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Evaluation of protein digestibility of fermented soybeans and changes in biochemical characteristics of digested fractions
Journal of Functional Foods 52 (2019) 640–647
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Evaluation of protein digestibility of fermented soybeans and changes in biochemical characteristics of digested fractions Sunantha Ketnawa, Yukiharu Ogawa
T
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Graduate School of Horticulture, Chiba University, 648, Matsudo, Matsudo 271-8510, Japan
ARTICLE INFO
ABSTRACT
Keywords: Natto Processing Amino acids TCA-soluble peptides In vitro anti-inflammatory
This study evaluated the protein digestibility of fermented soybeans inoculated with Bacillus subtilis var. natto using a simulated in vitro gastrointestinal digestion model. It was found that the total trichloroacetic acid (TCA)soluble peptide yield increased when the digestion stage progressed and compared to those of fermented soybeans before digestion. Most high molecular weight proteins disappeared after 4 h of digestion. All essential amino acids were found to increase remarkably (∼4%), especially Arg. Antioxidant amino acids such as Tyr, His and Phe were also found to increase. The highest antioxidant activity was found in the fractions after digestion corresponding to the antioxidant amino acids obtained. An increase in nitric oxide inhibition (9–29%) and a decrease in egg albumin denaturation activity (73–48%) were observed. Fermentation improved the protein digestibility of boiled soybeans by 45% and may benefit human health due to an improvement in essential amino acids and bioactivity through digestion.
1. Introduction A fermented soybean product, so-called ‘natto’, made by fermenting whole soybean seeds with pure culture of Bacillus subtilis subsp. natto, has traditionally been consumed in Japan for thousands of years (Weng, Yao, Sparks, & Wang, 2017). During fermentation, storage proteins of soybean can be degraded by microbial proteases. This can increase its nutritional value, reduce anti-nutritional factors and hydrolyse oligosaccharides (raffinose and stachyose) (Vernaza, Dia, Gonzalez de Mejia, & Chang, 2012). As a result, natto can be considered as a nutrition-rich food due to its high content of isoflavones, dietary fibre, vitamins, linoleic acid and some minerals. It also contains some functional compounds such as enzymes, bioactive peptides, nattokinase (a fibrinolytic agent), gamma-polyglutamic acid (γ-PGA) and so on (Sanjukta & Rai, 2016). Protein digestibility is an important factor in estimating the availability of protein for intestinal absorption after digestion, reflecting the efficiency of protein utilization in the diet (Almeida, Monteiro, CostaLima, Alvares, & Conte-Junior, 2015). A simulated in vitro gastrointestinal digestion model is a widely used method to determine protein digestibility. The method mimics the digestion process occurring in the human gastrointestinal tract through proteolytic enzymes (i.e. the pepsin–pancreatin enzyme system), measuring the percentage of protein content and peptide bonds which are hydrolysed by such enzymes
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(Hur, Lim, Decker, & McClements, 2011). Hydrolysis of higher molecular weight proteins into lower molecular weight oligopeptides, and the breakdown of peptide bonds to produce free amino acids, can be used as evidence of efficient digestion (Weng & Chen, 2010). Moreover, some small peptides generated from digestion have significant bioactive activity such as antioxidant activity or anti-inflammatory properties which can contribute to the suppression of some oxidation- and inflammation-related diseases in the human body (Liao, Huang, Sheen, & Chou, 2010; Udenigwe & Aluko, 2012). Currently, information on protein digestibility and the changes in biochemical characteristics of natto products during digestion through the human gastrointestinal tract is limited. The purpose of this study is to evaluate the effect of fermentation on soybeans, and the protein digestibility of fermented soybeans which were laboratory-scale inoculated with B. subtilis var. natto, using a simulated in vitro gastrointestinal digestion model. The digested fractions of fermented soybeans were investigated for biochemical characteristics such as total soluble nitrogen content, protein content, trichloroacetic acid (TCA)soluble peptides, molecular weight by SDS-PAGE, and free amino acids. Digested fractions of fermented soybeans were also analysed to determine their antioxidant properties. The in vitro anti-inflammatory activity of digested proteins of fermented soybeans was also investigated, by their ability to inhibit nitric oxide production and denature anti-albumin.
Corresponding author. E-mail address: [email protected] (Y. Ogawa).
https://doi.org/10.1016/j.jff.2018.11.046 Received 31 July 2018; Received in revised form 21 November 2018; Accepted 28 November 2018 1756-4646/ © 2018 Elsevier Ltd. All rights reserved.
Journal of Functional Foods 52 (2019) 640–647
S. Ketnawa, Y. Ogawa
2. Materials and methods
starting the experiment. The reactor was connected to a temperaturecontrolled water bath (NTT-20S; Eyela, Tokyo, Japan), and the liquid sample in the reactor was agitated continuously with a magnetic stirrer. The temperature of the reactor was maintained at 37 °C throughout the experiment, and the pH of the sample was adjusted to 1.20 with 3 M HCl before starting the gastric stage (symbolized as ‘G’). The gastric stage was initiated by addition of pepsin solution (19 mL), and the pH was readjusted to 1.20 ± 0.01 with 0.5 M HCl. After 120 min of the gastric stage, the pH of the sample was adjusted to 6.80 using 3 N NaOH to inactivate the pepsin. The small intestinal stage (symbolized as ‘I’) was initiated by addition of intestinal enzyme solution (23 mL), and the pH was then readjusted to 6.80 ± 0.01 with 0.5 M NaOH. The sample was maintained at the intestinal conditions for 120 min. The digestion was immediately adjusted pH to 7.0 using 1 M NaOH or 1 M HCl, and the digestive enzyme reaction terminated by heating the samples to 95 °C for 10 min using a temperature-controlled water bath with a Thermominder (SJ-10R; Taitec, Saitama, Japan). The centrifuge tubes containing aliquots of digested samples were centrifuged at 4000g at 4 °C for 10 min (Model 2800; Kubota, Tokyo, Japan). The supernatant was separated, collected and lyophilized for use in further analysis. Five sample sets were collected separately from each reactor and labelled as follows: (1) F for fermented samples before digestion, (2) G1 for samples from gastric digestion for 1 h, (3) G2 for samples from gastric digestion for 2 h, (4) G2I1 for samples after gastric digestion for 2 h and intestinal digestion for 1 h, and (5) G2I2 for samples after gastric digestion for 2 h and intestinal digestion for 2 h (after digestion).
2.1. Materials and reagents Soybeans (Glycine max cv. Enrei) were obtained from a local market in Ibaraki prefecture, Japan. Pepsin from porcine gastric mucosa (EC 3.4.23.1, activity of 800–2500 U/mg protein), pancreatin from porcine pancreas (EC 232-468-9, 8 × USP specifications), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ) and sodium nitroprusside (SNP) were all purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), FeSO4, 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p′-disulfonic acid monosodium salt hydrate (FerroZine), FeCl2, TCA and all other chemicals of analytical grade were purchased from Wako Pure Chemical Corporation (Tokyo, Japan). Double-deionized water was used as needed. 2.2. Raw material preparation Dehulled yellow-seeded soybeans (300 g) were washed using tap water and soaked in distilled water (soybeans/water ratio of 1:3, w/v) for 18 h at 20 °C. Soaked soybeans were collected by discarding the soaking water. Subsequently, the soybeans were washed again with tap water, boiled in fresh distilled water at the same ratio in a pressure cooker for 90 min, cooled and superficially dried at room temperature. Boiled soybeans (150 g) were transferred into a glass beaker and inoculated with 50 mL of diluted culture from a commercial natto product produced by S-903 Bacillus spp. natto (Takanofoods Co., Ltd, Tokyo, Japan). After inoculation, the soybeans (37.5 g) were packed into a paper cup (205 mL), the top surface covered with polyvinylidene chloride wrap film, and incubated at 40 °C for 18 h.
