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. 2014 Sep 22;2(6):768–785. doi: 10.1002/fsn3.171

Metabolite changes during natural and lactic acid bacteria fermentations in pastes of soybeans and soybean–maize blends

Tinna Austen Ng'ong'ola-Manani 1,2,✉, Hilde Marit Østlie 1, Agnes Mbachi Mwangwela 2, Trude Wicklund 1
PMCID: PMC4256583  PMID: 25493196

Abstract

The effect of natural and lactic acid bacteria (LAB) fermentation processes on metabolite changes in pastes of soybeans and soybean–maize blends was studied. Pastes composed of 100% soybeans, 90% soybeans and 10% maize, and 75% soybeans and 25% maize were naturally fermented (NFP), and were fermented by lactic acid bacteria (LFP). LAB fermentation processes were facilitated through back-slopping using a traditional fermented gruel, thobwa as an inoculum. Naturally fermented pastes were designated 100S, 90S, and 75S, while LFP were designated 100SBS, 90SBS, and 75SBS. All samples, except 75SBS, showed highest increase in soluble protein content at 48 h and this was highest in 100S (49%) followed by 90SBS (15%), while increases in 100SBS, 90S, and 75S were about 12%. Significant (P < 0.05) increases in total amino acids throughout fermentation were attributed to cysteine in 100S and 90S; and methionine in 100S and 90SBS. A 3.2% increase in sum of total amino acids was observed in 75SBS at 72 h, while decreases up to 7.4% in 100SBS at 48 and 72 h, 6.8% in 100S at 48 h and 4.7% in 75S at 72 h were observed. Increases in free amino acids throughout fermentation were observed in glutamate (NFP and 75SBS), GABA and alanine (LFP). Lactic acid was 2.5- to 3.5-fold higher in LFP than in NFP, and other organic acids detected were acetate and succinate. Maltose levels were the highest among the reducing sugars and were two to four times higher in LFP than in NFP at the beginning of the fermentation, but at 72 h, only fructose levels were significantly (P < 0.05) higher in LFP than in NFP. Enzyme activities were higher in LFP at 0 h, but at 72 h, the enzyme activities were higher in NFP. Both fermentation processes improved nutritional quality through increased protein and amino acid solubility and degradation of phytic acid (85% in NFP and 49% in LFP by 72 h).

Keywords: Amino acid, fermentation, lactic acid bacteria, phytic acid, protein, soybean paste

Introduction

Legumes, cereals, and their blends remain important in the diets of many people in developing countries. Legumes are the main source of protein because animal proteins are expensive. Soybeans contain up to 40% protein (Redondo-Cuenca et al. 2007) and when consumed together with maize, they provide a high-quality protein diet comparable to animal protein (Asgar et al. 2010). Soybeans and maize complement each other in terms of limiting amino acids. Cereals are deficient in lysine, but are rich in cysteine and methionine, whereas legumes are rich in lysine, but deficient in the sulfur-containing amino acids (Palanisamy et al. 2012). Therefore, by combining cereals with legumes, the overall protein quality of the diet is improved.

However, the biological utilization of nutrients from legumes is affected by the presence of antinutritional factors. Cereals, legumes, and their blends contain phytic acid, trypsin inhibitors, polyphenols, and flatulence causing oligosaccharides such as raffinose and stachyose (Mulimani and Devendra 1998; Sindhu and Khetarpaul 2001; Yoon and Hwang 2008). Trypsin inhibitor reduces digestibility of proteins by inhibiting protease activity of trypsin enzyme (Sindhu and Khetarpaul 2001), while α-galactosides (raffinose and stacchyose) are broken down by intestinal anaerobic microorganisms causing flatulence (Vidal-Valverde et al. 1993). Phytic acid forms complexes with proteins and minerals such as calcium, iron, magnesium, and zinc reducing their biological availability (Yoon et al. 1983; Chitra et al. 1996; Urbano et al. 2000). The presence of antinutritional factors along with disagreeable beany flavor has limited the consumption of soybean as a raw material (Wang et al. 2003). Several processing methods including fermentation reduce levels of antinutritional factors and hence they improve the nutritive value of processed foods (Golbitz 1995; Chitra et al. 1996; Wang and Murphy 1996; Palanisamy et al. 2012). Fermentation also improves flavors and textures of legumes (Deshpande and Salunkhe 2000) and other fermented products in general.

In Malawi, utilization of soybeans is limited to maize–soybean composite flour locally known as likuni phala which is used as a weaning food (Kalimbira et al. 2004; Maleta 2006). In an effort to increase utilization and consumption of soybeans by all age groups, solid state fermented pastes of soybeans and soybean–maize blends to be used as a side dish or meat alternative were developed (Ng'ong'ola-Manani et al. 2014). Many studies on solid state fermentation of soybeans and legumes have focused on natural fermentation which favors growth of Bacillus subtilis or molds. Bacillus-fermented soybean products include soy-dawadawa (Dakwa et al. 2005), Nepalese kinema (Sarkar and Tamang 1995), Japanese natto, Thai thua-nao (Dajanta et al. 2012), and Korean doenjang (Kim et al. 2010). The main metabolic activity of B. subtilis is proteolysis of proteins into amino acids and subsequent production of ammonia (Sarkar and Tamang 1995; Dakwa et al. 2005). High amount of ammonia in the fermented product results in a strong odor which some people find objectionable (Allagheny et al. 1996; Parkouda et al. 2009). On the other hand, lactic acid fermentation processes improve texture, flavor, and shelf life of traditional foods (Steinkraus 1997).

Cereal gruels such as ogi, koko, kenkey, and mahewu made from maize and/or sorghum (Sanni 1993), bushera from sorghum and millet (Muyanja et al. 2003), ben-saalga from pearl millet (Songré-Ouattara et al. 2008), and togwa from cassava, maize, sorghum, millet, or their blends (Mugula et al. 2003) are fermented by LAB. Like B. subtilis, some LAB degrade antinutritional factors like trypsin inhibitor, phytic acid, raffinose, and stachyose (Holzapfel 1997, 2002; Sindhu and Khetarpaul 2001). An additional advantage of lactic acid fermentation is the possibility of involvement of LAB with potential probiotic characteristics (Sindhu and Khetarpaul 2001) in addition to increased safety of the product. In this study, thobwa, a Malawian fermented cereal gruel prepared from maize flour and cofermented with malt flour from finger millet was used as a back-slopping material to facilitate LAB fermentation processes in LAB-fermented pastes (LFP).

Lactic acid bacteria (LAB)-fermented pastes were characterized by brown color, sourness, bitterness, saltiness, umami, burnt roasted soybean aroma, and maize aroma (Ng'ong'ola-Manani et al. 2014). Sensory properties that characterized naturally fermented pastes included higher pH, yellow color, fried egg-like appearance and aroma, sweetness, softness, roasted soybean aroma, rancid odor, and raw soybean odor (Ng'ong'ola-Manani et al. 2014). There was consumer segmentation in preference patterns of the fermented pastes and liking was biased toward naturally fermented pastes (Ng'ong'ola-Manani et al. 2014).

The fermented pastes were developed to serve as major sources of protein in maize-based diets, and a report on proximate composition of the pastes would give important nutrition information. Therefore, this study aimed at reporting and comparing metabolites and metabolite changes in pastes of soybeans and soybean–maize blends fermented naturally and by LAB. Particularly, changes in proteins, amino acids, organic acids, sugars, antinutritional factors, and enzyme activities during fermentation were investigated.

Materials and Methods

Preparation of pastes of soybeans and soybean–maize blends

Pastes of soybeans and soybean–maize blends were prepared in the laboratory according to Ng'ong'ola-Manani et al. (2014). Portions of 500 g pastes of soybeans and soybean–maize blends were naturally fermented or LAB fermented through back-slopping using thobwa. Thobwa was produced by making maize porridge containing 15% (w/v) maize flour and 80% water according to the protocol for togwa processed in the southern part of Tanzania (Kitabatake et al. 2003). The porridge was cooled to about 50–60°C before the addition of finger millet (Eleusine coracana) malt flour (5%, w/v). The porridge was left to ferment naturally at room temperature (23–28°C) for 18 h before being used as inocula in back-slopped samples. The quality of the thobwa was determined through monitoring continuous pH reduction during 18 h of thobwa fermentation. The LFP were back slopped with 10% (v/w) of the thobwa. The pH of the thobwa was around 4.5 with a LAB population of 108 cfu/mL.

Naturally fermented pastes (NFP) were designated as 100S, 90S, and 75S according to 100%, 90%, and 75% soybean composition in the pastes, the remaining proportions being maize. Similarly, back-slopped LFP were designated as 100SBS, 90SBS, and 75SBS. All treatments were fermented at 30°C for 72 h. The fermenting pastes were sampled at 0, 24, 48, and 72 h and samples were frozen at −20°C until analysis. Analyses were made from three independent experiments except in amino acids, organic acids, and sugars in which analyses were made from two experiments.

pH, titratable acidity, moisture content, and protein determination

AOAC (1990) methods were used to determine moisture content, pH, and titratable acidity. The pH was measured using a pH meter (WTW pH 525; D. Jurgens and Co., Bremen, Germany) fitted with a glass electrode (WTW SenTix 97T). Total proteins and water-soluble proteins were analyzed as total nitrogen and water soluble nitrogen, respectively by the Kjeldahl method according to Thiex et al. (2002). For total protein, samples were ground in a mortar with a pestle until they turned fine and homogenous, and 0.5 g of the sample was transferred into a digestion flask where 0.8 g CuSO4, 7.0 g of K2SO4, and 15 mL H2SO4 (98%) were added. The digestion was done on a Labconco microKjeldahl digestor (Model 60300-00; Kansas City) for 3 h. The digested material was distilled using a Kjeltec System 1002 distillation unit (Tecator, Hoganas, Sweden) with 4% boric acid containing a mixed indicator in the receiving flask. Samples for determination of water soluble nitrogen were prepared according to Sarkar and Tamang (1995) by homogenizing 2.0 g of sample with 100 mL of distilled water for 2 min in a Star Lab blender LB 400 (VWR, Fontenay Sous Bois Cedex, France) and centrifuging at 3500g for 10 min at 25°C. The supernatant was filtered through a Whatman No. 2 filter paper and the nitrogen content of a known volume was determined by the Kjeldahl method. A conversion factor of 6.25 was used to obtain percentage of protein (Dajanta et al. 2012).

Enzyme activities

Preparation of enzyme extract

Enzyme extracts of the fermenting pastes were prepared according to Dakwa et al. (2005) and Terlabie et al. (2006). Five grams of the sample was ground in 50 mL of 0.1 mol/L potassium hydrogen phosphate (Merck, KGaA, Damstadt, Germany) buffer, pH 6.5 as the extracting buffer. The suspension was washed with petroleum ether (Sigma-Aldrich, St Louis, MO) to extract the oil. The sample was centrifuged (Kokusan H-201 series; Kokusan Enshinki Co. Ltd., Tokyo, Japan) at 3500g for 5 min at 4°C. The supernatant constituting the crude enzyme was stored at −20°C until analysis.

Determination of α-amylase and α-galactosidase activities in fermenting pastes

Alpha-amylase activities were determined by the assay method of Bernfeld (1955). Two milliliters of the enzyme extract was mixed with 1 mL of 1% (w/v) starch (Merck) solution and was incubated for 1 h at 40°C. The reaction was stopped by adding 3 mL of dinitrosalisylic acid reagent (DNS; Alfa Aesar, Karlsruhe, Germany) before heating for 5 min. After cooling, the sample mixture was diluted with 18 mL of distilled water and the optical density was measured at 550 nm in a spectrophotometer (Jenway 6300; Bibby Scientific, Staffordshire, UK). A blank was prepared by adding DNS before the starch solution. The amount of reducing sugars formed was calculated from a standard curve prepared with known concentrations of maltose (Merck) according to Bernfeld (1955).

Alpha-galactosidase activities were determined according to Odunfa (1983). About 2 mL of the enzyme extract was mixed with 1 mL of 1% (w/v) melibiose monohydrate (Merck) solution before incubation for 2 h at 40°C. The reaction was stopped by adding 3 mL of DNS (Alfa Aesar) before boiling in a water bath for 5 min. The subsequent steps proceeded as in alpha-amylase determination.

