Chemical Changes during Thermal Processing of Unfermented and Fermented Red Kidney Beans (Phaseolus vulgaris) and effects on In Vitro Protein Digestibility

Background: Legumes are outstanding sources of macronutrients, micronutrients, phytochemicals, as well as antinutritional factors. These components present a complex system enabling interactions with different components within food matrices. The interactions result in insoluble complexes with reduced bioaccessibility of nutrients. The development of appropriate preparation technologies for use at the household and village-level become so imperative to facilitate processing and dietary availability of beans. Aim of the Study: This study aimed to evaluate the effect of thermal processing on the chemical contents of unfermented and fermented red kidney beans (Phaseolus vulgaris) and the effects of the resulting changes on the in vitro protein digestibility. This will enhance food security and Original Research Article Wulam et al.; EJNFS, 13(3): 124-136, 2021; Article no.EJNFS.70071 125 reduction in malnutrition. Methodology: Unfermented and fermented P. vulgaris were boiled using ordinary cooking pot and a pressure pot and the chemical contents were evaluated by standard methods. In vitro protein digestibility was carried out by pepsin digestion. Results: Fermentation resulted in a decrease in the traditional cooking time in the ordinary cooking pot by 40.32%. The protein content of the fermented sample increased by about 7%. The in vitro protein digestibility value was increased by more than 30% with greater percentage evident in fermented samples. Sulphur containing amino acids, methionine and cystein were the limiting amino acids but their contents appreciated by 6.64% and 10.92% respectively after fermentation. Total ash, crude fibre, crude fat contents of P. vulgaris were all affected by more than 20% during the open fermentation and cooking of unfermented beans. The antinutritional factors of P. vulgaris decreased most in boiled fermented samples compared with the other processing methods. There was overall improvement in the In vitro protein digestibility, reduction of cooking time and antinutritional factors when P. vulgaris was fermented and cooked. Conclusion: The outcome of the reserach justifies the fact that combining both fermentation and cooking results in the overall improvement in the nutritional value of P. vulgaris as against cooking without fermentation.


INTRODUCTION
Recent problems linked with meat consumption as source of protein have led to renewed interest in vegetation diets [1]. This phenomenon is reinforced by the fact that reserachers have emphasis that high intake of animal proteins meats may results to cardiovascular disesaes and some types of cancers. It is to this end, that intensive efforts are being made to find alternative sources of protein from the underutilizerd legumes plants in nutrition and in the formulation of new food products.
Red kidney beans are so named because of their shape which is very similar to that of the human kidney. Dry beans (Phaseolus spp. L.) are the most important grain legumes for human consumption [2]. Dry beans have been cultivated for thousands of years and have been reported to play an important role in the traditional diets of many regions throughout the world [3]. Beans are less significant in western diets compared to most of the developing countries [4] The daily per capital consumption of all bean products is 9g in the United States compared to about 110 g in Asia [5]. Phaseolus vulgaris originated from Central and South America, where it was cultivated as early as 6000 BC in Peru and 5000 BC in Mexico [6]. It was introduced to the old World by the Spaniards and Portuguese. It is now widespread and cultivated as a major food crop in many tropical, subtropical and temperate areas of the Americas, Europe, Africa and Asia [7].In Nigeria, red kidney bean is widely cultivated in Plateau State.
Kidney bean is a very good source of cholesterol-lowering fibre as are most other beans [8]. In addition to lowering cholesterol, kidney beans' high fibre content prevents blood sugar levels from rising too rapidly after a meal making these beans an especially good choice for individuals with diabetes, insulin resistance or hyperglycaemia [9].
Generally, legumes have been reported to have low nutritive value due to losw amounts of sulphur-containing amino acids, low protein digestibility and presence of anti-nutritional factors. Cooking is usually done before the use of legumes in a human diet. This improves the protein quality by destruction or inactivation of the heat-labile anti-nutritional factors [10]. However, cooking causes considerable losses in soluble solids especially vitamins and minerals [11]. Increasing the time and temperature of processing has been reported to reduce the nutritive value and available lysine of legumes [12]. Legumes are important sources of dietary protein for both human and animals, but the presence of relatively high concentration of toxins such as phytate, tannins and oxalate referred to anti-nutritive factors affects the nutritional quality by interacting with intestinal tract and also reduce protein digestibility and amino acid absorption. According to [13] unless these substances are destroyed by heat or other treatments, they can exert adverse physiological effects when ingested. The following methods, such as Soaking, cooking, germination, fermentation or irradiation treatments may be used to improve protein nutritional value. This research aimed to evaluate the effect of thermal processing on the chemical compositions of fermented red kidney beans (Phaseolus vulgaris) as well as the effects of these changes on in vitro protein digestibility.

Chemical and Reagents
The chemicals used for this research are: Pepsin, Catalyst and all other chemicals and reagents used were of analytical grades and were purchased from sigma.