2.5. Determination of total soluble nitrogen and protein content The total soluble nitrogen content was determined using a CN coder (MT-700 Mark 2; Yanako, Tokyo, Japan), based on the Dumas method principle by following the manufacturer’s instructions, using hippuric acid as the standard. The amount of protein present in the sample was calculated using the following formula:
2.3. Determination of moisture content The moisture content of samples (raw, soaked, boiled and fermented soybeans, and digested fractions from in vitro digestion before and after lyophilization) was determined by gravimetric determination according to AOAC (Horwitz & Latimer, 2000). An empty aluminium can and lid were dried in a hot-air dryer at 105 °C for 3 h and transferred to a desiccator to cool. The cooled empty can and lid were weighed. The sample (∼2 g) was put into the cooled aluminium can and dried at 105 °C for 16 h in a hot-air dryer (WFD-400; Eyela, Tokyo, Japan). After drying, the can and its dried sample were reweighed. The moisture content of the dried sample following each processing condition was expressed on a dry basis (d.b.), that is, as the dry matter mass in grams per 100 g of the total mass.
Protein content (%) = nitrogen (%) × factor
(1)
The in vitro digestibility was expressed as the percentage difference in protein content in the supernatant of the sample before digestion compared to that in the sample during digestion and after digestion. The degree of protein digestibility was calculated using the following formula:
Digestibility (%) = ( B
A)/ A× 100%
(2)
where A is the total protein content of the sample before digestion, and B is the residual protein content at each digestion stage. 2.6. TCA-soluble peptide yield (Ysp)
2.4. Simulated in vitro gastrointestinal digestion
Ysp was determined according to the method of Wang, Chi, Cheng, and Zhao (2018), with minor modifications. Each digested sample (100 μL) was added to 900 μL of 5% (w/w) TCA. Next, the mixture was kept for 1 h at 4 °C, and the unsolidified protein was then removed by centrifugation at 8000g for 10 min. The TCA-soluble peptide content in the supernatant was determined by measuring the absorbance at 280 nm using a UV–Visible spectrophotometer (V-630; Jasco, Tokyo, Japan). The content of TCA-soluble peptides was calculated as micromoles of tyrosine per gram of sample. Ysp was calculated using the following formula:
The static in vitro gastrointestinal digestion model described by Dartois, Singh, Kaur, and Singh (2010) that modified by Tamura, Singh, Kaur, and Ogawa (2016) was used, with minor adjustment. This step’s experiments were carried out in duplicate. Briefly, for the preparation of pepsin solution, 0.24 g pepsin from porcine gastric mucosa (activity of 800–2500 U/mg protein; Sigma-Aldrich, St. Louis, MO, USA) was dispersed in 50 mL gastric fluid buffer (adjusted to pH 1.20). The mixture was then mixed with a magnetic stirrer (Color Squid White, Ika Works, Wilmington, NC, USA) for 10 min. For the preparation of intestinal enzyme solution, 0.2 g pancreatin from porcine pancreas (Sigma-Aldrich), 0.015 g invertase and 4 mL amyloglucosidase (Megazyme, Co. Wicklow, Ireland) were prepared in the same manner as for the previous step but using 25 mL intestinal fluid buffer (adjusted to pH 6.80). The soybeans (15% protein, wet weight) (around 150 g) were blended with gastric buffer for 5 min at the highest speed and subsequently homogenized with a homogenizer (The Virtis Company Inc., Gardiner, NY) at 10,000g for 5 min, then added to a glass reactor before
Ysp (%) = (A/B) × 100%
(3)
where A is the peptide content of the supernatant, and B is the total protein content of the sample before digestion. 2.7. Soluble protein fractions and distribution by electrophoretic analysis The protein content of supernatants collected at different stages of 641
Journal of Functional Foods 52 (2019) 640–647
S. Ketnawa, Y. Ogawa
the simulated digestion was determined by the biuret method (Gornall, Bardawill, & David, 1949) using bovine serum albumin (BSA) as a standard. The samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to determine the protein patterns by using NuPAGE® Bis-Tris gradient precast gel (4–12% gradient, 10 × 10 cm2) in a Novex XCell Mini-Cell (Invitrogen, Thermo Scientific, Rockford, IL, USA). NuPAGE® MES SDS Running Buffer was used as the electrophoresis running buffer. According to the difference in protein content of a sample solution, the protein solution was diluted with deionized water to be the same concentration, and then mixed with 12.5 µL NuPAGE® LDS Sample Buffer and 5 µL NuPAGE® Reducing Agent. The mixture was heated at 70 °C for 10 min. The samples (all containing 20 μg of protein per well) and the protein standard markers (Thermo Scientific, Rockford, IL, USA) were loaded onto the gel. Electrophoresis was performed at 200 V for 35 min. Proteins were visualized by staining with SimplyBlue™ SafeStain (Thermo Scientific, Rockford, IL, USA) overnight then de-staining with distilled water for 1 h. The water used for washing was changed every hour until the background was clear, at which point gels were then dried.
method of Ketnawa et al. (2017), with slight modification. ABTS radicals (ABTS%+) were produced in a reaction of 7 mM ABTS dissolved in 2.45 mM potassium persulfate, allowing the mixture to react in the dark at room temperature for 12 h before use. The ABTS+ solution was diluted with distilled water to obtain an absorbance of 0.7xx at 734 nm. To initiate the reaction, 0.02 mL of the sample was mixed with 0.98 mL of diluted ABTS%+ solution. The extent to which the ABTS%+ was quenched was measured at 734 nm after 10 min incubation at 30 °C in the dark. An ascorbic acid standard curve (0–0.1 mg/mL) was prepared. Distilled water was used instead of the sample and prepared in the same manner to obtain the control. ABTS+ radical scavenging activity was expressed as milligrams of ascorbic acid equivalent (AA) per gram of protein (dry matter). 2.12. Ferric reducing antioxidant power (FRAP) activity The capacity of digested fractions to reduce ferric–tripyridyltriazine complex was evaluated by the FRAP assay, as described by Ketnawa et al. (2017), with a slight modification. Freshly prepared FRAP reagent (10 mM TPTZ solution in 40 mM HCl plus 20 mM FeCl3·6H2O solution and 300 mM acetate buffer, pH 3.6, at a ratio of 1:1:10 (v/v/v)) (1.30 mL) was incubated at 37 °C before being mixed with 0.2 mL of the sample. The mixture was incubated at the same temperature for 30 min in the dark. Absorbance at 595 nm was recorded after 30 min of reaction. The control was prepared in the same manner as previously described, with distilled water instead of the sample. FRAP was calculated from the FeSO4 standard curve (0–100 μmol/L) and expressed as micromoles of FeSO4 equivalent per gram of protein (dry matter).
2.8. Free amino acid analysis Free amino acid content was analysed using an automatic amino acid analyser (JLC-500/V2 equipped with an ion exchange column; Jeol, Tokyo, Japan). In summary, freeze-dried powder of each sample was dissolved in distilled water (20 mg/mL protein content), then a 0.2 mL aliquot was added to 1.8 mL of 5% (w/v) TCA solution, mixed and centrifuged at 10000g for 10 min at 10 °C. The supernatant was diluted to an appropriate range, adjusted to pH 2 to 3 by 0.02 N HCl and filtrated through a 0.45-µM filter (polytetrafluoroethylene (PTFE), Advantec® Dismic®-13HP; Toyo Roshi Kaisha, Ltd, Chiba, Japan). Finally, the collected supernatant was subjected to an amino acid analyser, and open Type AN-II and Type B (Wako Pure Chemical, Ltd, Osaka, Japan) were used as reference amino acid standards. Free amino acids were separated in high separation mode (110 min) with a lithium citrate buffer system and measured by photometric detection after derivatization with ninhydrin.