Amino acids

Total amino acids

Total amino acids were determined according to the method of Official Journal of the European Communities (1998). Amino acids were extracted from a weighed (116.5–190.2 mg) well homogenized freeze-dried sample. A closed hydrolysis was done to extract the amino acids, and the procedure for hydrolysis was amino acid dependent. For instance, cysteine and methionine were oxidized to cysteic acid and methionine sulfone, respectively, prior to hydrolysis. Asparagine and glutamine were converted to aspartic acid and glutamic acid before hydrolysis, while tyrosine was analyzed separately from the rest of the amino acids using basic hydrolysis and high-performance liquid chromatography (HPLC)/fluorescence detection. Different optimal times for hydrolysis for each amino acid were used. The pH of the hydrolysates was adjusted to 2.20 using an autotitrator. The hydrolysates were then run on a Biochrom 30 amino acid analyzer (Biochrom Co, Cambridge, UK), equipped with a sodium high-performance oxidized column (Biochrom). The UV-signals were read after postcolumn derivatization with ninhydrin at 570 and 440 nm using Chromeleon software (Dionex, Sunnyvale, CA). Cysteic acid, methionine sulfone, lysine, threonine, alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, leucine, phenylalanine, proline, serine, tyrosine, and valine standards were used in the analysis and were obtained from Sigma-Aldrich.

Free amino acids

Free amino acids were extracted from a 1.00 g freeze-dried homogenized sample which was weighed into a 15-mL centrifuge tube. To each sample, 5 mL of 0.1 mol/L HCl standard solution containing 0.4 μmol/mL l-norvaline and piperidine-4-carboxylic acid was added. The sample and the standard solution were thoroughly mixed on a vortex. The sample mixture was put on ultra sound water bath (Branson 2510, Soest, Netherland) at room temperature for 30 min. Sonication was followed by centrifugation at 3000g (Beckman J2-MC; GMI Inc, Ramsey, MN) for 40 min at 4°C. From the supernatant, 1 mL of extract was transferred into a 2-mL Eppendorf tube to which 1 mL of 4% trichloroacetic acid (Merck) was added. The rest of the procedure was done according to Bütikofer and Ardö (1999).

Organic acids and sugars

To 1.0 g of freeze-dried homogenized sample, 5 mL of milliQ water was added and mixed thoroughly. Then 1.00 g of the sample mixture was transferred to another tube to which 2.5 mL of milliQ water, followed by 0.2 mL of 0.5 mol/L H2SO4 (Merck) and 8 mL of acetonitrile (Merck) were added. Mixing was done for 30 min on a MultiRS-60 BIOSAN rotator (Nerlien, Oslo, Norway). The rest of the procedure was done according to Narvhus et al. (1998). Organic acids, glucose, fructose, and maltose levels were analyzed by HPLC. The organic acids were detected with a UV detector set at 210 nm and the sugars were determined using a refractive index detector (Perkin Elmer series 200, Norwalk, CT). Organic acids were identified based on comparison of their retention times with standard solutions of citrate, orotic acid, pyruvate, succinate, dl-lactate, uric acid, dl-pyroglutamate, propionate, α-ketoglutaric acid, oxalic acid, acetate, and formate (Merck). Identification of sugars was also based on retention times of standard solutions of maltose, lactose, galactose, fructose, and glucose (Merck). Quantification was done using external calibration curves of mixed standards in deionized water.

Antinutritional factors (phytic acid and trypsin inhibitor)

Phytic acid was extracted from 0.5 g samples in 25 mL of 0.2 N HCl for 3 h with continuous shaking, according to Erdal et al. (2002). The extracts were centrifuged at 3500g for 10 min at 4°C and the supernatants were used for analysis. The extracted phytate was assayed according to the method described by Haug and Lantzsch (1983). Trypsin inhibitors were measured by the method of Kakade et al. (1974) as modified by Hamerstrand et al. (1980).

Statistical analysis

Analysis of variance (ANOVA) at P = 0.05 was performed in SPSS 15.0 (SPSS Inc., Chicago, IL) and least squares difference test was used to separate means.

Results and Discussion

Proximate composition

The initial pH and titratable acidity were almost the same for all samples, despite LFP being inoculated with a LAB-fermented product (Table1). The pH for LFP decreased faster than for NFP. The relatively fast drop in pH as in LFP to about 4.0 at 24 h would be desirable to prevent growth of pathogens and spoilage bacteria. The slow drop in pH in NFP indicated cofermentation by LAB and other microorganisms. Nevertheless, the gradual decline in pH in NFP suggested a bias toward LAB fermentation as opposed to alkaline fermentation, reported in natural fermentation processes of soybeans (Sarkar et al. 1994, 2002; Dakwa et al. 2005; Parkouda et al. 2009; Dajanta et al. 2011). The lactic acid fermentation could be attributed to limited oxygen during fermentation in the jars which could have favored growth of microaerophiles while limiting growth of spore formers, eventually reducing ammonia production with no increase in pH (Allagheny et al. 1996; Parkouda et al. 2009). Significant increases in the amount of titratable acidity were observed in all samples (except in 100S) from 0 to 24 h (Table1) and thereafter continuous increases throughout fermentation were observed, although some of them were not significant. Continuous increases in titratable acidity in alkaline fermentation processes have been reported previously (Sarkar and Tamang 1995).

Table 1.

Changes in pH, acidity, moisture content, protein content, and enzyme activities of the pastes during fermentation

Parameter Treatment 0 h 24 h 48 h 72 h
pH 100S 6.95 ± 0.13a 6.74 ± 0.20a 5.93 ± 0.50b 5.81 ± 0.59b
90S 6.98 ± 0.16a 6.15 ± 0.25b 5.80 ± 0.20c 5.36 ± 0.14d
75S 6.88 ± 0.14a 6.61 ± 0.32a 6.09 ± 0.27b 5.41 ± 0.18c
100SBS 6.46 ± 0.57a 4.64 ± 0.37b 4.47 ± 0.34b 4.26 ± 0.28b
90SBS 6.45 ± 0.48a 4.36 ± 0.20b 4.11 ± 0.36b 4.01 ± 0.31b
75SBS 6.44 ± 0.40a 4.20 ± 0.24b 4.02 ± 0.39b 3.91 ± 0.29b
Titratable acidity (g lactic acid/100 g sample) 100S 0.10 ± 0.05a 0.16 ± 0.03a 0.40 ± 0.23b 0.58 ± 0.31b
90S 0.10 ± 0.03a 0.25 ± 0.05b 0.28 ± 0.12bc 0.37 ± 0.08c
75S 0.09 ± 0.02a 0.17 ± 0.07ab 0.27 ± 0.14b 0.50 ± 0.18c
100SBS 0.16 ± 0.09a 0.44 ± 0.16b 0.48 ± 0.13b 0.56 ± 0.13b
90SBS 0.16 ± 0.09a 0.46 ± 0.13bc 0.57 ± 0.12cd 0.68 ± 0.16d
75SBS 0.20 ± 0.09a 0.53 ± 0.15bc 0.64 ± 0.18cd 0.85 ± 0.24c
Moisture (%) 100S 68.20 ± 3.94a 68.06 ± 2.87a 71.01 ± 0.92b 71.05 ± 1.98b
90S 69.12 ± 3.49a 68.66 ± 3.24a 70.19 ± 1.02a 69.89 ± 1.23a
75S 66.87 ± 2.04a 66.10 ± 4.24a 66.40 ± 2.98a 66.59 ± 3.89a
100SBS 71.40 ± 4.57a 70.99 ± 4.56a 70.01 ± 1.44a 70.74 ± 1.30a
90SBS 68.76 ± 3.05a 70.01 ± 5.45ab 72.28 ± 1.64b 70.56 ± 1.62ab
75SBS 67.70 ± 4.36a 67.80 ± 4.32a 67.36 ± 3.29a 67.95 ± 2.83a
Total protein (%) 100S 43.94 ± 5.38a 39.74 ± 6.11a 42.83 ± 5.45a 44.74 ± 3.42a
90S 40.15 ± 3.50a 36.67 ± 2.71a 39.16 ± 2.38a 39.27 ± 4.82a
75S 26.36 ± 5.0a 27.77 ± 1.97a 26.42 ± 2.72a 27.99 ± 1.44a
100SBS 42.47 ± 4.96a 42.19 ± 6.44a 41.82 ± 4.40a 34.82 ± 1.53a
90SBS 33.65 ± 6.68a 29.55 ± 4.22a 35.66 ± 6.63a 36.52 ± 2.58a
75SBS 26.15 ± 5.22a 27.11 ± 4.69a 28.10 ± 5.92a 24.99 ± 5.16a
Soluble protein (%) 100S 9.72 ± 1.18a 8.52 ± 1.38a 14.49 ± 2.38b 8.14 ± 5.56a
90S 11.48 ± 3.01a 11.35 ± 4.87a 12.85 ± 2.81a 10.84 ± 3.68a
75S 8.80 ± 1.10a 10.37 ± 2.26a 9.92 ± 1.72a 11.21 ± 1.36a
100SBS 10.82 ± 2.44ab 11.00 ± 4.62b 12.12 ± 3.74c 8.28 ± 0.69a
90SBS 9.45 ± 1.61a 8.84 ± 3.09a 10.90 ± 3.48a 8.85 ± 1.47a
75SBS 9.64 ± 1.82a 9.81 ± 2.92a 6.96 ± 1.60b 7.19 ± 2.20b
α-Amylase (mg maltose/mL) 100S 0.41 ± 0.15a 0.43 ± 0.16a 1.29 ± 0.14b 1.07 ± 0.56ab
90S 0.37 ± 0.24a 0.72 ± 0.41b 0.55 ± 0.32ab 1.05 ± 0.33b
75S 0.83 ± 0.39a 0.70 ± 0.34a 2.20 ± 1.17b 1.52 ± 0.39c
100SBS 0.77 ± 0.31a 0.58 ± 0.24ab 0.37 ± 0.11b 0.30 ± 0.24b
90SBS 0.93 ± 0.37ab 0.74 ± 0.55a 0.66 ± 0.21a 1.57 ± 0.66b
75SBS 1.30 ± 0.86ab 0.98 ± 0.35ac 2.01 ± 1.45b 0.56 ± 0.30c
α-Galactosidase (mg maltose/mL) 100S 0.39 ± 0.22a 1.06 ± 0.22b 1.61 ± 0.87b 1.83 ± 0.82b
90S 0.77 ± 0.36a 0.99 ± 0.58ab 1.39 ± 0.64b 1.13 ± 0.55b
75S 1.32 ± 0.71a 1.18 ± 0.42a 2.48 ± 0.91b 1.16 ± 0.69a
100SBS 1.61 ± 0.72a 1.21 ± 0.29ab 1.21 ± 0.50ab 0.90 ± 0.62b
90SBS 1.12 ± 0.46ab 1.48 ± 0.59b 1.04 ± 0.72a 0.99 ± 0.38a
75SBS 1.58 ± 0.29a 1.02 ± 0.39ab 1.75 ± 0.20a 0.50 ± 0.12b
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Means in a row not sharing superscripts are significantly different (P < 0.05). Samples coded 100S, 90S, and 75S represent naturally fermented pastes, while samples coded 100SBS, 90SBS, and 75SBS represent lactic acid-fermented pastes. Pastes are designated according to 100%, 90%, and 75% soybean composition, the remaining proportions being maize.

Moisture content was not affected by fermentation time except in 100S where significant differences were observed between early stages and late stages of fermentation. In 90SBS, differences were observed between 0 and 48 h (Table1). During fermentation of kinema, no appreciable changes in moisture content were reported (Sarkar and Tamang 1995; Yang et al. 2011). Samples in this study had higher moisture content than in kinema (Yang et al. 2011).

Composition of the samples influenced the amount of total protein, with an increased amount of maize resulting in a reduced amount of total protein. Fermentation time had no significant influence on the amount of total protein in all the treatments, although fluctuations were observed (Table1). Other studies in fermentation of soybeans, pearl millet, and maize concluded that fermentation does not seem to be a viable means of increasing total protein content (Khetarpaul and Chauhan 1989; Mohiedeen et al. 2010; Yang et al. 2011) because no significant changes were observed. Khetarpaul and Chauhan (1989) and Visessanguan et al. (2005) suggested that decreases in protein content were due to protein degradation by proteolytic activities of microorganisms, while Mohiedeen et al. (2010) attributed the slight gains to protein synthesis during microbial growth.