Open Fermentation
Red kidney beans sample was rinsed with distilled water and dried in an oven at 55 o C for 24 hours. The rinsed beans were placed into a transparent plastic container and three cups of cold water for every one cup of dried red kidney beans was added. The beans were then allowed to soak for three (3) days uncovered and was fermented by atmospheric microorganisms [14], during which the seed coat remains intact. After fermentation, the microbial growth was terminated by drying at 55 o C in an oven for 24 hours [15].

Thermal Processing
The unfermented and fermented bean samples were boiled using ordinary cooking pot and pressure cooker. Boiling of unfermented bean was for 143.42 and 40.32 minutes using ordinary cooking and pressure pots respectively while 84.91 and 39.27 minutes were for fermented bean samples using ordinary pot and pressure cooking pot respectively after which it was filtered and rinsed with water in each case. The boiled beans were then dried in an oven at 55 o C for 24h after which the bean sample was ground in a laboratory bench mill and kept in a cool dry rubber container for subsequent analysis.

Determination of Proximate Composition 2.5.1 Determination of moisture
The method described by AOAC, [16]

Ash content determination
Determination of ash content was done according to the method described by AOAC, [17]

Determination of lipid content
The lipid content of each sample was determined by the procedure described by AOAC, [18]

Determination of crude fibre
Crude fibre was determined by the method of AOAC, [19].

Determination of nitrogen content and crude protein
Proteins are major compounds containing nitrogen primarily in the form of amino acids which are their building blocks. Nitrogen is used as an index termed crude protein as distinct from true protein. The Kjedahl method of AOAC [20] was used for the crude protein determination.

Ddetermination of carbohydrate content
The percentage carbohydrate was obtained by difference.

6 Determination of Amino Acid Profile
The amino acid profile in the sample was determined using the method described by Adeyeye and Afolabi, [21]. The sample was dried to constant weight, defatted, hydrolyzed, evaporated in a rotary evaporator and loaded into the Technicon sequential Multi-Sample Amino Acid Analyzer (TSM).

Determination of Mineral Contents
Magnesium, calcium, zinc and iron were determined using the atomic absorption spectrophotometry as described by AOAC [22].

Determination of cyanide
The cyanide content was determined according to the method of AOAC [23]

Determination of phytic acid
The phytic acid was determined using the procedure described by Lucas and Markakas [24].

Determination of alkaloids
The gravimetric method as described by AOAC [25] was adopted.

Determination of oxalates
Oxalate was determined using the method of Oke [26].

Determination of tannins
The tannin content of each sample was determined using the method described by Krishnaiah et al., [27].

Determination of in vitro Protein Digestibility
The in vitro protein digestibility was carried out according to the method of [28]. Two hundred (200) milligramme of the powdered bean sample was dispersed in 20cm 3 of pepsin reagent (1.5mg/ml in 0.1M phosphate buffer of pH 2.0) and shaken vigorously. All the tubes were kept in a water bath at 37 o C for three hours with constant shaking at 15 minutes' interval. After three hours, the digestion was stopped by removing the tubes from the water bath and placing them in ice bath for 30 minutes. The samples were then filtered through whatman No.1 filter paper and the residue washed with buffer and dried at 80 o C for 2 hours. The dried residue was placed in a 50cm 3 mico-kjedahl flask and analysed for nitrogen by mico-kjedahl digestion. The indigestible nitrogen was subtracted from total nitrogen of the sample to obtain digestible nitrogen using the following: % in vitro digestibility = Digestible protein/ Total protein in sample × 100

Statistical Analysis
Data obtained is expressed as mean ± standard deviation (SD). Statistical analysis was done by the One Way Analysis of Variance (ANOVA) and Paired Sample T-test using SPSS (version 20). Duncan Multiple Range Test was used to determine the source of variance at P<0.05.

Effects of the Different Processing Methods on Cooking Time of Red Kidney Beans (Phaseolus vulgaris)
Fig . 1 shows the various cooking time for the different processing methods viz: Unfermented beans boiled with ordinary cooking pot and pressure pot and fermented beans boiled with ordinary cooking pot and pressure pot. Based on these, there was a significant decrease in the cooking time of fermented beans cooked with the ordinary cooking pot compared with the unfermented beans cooked with the ordinary cooking pot.

Effects of Open Fermentation of Whole Grains on the Amino Acid Profile of Red Kidney Beans (Phaseolus vulgaris).
The seventeen (17) amino acids determined were observed to be insignificantly (P>0.05) higher in fermented bean samples when compared to the raw bean samples. However, threonine, glycine and cysteine in fermented bean samples showed more than 10% increase when compared to the raw bean samples (Table  .4)

Mineral Contents of the Different Processing Methods
Table, .5, .6 and .7 shows the mineral composition of different processed P. vulgaris.