2.13. Metal chelating activity (MIC) The chelating activity on Fe2+ was measured using the method of Ketnawa et al. (2017), with a slight modification. Diluted sample (0.8 mL) was mixed with 2 mM FeSO4 (0.01 mL) and 5 mM FerroZine® (0.02 mL). The reaction mixture was allowed to stand for 10 min at room temperature. The absorbance was then measured at 562 nm. The blank was prepared in the same manner but distilled water was used instead of the sample. A standard curve for EDTA (0–10 mmol/L) was prepared and used as the positive control. Ferrous chelating activity was expressed as micromoles of EDTA equivalent per gram of protein (dry matter).
2.9. Total phenolic content (TPC) TPC was determined according to the method of Donlao and Ogawa (2018). Briefly, 0.2 mL of the test solution was mixed with 1 mL of 10% (v/v) Folin–Ciocalteu solution and 0.8 mL of 7.5% (w/v) sodium carbonate solution. The mixture was incubated for 1 h at room temperature, and absorbance was then measured using a UV–Vis spectrophotometer (V-630; Jasco, Tokyo, Japan) at 765 nm; distilled water was used as a blank. TPC was reported as the milligrams of gallic acid equivalent (GAE) per gram of protein (dry matter).
2.14. Determination of nitric oxide (NO) inhibition activity The compound sodium nitroprusside (SNP) is known to decompose in aqueous solution at physiological pH (7.2), producing NO radicals (NO%). Under aerobic conditions, NO% reacts with oxygen to produce stable products (nitrate and nitrite), the quantities of which can be determined using Griess reagent. The scavenging effect of the plant extract on NO was measured according to the modified method of Marcocci, Maguire, Droylefaix, and Packer (1994). One millilitre of sample at different concentrations (0–1000 µg/mL protein) was added to test tubes containing 1 mL of SNP solution (100 mM), and the tubes were incubated at 29 °C for 2.5 h. An aliquot of 1 mL of the incubation solution was removed and diluted with 1 mL of Griess reagent by mixing equal amounts of 1% sulfanilamide and 0.1% N-1-naphthylethylenediamine dihydrochloride in 2% H3PO4. The absorbance of the chromophore (purple azo dye) that formed during the diazotization of nitrite ions with sulfanilamide and subsequent coupling with naphthylethylenediamine dihydrochloride was immediately read at 540 nm. The percentage nitrite radical scavenging activity of the sample and gallic acid (0–1000 µg/mL) was calculated using the following formula:
2.10. DPPH radical scavenging activity The DPPH radical scavenging activity of the sample was determined according to the method described by Ketnawa, Benjakul, MartínezAlvarez, and Rawdkuen (2017). Sample solution (0.5 mL) was added to 0.1 mM DPPH in 95% ethanol (0.5 mL). The mixture was vigorously mixed and allowed to stand for 30 min in the dark at room temperature. The absorbance of the resulting solution was measured at 517 nm. The DPPH blank is the value for 0.5 mL of 95% ethanol mixed with 0.5 mL of 0.1 mM DPPH in 95% ethanol. The DPPH radical scavenging activity was calculated by a Trolox standard curve (0–1000 μmol/L) and expressed as micromoles of Trolox equivalent (TE) per gram of protein (dry matter). 2.11. ABTS radical scavenging activity
NO inhibition activity (%) =
ABTS radical scavenging activity was determined according to the 642
Acontrol
Asample
Acontrol
× 100
(4)
Journal of Functional Foods 52 (2019) 640–647
S. Ketnawa, Y. Ogawa
where Acontrol is the absorbance of the control without the tested samples, and Asample is absorbance in the presence of the samples or standards. 2.15. Inhibition of egg albumin denaturation In vitro anti-inflammatory activity of digested proteins during simulated digestion was evaluated with the protein denaturation method of Mizushima and Kobayashi (1968) as modified by Osman, Sidik, Awal, Adam, and Rezali (2016). Diclofenac sodium, a powerful nonsteroidal anti-inflammatory drug, was used as a standard. The reaction mixture consisting of 1 mL of different concentrations of protein isolate (125–500 μg/mL) or standard diclofenac sodium at a final concentration of 125–1000 µg/mL and 1.4 mL of phosphate-buffered saline (pH 6.4) was mixed with 0.2 mL of egg albumin (from fresh hen egg) and incubated at 37 °C for 15 min. Denaturation was induced by keeping the reaction mixture at 57 °C in a water bath for 20 min. After cooling, the absorbance was measured at 660 nm using distilled water as a blank. Increments in the absorbance of test samples with respect to the control indicated stabilization of the protein, i.e. inhibition of heat-induced protein (albumin) denaturation by the test sample and reference drug diclofenac sodium. Each experiment was done in triplicate, and the average was taken. The percentage inhibition of protein denaturation was calculated using following formula:
Inhibition(%) = 1
Asample A control
× 100
Fig. 1. Changes in protein content, nitrogen content, trichloroacetic acid (TCA)-soluble peptide yield and protein digestibility of the fermented soybeans during stimulated in vitro digestion, calculated and reported as percentages (%). Bars represent the standard deviation from triplicate determinations. Significant differences were found in all digestion stage compared to before digestion (F). Non-significant differences are highlighted with an asterisk as a function of digestion stage (P > 0.05).
compounds contained in the soybean samples are also known to inhibit protein absorption. This has been mainly described for high concentrations of tannins that nonspecifically bind and precipitate proteins and therefore, are considered as anti-nutritional compounds (MartinezGonzalez et al., 2017). Previous studies have shown that fermentation of soybeans using several Bacillus spp. results in major biochemical changes in the substrate, leading to an increase in soluble material and material dialysable into water-soluble low molecular weight peptides, oligosaccharides and monosaccharides (Sanjukta, Rai, Muhammed, Jeyaram, & Talukdar, 2015; Weng & Chen, 2010, 2011). It was shown that Bacillus spp. are able to degrade soybean macromolecules to a large extent, resulting in water-soluble low molecular weight compounds as observed by the difference between protein patterns in boiled soybeans (B, lane 3) and fermented soybeans (F, lane 4), as shown in Fig. 2 corresponding to the higher protein digestibility number in Fig. 1. In addition, simulated digestion of fermented soybeans using digestive enzymes also increased the amount of dialysable matter, which clearly demonstrated the beneficial effect of Bacillus fermentation on food nutrient availability in stimulated digestion (Sanjukta et al., 2015). The increase in protein digestion improved the nutritional quality of the soybean proteins by favouring hydrolysis and producing short-chain peptides, which is essential to human metabolism as can be seen from the amino acid composition in Table 1. The progress of hydrolysis was also confirmed by the determination of TCA-soluble peptide content and is shown in Fig. 1. TCA-soluble protein means the amount of digestible soybean protein that dissolves in TCA; it measures the portion of protein soluble in TCA which contains polypeptides of fewer than 10 amino acids and free amino acids whereas nondegraded proteins are precipitated by TCA (Chen, Chiou, & Yu, 2010). TCA-soluble protein content after digestion (59.83%; G2I2) was significantly higher than before digestion (4%; F) (P < 0.05). The higher Ysp is due to the large amount of soluble peptides initially present in the fermented soybeans in cooperation with hydrolysis by digestive enzymes (Chen et al., 2013). The change in TCA-soluble peptides during digestion shows that the protein of fermented soybeans was digested and that protein utilization was significantly improved. Previous research has also reported an increase in TCA-soluble peptides in fermented soybeans compared to raw material or protein flour (Weng & Chen, 2010). The results suggest that digested fermented soybeans can improve the absorption of oligopeptides through simulated digestion.