Nevertheless, there were significant changes (P < 0.05) in total soluble protein content at 48 h in 100S (Table1) and from 24 to 48 h in 100SBS. At 24 h, soluble protein content of 75S increased by 17% and slight increases were observed in 100SBS and 75SBS. On the other hand, 100S had the highest percentage loss (12%) amongst all samples at 24 h but had the highest percentage gain at 48 h. In fact, net increases in soluble protein content from the initial were highest at 48 h and all samples showed soluble protein gains at this time except in 75SBS in which a 27% loss was observed. The highest soluble protein increase was in 100S (49%), followed by 90SBS (15%), while the increases in 100SBS, 90S, and 75S were about 12%. At 72 h, net gain from the initial was only observed in 75S (27%) while soluble protein losses were observed in all the remaining samples. In fermentation of soybeans to produce kinema, Sarkar and Tamang (1995) reported a 47% increase in soluble nitrogen between 6 and 9 h of fermentation. Visessanguan et al. (2005) attributed the increases in soluble nitrogen and free amino acids to hydrolysis of soy proteins and suggested the presence of proteolytic activity during fermentation. Sripriya et al. (1997) reported a 10-fold increase in soluble protein during fermentation of finger millet. They attributed the increases to microbial enzyme activity and protein hydrolysis. Increase in soluble protein improves digestibility of soybeans by increasing the amount of protein that could be readily absorbed.

At the beginning of fermentation, higher enzyme activities were observed in LFP because of the back-slopping material, which was made by adding finger millet malt flour to a maize porridge. Malting or sprouting increases activities of starch hydrolyzing enzymes (amylase activities) and galactosidases (Malleshi et al. 1986). At the beginning of fermentation, α-amylase activities were significantly higher in LFPs and 75S than in 100S and 90S (Table1). After 24 h, different trends in enzyme activities were observed according to the type of fermentation and composition of the paste. There was a lag phase in 100S before a significant increase of α-amylase activity was observed at 48 h. Fluctuations were observed in 90S with significant increases at 24 and 72 h and a significant decrease at 48 h. Trends in α-amylase activities were similar in 75S and 75SBS; decreases at 24 h were followed by significant increases at 48 h which were followed by decreases. The α-amylase activities in these two samples were comparatively higher probably because of the higher starch content. There were continuous decreases in α-amylase and α-galactosidase activities in 100SBS throughout fermentation, while continuous increases in α-amylase and α-galactosidase activities in 100S were observed except at 72 h where a decrease in α-amylase was seen (Table1). The α-galactosidase activities significantly increased at 48 h in NFP, while in 90SBS, though not significant, an increase was noticed at 24 h. With the exception of the α-amylase activity of 90SBS, α-amylase and α-galactosidase activities were higher in NFP than in LFP at 72 h.

Production of α-amylase and α-galactosidase by Lactobacillus fermentum and Lactobacillus planturum (Songré-Ouattara et al. 2008) has been documented. In this study, Lb. fermentum was among the dominant LAB microflora involved in fermentation (data not shown). In B. subtilis-dominated fermentation, increases in α-amylase activities (Dakwa et al. 2005; Terlabie et al. 2006) and degradation of oligosaccharides (Sarkar et al. 1997a) were reported. The importance of high amylase activities and their starch-hydrolyzing capacity in cereal and legume-based foods is the possibility of increasing energy density in fermented foods since dietary bulkiness is reduced and hence more raw material can be used (Mosha and Svanberg 1983; Hansen et al. 1989; Lorri and Svanberg 1993). This could eventually address low energy and nutrient density intake, a nutritional problem recognized in most African countries (Lorri and Svanberg 1993; Maleta 2006). The main oligosaccharides in mature soybeans are stachyose (14–41 g/kg dry weight) and raffinose (1–9 g/kg dry weight). These two flatulence-causing sugars contain both β-fructosidic and α-galactosidic linkages (Lan et al. 2007). Mammals do not synthesize α-galactosidase enzymes required to hydrolyze α-galactosidic linkages (Medic et al. 2014). Therefore, increases in α-galactosidase activities could imply a possible degradation of flatulence-causing oligosaccharides. This could in turn improve the acceptance and utilization of soybeans.

Amino acids

Seventeen total amino acids including cyst(e)ine (Cys), methionine (Met), aspartic acid (Asp), threonine (Thr), serine (Ser), glutamic acid (Glu), proline (Pro), glycine (Gly), alanine (Ala), valine (Val), isoleucine (Ile), leucine (Leu), tyrosine (Tyr), phenylalanine (Phe), histidine (His), lysine (Lys), and arginine (Arg) were identified. Fluctuations in amino acids were observed and in most cases the changes were not significant (Table2). Significant increases (P < 0.05) throughout fermentation were only observed in Cys in 100S and 90S, and Met in 90S, while significant increases during 48 h of fermentation were observed in Cys in all LFP and in Met in 75SBS. Significant increases at 48 h followed by decreases at 72 h were observed in Cys in LFP and in Met, Asp, Ser, and Arg in 75SBS. In 100S, significant decreases at 48 h in Asp, Glu, Pro, Val, Phe, His, Arg were observed, and levels of many amino acids decreased in 100SBS at 48 h.

Table 2.

Changes in levels of total amino acids in naturally fermented pastes during fermentation

g per kg sample
Sample 100S
 90S
 75S
Amino acid 0 h 48 h 72 h 0 h 48 h 72 h 0 h 48 h 72 h
Cys 5.96 ± 0.10a 6.76 ± 0.058b 6.82 ± 0.16b 5.61 ± 0.03a 6.04 ± 0.03b 6.11 ± 0.06b 5.01 ± 0.01a 5.29 ± 0.07a 5.21 ± 0.14a
Met 5.65 ± 0.07a 5.69 ± 0.34a 5.88 ± 0.11a 4.85 ± 0.06a 5.27 ± 0.17b 5.21 ± 0.06b 4.64 ± 0.22a 4.56 ± 0.28a 4.48 ± 0.14a
Asp 53.85 ± 0.57a 50.54 ± 0.64b 51.95 ± 1.61ab 45.21 ± 0.12ab 45.80 ± 0.31b 44.26 ± 0.51a 39.45 ± 1.06a 37.99 ± 0.59a 37.29 ± 0.69a
Thr 18.73 ± 0.39a 17.72 ± 0.62a 18.49 ± 0.72a 16.03 ± 0.01a 16.22 ± 0.03a 15.80 ± 0.44a 14.15 ± 0.39a 13.83 ± 0.23a 13.37 ± 0.27a
Ser 24.89 ± 0.13a 23.33 ± 0.34a 24.34 ± 1.03a 21.16 ± 0.05a 21.64 ± 0.12a 20.82 ± 0.46a 18.84 ± 0.50a 18.51 ± 0.06a 18.04 ± 0.35a
Glu 93.29 ± 0.44a 86.30 ± 0.10b 87.69 ± 2.86b 79.56 ± 0.26a 79.01 ± 0.58a 76.83 ± 0.12b 70.41 ± 1.78a 66.36 ± 0.18b 65.85 ± 1.43b
Pro 21.73 ± 0.26a 19.94 ± 0.25b 21.03 ± 0.96a 19.14 ± 0.28a 18.89 ± 0.60a 18.17 ± 0.09 17.61 ± 0.56a 16.45b 16.74 ± 0.30ab
Gly 17.20 ± 0.07a 16.09 ± 0.01a 16.52 ± 0.64a 14.57 ± 0.05a 14.47 ± 0.09a 14.03 ± 0.01b 12.69 ± 0.21a 12.21 ± 0.03a 12.07 ± 0.19b
Ala 17.71 ± 0.07a 17.03 ± 0.05a 17.84 ± 0.86a 15.74 ± 0.09a 15.76 ± 0.13a 15.18 ± 0.07a 14.10 ± 0.06a 13.91 ± 0.17a 13.84 ± 0.07a
Val 20.79 ± 0.48a 18.91 ± 0.21b 20.71 ± 0.85a 18.07 ± 0.03a 17.84 ± 0.14a 16.96 ± 0.22b 15.48 ± 0.36a 15.10 ± 0.13a 14.93 ± 0.15a
Ile 21.63 ± 0.70a 20.40 ± 0.16a 21.59 ± 1.38a 18.87 ± 0.28a 18.47 ± 0.27ab 17.82 ± 0.07b 15.91 ± 0.46a 15.66 ± 0.02a 15.33 ± 0.53a
Leu 36.13 ± 0.57a 34.07 ± 0.37a 35.99 ± 1.77a 31.91 ± 0.14a 31.47 ± 0.05b 30.52 ± 0.12c 28.59 ± 0.56a 27.83 ± 0.19a 27.65 ± 1.06a
Tyr 17.00 ± 0.26a 16 ± 0.13a 16.44 ± 0.86a 14.50 ± 0.45a 14.67 ± 0.19a 14.32 ± 0.04a 12.70 ± 0.15a 12.40 ± 0.02a 12.26 ± 0.28a
Phe 24.83 ± 0.07a 22.47 ± 0.37b 23.57 ± 1.04ab 20.81 ± 1.13a 20.76 ± 0.48a 20.37 ± 0.12a 18.22 ± 0.56a 17.28 ± 0.03a 17.25 ± 0.07a
His 14.18 ± 0.18a 13.15 ± 0.05b 13.47 ± 0.55ab 12.11 ± 0.02a 12.05 ± 0.14a 11.70 ± 0.10b 10.65 ± 0.20a 10.29 ± 0.06a 10.11 ± 0.21a
Lys 29.45 ± 0.06a 28.4 ± 0.62a 28.76 ± 0.83a 25.09 ± 0.29a 25.22 ± 0.22a 24.41 ± 0.19b 21.50 ± 0.38a 20.81 ± 0.03a 20.76 ± 0.54a
Arg 35.88 ± 0.19a 30.64 ± 1.77b 32.78 ± 1.48ab 29.67 ± 0.01a 29.24 ± 0.12a 29.51 ± 2.04a 25.54 ± 0.61a 24.36 ± 0.06ab 23.80 ± 0.86b
SumAA 458.9 ± 3.39a 427.45 ± 2.19b 443.8 ± 17.68ab 392.85 ± 1.91a 392.8 ± 2.55a 382 ± 4.10b 345.5 ± 7.92a 332.85 ± 0.92a 328.95 ± 6.86a
(g per kg sample)
Sample 100SBS
 90SBS
 75SBS
Amino acid 0 h 48 h 72 h 0 h 48 h 72 h 0 h 48 h 72 h
Cys 5.95 ± 0.11a 6.43 ± 0.18b 6.36 ± 0.15ab 5.35 ± 0.24a 6.11 ± 0.06b 6.02 ± 0.09b 4.66 ± 0.09a 5.46 ± 0.02b 5.28 ± 0.01b
Met 5.41 ± 0.38a 5.56 ± 0.20a 5.53 ± 0.11a 4.90 ± 0.09a 5.02 ± 0.19a 5.13 ± 0.28a 4.04 ± 0.15a 4.55 ± 0.03b 4.45 ± 0.10b
Asp 51.88 ± 1.13a 47.92 ± 0.08b 47.86 ± 0.33b 43.02 ± 0.63a 44.19 ± 0.46a 44.36 ± 0.78a 37.2 ± 0.14a 38.83 ± 0.75b 37.07 ± 0.60a
Thr 18.28 ± 0.78a 16.98 ± 0.22a 17.07 ± 0.19a 15.32 ± 0.34a 15.72 ± 0.39a 15.83 ± 0.28a 13.28 ± 0.23a 14.21 ± 0.15a 13.40 ± 0.50a
Ser 24.14 ± 0.78a 22.48 ± 0.14b 22.49 ± 0.17b 20.02 ± 0.51a 20.85 ± 0.24ab 21.18 ± 0.23b 17.63 ± 0.27a 18.82 ± 0.11b 17.67 ± 0.54a
Glu 89.87 ± 1.42a 81.80 ± 1.11b 80.99 ± 0.28b 75.94 ± 1.05a 76.52 ± 0.23a 75.97 ± 1.11a 68.06 ± 1.42a 68.98 ± 1.07a 68.25 ± 1.41a
Pro 21.27 ± 1.08a 19.87 ± 0.70a 19.02 ± 0.38a 18.40 ± 0.62a 18.43 ± 0.01a 18.53 ± 0.18a 17.03 ± 0.23a 16.87 ± 0.07a 17.29 ± 0.48a
Gly 16.38 ± 0.24a 15.14 ± 0.01b 15.31 ± 0.04b 13.81 ± 0.11a 14.12 ± 0.12a 14.04 ± 0.22a 12.32 ± 0.24a 12.62 ± 0.30a 12.17 ± 0.12a
Ala 17.14 ± 0.34a 16.17 ± 0.16b 16.80 ± 0.32ab 15.28 ± 0.11a 15.59 ± 0.08a 15.55 ± 0.09a 13.80 ± 0.34a 14.53 ± 0.31a 14.15 ± 0.13a
Val 20.01 ± 0.47a 18.23 ± 0.19b 19.03 ± 0.29ab 17.17 ± 0.08a 16.81 ± 0.13b 16.94 ± 0.12ab 15.18 ± 0.27a 15.52 ± 0.35a 14.76 ± 0.08a
Isoleu 20.91 ± 0.32a 19.59 ± 0.41a 20.35 ± 0.54a 17.84a 17.58 ± 0.34a 18.07 ± 0.19a 15.45 ± 0.13a 16.09 ± 0.22a 15.53 ± 0.29a
Leu 34.83 ± 0.67a 32.82 ± 1.02a 33.68 ± 0.82a 30.91 ± 0.14a 30.49 ± 0.39a 30.83 ± 0.20a 28.09 ± 0.37a 28.24 ± 0.69a 28.11 ± 0.70a
Tyr 16.40 ± 0.03a 15.43 ± 0.20b 14.69 ± 0.11c 13.89 ± 0.31a 14.47 ± 0.28a 14.29 ± 0.21a 12.41 ± 0.05a 12.79 ± 0.10a 12.44 ± 0.10a
Phe 23.22 ± 0.13a 21.67 ± 0.04b 21.32 ± 0.22b 19.76 ± 0.05a 20.36 ± 0.32a 20.26 ± 0.35a 17.87 ± 0.045ab 18.03 ± 0.12b 17.55 ± 0.14a
His 13.66 ± 0.20a 12.39 ± 0.01b 12.48 ± 0.05b 11.57 ± 0.05a 11.65 ± 0.03a 11.59 ± 0.14a 10.36 ± 0.22a 10.53 ± 0.09a 10.30 ± 0.08a
Lys 28.19 ± 0.36a 26.55 ± 0.05b 26.55 ± 0.15b 23.46 ± 0.12a 24.33 ± 0.04b 24.42 ± 0.19b 20.34 ± 0.23a 21.32 ± 0.71a 20.48 ± 0.41a
Arg 34.47 ± 0.62a 30.30 ± 0.10b 30.38 ± 0.11b 28.26 ± 0.52a 28.22 ± 0.05a 28.09 ± 0.83a 25.11 ± 0.54ab 26 ± 2.78b 23.53 ± 0.31a
SumAA 442 ± 8.77a 409.3 ± 3.11b 409.9 ± 0.99b 374.85 ± 3.46a 380.45 ± 3.04a 381.1 ± 4.81a 332.8 ± 3.54a 343.4 ± 7.78a 332.45 ± 0.21a
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Means in a row and within a sample not sharing superscripts are significantly different (P < 0.05). Samples coded 100S, 90S, and 75S represent naturally fermented pastes, while samples coded 100SBS, 90SBS, and 75SBS represent lactic acid-fermented pastes. Pastes are designated according to 100%, 90%, and 75% soybean composition, the remaining proportions being maize.

