with that in the control group . furthermore , the digestion absorption rate of crude lipid was 78 . 3 % in the control group and 81 . 6 - 84 . 3 % in the addition groups , showing statistically significant results in the addition groups . from these results , it was confirmed that the additive of the present invention brought about improved digestion and absorption of feed . 2 . field studies of the assorted feed supplemented with the additive of the present invention for a young yellowtail ( test period : november to february ) in a polyethylene crawl connected to a raft which is divided into small sections at the surface of sea , the young yellowtail of 50 fishes each , having an average body weight of 709 g , were grown , and tests were carried out . feed used for tests were ( a ) raw food ( sardines ), ( b ) assorted feed : the combination of compound , water , and squid oil ; and the combination of compound , raw food ( any one of the mackerel , sand eel , and sardine ), and squid oil , ( c ) the feed plus the additive of the present invention : the combination of compound , raw food ( sardines ), squid oil , and the additive ( 1 . 5 %). ( b ) and ( c ) were given in the form of moist pellet . at the beginning , middle , and end of the test , all fish in each group were weighed , and the state of the remnant of feed was observed every day . in the last day of the test , a sensuous test was carried out by 12 persons concerning the color of fish , hardness in the muscle , the degree of fat , and taste . the state of ingestion was observed to be extremely good in both the additive group ( c ) and raw food group ( a ), but bad in the assorted feed group ( b ). increase in the body weight was observed in the raw food group ( a ), the additive group ( c ), and the assorted feed plus mackerel group ( b - mackerel ); feed efficiency estimated in dried substance were 32 %, 18 %, and 14 %, respectively . dead fish throughout the test period was 1 in the raw food group ( a ), 5 in the additive group ( c ), 12 in the assorted feed plus mackerel group ( b - mackerel ), and 22 to 27 in the assorted feed plus others group ( b - others ). in the sensuous test , the sum of &# 34 ; delicious &# 34 ; and &# 34 ; a little delicious &# 34 ; taste was 67 % in the additive group ( c ) and was in the order of the assorted feed plus sardine group ( b - sardine ; 60 %) and the raw food group ( a ; 54 %), winning the reputation of the feed plus raw food , sardine , group . furthermore , the digestion - absorption rate of protein was determined in the contents of the intestine of a young yellowtail cultivated with the combined feed of compound , raw food ( a sardine ), and squid oil and with those plus 1 % of additive , and results were 76 . 3 % in the additive group and 69 . 1 % in the control group , showing a rise of 7 % of the digestion - absorption rate by the addition of additive . in these tests , some improvements were observed on the ingestion , growth of fish , and mortality . they are considered to be the result of the improvement of the texture of the feeds and the digestion absorption rate of feeds by addition of the additive of the present invention . 3 . preventive effect of the additive on the pollution of cultivating water by fish feces ( test period : october to november ) in group ( a ) the rainbow trout , at 9 months after hatching , were divided into 3 groups , 150 fish per group , and tests were made ( the average body weight was about 100 g ). spring water , having a temperature of 12 ° c ., was flowed at a rate of 202 l / min . the quantity of the feed supplied was 1 . 1 % of the body weight a day on the basis of the table of feed supply rate by leitritz et al ., and the feed was given twice , 10 and 15 hours . in group ( b ) the rainbow trout , at 7 months after hatching , were divided into 3 groups , 960 fish per group , and tests were made ( the average body weight was 20 . 8 g ). spring water , 11 . 5 ° c ., was flowed at a rate of 202 l / min . the quantity of feed supplied was 2 . 1 % of the body weight a day on the basis of the table of feed supply rate by leitritz et al ., and the feed was given twice , 10 and 15 o &# 39 ; clock . as a control feed , the feed not containing the additive was used . as a test feed , feeds containing the additive in an amount of 0 . 