(5)
where Acontrol is the absorbance of the control without the tested samples, and Asample is absorbance in the presence of the samples or standards. 2.16. Statistical analysis Simulated digestion was done in duplicate for fermented samples. The other experiments were carried out in triplicate. Data were subjected to analysis of variance, and mean comparisons were performed using Duncan’s multiple range test. Statistical analysis was carried out using the Statistical Package for the Social Sciences (SPSS for Windows, SPSS, Inc., Chicago, IL). Differences were considered to be significant at P < 0.05. 3. Results and discussion 3.1. Parameters for in vitro protein digestibility Four parameters were used to evaluate protein digestibility during a two-stage (1–2 h of gastric enzymes, pepsin, pH 1.2, 37 °C: G1 and G2, followed by 1–2 h of intestinal enzymes, a mixture of pancreatin, invertase and α-amyloglucosidase, pH 6.8, 37 °C: G2I1 and G2I2) simulated gastro-small intestinal digestion compared to that in the fermented soybean samples before digestion (F). TCA-soluble peptides, total soluble nitrogen, SDS-PAGE profile and amino acid content were determined. Fig. 1 shows the variability in protein and nitrogen content, in vitro protein digestibility (expressed as the percentage protein content compared to that in F) and Ysp. Protein and nitrogen content increased approximately 1.52-fold during digestion. Protein digestibility for G1 was 30.86%, and increased to 47.29% (G2), 49.19% (G2I1) and 48.99% (G2I2). So, digestibility increased around 1.59-fold (Fig. 1). This increase shows the ability of digestive enzymes in the gastric digestion stage to digest protein in the fermented samples. The digestibility in the intestinal stage was quite stable may be due to trypsin and α-chymotrypsin inhibitors present in the samples (Chen, Zhao, & Sun, 2013). Weng and Chen (2011) and Weng and Chen (2010) reported levels of trypsin and α-chymotrypsin inhibitors of 46 ± 6 and 2.7 ± 1.6 IU/10 g natto, respectively. Moreover, some phenolic 643
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3.2. Soluble protein fractions and distribution by SDS-PAGE The soluble protein distribution profile of different stages of simulated digestion is presented by Bis-Tris-SDS-PAGE analysis (Fig. 2). The SDS-PAGE technique has originally been used to study soybean protein subfractions and to evaluate the degradation rate of individual digested fractions. The protein subfractions were divided into three parts according to molecular weight. The protein bands of 55 kDa and above were grouped as large-size fractions, 16–55 kDa as medium-size fractions, and smaller than 16 kDa as small-size fractions. Fig. 2 shows a typical protein profile of several polypeptides ranging from 140 to < 10 kDa. Soaked soybeans, S (lane 2, Fig. 2), contained several major storage proteins including α, α′ and β subunits of β-conglycinin and acidic and basic subunits of glycinin, prominent among which were 90, 67, 63, 48, 40, 36, 30 and 15 kDa proteins probably corresponding to α, α′ and β subunits of β-conglycinin (67–48 kDa) and acidic and basic subunits of glycinin (38.5–20.5 kDa). In the electrophoretic patterns for the boiled soybeans, B (lane 3, Fig. 2), a total of seven major protein bands having molecular weights around 120, 75, 70, 55, 40, 36, 25, 17 and 15 kDa were observed. These results agree with previous studies of raw soybean protein profiles (González-Montoya, Hernández-Ledesma, Silván, Mora-Escobedo, & Martínez-Villaluenga, 2018; Liu, Zhou, Tian, & Gai, 2007; Mujoo, Trinh, & Ng, 2003). During pepsin digestion (G1–G2), the intensities of the protein bands corresponding to 7S and 11S fractions were decreased, and many peptide bands appeared at 11–15 kDa, indicating protein hydrolysis. Enzymatic hydrolysis led to gradual breakdown of these two subunits of storage proteins into several subunits, in a hydrolysis time-dependent manner. Enzymatic hydrolysis produced a loss of α′-, α and β-7S subunits, and total disappearance of bands corresponding to these subunits of 7S. Simulated digestion of fermented soybeans caused complete degradation of polypeptides > 20 kDa and increased the abundance of oligopeptides with molecular weight < 10 kDa (G2I2; lane 8, Fig. 2). After simulated digestion, the ratio of small protein fractions in G2I2 was increased. These results show that some peptides with high molecular weight were degraded during simulated digestion, and peptides with low molecular weight were formed. This also indicates that pepsin broke down the intra-chain peptide bonds and mainly produced relatively larger peptide fragments. After subsequent intestinal digestion with pancreatin, the larger peptide fractions were further degraded into oligopeptides and amino acids. Zhang et al. (2018), from their study on simulated digestion of Alcalase–soybean protein hydrolysates, reported that after the first 2 h of gastric digestion with pepsin, the larger fraction with molecular weight > 10 kDa was significantly reduced (P < 0.05), while the smaller fractions with molecular weight of 5–10 kDa and 1–5 kDa were significantly increased (P < 0.05). The evidence from this study showed mainly that proteins are digested to smaller molecular weight sizes by digestive enzymes, which could be an important contributing factor to good bioavailability and benefits to human health.
Fig. 2. Change in sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) electrophoretograms for soybean protein at various process and fermented soybean protein at different digestion stages. Upper left is the original gel image, and right is the image of a portion extracted from the original for easier comparison of each lane. Lane 1 (MK) for standard molecular markers, lane 2 (S) for soaked soybeans, lane 3 (B) for boiled soybeans, lane 4 (F) for fermented soybeans, lane 5 (G1) for the fraction at the gastric digestion stage for 1 h, lane 6 (G2) for the fraction at the gastric digestion stage for 2 h, lane 7 (G2I1) for the fraction at the gastric digestion stage for 2 h and intestinal digestion stage for 1 h, and lane 8 (G2I2) for the fraction at the gastric digestion stage for 2 h and intestinal digestion stage for 2 h.
Table 1 Changes in free amino acid content in fermented soybeans during simulated in vitro digestion. Amino acid
Thr Ser Asp Glu Gly Ala Val Met Cysta*** Ile Leu Tyr Phe His Lys Arg Pro Total
Increment (fold)*
Total increment (fold)**
Before digestion
After digestion
3.32 ± 0.41 3.61 ± 0.49 3.07 ± 0.30 6.10 ± 0.61 8.99 ± 0.80 2.02 ± 0.20 2.61 ± 0.30 10.49 ± 1.19 2.44 ± 1.06 16.14 ± 3.35 39.52 ± 2.84 21.48 ± 2.22 37.96 ± 0.54 6.80 ± 0.78 18.54 ± 1.82 0.30 ± 0.02 2.56 ± 0.00 5.96 ± 0.43
8.02 ± 1.07 7.67 ± 0.75 7.13 ± 1.54 10.15 ± 1.20 15.35 ± 1.65 3.68 ± 0.40 4.79 ± 0.59 24.46 ± 2.62 4.54 ± 0.28 36.79 ± 4.05 144.13 ± 0.15 83.52 ± 0.35 125.48 ± 0.54 10.80 ± 1.14 72.78 ± 8.84 14.84 ± 1.76 2.46 ± 0.00 21.10 ± 2.39
2.49 ± 0.63 2.19 ± 0.51 2.40 ± 0.73 1.70 ± 0.37 1.74 ± 0.34 1.86 ± 0.39 1.88 ± 0.44 2.39 ± 0.52 2.36 ± 1.14 2.44 ± 0.76 3.64 ± 0.13 3.98 ± 0.85 3.30 ± 0.31 1.63 ± 0.36 4.01 ± 0.85 49.43 ± 2.86 0.96 ± 0.00 3.59 ± 0.66
3.3. TPC Changes in TPC for different stages during simulated digestion of fermented soybeans are shown in Fig. 1. A significant increment was found for digested fractions. The level of free phenolics in unfermented soybeans was 21.16 ± 0.31 mg GAE/g protein, and increased dramatically to 10.41–25.65 mg GAE/g protein in the digested fractions (Fig. 3). This may be due to the digestive enzymes playing a role in exposing water-soluble polyphenols from the structure because more water-soluble compounds were free. The effectiveness of polyphenols is related to their antioxidant potential. The increase in free phenolic acids in the fermented soybeans is probably due to β-glucosidase production during fermentation (Sanjukta et al., 2015). An increase in free phenolics is expected to improve their bioavailability in the intestine, which will further improve the beneficial effects associated with them.