The sums of the total amino acids were higher in NFP than in LFP except in 75S at 48 and 72 h. Although not significant, slight percent increases in sums of total amino acids were observed in 90SBS and 75SBS (48 h). At 48 and 72 h, 90SBS showed 1.5% and 1.7% increases, respectively. In 75SBS, a 3.2% increase was noted at 48 h. On the other hand, reductions were noted in all NFP at 48 h (from 0.01% in 90S to 6.8% in 100S) and in 100SBS. Higher decreases were noted at 72 h in 75S (4.7%), at 48 h in 100S (6.8%), and at 48 and 72 h in 100SBS (7.4%). In 75S, there were decreases throughout fermentation.

In all samples, Glu was the most abundant amino acid followed by Asp, while Cys and Met were the limiting amino acids. Similar results were reported in fermentation of kinema by B. subtilis (Sarkar et al. 1997b). In fermentation of doenjang by B. subtilis, increases in Leu, Phe, Lys, and Ala were up to three times greater after 40 and 100 days of fermentation than the initial levels (Namgung et al. 2010). In cheonggukjang fermented for 3 days with Bacillus spp., total amino acids significantly (P < 0.05) increased between 24 and 48 h (Baek et al. 2010). In their study, Baek et al. (2010) identified Ala, Glu, Phe, and Trp as major amino acids (above one related peak area) during the initial stages of fermentation. In this study, Glu, Asp, Leu, Arg, Lys, Ser, and Phe were considered the main amino acids (>20 g per kg sample) throughout fermentation. In kinema, Glu, Asp, Leu, Arg and Lys were major amino acids (Sarkar et al. 1997b), while in soy-dawadawa, Glu and Ser were not among the major amino acids (Dakwa et al. 2005).

A total of 21 free amino acids (Table3) including cyst(e)ine, methionine, aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, valine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, arginine, glutamine (Gln), asparagine (Asn), citrulline (Cit), γ-aminobutyric acid (GABA), ornithine (Orn), and tryptophan (Trp) were identified. Fluctuations in free amino acids were also observed. The fluctuations reflected the conversion of peptides to free amino acids and the subsequent utilization of these amino acids. Peptide conversion into free amino acids is a central metabolic activity in LAB (Christensen et al. 1999). Increases throughout fermentation were observed in Glu (all samples), Ala (all LFP), GABA and Lys (100SBS and 90SBS) and Asp (90S). Decreases at 24 h followed by increases at 48 and 72 h were observed in NFP in Ala, Val, Ile, and Leu; and in LFP in Asn and Leu. These changes were also observed in 100S in Asn and Gly and in 100SBS in Val. At the end of the fermentation, the following amino acids were significantly higher than at the beginning of the fermentation: Glu, Ala, Lys in all samples; Leu in NFP; Gln in LFP, 100S and 90S; Thr and GABA in 100SBS and 90SBS; Asn, Cit, and Ile in 100S; Gly in 100S, 90S, and 100SBS; Phe in 100S and 90S; and Val in 90S and 100SBS (Table3). Sarkar et al. (1997b) reported significant increases in free amino acids during 48 h of fermentation in kinema. They also reported net decreases in some amino acids and suggested that the amino acids were metabolized to a greater extent than they were replaced by proteolytic activities. In cheonggukjang fermented for 2–3 days, fluctuations in amino acids were also observed and the levels of most amino acids decreased in the early stages of fermentation and increased in the late stages of fermentation (Park et al. 2010). Increases in free amino acids would be desirable to improve digestibility of soybean proteins.

Table 3.

Changes in levels of free amino acids in naturally fermented pastes during fermentation