5 % and 2 . 0 % were prepared . as the basic food in each feed , the assorted feed for cultivating rainbow trout , which meets standard requirements , was employed . chrome oxide was added to each feed in an amount of 0 . 5 % as an indicator . the feces were diluted with a diluent ( spring water exposed to air in the pond ) by 50 , 000 , 100 , 000 and 300 , 000 times and the quantity of dissolving oxygen prior to and after storage at a constant temperature ( 20 ° c ., for 5 days ) was determined and bod was calculated . chrome oxide was oxidized by nitric acid or perchloric acid and the optical density at 375 nm was determined . in group ( a ), bod value per 1 kg of feed in the additive group reduced by 56 . 1 to 59 . 8 % compared with that in the control group . from these results , it is proved that the additive of the present invention promotes digestion and absorption of feeds , reduces the pollution of water for cultivation derived from the feces excreted from fish , and offers better circumstances for cultivating healthy fish . ______________________________________active principle of lb ( note 1 ) 10 g ( 7 × 10 . sup . 8 bacteria / g ) active principle of sb ( spore ) ( note 2 ) 50 g ( 5 × 10 . sup . 8 / g ) active principle of bb ( spore ) ( note 3 ) 75 g ( 8 × 10 . sup . 7 / g ) proteinase ( note 4 ) 130 , 000 unitslipase ( note 5 ) 350 , 000 unitsyeast ( a nutrition supplement ) proper quantitycorn starch ( a nutrition supplement proper quantityand vehicle ) calcium carbonate ( a vehicle ) proper quantity______________________________________ ( note 1 ) bifidobacterium bifidum was used . ( note 2 ) bacillus mesentericus was used . ( note 3 ) clostridium butyricum was used . ( note 4 ) an alkaline proteinase yielded from the bacteria belonging to genus aspergillus . ( note 5 ) an alkaline lipase yielded from the bacteria belonging to genus rhizopus . the effect of the feed additive of the present invention on the growth of eel the eel , anguilla japonica , were divided for study into 2 groups , a young eel group ( 67 - 83 g ). the water tank for the young eel group was 6 m × 6 m × 1 m ( depth ) in size and 10 m × 10 m × 1 m ( depth ) for the adult eel group , and an additive addition group and additive free group ( control group ) were made in each group . spring water , 25 ° c . to 33 ° c ., was supplied at a water exchange rate of 4 . 5 to 6 . 0 %. the assorted feed consisting of animal feed 67 %, wheat 23 %, plant oil grounds 3 %, and others 7 % was used as a basic feed , to which the additive of the present invention was added in an amount of 2 . 5 %. the quantity of feed supplied was equal to 1 . 0 - 3 . 5 % of the body weight and adjusted after observing the degree of ingestion . the culturing period was 35 days in the young eel group and 80 days in the adult eel group . in both groups , the of the body weight and the feed efficiency were improved by adding the additive . this finding proved that the additive had a good effect on digestion and absorption of feeds . the number of dead eel was also observed to be statistically significant . ______________________________________test result of eel cultivation young eel adult eel addition control addition control______________________________________total weight ( kg ) 540 570 1 , 700 1 , 750at the beginningnumber of fish 54 , 000 57 , 000 20 , 400 26 , 250at the beginningaverage body weight 10 10 83 67 ( g ) at the beginningnumber of the dead 9 25 5 17total weight ( kg ) 1 , 172 1 , 117 2 , 447 2 , 275at the endnumber of fish 53 , 991 56 , 975 20 , 395 26 , 233at the endaverage body weight 21 . 7 19 . 6 120 87 ( g ) at the endquantity of feed ( kg ) 826 763 779 624increase rate of 217 196 144 130body weight (%) feed efficiency (%) 76 . 5 71 . 7 95 . 9 84 . 1______________________________________
a feed additive for fish cultivation , which contains , as effective components , a proteinase , lipase , and mixture of three kinds of bacteria consisting of lactic acid producing microbe , saccarificating microbe , and butyric acid producing microbe .