* The increment of amino acids was calculated by dividing the quantity (nmol/mL) of each individual amino acid by the concentration of each amino acid in soaked soybeans (data not shown). Values are expressed as increment folds and are means of duplicate measurements. ** The increment of amino acids was calculated by dividing the quantity (nmol/ml) of each individual amino acid by the concentration of each amino acid in fermented soybeans before and after digestion. Values are expressed as increment folds and are means of duplicate measurements. *** Cysteine (Cys) was determined in the form of cystathionine (Cysta). Cysta is a dipeptide formed by serine and homocysteine; the trans-sulfuration of methionine yields homocysteine which combines with serine to form cystathionine. Tryptophan (Trp) cannot be reported because it is unstable and produces ammonia, so it was not contained in the amino acid standard solution. 644
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(1.72 ± 0.02 mg AA/g protein) compared to F (0.84 ± 0.03 mg AA/g protein). When the pepsin digest was hydrolysed with pancreatin, additional peptide-bond cleavage led to the accumulation of shorter peptides (tri- and di-peptides) and free amino acids, thus becoming more hydrophilic. Digests with increased polarity (amino acids, small peptides) can readily react with water-soluble ABTS%+ (Zhu, Chen, Tang, & Xiong, 2008). The conceivable structural changes resulting from pepsin digestion may also favour trapping of free radicals like DPPH and ABTS, thus further enhancing quenching by the sample digest. Furthermore, reducing power (FRAP) continuously increased during simulated digestion, up to 1.72-fold for the gastric stage (18.80 ± 0.18 µmol FeSO4/g protein) and 1.93-fold for G2I1 and G2I2 (21.10 ± 0.05 µmol FeSO4/g protein) (P < 0.05). This increase in the reducing power of digested samples shows that the proteins of fermented soybeans can be more effective hydrogen or electron donors after simulated digestion (Fig. 3). The reducing power of fermented soybeans increased with increasing digestion time (0–4 hrs). Besides that, increasing MIC activity during the simulated digestion was also observed; the value was increased approximately 1.98-fold for the gastric stage (5.47 ± 0.02 µmol EDTA)/g protein) and 2.21-fold for the intestinal stage (6.10 ± 0.03 µmol EDTA)/g protein) (P < 0.05). The activity changes for DPPH, FRAP, Fe2+ chelation and ABTS%+ scavenging followed a similar trend during simulated digestion, suggesting that the two might be related. It is possible that treatment with pepsin disrupted the spatial structure of fermented soybean peptides, conducive to binding and trapping of Fe2+ and ABTS%+ and resulting in reduced chelation capability. On the other hand, as high-affinity metal-binding groups became fully exposed or newly formed by pancreatic digestion, for example, the imidazole and carboxylic groups and electrostatic and ionic interactions with Fe2+ were probably enforced. So, digested protein of fermented soybeans showed capability to bind metal ions. Sanjukta et al. (2015) reported that soybeans fermented with B. subtilis MTCC5480 showed a decrease in antioxidant activity on pepsin digestion, followed by an increase in activity on pancreatin digestion. The results clearly indicate that the protein of fermented soybeans should be resistant to gastrointestinal digestion to reach the target tissue in an active form. In addition, the digested fraction of fermented soybeans after the digestion stage (G2I2) showed the highest activity because the resulting short-chain peptides and smaller sizes led to higher activity than for the parental proteins, containing peptides, and are electron donors and can react with free radicals to convert them into more stable products and terminate the radical chain reaction (Aluko, 2015). From Fig. 2, it can be seen that the smaller sizes presented in SDS-PAGE may support the antioxidant activity. In the present study, in the final digest of fermented soybeans (G2I2), which exhibited the highest overall antioxidant potential, larger molecular weight fractions disappeared.
Fig. 3. Total phenolic content (TPC) expressed as “mg of gallic acid equivalents/g protein”, DPPH radical scavenging capacity (DPPH) expressed as “μmol Trolox equivalents/g protein”, ABTS radical scavenging capacity (ABTS) expressed as “mg of ascorbic acid equivalents/g protein”, ferric reducing antioxidant power activity (FRAP) expressed as “mmol FeSO4·7H2O equivalents/g protein”, and metal ion chelating activity (MIC) expressed as “μmol EDTA equivalents/g protein” of the fermented soybeans at each digestion stage. F represents fermented soybeans before digestion, G1 represents the gastric digestion stage for 1 h, G2 represents the gastric digestion stage for 2 h, G2I1 represents the gastric digestion stage for 2 h and intestinal digestion stage for 1 h, and G2I2 represents the gastric digestion stage for 2 h and intestinal digestion stage for 2 h. Bars represent the standard deviation from triplicate determinations. Significant differences were found in all digestion stage compared to before digestion (F). Non-significant differences are highlighted with an asterisk as a function of digestion stage (P > 0.05).
Many researchers have suggested that the increase in antioxidant activity is due to an increase in free polyphenols. Watanabe, Fujimoto, and Aoki (2007) suggested that antioxidant activity in the water-soluble fraction of Rhizopus-fermented tempeh may be due to the amino acids and peptides formed during fermentation. 3.4. Antioxidant activity The antioxidant activity of protein foods or hydrolysed proteins and peptides has been well documented in the literature, but relatively few studies have been conducted to evaluate their activity and fate in the human digestive system. Because of the unique peptide-bond specificity of digestive proteases, the products from pepsin and pancreatin (trypsin and chymotrypsin) digestion will depend on the characteristics of the peptides ingested. Antioxidant activity is enhanced, contributed by the increase of short-chain peptides and phenolic compounds (Möller, Scholz-Ahrens, Roos, & Schrezenmeir, 2008). The antioxidant activity of fermented soybeans came from both phenolic compounds and bioactive peptides. From preliminary tests for the digestion of raw soybeans and boiled soybeans, TPC not only plays a part in antioxidant properties but peptides released during digestion also improve antioxidant activity (Fig. 3). In addition, Sanjukta et al. (2015) suggested that antioxidant activity in the water-soluble fraction of B. subtilis MTCC5480-fermented soybeans is due to the amino acids and peptides formed during fermentation as well as antioxidant peptides that are released from gastrointestinal digestion. The increment in DPPH, ABTS, FRAP and MIC was 2.85-, 4.32-, 1.93- and 2.21-fold, respectively. The biggest change (4.32-fold) was observed for ABTS. Strong ABTS%+ scavenging activity for the watersoluble ABTS%+ free radicals, expressed as L-ascorbic acid equivalent antioxidant capacity (AA), was demonstrated by digested samples (Fig. 3). Following gastric (or pepsin) digestion (G1 and G2), ABTS increased (P < 0.05) around 2.05-fold in the gastric stage
3.5. Changes in free amino acid composition Pepsin plays an important role in the primary digestion of food proteins, resulting in long-chain peptides, followed by secondary digestion by intestinal enzymes, resulting in the formation of short-chain peptides (Goodman, 2010). The nutritional quality of a protein is primarily related to its amino acid composition. In addition, amino acid availability is also a key factor in protein quality. Bioavailability depends on the process of digestion, which may be affected by many factors such as the characteristics of the protein itself. Fermented soybeans and their digested fractions were analysed for their free amino acid profile, responsible for nutritional aspects and bioactive properties, divided by groups of amino acids. The change in free amino acid profile was reported in terms of the free amino acid increment between F and G2I2 and is shown in Tables 1 and 2. When compared to that for the raw material (raw soaked soybeans, S), the total free amino acid composition of fermented soybeans was 645
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Table 2 Increment of selected amino acid groups of fermented soybeans during simulated in vitro gastrointestinal digestion. Amino acid group
Increment (fold)*
EAA HAA AAA AXA
4.13 3.01 3.20 2.99
± ± ± ±
Table 3 In vitro anti-inflammatory activity of fermented soybeans at different stages of simulated in vitro digestion.