  (μmol/mL)
Sample 100S
 90S
 75S
Amino acid 0 h 24 h 48 h 72 h 0 h 24 h 48 h 72 h 0 h 24 h 48 h 72 h
Asp 0.58 ± 0.04a 0.36 ± 0.33a 0.55 ± 0.5a 0.79 ± 0.50a 0.56 ± 0.014a 0.64 ± 0.36a 0.84 ± 0.08a 1.23 ± 1.05a 0.61 ± 0.02a 0.71 ± 0.46a 0.85 ± 0.71a 0.78 ± 0.37a
Glu 0.8 ± 0.03a 1.12 ± 0.45a 2.92 ± 1.43ab 4.84 ± 0.48b 0.87 ± 0.02a 0.92 ± 0.35a 2.16 ± 0.91b 3.71 ± 2.64b 0.82 ± 0.01a 0.99 ± 0.13a 1.36 ± 0.09ab 2.38 ± 0.72b
Asn 0.6a 0.03b 0.13bc 0.2 ± 0.08c 0.51 ± 0.01a 0.17 ± 0.03ab 0.09 ± 0.08b 0.15 ± 0.15ab 0.63 ± 0.03a 0.19 ± 0.14b 0.05 ± 0.03b 0.12 ± 0.09b
Ser 0.12 ± 0.01a 0.35 ± 0.01b 0.65 ± 0.09c 0.63 ± 0.07c 0.10 + 0.01a 0.11 ± 0.06a 0.27 ± 0.22a 0.29 ± 0.3a 0.1a 0.1 ± 0.02a 0.22 ± 0.09a 0.3 ± 0.17a
Gln 0.01a 0.03 ± 0.01a 0.06 ± 0.04ab 0.12 ± 0.03b 0.01a 0.04 ± 0.01a 0.06 ± 0.03ab 0.13 ± 0.12b 0.01a 0.04 ± 0.01a 0.04 ± 0.01a 0.07 ± 0.04a
His 0.29 ± 0.04a 0.09 ± 0.03b 0.06 ± 0.02b 0.38c 0.19 ± 0.01a 0.12 ± 0.01a 0.12 ± 0.09a 0.24a 0.16 ± 0.02a 0.15 ± 0.02a 0.14 ± 0.06a 0.07 ± 0.06a
Gly 0.22ab 0.15 ± 0.08b 0.25 ± 0.13ab 0.47 ± 0.09c 0.20a 0.19 ± 0.04a 0.41 ± 0.18b 0.57 ± 0.30b 0.23 ± 0.01a 0.18 ± 0.04a 0.18 ± 0.08a 0.29 ± 0.16a
Thr 0.08 ± 0.01a 0.03 ± 0.01a 0.10 ± 0.11a 0.13 ± 0.08a 0.08 ± 0.01a 0.03 ± 0.01a 0.12 ± 0.06a 0.20 ± 0.24a 0.08 ± 0.01a 0.04 ± 0.01b 0.07 ± 0.01a 0.07 ± 0.01a
Cit 0.01a 0.19b 0.13 ± 0.16b 0.37c 0.01a 0.06 ± 0.07a 0.07 ± 0.06a 0.07 ± 0.07a n.d. 0.07 0.10 ± 0.06 0.06 ± 0.01
Arg 5.82 ± 0.01a 1.45 ± 1.49b 0.05 ± 0.02c 0.06c 3.16 ± 0.02a 1.94 ± 1.46ab 0.11 ± 0.12b 0.07 ± 0.01b 2.82 ± 0.04a 1.31 ± 0.24b 0.04 ± 0.01c 0.04 ± 0.02c
Ala 0.67ac 0.35 ± 0.11ab 1.79 ± 0.66c 3.63 ± 0.46d 0.59 ± 0.01a 0.30 ± 0.18a 1.15 ± 1.03b 2.63 ± 2.32c 0.64 ± 0.01a 0.37 ± 0.12a 0.61 ± 0.37a 1.43 ± 0.41b
GABA 0.13 ± 0.14a 0.14 ± 0.02a 0.13 ± 0.04a 0.12a 0.11a 0.10a 0.12 ± 0.01a 0.23 ± 0.13a 0.15 ± 0.01a 0.13 ± 0.01a 0.13 ± 0.01a 0.21 ± 0.09a
Tyr 0.11 ± 0.01a 0.15 ± 0.15a 0.12 ± 0.11a 0.07 ± 0.06a 0.12a 0.10a 0.19 ± 0.23a 0.18 ± 0.03a 0.14 ± 0.01a 0.11 ± 0.11a 0.13 ± 0.16a 0.08 ± 0.06a
Val 0.17 ± 0.01a 0.06a 0.57 ± 0.11ab 1.0 ± 0.37b 0.13 ± 0.01a 0.05 ± 0.05a 0.42 ± 0.52ab 0.89 ± 1.17b 0.13 ± 0.01a 0.05 ± 0.03a 0.21 ± 0.24a 0.50 ± 0.43a
Met 0.10 ± 0.01a 0.01a 0.02 ± 0.01a 0.03 ± 0.02a 0.07a 0.05a 0.05a 0.05a 0.06 0.03 0.02 n.d.
Ile 0.16 ± 0.01a 0.01a 0.26 ± 0.13ab 0.55 ± 0.19b 0.12a 0.04 ± 0.04a 0.21 ± 0.26a 0.65 ± 0.88a 0.10 ± 0.01a 0.05 ± 0.04a 0.15 ± 0.17a 0.32 ± 0.26a
Phe 0.2 ± 0.03a 0.25 ± 0.17a 1.13 ± 0.31b 1.66 ± 1.12b 0.2a 0.15a 0.84 ± 1.06b 1.61 ± 2.15b 0.15 ± 0.01a 0.12 ± 0.11a 0.44 ± 0.56a 0.67 ± 0.71a
Trp 0.29 ± 0.04a 0.27 ± 0.11a 0.33 ± 0.07a 0.33 ± 0.05a 0.28 ± 0.021a 0.29 ± 0.01a 0.25 ± 0.06a 0.24 ± 0.04a 0.22 ± 0.01a 0.24 ± 0.05a 0.25 ± 0.01a 0.21a
Leu 0.20 ± 0.01a 0.09 ± 0.04a 1.06 ± 0.21ab 2.19 ± 1.0b 0.16 + 0.01a 0.05 ± 0.05a 1.12 ± 1.47b 2.59 ± 3.49b 0.13a 0.06 ± 0.03a 0.42 ± 0.52a 1.11 ± 0.95b
Orn 1.05 ± 0.20ab 1.91 ± 0.61a 0.82 ± 0.67ab 0.42 ± 0.34b 0.72 ± 0.08a 0.75 ± 0.21a 1.01 ± 0.12a 0.25 ± 0.08b 0.61 ± 0.09a 0.83 ± 0.24a 1.38 ± 0.25b 0.56 ± 0.16a
Lys 0.33 ± 0.01a 0.35 ± 0.06ab 0.58 ± 0.13b 0.95 ± 0.08c 0.31 ± 0.10a 0.25 ± 0.01a 0.65 ± 0.45ab 0.92 ± 0.65b 0.3a 0.37 ± 0.12a 0.34 ± 0.01a 0.74 ± 0.30a
  (μmol/mL)
Sample 100SBS
 90SBS
 75SBS
Amino acid 0 h 24 h 48 h 72 h 0 h 24 h 48 h 72 h 0 h 24 h 48 h 72 h
Asp 0.55 ± 0.04a 0.99 ± 0.41a 0.74 ± 0.13a 0.78 ± 0.12a 0.66 ± 0.11a 0.84 ± 0.3a 1.05 ± 0.03a 0.90 ± 0.04a 0.89 ± 0.12a 1.13 ± 0.39a 0.83 ± 0.11a 0.72 ± 0.14a
Glu 0.87 ± 0.01a 2.79 ± 0.1b 2.33 ± 0.54b 2.55 ± 0.38b 0.98 ± 0.1a 2.56 ± 0.1b 3.37 ± 0.1c 3.07 ± 0.35c 0.98 ± 0.06a 2.37 ± 0.05ab 2.94 ± 0.42b 3.14 ± 1.30b
Asn 0.50 ± 0.05a 0.15 ± 0.03a 0.25 ± 0.21a 0.36 ± 0.19a 0.50 ± 0.08a 0.12 ± 0.01b 0.33 ± 0.10ab 0.35 ± 0.10a 0.85 ± 0.11a 0.11 ± 0.01b 0.21 ± 0.04b 0.25 ± 0.14b
Ser 0.14a 0.09 ± 0.05a 0.11 ± 0.01a 0.18 ± 0.05a 0.18 ± 0.01a 0.08 ± 0.04b 0.12 ± 0.04ab 0.19 ± 0.01a 0.20 ± 0.01a 0.07 ± 0.02b 0.11ab 0.18 ± 0.01a
Gln 0.01a 0.12 ± 0.01b 0.22 ± 0.03c 0.33 ± 0.06d 0.02a 0.16 ± 0.03ab 0.42 ± 0.16b 0.5 ± 0.21b 0.03 ± 0.01a 0.18 ± 0.01b 0.39 ± 0.03c 0.43 ± 0.08c
His 0.14 ± 0.08 n.d. n.d. n.d. 0.11 ± 0.06 n.d. n.d. n.d. 0.17 ± 0.09 n.d. n.d. n.d.
Gly 0.25 ± 0.01a 0.46 ± 0.03ab 0.79 ± 0.14ab 1.07 ± 0.47b 0.27 ± 0.01a 0.51 ± 0.02ab 0.75 ± 0.18b 1.06 ± 0.06c 0.32 ± 0.02a 0.49 ± 0.01b 0.71 ± 0.08c 0.73 ± 0.01c
Thr 0.07 ± 0.02a 0.13 ± 0.06a 0.11 ± 0.02a 0.24 ± 0.01b 0.10 ± 0.05a 0.11 ± 0.04a 0.29 ± 0.09b 0.29 ± 0.05b 0.11 ± 0.04a 0.13 ± 0.04a 0.19 ± 0.09a 0.31 ± 0.11a
Cit 0.01a 0.28 ± 0.36a 0.13 ± 0.15a 0.04 ± 0.02a 0.01a 0.29 ± 0.36a 0.27 ± 0.36a 0.15 ± 0.18a 0.02 ± 0.01a 0.37 ± 0.39a 0.31 ± 0.38a 0.19 ± 0.23a
Arg 5.09 ± 0.03a 0.06 ± 0.04b 0.06 ± 0.01b 0.07 ± 0.01b 3.12 ± 0.02a 0.06 ± 0.04b 0.05 ± 0.01b 0.05 ± 0.01b 4.20 ± 0.05a 0.08 ± 0.05b 0.09 ± 0.06b 0.10 ± 0.04b
Ala 0.70 ± 0.01a 1.34 ± 0.43a 2.67 ± 0.66ab 3.84 ± 1.53b 0.72 ± 0.02a 1.25 ± 0.34ab 2.32 ± 0.5bc 3.27 ± 0.66c 0.89 ± 0.01a 1.19 ± 0.19ab 2.00 ± 0.53ab 2.54 ± 0.81b
GABA 0.17 ± 0.01a 0.51 ± 0.42a 2.49 ± 2.77b 3.75 ± 3.75b 0.21 + 0.03a 0.35 + 0.13a 1.07 ± 0.86b 2.20 ± 1.96b 0.27 ± 0.04a 0.31 ± 0.06a 0.41 ± 0.03a 0.43 ± 0.15a
Tyr 0.13 ± 0.01a 0.06 ± 0.02ab 0.03b 0.06 ± 0.03ab 0.15a 0.04 + 0.02b 0.03 ± 0.01b 0.04 ± 0.01b 0.16a 0.03 ± 0.02b 0.02 ± 0.01b 0.04 ± 0.03b
Val 0.20 ± 0.01ab 0.05 ± 0.01a 0.25 ± 0.21ab 0.53 ± 0.26b 0.21 ± 0.01a 0.03 ± 0.01a 0.13 ± 0.13a 0.26 ± 0.18a 0.24 ± 0.01a 0.02b 0.06 ± 0.03b 0.18 ± 0.12ab
Met 0.10 ± 0.01 n.d. 0.03 0.04 + 0.03 0.09 n.d. n.d. 0.02 0.09 n.d. n.d. 0.01
Ile 0.14 ± 0.02a 0.10 ± 0.01a 0.03 ± 0.03a 0.13 ± 0.07a 0.12 + 0.01a 0.11 ± 0.01a 0.07 ± 0.07a 0.04 ± 0.03a 0.12a 0.11 ± 0.01a 0.12 ± 0.02a 0.10 ± 0.08a
Phe 0.20a 0.14 ± 0.08a 0.20 ± 0.15a 0.38 ± 0.25a 0.20 ± 0.02a 0.10 ± 0.05a 0.15 ± 0.08a 0.22 ± 0.15a 0.18 ± 0.01a 0.09 ± 0.06a 0.08 ± 0.01a 0.17 ± 0.12a
Trp 0.29 ± 0.02a 0.26 ± 0.07a 0.16 ± 0.08a 0.13 ± 0.07a 0.27 ± 0.01a 0.22 ± 0.03ab 0.16 ± 0.07ab 0.12 ± 0.04b 0.23 ± 0.01a 0.19 ± 0.01a 0.12 ± 0.06a 0.09 ± 0.08a
Leu 0.22 ± 0.01a 0.07 ± 0.05a 0.4 ± 0.05a 0.96 ± 0.87a 0.23 ± 0.01a 0.05 ± 0.06a 0.22 ± 0.28a 0.42 ± 0.49a 0.22 ± 0.01a 0.03 ± 0.01a 0.08 ± 0.09a 0.28 ± 0.33a
Orn 0.98 ± 0.06a 1.63 ± 1.98a 0.21 ± 0.03a 0.25 ± 0.01a 0.74a 1.01 ± 1.09a 0.71 ± 0.71a 0.35 ± 0.16a 0.84 + 0.01a 2.77 ± 0.01b 0.99 ± 0.94a 0.62 ± 0.53a
Lys 0.30 ± 0.10a 0.67 ± 0.04a 1.00 ± 0.5ab 1.49 ± 0.77b 0.28 ± 0.06a 0.50 ± 0.08a 0.98 ± 0.09b 1.47 ± 0.26c 0.35 ± 0.01a 0.57 ± 0.03ab 0.84 ± 0.16b 0.78 ± 0.23b
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Means in a row and within a sample not sharing superscripts are significantly different (P < 0.05). n.d., not detected. Samples coded 100S, 90S, and 75S represent naturally fermented pastes, while samples coded 100SBS, 90SBS, and 75SBS represent lactic acid-fermented pastes. Pastes are designated according to 100%, 90%, and 75% soybean composition, the remaining proportions being maize.

In LFP, His was not detected beyond 24 h while Met was not detected at 24 h but was detected at 48 and/or 72 h. The absence of His and Met during further fermentation suggested degradation of the amino acids. The breakdown of His to the biogenic amine, histamine has received attention due to food safety concerns since histamine can result in food poisoning (Christensen et al. 1999; Fernandez and Zuniga 2006). The physiological roles of His decarboxylation in LAB include regulation of intracellular pH and generation of metabolic energy (Christensen et al. 1999). On the other hand, Met degradation is associated with aroma compounds in cheese (Fernandez and Zuniga 2006). In all samples, Arg decreased between 0 and 24 h and the decreases were more pronounced in LFP. Sarkar et al. (1997b) attributed Arg's pronounced decreases to its preferential uptake by B. subtilis. In addition, Arg provides energy in LAB via substrate-level phosphorylation (Christensen et al. 1999). Arg can also be converted to Orn via the arginine-deiminase pathway by several lactobacilli. This pathway contributes to the acid tolerance of lactobacilli (Gänzle et al. 2007).

The main free amino acids at the beginning of the fermentation in NFP and LFP were Asp, Glu, Arg, Ala, Orn, and Asn. At 72 h, major amino acids in NFP were Asp, Glu, Ala, Orn, Val, Ile, Phe, Leu and Lys while major amino acids in LFP were Asp, Glu, Gly, GABA, Val and Lys. Gly was one of the major amino acids in 90S and LFP, Leu in 100SBS, GABA in 100SBS, and 90SBS and Orn in 75SBS. High quantities of Ser were also observed in 100S. GABA, a nonprotein amino acid abundant in nature and present in soybeans (Namgung et al. 2010; Park et al. 2010), significantly increased at 48 and 72 h in 100SBS and 90SBS. GABA is produced by decarboxylation of l-Glu catalyzed by glutamate decarboxylase and has diverse physiological functions in humans including hypotensive effects and regulation of cardiovascular functions (Park and Oh 2007; Park et al. 2010).

Amino acids play important roles in aroma and taste development in food (Dajanta et al. 2011) as they are involved in Maillard reactions and Strecker degradation (Park et al. 2010). For instance, Orn is a precursor for a key flavor compound of wheat bread crust that intensifies the roasty note of the crust odor (Gänzle et al. 2007). During fermentation, Orn was highest at 24 h in most samples and by 72 h, the highest content was in 75SBS. Glu elicits the savory taste sensation of umami in humans (Zhao et al. 2003). At the end of the fermentation, Glu was three to six times greater in NFP and about three times greater in LFP than at the start of the fermentation and Glu was highest in 100S. Glu was reported as the most abundant amino acid in soybean paste during ripening and storage (Namgung et al. 2010). Amino acids associated with bitterness were high in 100S (Val and Leu) and 90S (Ile, Leu and Phe). Amino acids associated with sweetness such as Gly, Ala, and Lys were mostly high in LFP and were highest in 100SBS, while other sweet amino acids such as Ser and Ala were high in 100S. The higher levels of total and free Asp and Glu in NFP would suggest that NFP would have higher umami intensities, while the higher levels of amino acids associated with sweetness in LFP would suggest high sweetness intensities in LFP. On the contrary, sensory perception by descriptive panel rated LFP higher in umami intensities and NFP higher in sweetness intensities. This was explained in terms of interaction effects with other tastants including organic acids that were responsible for high sourness intensities in LFP (Ng'ong'ola-Manani et al. 2014).