DISCUSSION
There was significant decrease in the cooking time of fermented beans with pressure pot and ordinary cooking pot as well as that of unfermented beans cooked with pressure pot (Fig .1). The decrease in cooking time of fermented beans cooked with both pressure pot and an ordinary cooking pot could be as a result of soaking of the beans during the fermentation period resulting in its softening and probably the shorter period of time required by the beans to absorb water during the cooking period.
The results showed that ash, fat and crude fibre contents significantly (P<0.05) decreased during the combined processing methods of fermentation and cooking. Crude protein also decreased significantly during cooking with the ordinary cooking pot but increased significantly during fermentation. Carbohydrate was observed to significantly increase during all the processing methods. The decrease in fat during fermentation agrees with the findings of [29] who worked on the effects of different processing methods on the chemical compositions of different sorghum cultivars. This decrease in fat during fermentation also agrees with the findings of Babalola and Giwa [30] who worked on the effects of fermentation on the nutritional and antinutritional properties of fermenting soybeans. The decrease in the crude fibre contents during fermentation could be as a result of the increase the crude fibre which could be due to the activities of methobolising organisms using fibre as energy source hence decreasing the fibre contents [31].
Open fermentation of whole grains of red kidney beans has no significant (P<0.05) effect on the amino acid compositions of red kidney beans (Table 4). Like other legumes sulphur containing amino acids, cystein and methionine were the limiting amino acids [32]. When combined with other protein sources, P. vulgaris could serve as a good source of amino acids. Red kidney bean was also found to contain high amount of Aspartic acid, glutamic acids, leucine and lysine in comparism to the other amino acids contained. This slight increase in the amino acid contents could be due to the increase in protein solubility [33].
The minerals determined in this research include: sodium, potassium, calcium, magnesium, iron and zinc. Based on the results obtained, there was a significant (P<0.05) decrease in the calcium, zinc, iron and potassium contents of the beans during the open fermentation of whole grains while there was insignificant decrease for sodium and magnesium. This decrease in minerals agrees with the finding s of , [34] who attributed it to the leaching of these minerals in the fermenting water.
Phytic acids, alkaloids, oxalic acids, cyanides and tannins were evaluated during this research. During the open fermentation of the whole grains, phytic acids, alkaloids, cyanogenic glycosides and tannins all significantly (P<0.05) decreased. This is in agreement with the findings of [35] who attributed this decrease to degradative actions of fermenting micro organisms ( Table 8).
The result of this research shows that cooking significantly (P<0,05) increased the digestibility of both unfermented and fermented red kidney beans (Table 11). This finding agrees with that of [36] that attributed to the low digestibility values to the high tannins and phytate contents which bind to the proteins and thus limiting their solubility and reducing the binding of the proteases to the proteins. The digestibility of boiled bean samples (either cooked with the pressure pot or the ordinary cooking pot) was found to significantly increase [37]. However, reported a decrease in the digestibility values of protein in lentils and faba beans during cooking of the beans in contrast to [38] who reported an improvement in the in vitro protein digestibility of a 'k131 bean variety' during cooking especially when it was dehulled [39]. Attributed this increase in protein digestibility of the beans to reduction of phytate and tannin levels beyond detection as a result of dehulling.
The digestibility values of fermented bean samples (either boiled or unboiled) was found to significantly increase more than those of the unfermented boiled bean samples. Fermentation thus, improved the in vitro protein digestibility. This finding agrees with [40] report. This improvement in the digestibility of the fermented sample could be as a result of decrease in the phytate and tannin contents which could improve the solubility of the protein hence enhancing its digestibility. This improvement in protein digestibility could also be attributed to the degradation of complex molecules like fibre during fermentation hence increasing access of substrates to the active sites of digestive enzymes. Cooking and fermentation have been reported to result in the break down tanninenzyme and protein-tannin complexes and released free tannins which subsequently leached out the products [41], hence, increasing the access to substrates to the active sites of digestive enzymes.
This research result showed significant (P<0.05) reduction in cooking time of P. vulgaris. This could be of economic importance as it will help reduce the cost of processing through reduction in the amount of cooking fuel used hence encouraging increase in consumption of P. vulgaris. Although, the fermentation period of three (3) days might be discouraging, P. vulgaris can be fermented in large quantity after harvest and kept for future use as this helps to disrupt the seed coats hence, softening the seed coats.although there was significant reduction in the ash, fat and crude fibre content during these processing methods, the combined method of fermentation and cooking can be employed and alternate sources of these nutrients be used to replenish these nutrients when P. vulgaris is processed for consumption. Despite the significant reduction in protein contents when unfermented P. vulgaris is cooked with the ordinary cooking pot (BUC}, there was overall improvement in protein content and protein quality when the combined processing method of fermentation and cokking was employed. The amino acid contents and the protein digestibility value increased during these processes.

CONCLUSION
There was overall improvement in the in vitro protein digestibility, reduction of cooking time and antinutritional factors when P. vulgaris was fermented and cooked. This justifies the fact that combining both fermentation and cooking results in the overall improvement in the nutritional value of P. vulgaris as against cooking without fermentation.