0.47a 0.33b 0.35b 0.32b
EAA = essential amino acids: Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Try and Val; HAA = hydrophobic amino acids: Ala, Val, Ile, Leu, Tyr, Phe, Trp, Pro, Met and Cys; AAA = aromatic amino acids: Phe, Trp, Tyr and His; AXA = antioxidant amino acids: Trp, Tyr, Met, Cys, His, Phe and Pro. * The increment of amino acids was calculated by dividing the quantity (nmol/ml) of each individual amino acid by the concentration of each amino acid in fermented soybeans before and after simulated digestion. Values are expressed as increment folds and are means of duplicate measurements. Different letters in the same column indicate a significant difference (P < 0.05) among different amino acid groups.
Digestion stage
Inhibition of nitric oxide production (%)
Inhibition of egg albumin denaturation (%)
F G1 G2 G2I1 G2I2
9.01 ± 0.29e 13.66 ± 0.62d 17.41 ± 0.66c 23.58 ± 0.87a 28.54 ± 0.38a
73.09 73.53 67.47 57.81 48.08
± ± ± ± ±
1.69a 1.39a 0.00b 1.28c 1.15d
Different small letters in the same column indicate a significant difference (P < 0.05) among different digestion stages. Digestion stage captions are described in Fig. 3.
(Udenigwe, Je, Cho, & Yada, 2013). The assay is based on the principle that sodium nitroprusside (SNP) in aqueous solution at physiological pH spontaneously generates NO which interacts with oxygen to produce nitrite ions that can be estimated using Griess reagent. NO released from SNP has a strong NO character which can alter the structure and function of many cellular components. Scavengers of N compete with oxygen, leading to reduced production of nitrite ions (GonzálezMontoya et al., 2018). In this way, the digested fraction of fermented soybeans at different stages showed anti-inflammatory activity by inhibiting NO production, reported as a percentage and shown in Table 3. The inhibition increased with an increase in digestion time. Before digestion, F showed inhibition of NO production of 9.01 ± 0.29%; inhibition activity gradually increased up to 28.54 ± 0.38% in G2I2. From the results, we assume that the peptides released during gastrointestinal digestion of fermented soybean proteins have potential bioactivity against inflammation. Another in vitro anti-inflammatory effect of digested proteins of fermented soybeans was evaluated: denaturation of bovine/egg albumin. Denaturation of proteins is a well-documented cause of inflammation. The present findings were that there was a slight decrease in the inhibition of egg albumin denaturation during digestion and are summarized in Table 3. At the protein concentration used of 1.25 mg/ mL, the inhibition of egg albumin denaturation decreased from 73.09 ± 1.69% to 48.08 ± 1.15% before and after digestion, respectively. Non-steroidal anti-inflammatory drugs have the ability to stabilize (prevent denaturation) heat-treated albumin at physiological pH (6.2–6.5). Meanwhile, the non-steroidal anti-inflammatory drug (NSAID) used, diclofenac sodium, exhibited a relatively higher inhibition capacity of 4.71 ± 0.06% at the same test concentration (1.25 mg/mL). It is suggested that the regular intake of soymilk or fermented soymilk may provide protection against cardiovascular diseases and chronic inflammatory diseases in LPS-induced macrophages (Liao et al., 2010). However, not only proteins can play an anti-inflammatory role: the major phytochemicals (from high to low abundance) in soybeans, phytic acid (1.0–2.2%), sterols (0.23–0.46%), saponins (0.17–6.16%), isoflavones (0.1–0.3%) and lignans (0.02%), also show a strong anti-inflammatory effect and display a wide range of other bioactivities, including anti-cancer, antioxidative, anti-viral, cardiovascular protective and hepatoprotective actions (Sanjukta & Rai, 2016). Another point to consider is that a decrease in in vitro anti-inflammatory ability depends on molecular weight as well; higher antiinflammatory potential of peptides with molecular weight from 5 to 10 kDa was observed in germinated soybean peptides by GonzálezMontoya et al. (2018). Udenigwe et al. (2013) also reported that a high molecular weight almond protein fraction (> 5 kDa) showed the best inhibitory activity for NO production.
higher, 5.96-fold and 21.10-fold before and after simulated digestion, respectively. The content of all other amino acids also increased; the highest increment was observed for the fermented soybeans after simulated digestion (Table 1). When comparing F and G2I2 (after simulated digestion), the total increment was 3.59-fold. Most represented amino acids were increased significantly, around 1.5- to 3.5-fold. Table 2 shows the changes in free amino acid content by different groups. The maximum change was found for Arg (49.43-fold) while Pro was found to slightly decrease (0.95-fold). Considering amino acids divided by group, we found the maximum change (4.59-fold) in the essential amino acid group (EAA: Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Try and Val). As mentioned, Arg was increased the most, while His was increased the least. This could imply that simulated digestion improves the generation of EAA. Apart from EAA, we also found an increment in other groups of amino acids, for example, hydrophobic amino acids (HAA: Ala, Val, Ile, Leu, Tyr, Phe, Trp, Pro, Met and Cys), aromatic amino acids (AAA: Phe, Trp, Tyr and His) and antioxidant amino acids (AXA: Trp, Tyr, Met, Cys, His, Phe and Pro), of around 3-fold. Pepsin is most effective at cleaving peptide bonds between HAA and preferably AAA such as Phe, Trp and Try. Pancreatin exhibits the activity of trypsin (cleavage of peptide bonds at Arg and Lys sites), chymotrypsin (cleavage of peptide bonds at Phe, Trp, Tyr and Leu sites) and elastase (cleavage of peptide bonds at Ala and other aliphatic amino acids) (Udenigwe & Aluko, 2012). Beyond its effect on nutritional quality, the increase in free amino acid content is responsible for the remarkable increase in antioxidant properties shown in Fig. 1. The results for amino acid content showed the same trend as in a study of the amino acid content of soybean meal fermented with B. subtilis; Glu, Val, Tyr, Leu and Phe were higher (Song, Frias, Martínez-Villaluenga, VidalValdeverde, & de Mejia, 2008). From the increase in amino acids in this study, we infer that the digested fraction of fermented soybeans could increase the nutritional value and physiological functions in humans. 3.6. In vitro anti-inflammatory activity In vitro anti-inflammatory activity was evaluated using NO production inhibition and albumin denaturation assays. NO is a signalling molecule that plays a key role in the pathogenesis of inflammation. It gives an anti-inflammatory effect under normal physiological conditions. On the other hand, NO is considered as a pro-inflammatory mediator that induces inflammation due to overproduction in abnormal situations (Sharma, Al-Omran, & Parvathy, 2007). Various inflammatory diseases are related to the overproduction of NO
4. Conclusions Fermented soybeans digested through simulated in vitro gastrointestinal digestion had improved protein digestibility, leading to an 646
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improvement in protein utilization. Besides that, antioxidant properties and NO inhibition improved after simulated digestion, due to leaching out of bioactive peptides during digestion. These activities of fermented soybeans were retained or improved following stepwise enzyme digestion simulating the human digestive tract. The concentration of free amino acids as well as of polyphenols appears to collectively contribute to higher nutritional value by increment of EAA and the strong bioactivity of the protein digests. Therefore, the benefits of fermented soybean, ‘natto’, could be extended beyond its role as a good nutritional and digestible food. Our results suggest that the Bacillus fermentation process could be a potential functional processing method with good bioavailability characteristics due to antioxidant and anti-inflammatory activity.