Organic acids and sugars

Citric, orotic, succinic, dl-lactic, uric, dl-pyroglutamic, propionic, α-ketoglutaric, oxalic, acetic and formic acids, and pyruvate were analyzed in the samples. However, detectable levels were only found in lactic, succinic, and acetic acids (Fig1). More lactic acid was produced in both NFP and LFP compared to acetic and succinic acids. Higher lactic acid productions implied lactic acid as the major end product of fermentation, a characteristic of LAB metabolism (Kandler 1983; Axelsson 1998; Klein et al. 1998; Holzapfel et al. 2001). Lactic acid in LFP was 2.5 to 3.5-fold higher than in NFP (Fig1A). Significantly high lactic acid production in LFP could mean higher LAB numbers in LFP resulting in dominant LAB metabolism compared to NFP. Alternatively, mixed fermentation processes of LAB and other microflora could be suggested for NFP. At 72 h, production of lactic acid was five to 16-fold and 19- to 30-fold higher than of succinic acid in NFP and LFP, respectively, while at the same time, lactic acid was one to twofold and 10- to 11-fold higher than acetic acid in NFP and LFP, respectively. The presence of acetic acid suggested heterofermentation in both LFP and NFP. Heterofermentative LAB produce acetic acid, ethanol, and CO2 in addition to lactic acid as products of fermentation (Kandler 1983). Heterofermentative and homofermentative LAB were identified in both the fermentation processes, and the former were dominant (data not shown).

Figure 1.

Figure 1

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Changes in organic acids during fermentation. Samples coded 100S, 90S, and 75S represent naturally fermented pastes, while samples coded 100SBS, 90SBS, and 75SBS represent lactic acid-fermented pastes. Pastes are designated according to 100%, 90%, and 75% soybean composition, the remaining proportions being maize.

At 24 h, there were no significant differences in acetic acid production in all samples except 75S (Fig1C) which had a significantly (P < 0.05) lower acetic acid level. At 48 h, 90S and 100S contained more acetic acid than all LFP and 75S. At the end of the fermentation, highest acetic acid amount was produced in 100S followed by 90S and 75S although contents in 90S and 75S and all LFP were not significantly different (P > 0.05). High acetic acid production in NFP could be attributed to natural fermentation probably by Bacillus spp. because acetic acid is a major product of carbohydrate metabolism in B. subtilis (Moat et al. 2002). The presence of succinic acid confirmed heterofermentation (Axelsson 2004) and could also mean that pyruvate entered alternative pathways (Moat et al. 2002). No particular trend in succinic acid was observable except that there was a continual increase in production throughout fermentation in 75SBS, 90SBS, 90S, and 100S, while increases were followed by sharp decreases at 48 h in 75S and 100SBS (Fig1B).

Organic acids in fermented soybean pastes like doenjang are used as quality indicators. They affect the flavor of the pastes through increases in acidity and development of unpleasant odors. Lactic and succinic acids for instance are related to sourness (Namgung et al. 2010). Likewise, the sourness intensities of LFP were higher than those of NFP (Ng'ong'ola-Manani et al. 2014). Acetic acid is considered to provide an unpleasant flavor in fermented soy foods (Namgung et al. 2010).

Soybeans contain about 9.94% carbohydrates in the form of polysaccharides and sugars. Fermentable sugars such as glucose and galactose ranging from 3.29 to 4.44/100 g and 2.91 to 3.36/100 g, respectively, were reported as part of the total dietary fiber (Redondo-Cuenca et al. 2007). Iheanacho (2010) reported 5.13% of maltose and 14.05% of fructose in soybeans. In this study, there were more sugars in LFP than in NFP at 0 h (Fig2) probably because of the back-slopping material which had been previously fermented and was made using malt flour. Malting increases sugar (glucose, fructose, or maltose) content due to amylolytic activities (Malleshi et al. 1986). Fermentation leads to increases and decreases in sugar content in cereal-based products (Palanisamy et al. 2012). Rapid decreases in maltose in 100SBS and 90SBS (Fig2A) could be due to its utilization as energy source and subsequent conversion into organic acids and other metabolites. The catabolism of maltose begins with its phosphorylatic cleavage catalyzed by maltose phosphorylase, yielding glucose and glucose-1-phosphate (Axelsson 2004; Gänzle et al. 2007). Homofermentative and heterofermentative LAB convert glucose-1-phosphate to glucose-6-phosphate, which is further metabolized via glycolysis to lactic acid or via phosphogluconate pathway to yield lactic acid, carbon dioxide, and ethanol/acetic acid, respectively. Glucose can also be phosphorylated by homofermentative LAB and follow the glycolytic pathway or it can be converted to glucose-6-phosphate and follow the phosphogluconate pathway by heterofermentative LAB (Vogel et al. 1999; De Vuyst et al. 2002; Axelsson 2004; Gänzle et al. 2007).

Figure 2.

Figure 2

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Changes in sugars during fermentation. Samples coded 100S, 90S, and 75S represent naturally fermented pastes, while samples coded 100SBS, 90SBS, and 75SBS represent lactic acid-fermented pastes. Pastes are designated according to 100%, 90%, and 75% soybean composition, the remaining proportions being maize.

Rapid decreases in glucose in LFP between 0 and 24 h (Fig2B) could be due to its utilization in the generation of energy via glycolysis or the phosphogluconate pathway (Vogel et al. 1999; Axelsson 2004) to support growth of a higher microbial population in LFP at the beginning of the fermentation. Although fructose content was higher throughout fermentation in LFP than in NFP (Fig2C), it followed similar trends. Increases between 0 and 24 h in all samples, except 100S, and thereafter gradual decreases throughout fermentation in all samples, except 75SBS, were observed. The presence and increases of fructose could have been due to accumulation as a result of metabolism of other sugars like sucrose while decreases could have been due to utilization as carbon source or bioconversion of the sugar. Fructose accumulation can also be explained in terms of preferential carbohydrate utilization of LAB (Gänzle et al. 2007). All microorganisms important in foods can metabolize glucose, but vary greatly in their ability to utilize other fermentable sugars including fructose (Ray 2003). Fructose and glucose can be released from sucrose fermentation which starts with the cleavage of the sugar by sucrose hydrolase into the two monosaccharide units. The two sugars then enter the major fermentation pathways (Axelsson 2004). Heterofermentative LAB can assimilate fructose via the 6-phosphogluconate/phosphoketolase pathway for hexose (Wisselink et al. 2002). Fructose may also be used as an alternative electron acceptor in LAB fermentation processes to increase LAB energy yield, resulting in its reduction to mannitol (Chen et al. 1983; Kandler 1983; Vogel et al. 1999; Gänzle et al. 2007). By 72 h, there were no significant differences in maltose and glucose levels between LFP and NFP and these two sugars were nearly all used up, while there were significant differences in the fructose content.

Antinutritional factors

Phytic acid

The levels of phytic acid content at the beginning of the fermentation processes varied among the pastes and did not seem to be influenced by sample composition. However, after 48 and 72 h, significant reductions were observed in all samples and levels of degradation depended on the type of fermentation. Overall, natural fermentation was more effective in reducing phytic acid levels (Table4). A 33–54% reduction was achieved by natural fermentation at 24 and by 72 h, 85% reduction was noted while in some samples the phytate could not be detected. On the contrary, only 18–32% reduction was achieved in LFP at 24 h and 37–49% reduction was achieved by 72 h. Bacillus subtilis (Shimizo 1992; Kerovuo et al. 1998) and LAB species (Songré-Ouattara et al. 2008; Khodaii et al. 2013) with phytase activities have been reported previously.

Table 4.

Phytic acid content at different times of fermentation of the pastes

Phytic acid (g/100 g sample dry matter)
Sample 0 h 24 h 48 h 72 h
100S 0.314 ± 0.037a 0.183 ± 0.112ab n.d. n.d.
90S 0.274 ± 0.036a 0.125 ± 0.094b 0.132 ± 0.087ab 0.040 ± 0.215c
75S 0.319 ± 0.063a 0.213 ± 0.05b 0.079 ± 0.06bc n.d.
100SBS 0.322 ± 0.019a 0.226 ± 0.072b 0.199 ± 0.053c 0.202 ± 0.048bc
90SBS 0.234 ± 0.080a 0.191 ± 0.128a 0.194 ± 0.043a 0.138 ± 0.062b
75SBS 0.276 ± 0.047a 0.186 ± 0.030b 0.160 ± 0.010bc 0.141 ± 0.025c
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Means not sharing superscript letter(s) are significantly different (P < 0.05) within a row. n.d., not detected. Samples coded 100S, 90S, and 75S represent naturally fermented pastes, while samples coded 100SBS, 90SBS, and 75SBS represent lactic acid-fermented pastes. Pastes are designated according to 100%, 90%, and 75% soybean composition, the remaining proportions being maize.

The differences in the extent of phytic acid degradation between LFP and NFP can probably be explained in terms of the complexity of the physiological and environmental factors that affect the production and activity of phytases; and also in terms of the types of microflora in the pastes. Phytase activities in Bacillus spp. are optimal at a wide pH range of 4.5–8.5 (Shimizo 1992; Kim et al. 1998; Choi et al. 2001). In sourdough LAB, pH 4.0 was optimum for phytase activity and the activity rapidly decreased at pH 3.5 or pH 4.5 (De Angelis et al. 2003). Palacios et al. (2005) reported an optimum pH of 5.0 and 50% retention of optimum phytase activity at pH 4.5 and 5.5, while only 20% retention at pH 4.0 and 6.0 were reported for various LAB strains. Extracellular phytase activities in Bacillus spp. are known (Shimizo 1992; Kim et al. 1998; Choi et al. 2001) while in LAB only intracellular activities have been detected (De Angelis et al. 2003; Palacios et al. 2005). Further, Palacios et al. (2005) purified and characterized an acid phosphatase (produced by LAB strains) with broad specificity that hydrolyzed monophosphorylated substrates and also phytic acid. This could suggest the possibility of phytic acid degradation activity by LAB due to nonspecific acid phosphatase with residual activity on phytic acid (Haros et al. 2008). On the contrary, enzymes with high specificity for sodium phytate have been isolated and purified from Bacillus spp. (Shimizo 1992; Kim et al. 1998). Finally, the synthesis of phytase in lactobacilli strains responded to limiting concentrations of carbon source (Palacios et al. 2005). Nevertheless, phytic acid degradation in both LFP and NFP fermentation processes is essential to improve bioavailability of minerals such as Ca and Zn (Kim et al. 2010).

Trypsin inhibitor

In this study, heating during paste preparation was the most effective way of reducing trypsin inhibitor. This was in agreement with results reported by Egounlety and Aworh (2003). The content of trypsin inhibitor in raw soybeans was 19 mg/g sample, but after boiling, trypsin inhibitor could not be detected in 100S while the highest trypsin inhibitor at 0 h was 0.169 mg/g sample signifying a 99% reduction (Table5). Higher levels of trypsin inhibitor in LFP could be due to the back-slopping material which was made using finger millet malt that was added after cooling the porridge to 50–60°C. Although reductions were observed in both types of fermentation processes, fluctuations were observed in 100SBS and 90SBS in which marked increases were observed at 24 h. Higher trypsin inhibitor levels at 24 h in 100SBS and 90SBS could be due to release of bound trypsin inhibitors. Wang et al. (1972) and Egounlety and Aworh (2003) reported increases in levels of trypsin-inhibiting activities of heated soybeans fermented with Rhizopus oligosporus. According to Wang et al. (1972), various proteases produced by the mold were responsible for releasing bound trypsin inhibitor from the soybean substrate. Release of bound trypsin inhibitors by gastric digestion has also been suggested (Wang et al. 1972).

Table 5.

Trypsin inhibitor at different times of fermentation of the pastes

Trypsin inhibitor (mg/g sample dry matter)
Sample 0 h 24 h 48 h 72 h
100S n.d. 0.002 ± 0.15 n.d. n.d.
90S 0.156 ± 0.08 n.d. n.d. n.d.
75S 0.013 ± 0.17 n.d. n.d. n.d.
100SBS 0.160 ± 0.20 0.293 ± 0.43 n.d. n.d.
90SBS 0.069 ± 0.09 0.112 ± 0.33 n.d. n.d.
75SBS 0.169 ± 0.10 n.d. n.d. n.d.
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Samples coded 100S, 90S, and 75S represent naturally fermented pastes, while samples coded 100SBS, 90SBS, and 75SBS represent lactic acid-fermented pastes. Pastes are designated according to 100%, 90%, and 75% soybean composition, the remaining proportions being maize. n.d., not detected.