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5. Ethics statements Our research did not include any human subjects and animal experiments. Acknowledgements The authors are grateful for the JSPS Postdoctoral Fellowship for Research in Japan awarded to Dr. Sunantha Ketnawa. We would like to thank The Tojuro Iijima Foundation for Food Science and Technology. We would also like to thank Assoc. Prof. Dr. Terumasa Takahashi, Graduate School of Horticulture, Chiba University, and Dr. Tamaki Hirose, Research Facility Centre for Science and Technology, University of Tsukuba, for technical support for measurement of crude protein content and free amino acid analysis, respectively. Conflicts of interest The authors declare no conflict of interest. The project was funded by JSPS and The Tojuro Iijima Foundation for Food Science and Technology, but they had no role in study design, data collection or analysis. The authors alone are responsible for the content and writing of the paper. References Almeida, C. C., Monteiro, M. L. G., Costa-Lima, B. R. C. D., Alvares, T. S., & Conte-Junior, C. A. (2015). In vitro digestibility of commercial whey protein supplements. LWT Food Science and Technology, 61(1), 7–11. https://doi.org/10.1016/j.lwt.2014.11.038. Aluko, R. E. (2015). Amino acids, peptides, and proteins as antioxidants for food preservation. In F. Shahidi (Ed.). Handbook of antioxidants for food preservation (pp. 105– 140). Amsterdam: Woodhead Publishing. Chen, C., Chiou, P., & Yu, B. (2010). Evaluating nutritional quality of single stage- and two stage-fermented soybean meal. Asian-Australasian Journal of Animal Sciences, 23(5), 598. Chen, N., Zhao, M., & Sun, W. (2013). Effect of protein oxidation on the in vitro digestibility of soy protein isolate. Food Chemistry, 141(3), 3224–3229. https://doi.org/10. 1016/j.foodchem.2013.05.113. Dartois, A., Singh, J., Kaur, L., & Singh, H. (2010). Influence of Guar Gum on the in vitro starch digestibility-rheological and microstructural characteristics. Food Biophysics, 5(3), 149–160. https://doi.org/10.1007/s11483-010-9155-2. Donlao, N., & Ogawa, Y. (2018). Impacts of processing conditions on digestive recovery of polyphenolic compounds and stability of the antioxidant activity of green tea infusion during in vitro gastrointestinal digestion. LWT Food Science and Technology, 89, 648–656. https://doi.org/10.1016/j.lwt.2017.11.051. González-Montoya, M., Hernández-Ledesma, B., Silván, J. M., Mora-Escobedo, R., & Martínez-Villaluenga, C. (2018). Peptides derived from in vitro gastrointestinal digestion of germinated soybean proteins inhibit human colon cancer cells proliferation and inflammation. Food Chemistry, 242, 75–82. https://doi.org/10.1016/j.foodchem. 2017.09.035. Goodman, B. E. (2010). Insights into digestion and absorption of major nutrients in humans. Advances in Physiology Education, 34(2), 44–53. Gornall, A. G., Bardawill, C. J., & David, M. M. (1949). Determination of serum proteins by means of the biuret reaction. Journal of Biological Chemistry, 177(2), 751–766. Horwitz, W., & Latimer, G. (2000). Official methods of analysis of AOAC International. Gaithersburg, MA, USA: Association of Official Analytical Chemists. Hur, S. J., Lim, B. O., Decker, E. A., & McClements, D. J. (2011). In vitro human digestion models for food applications. Food Chemistry, 125(1), 1–12. https://doi.org/10.1016/
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Evaluation of protein digestibility of fermented soybeans and changes in biochemical characteristics of digested fractions Sunantha Ketnawa, Yukiharu Ogawa
T
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Graduate School of Horticulture, Chiba University, 648, Matsudo, Matsudo 271-8510, Japan
ARTICLE INFO
ABSTRACT
Keywords: Natto Processing Amino acids TCA-soluble peptides In vitro anti-inflammatory
This study evaluated the protein digestibility of fermented soybeans inoculated with Bacillus subtilis var. natto using a simulated in vitro gastrointestinal digestion model. It was found that the total trichloroacetic acid (TCA)soluble peptide yield increased when the digestion stage progressed and compared to those of fermented soybeans before digestion. Most high molecular weight proteins disappeared after 4 h of digestion. All essential amino acids were found to increase remarkably (∼4%), especially Arg. Antioxidant amino acids such as Tyr, His and Phe were also found to increase. The highest antioxidant activity was found in the fractions after digestion corresponding to the antioxidant amino acids obtained. An increase in nitric oxide inhibition (9–29%) and a decrease in egg albumin denaturation activity (73–48%) were observed. Fermentation improved the protein digestibility of boiled soybeans by 45% and may benefit human health due to an improvement in essential amino acids and bioactivity through digestion.
1. Introduction A fermented soybean product, so-called ‘natto’, made by fermenting whole soybean seeds with pure culture of Bacillus subtilis subsp. natto, has traditionally been consumed in Japan for thousands of years (Weng, Yao, Sparks, & Wang, 2017). During fermentation, storage proteins of soybean can be degraded by microbial proteases. This can increase its nutritional value, reduce anti-nutritional factors and hydrolyse oligosaccharides (raffinose and stachyose) (Vernaza, Dia, Gonzalez de Mejia, & Chang, 2012). As a result, natto can be considered as a nutrition-rich food due to its high content of isoflavones, dietary fibre, vitamins, linoleic acid and some minerals. It also contains some functional compounds such as enzymes, bioactive peptides, nattokinase (a fibrinolytic agent), gamma-polyglutamic acid (γ-PGA) and so on (Sanjukta & Rai, 2016). Protein digestibility is an important factor in estimating the availability of protein for intestinal absorption after digestion, reflecting the efficiency of protein utilization in the diet (Almeida, Monteiro, CostaLima, Alvares, & Conte-Junior, 2015). A simulated in vitro gastrointestinal digestion model is a widely used method to determine protein digestibility. The method mimics the digestion process occurring in the human gastrointestinal tract through proteolytic enzymes (i.e. the pepsin–pancreatin enzyme system), measuring the percentage of protein content and peptide bonds which are hydrolysed by such enzymes
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(Hur, Lim, Decker, & McClements, 2011). Hydrolysis of higher molecular weight proteins into lower molecular weight oligopeptides, and the breakdown of peptide bonds to produce free amino acids, can be used as evidence of efficient digestion (Weng & Chen, 2010). Moreover, some small peptides generated from digestion have significant bioactive activity such as antioxidant activity or anti-inflammatory properties which can contribute to the suppression of some oxidation- and inflammation-related diseases in the human body (Liao, Huang, Sheen, & Chou, 2010; Udenigwe & Aluko, 2012). Currently, information on protein digestibility and the changes in biochemical characteristics of natto products during digestion through the human gastrointestinal tract is limited. The purpose of this study is to evaluate the effect of fermentation on soybeans, and the protein digestibility of fermented soybeans which were laboratory-scale inoculated with B. subtilis var. natto, using a simulated in vitro gastrointestinal digestion model. The digested fractions of fermented soybeans were investigated for biochemical characteristics such as total soluble nitrogen content, protein content, trichloroacetic acid (TCA)soluble peptides, molecular weight by SDS-PAGE, and free amino acids. Digested fractions of fermented soybeans were also analysed to determine their antioxidant properties. The in vitro anti-inflammatory activity of digested proteins of fermented soybeans was also investigated, by their ability to inhibit nitric oxide production and denature anti-albumin.