Conclusions

LAB fermentation and natural fermentation improved the nutritional quality of pastes of soybeans and soybean–maize blends through increases in soluble protein, increases in some total and free amino acids, and degradation of antinutritional factors. Increases in α-amylase activities in NFP and 75SBS could suggest an increased starch digestibility and possibility of reduced dietary bulkiness providing room for increasing energy density. Both types of fermentation processes resulted in nonsignificant changes in most of the total amino acids, although the fermentation processes increased the levels of the sulfur-containing amino acids, cysteine, and methionine, which are limiting in legumes. In this study, Glu, Asp, Leu, Arg, Lys, Ser, and Phe were considered the main total amino acids throughout fermentation. Amino acid metabolism and proteolytic activities in the fermentation processes resulted in differences in major free amino acids. In NFP, these were Asp, Glu, Ala, Val, Phe, Leu, and Lys, while in LFP, these were Asp, Glu, Gly, Ala, GABA, Leu, and Lys. The free amino acids together with the organic acids would influence the taste of the pastes. High lactic acid production in LFP could mean an increased shelf life, a better microbial safety, and an increased sour taste. A comparative advantage of natural fermentation over lactic acid fermentation in this study was the higher degradation of the antinutrient, phytic acid in natural fermentation.

Acknowledgments

We acknowledge financial support from the Norwegian Programme for Development, Research and Education (NUFU) and Norwegian State Education Fund (Lånekassen). This research was also partly financed by the International Foundation for Science (IFS) through grant no. E/4889-1 awarded to Tinna A. Ng'ong'ola-Manani. We also acknowledge Kari Olsen of the Norwegian University of Life Sciences for assistance in HPLC measurements of free amino acids, organic acids, and sugars.

Conflict of Interest

None declared.

References

  1. Allagheny N, Obanu ZA, Campbell-Platt G. Owens JD. Control of ammonia formation during Bacillus subtilis fermentation of legumes. Int. J. Food Microbiol. 1996;29:321–333. doi: 10.1016/0168-1605(95)00069-0. [DOI] [PubMed] [Google Scholar]
  2. AOAC. Official methods of analysis of the Association of Official Chemists. Washington, D.C: Association of Official Chemists; 1990. [Google Scholar]
  3. Asgar MA, Fazilah A, Huda N, Bhat R. Karim AA. Nonmeat protein alternatives as meat extenders and meat analogs. Compr. Rev. Food Sci. Food Saf. 2010;9:513–529. doi: 10.1111/j.1541-4337.2010.00124.x. [DOI] [PubMed] [Google Scholar]
  4. Axelsson L. Lactic acid bacteria: classification and physiology. New York, NY: Marcel Dekker Inc; 1998. [Google Scholar]
  5. Axelsson L. Lactic acid bacteria: classification and physiology. In: Salminen S, Von Wright A, editors. Lactic acid bacteria: microbiological and functional aspects. 3rd. New York, NY: Marcel Dekker Inc; 2004. pp. 1–66. edn (Revised & Expanded). [Google Scholar]
  6. Baek JG, Shim SM, Kwon DY, Choi HK, Lee CH. Kim YS. Metabolite profiling of Cheonggukjang, a fermented soybean paste, inoculated with various Bacillus strains during fermentation. Biosci. Biotechnol. Biochem. 2010;74:1860–1868. doi: 10.1271/bbb.100269. [DOI] [PubMed] [Google Scholar]
  7. Bernfeld P. Amylases a and ß. New York, NY: Academic Press; 1955. [Google Scholar]
  8. Bütikofer U. Ardö Y. Quantitative determination of free amino acids in cheese. Bull. Int. Dairy Fed. 1999;337:24–32. [Google Scholar]
  9. Chen KH, Mcfeeters RF. Fleming HP. Fermentation characteristics of heterolactic acid bacteria in green bean juice. J. Food Sci. 1983;48:962–966. [Google Scholar]
  10. Chitra U, Singh U. Venkateswara Rao P. Phytic acid, in vitro protein digestibility, dietary fiber, and minerals of pulses as influenced by processing methods. Plant Foods Hum. Nutr. 1996;49:307–316. doi: 10.1007/BF01091980. [DOI] [PubMed] [Google Scholar]
  11. Choi YM, Suh HJ. Kim JM. Purification and properties of extracellular phytase from Bacillus sp. KHU-10. J. Protein Chem. 2001;20:287–292. doi: 10.1023/a:1010945416862. [DOI] [PubMed] [Google Scholar]
  12. Christensen J, Dudley E, Pederson J. Steele J. Peptidases and amino acid catabolism in lactic acid bacteria. Antonie Van Leeuwenhoek. 1999;76:217–246. [PubMed] [Google Scholar]
  13. Dajanta K, Apichartsrangkoon A, Chukeatirote E. Frazier RA. Free-amino acid profiles of thua nao, a Thai fermented soybean. Food Chem. 2011;125:342–347. [Google Scholar]
  14. Dajanta K, Chukeatirote E. Apichartsrangkoon A. Improvement of thua nao production using protein-rich soybean and Bacillus subtilis TN51 starter culture. Ann. Microbiol. 2012;62:785–795. [Google Scholar]
  15. Dakwa S, Sakyi-Dawson E, Diako C, Annan NT. Amoa-Awua WK. Effect of boiling and roasting on the fermentation of soybeans into dawadawa (soy-dawadawa) Int. J. Food Microbiol. 2005;104:69–82. doi: 10.1016/j.ijfoodmicro.2005.02.006. [DOI] [PubMed] [Google Scholar]
  16. De Angelis M, Gallo G, Corbo MR, McSweeney PLH, Faccia M, Giovine M, et al. Phytase activity in sourdough lactic acid bacteria: purification and characterization of a phytase from Lactobacillus sanfranciscensis CB1. Int. J. Food Microbiol. 2003;87:259–270. doi: 10.1016/s0168-1605(03)00072-2. [DOI] [PubMed] [Google Scholar]
  17. De Vuyst L, Schrijvers V, Paramithiotis S, Hoste B, Vancanneyt M, Swings J, et al. The biodiversity of lactic acid bacteria in Greek traditional wheat sourdoughs is reflected in both composition and metabolite formation. Appl. Environ. Microbiol. 2002;68:6059–6069. doi: 10.1128/AEM.68.12.6059-6069.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Deshpande SS. Salunkhe DK. Grain legumes, seeds and nuts: rationale for fermentation. In: Deshpande SS, editor; Fermented grain legumes, seeds and nuts: a global perspective. Rome, Italy: Food and Agriculture Organization of the United Nations; 2000. pp. 1–32. [Google Scholar]
  19. Egounlety M. Aworh OC. Effect of soaking, dehulling, cooking and fermentation with Rhizopus oligosporus on the oligosaccharides, trypsin inhibitor, phytic acid and tannins of soybean (Glycine max Merr.), cowpea (Vigna unguiculata L. Walp) and groundbean (Macrotyloma geocarpa Harms) J. Food Eng. 2003;56:249–254. [Google Scholar]
  20. Erdal I, Yilmaz A, Taban S, Eker S, Torun B. Cakmak I. Phytic acid and phosphorus concentrations in seeds of wheat cultivars grown with and without zinc fertilization. J. Plant Nutr. 2002;25:113–127. [Google Scholar]
  21. Fernandez M. Zuniga M. Amino acid catabolic pathways of lactic acid bacteria. Crit. Rev. Microbiol. 2006;32:155–183. doi: 10.1080/10408410600880643. [DOI] [PubMed] [Google Scholar]
  22. Gänzle MG, Vermeulen N. Vogel RF. Carbohydrate, peptide and lipid metabolism of lactic acid bacteria in sourdough. Food Microbiol. 2007;24:128–138. doi: 10.1016/j.fm.2006.07.006. [DOI] [PubMed] [Google Scholar]
  23. Golbitz P. Overview of soybean processing and production. Traditional soyfoods: processing and products. J. Nutr. 1995;125:570S–572S. doi: 10.1093/jn/125.suppl_3.570S. [DOI] [PubMed] [Google Scholar]
  24. Hamerstrand GE, Black LT. Glover JD. Trypsin inhibitors is soy products: modification of the standard analytical procedure. Cereal Chem. 1980;58:42–45. [Google Scholar]
  25. Hansen M, Pedersen B, Munck L. Eggum BO. Weaning foods with improved energy and nutrient density prepared from germinated cereals. 1. Preparation and dietary bulk of gruels based on barley. Tokyo, Japan: United Nations University; 1989. [Google Scholar]
  26. Haros M, Bielecka M, Honke J. Sanz Y. Phytate-degrading activity in lactic acid bacteria. Polish J. Food Nutr. Sci. 2008;58:33–40. [Google Scholar]
  27. Haug W. Lantzsch H-J. Sensitive method for the rapid determination of phytate in cereals and cereal products. J. Sci. Food Agric. 1983;34:1423–1426. [Google Scholar]
  28. Holzapfel W. Use of starter cultures in fermentation on a household scale. Food Control. 1997;8:241–258. [Google Scholar]
  29. Holzapfel WH. Appropriate starter culture technologies for small-scale fermentation in developing countries. Int. J. Food Microbiol. 2002;75:197–212. doi: 10.1016/s0168-1605(01)00707-3. [DOI] [PubMed] [Google Scholar]
  30. Holzapfel WH, Haberer P, Geisen R, Björkroth J. Schillinger U. Taxonomy and important features of probiotic microorganisms in food and nutrition. Am. J. Clin. Nutr. 2001;73:365s–373s. doi: 10.1093/ajcn/73.2.365s. [DOI] [PubMed] [Google Scholar]
  31. Iheanacho KME. Comparative studies of the nutritional composition of soy bean (Glycine max) and lima bean (Phaseolus lunatus. Sci. Afr. 2010;9:29–35. [Google Scholar]
  32. Kakade ML, Rackis JJ, McGhee JE. Puski G. Determination of trypsin inhibitor activity of soy products: a collaborative analysis of an improved procedure. Cereal Chem. 1974;51:376–382. [Google Scholar]
  33. Kalimbira AA, Mtimuni BM. Mtimuni JP. Effect of incorporating legumes on nutritive value of cassava-based complementary foods. Bunda J. Agric. Environ. Sci. Technol. 2004;2:13–21. [Google Scholar]
  34. Kandler O. Carbohydrate metabolism in lactic acid bacteria. Antonie Van Leeuwenhoek. 1983;49:209–224. doi: 10.1007/BF00399499. [DOI] [PubMed] [Google Scholar]
  35. Kerovuo J, Lauraeus M, Nurminen P, Kalkkinen N. Apajalahti J. Isolation, characterization, molecular gene cloning, and sequencing of a novel phytase from Bacillus subtilis. Appl. Environ. Microbiol. 1998;64:2079–2085. doi: 10.1128/aem.64.6.2079-2085.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Khetarpaul N. Chauhan BM. Effect of fermentation on protein, fat, minerals and thiamine content of pearl millet. Plant Foods Hum. Nutr. 1989;39:169–177. doi: 10.1007/BF01091897. [DOI] [PubMed] [Google Scholar]
  37. Khodaii Z, Mehrabani Natanzi M, Naseri MH, Goudarzvand M. Dodsons H. Phytase activity of lactic acid bacteria isolated from dairy and pharmaceutical probiotic products. Int. J. Ent. Pathog. 2013;1:12–16. [Google Scholar]
  38. Kim Y-O, Kim H-K, Bae K-S, Yu J-H. Oh T-K. Purification and properties of a thermostable phytase from Bacillus sp. DS11. Enzyme Microb. Technol. 1998;22:2–7. doi: 10.1111/j.1574-6968.1998.tb12997.x. [DOI] [PubMed] [Google Scholar]
  39. Kim T, Lee J-H, Park M-H. Kim H-Y. Analysis of bacterial and fungal communities in Japanese- and Chinese-fermented soybean pastes using nested PCR–DGGE. Curr. Microbiol. 2010;60:315–320. doi: 10.1007/s00284-009-9542-4. [DOI] [PubMed] [Google Scholar]
  40. Kitabatake N, Gimbi DM. Oi Y. Traditional non-alcoholic beverage, Togwa, in East Africa, produced from maize flour and germinated finger millet. Int. J. Food Sci. Nutr. 2003;54:447–455. doi: 10.1080/09637480120092053. [DOI] [PubMed] [Google Scholar]
  41. Klein G, Pack A, Bonaparte C. Reuter G. Taxonomy and physiology of probiotic lactic acid bacteria. Int. J. Food Microbiol. 1998;41:103–125. doi: 10.1016/s0168-1605(98)00049-x. [DOI] [PubMed] [Google Scholar]
  42. Lan Y, Williams BA, Verstegen MWA, Patterson R. Tamminga S. Soy oligosaccharides in vitro fermentation characteristics and its effect on caecal microorganisms of young broiler chickens. Anim. Feed Sci. Technol. 2007;133:286–297. [Google Scholar]
  43. Lorri W. Svanberg U. Lactic acid-fermented cereal gruels: viscosity and flour concentration. Int. J. Food Sci. Nutr. 1993;44:207–213. [Google Scholar]
  44. Maleta K. Undernutrition. Malawi Med. J. 2006;18:191–207. [PMC free article] [PubMed] [Google Scholar]
  45. Malleshi NG, Desikachar HSR. Tharanathan RN. Free sugars and non-starchy polysaccharides of finger millet (Eleusine coracana), pearl millet (Pennisetum typhoideum), foxtail millet (Setaria italica) and their malts. Food Chem. 1986;20:253–261. [Google Scholar]
  46. Medic J, Atkinson C. Hurburgh C., Jr Current knowledge in soybean composition. J. Am. Oil Chem. Soc. 2014;91:363–384. [Google Scholar]
  47. Moat AG, Foster JW. Spector MP. Microbial physiology. New York, NY: John Wiley & Sons Inc; 2002. 4th ed. [Google Scholar]
  48. Mohiedeen IE, Tinay AHE, Elkhalifa AEO, Babiker EE. Mallasiy LO. Effect of fermentation on in vitro protein digestibility, protein fractions and amino acids composition of maize (Zea mays Linnaeus) cultivars. Electronic J. Environ. Agric. Food Chem. 2010;9:838–847. [Google Scholar]
  49. Mosha AC. Svanberg UH. Preparation of weaning foods with high nutrient density using flour of germinated cereals. Food Nutr. Bull. 1983;5:10–14. [Google Scholar]
  50. Mugula JK, Nnko SAM, Narvhus JA. Sørhaug T. Microbiological and fermentation characteristics of togwa, a Tanzanian fermented food. Int. J. Food Microbiol. 2003;80:187–199. doi: 10.1016/s0168-1605(02)00141-1. [DOI] [PubMed] [Google Scholar]
  51. Mulimani VH. Devendra S. Effect of soaking, cooking and crude α-galactosidase treatment on the oligosaccharide content of red gram flour. Food Chem. 1998;61:475–479. [Google Scholar]
  52. Muyanja CMBK, Narvhus JA, Treimo J. Langsrud T. Isolation, characterisation and identification of lactic acid bacteria from bushera: a Ugandan traditional fermented beverage. Int. J. Food Microbiol. 2003;80:201–210. doi: 10.1016/s0168-1605(02)00148-4. [DOI] [PubMed] [Google Scholar]
  53. Namgung H-J, Park H-J, Cho IH, Choi H-K, Kwon D-Y, Shim S-M, et al. Metabolite profiling of doenjang, fermented soybean paste, during fermentation. J. Sci. Food Agric. 2010;90:1926–1935. doi: 10.1002/jsfa.4036. [DOI] [PubMed] [Google Scholar]
  54. Narvhus JA, Østeraas K, Mutukumila T. Abrahamsen RK. Production of fermented milk using malty compound-producing strain of Lactococcus lactis subsp. lactis biovar. diacetylactis isolated from Zimbabwe naturally fermented milk. Int. J. Food Microbiol. 1998;41:73–80. doi: 10.1016/s0168-1605(98)00036-1. [DOI] [PubMed] [Google Scholar]
  55. Ng'ong'ola-Manani TA, Mwangwela AM, Schüller RB, Østlie HM. Wicklund T. Sensory evaluation and consumer acceptance of naturally and lactic acid bacteria-fermented pastes of soybeans and soybean–maize blends. Food Sci. Nutr. 2014;2:114–131. doi: 10.1002/fsn3.82. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Odunfa SA. Carbohydrate changes in fermenting locust bean (Parkia filicoidea) duringiru preparation. Plant Foods Hum. Nutr. 1983;32:3–10. [Google Scholar]
  57. Official Journal of the European Communities. Establishing Community methods of analysis for the determination of amino acids, crude oils and fats, and olaquindox in feedingstuffs and amending Directive 71/393/EEC. Commission Directive 98/64/EC. 1998:L 257/14–L 257/28. [Google Scholar]
  58. Palacios MC, Haros M, Rosell CM. Sanz Y. Characterization of an acid phosphatase from Lactobacillus pentosus: regulation and biochemical properties. J. Appl. Microbiol. 2005;98:229–237. doi: 10.1111/j.1365-2672.2004.02447.x. [DOI] [PubMed] [Google Scholar]
  59. Palanisamy BD, Rajendran V, Sathyaseelan S, Bhat R. Venkatesan BP. Enhancement of nutritional value of finger millet-based food (Indian dosa) by co-fermentation with horse gram flour. Int. J. Food Sci. Nutr. 2012;63:5–15. doi: 10.3109/09637486.2011.591367. [DOI] [PubMed] [Google Scholar]
  60. Park K-B. Oh S-H. Production of yogurt with enhanced levels of gamma-aminobutyric acid and valuable nutrients using lactic acid bacteria and germinated soybean extract. Bioresour. Technol. 2007;98:1675–1679. doi: 10.1016/j.biortech.2006.06.006. [DOI] [PubMed] [Google Scholar]
  61. Park MK, Cho IH, Lee S, Choi H-K, Kwon D-Y. Kim Y-S. Metabolite profiling of Cheonggukjang, a fermented soybean paste, during fermentation by gas chromatography-mass spectrometry and principal component analysis. Food Chem. 2010;122:1313–1319. [Google Scholar]
  62. Parkouda C, Nielsen DS, Azokpota P, Ivette Irène Ouoba L, Amoa-Awua WK, Thorsen L, et al. The microbiology of alkaline-fermentation of indigenous seeds used as food condiments in Africa and Asia. Crit. Rev. Microbiol. 2009;35:139–156. doi: 10.1080/10408410902793056. [DOI] [PubMed] [Google Scholar]
  63. Ray B. Fundamental food microbiology. 3rd ed. New York, NY: CRC Press; 2003. [Google Scholar]
  64. Redondo-Cuenca A, Villanueva-Suárez MJ, Rodríguez-Sevilla MD. Mateos-Aparicio I. Chemical composition and dietary fibre of yellow and green commercial soybeans (Glycine max. Food Chem. 2007;101:1216–1222. [Google Scholar]
  65. Sanni AI. The need for process optimization of African fermented foods and beverages. Int. J. Food Microbiol. 1993;18:85–95. doi: 10.1016/0168-1605(93)90213-z. [DOI] [PubMed] [Google Scholar]
  66. Sarkar PK. Tamang JP. Changes in the microbial profile and proximate composition during natural fermentations of soybeans to produce kinema. Food Microbiol. 1995;12:317–325. [Google Scholar]
  67. Sarkar PK, Tamang JP, Cook PE. Owens JD. Kinema — a traditional soybean fermented food: proximate composition and microflora. Food Microbiol. 1994;11:47–55. [Google Scholar]
  68. Sarkar PK, Jones LJ, Craven GS. Somerset SM. Oligosaccharide profiles during kinema production. Lett. Appl. Microbiol. 1997a;24:337–339. [Google Scholar]
  69. Sarkar PK, Jones LJ, Craven GS, Somerset SM. Palmer C. Amino acid profiles of kinema, a soybean-fermented food. Food Chem. 1997b;59:69–75. [Google Scholar]
  70. Sarkar PK, Hasenack B. Nout MJR. Diversity and functionality of Bacillus and related genera isolated from spontaneously fermented soybeans (Indian kinema) and locust beans (African soumbala. Int. J. Food Microbiol. 2002;77:175–186. doi: 10.1016/s0168-1605(02)00124-1. [DOI] [PubMed] [Google Scholar]
  71. Shimizo M. Purification and characterization of phytase from Bacillus subtilisnatto) N-77. Biosci. Biotechnol. Biochem. 1992;56:1266–1269. [Google Scholar]
  72. Sindhu SC. Khetarpaul N. Probiotic fermentation of indigenous food mixture: effect on antinutrients and digestibility of starch and protein. J. Food Compos. Anal. 2001;14:601–609. [Google Scholar]
  73. Songré-Ouattara LT, Mouquet-Rivier C, Icard-Vernière C, Humblot C, Diawara B. Guyot JP. Enzyme activities of lactic acid bacteria from a pearl millet fermented gruel (ben-saalga) of functional interest in nutrition. Int. J. Food Microbiol. 2008;128:395–400. doi: 10.1016/j.ijfoodmicro.2008.09.004. [DOI] [PubMed] [Google Scholar]
  74. Sripriya G, Antony U. Chandra TS. Changes in carbohydrate, free amino acids, organic acids, phytate and HCl extractability of minerals during germination and fermentation of finger millet (Eleusine coracana. Food Chem. 1997;58:345–350. [Google Scholar]
  75. Steinkraus KH. Classification of fermented foods: worldwide review of household fermentation techniques. Food Control. 1997;8:311–317. [Google Scholar]
  76. Terlabie NN, Sakyi-Dawson E. Amoa-Awua WK. The comparative ability of four isolates of Bacillus subtilis to fermemt soybeans into dawadawa. Int. J. Food Microbiol. 2006;106:145–152. doi: 10.1016/j.ijfoodmicro.2005.05.021. [DOI] [PubMed] [Google Scholar]
  77. Thiex NJ, Manson H, Anderson S. Persson J-Å. Determination of crude protein in animal feed, forage, grain, and oilseeds by using block digestion with a copper catalyst and steam distillation into boric acid: collaborative study. J. AOAC Int. 2002;85:309–317. [PubMed] [Google Scholar]
  78. Urbano G, López-Jurado M, Aranda P, Vidal-Valverde C, Tenorio E. Porres J. The role of phytic acid in legumes: antinutrient or beneficial function? J. Physiol. Biochem. 2000;56:283–294. doi: 10.1007/BF03179796. [DOI] [PubMed] [Google Scholar]
  79. Vidal-Valverde C, Frías J. Valverde S. Changes in the carbohydrate composition of legumes after soaking and cooking. J. Am. Diet. Assoc. 1993;93:547–550. doi: 10.1016/0002-8223(93)91814-7. [DOI] [PubMed] [Google Scholar]
  80. Visessanguan W, Benjakul S, Potachareon W, Panya A. Riebroy S. Accelerated proteolysis of soy proteins during fermentation of thua-nao inoculated with Bacillus subtilis. J. Food Biochem. 2005;29:349–366. [Google Scholar]
  81. Vogel R, Knorr R, Müller MA, Steudel U, Gänzle M. Ehrmann M. Non-dairy lactic fermentations: the cereal world. Antonie Van Leeuwenhoek. 1999;76:403–411. [PubMed] [Google Scholar]
  82. Wang H-J. Murphy PA. Mass balance study of isoflavones during soybean processing. J. Agric. Food Chem. 1996;44:2377–2383. [Google Scholar]
  83. Wang HL, Vespa JB. Hesseltine W. Release of bound trypsin inhibitors in soybeans by Rhizopus oligosporus. J. Nutr. 1972;102:1495–1500. doi: 10.1093/jn/102.11.1495. [DOI] [PubMed] [Google Scholar]
  84. Wang Y-C, Yu R-C, Yang H-Y. Chou C-C. Sugar and acid contents in soymilk fermented with lactic acid bacteria alone or simultaneously with bifidobacteria. Food Microbiol. 2003;20:333–338. [Google Scholar]
  85. Wisselink HW, Weusthuis RA, Eggink G, Hugenholtz J. Grobben GJ. Mannitol production by lactic acid bacteria: a review. Int. Dairy J. 2002;12:151–161. [Google Scholar]
  86. Yang HJ, Park S, Pak V, Chung KR. Kwon DY. Fermented soybean products and their bioactive compounds. In: El-Shemy PH, editor; Soybean and health. Croatia: InTech Europe; 2011. pp. 21–59. [Google Scholar]
  87. Yoon MY. Hwang H-J. Reduction of soybean oligosaccharides and properties of α-d-galactosidase from Lactobacillus curvatus R08 and Leuconostoc mesenteriodes JK55. Food Microbiol. 2008;25:815–823. doi: 10.1016/j.fm.2008.04.008. [DOI] [PubMed] [Google Scholar]
  88. Yoon JH, Thompson LU. Jenkins DJ. The effect of phytic acid on in vitro rate of starch digestibility and blood glucose response. Am. J. Clin. Nutr. 1983;38:835–842. doi: 10.1093/ajcn/38.6.835. [DOI] [PubMed] [Google Scholar]
  89. Zhao GQ, Zhang Y, Hoon MA, Chandrashekar J, Erlenbach IE, Ryba NJP, et al. The receptors for mammalian sweet and umami taste. Cell. 2003;115:255–266. doi: 10.1016/s0092-8674(03)00844-4. [DOI] [PubMed] [Google Scholar]

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