Corresponding author. E-mail address: [email protected] (Y. Ogawa).
https://doi.org/10.1016/j.jff.2018.11.046 Received 31 July 2018; Received in revised form 21 November 2018; Accepted 28 November 2018 1756-4646/ © 2018 Elsevier Ltd. All rights reserved.
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2. Materials and methods
starting the experiment. The reactor was connected to a temperaturecontrolled water bath (NTT-20S; Eyela, Tokyo, Japan), and the liquid sample in the reactor was agitated continuously with a magnetic stirrer. The temperature of the reactor was maintained at 37 °C throughout the experiment, and the pH of the sample was adjusted to 1.20 with 3 M HCl before starting the gastric stage (symbolized as ‘G’). The gastric stage was initiated by addition of pepsin solution (19 mL), and the pH was readjusted to 1.20 ± 0.01 with 0.5 M HCl. After 120 min of the gastric stage, the pH of the sample was adjusted to 6.80 using 3 N NaOH to inactivate the pepsin. The small intestinal stage (symbolized as ‘I’) was initiated by addition of intestinal enzyme solution (23 mL), and the pH was then readjusted to 6.80 ± 0.01 with 0.5 M NaOH. The sample was maintained at the intestinal conditions for 120 min. The digestion was immediately adjusted pH to 7.0 using 1 M NaOH or 1 M HCl, and the digestive enzyme reaction terminated by heating the samples to 95 °C for 10 min using a temperature-controlled water bath with a Thermominder (SJ-10R; Taitec, Saitama, Japan). The centrifuge tubes containing aliquots of digested samples were centrifuged at 4000g at 4 °C for 10 min (Model 2800; Kubota, Tokyo, Japan). The supernatant was separated, collected and lyophilized for use in further analysis. Five sample sets were collected separately from each reactor and labelled as follows: (1) F for fermented samples before digestion, (2) G1 for samples from gastric digestion for 1 h, (3) G2 for samples from gastric digestion for 2 h, (4) G2I1 for samples after gastric digestion for 2 h and intestinal digestion for 1 h, and (5) G2I2 for samples after gastric digestion for 2 h and intestinal digestion for 2 h (after digestion).
2.1. Materials and reagents Soybeans (Glycine max cv. Enrei) were obtained from a local market in Ibaraki prefecture, Japan. Pepsin from porcine gastric mucosa (EC 3.4.23.1, activity of 800–2500 U/mg protein), pancreatin from porcine pancreas (EC 232-468-9, 8 × USP specifications), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ) and sodium nitroprusside (SNP) were all purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), FeSO4, 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p′-disulfonic acid monosodium salt hydrate (FerroZine), FeCl2, TCA and all other chemicals of analytical grade were purchased from Wako Pure Chemical Corporation (Tokyo, Japan). Double-deionized water was used as needed. 2.2. Raw material preparation Dehulled yellow-seeded soybeans (300 g) were washed using tap water and soaked in distilled water (soybeans/water ratio of 1:3, w/v) for 18 h at 20 °C. Soaked soybeans were collected by discarding the soaking water. Subsequently, the soybeans were washed again with tap water, boiled in fresh distilled water at the same ratio in a pressure cooker for 90 min, cooled and superficially dried at room temperature. Boiled soybeans (150 g) were transferred into a glass beaker and inoculated with 50 mL of diluted culture from a commercial natto product produced by S-903 Bacillus spp. natto (Takanofoods Co., Ltd, Tokyo, Japan). After inoculation, the soybeans (37.5 g) were packed into a paper cup (205 mL), the top surface covered with polyvinylidene chloride wrap film, and incubated at 40 °C for 18 h.
2.5. Determination of total soluble nitrogen and protein content The total soluble nitrogen content was determined using a CN coder (MT-700 Mark 2; Yanako, Tokyo, Japan), based on the Dumas method principle by following the manufacturer’s instructions, using hippuric acid as the standard. The amount of protein present in the sample was calculated using the following formula:
2.3. Determination of moisture content The moisture content of samples (raw, soaked, boiled and fermented soybeans, and digested fractions from in vitro digestion before and after lyophilization) was determined by gravimetric determination according to AOAC (Horwitz & Latimer, 2000). An empty aluminium can and lid were dried in a hot-air dryer at 105 °C for 3 h and transferred to a desiccator to cool. The cooled empty can and lid were weighed. The sample (∼2 g) was put into the cooled aluminium can and dried at 105 °C for 16 h in a hot-air dryer (WFD-400; Eyela, Tokyo, Japan). After drying, the can and its dried sample were reweighed. The moisture content of the dried sample following each processing condition was expressed on a dry basis (d.b.), that is, as the dry matter mass in grams per 100 g of the total mass.
Protein content (%) = nitrogen (%) × factor
(1)
The in vitro digestibility was expressed as the percentage difference in protein content in the supernatant of the sample before digestion compared to that in the sample during digestion and after digestion. The degree of protein digestibility was calculated using the following formula:
Digestibility (%) = ( B
A)/ A× 100%
(2)
where A is the total protein content of the sample before digestion, and B is the residual protein content at each digestion stage. 2.6. TCA-soluble peptide yield (Ysp)
2.4. Simulated in vitro gastrointestinal digestion
Ysp was determined according to the method of Wang, Chi, Cheng, and Zhao (2018), with minor modifications. Each digested sample (100 μL) was added to 900 μL of 5% (w/w) TCA. Next, the mixture was kept for 1 h at 4 °C, and the unsolidified protein was then removed by centrifugation at 8000g for 10 min. The TCA-soluble peptide content in the supernatant was determined by measuring the absorbance at 280 nm using a UV–Visible spectrophotometer (V-630; Jasco, Tokyo, Japan). The content of TCA-soluble peptides was calculated as micromoles of tyrosine per gram of sample. Ysp was calculated using the following formula:
The static in vitro gastrointestinal digestion model described by Dartois, Singh, Kaur, and Singh (2010) that modified by Tamura, Singh, Kaur, and Ogawa (2016) was used, with minor adjustment. This step’s experiments were carried out in duplicate. Briefly, for the preparation of pepsin solution, 0.24 g pepsin from porcine gastric mucosa (activity of 800–2500 U/mg protein; Sigma-Aldrich, St. Louis, MO, USA) was dispersed in 50 mL gastric fluid buffer (adjusted to pH 1.20). The mixture was then mixed with a magnetic stirrer (Color Squid White, Ika Works, Wilmington, NC, USA) for 10 min. For the preparation of intestinal enzyme solution, 0.2 g pancreatin from porcine pancreas (Sigma-Aldrich), 0.015 g invertase and 4 mL amyloglucosidase (Megazyme, Co. Wicklow, Ireland) were prepared in the same manner as for the previous step but using 25 mL intestinal fluid buffer (adjusted to pH 6.80). The soybeans (15% protein, wet weight) (around 150 g) were blended with gastric buffer for 5 min at the highest speed and subsequently homogenized with a homogenizer (The Virtis Company Inc., Gardiner, NY) at 10,000g for 5 min, then added to a glass reactor before
Ysp (%) = (A/B) × 100%
(3)
where A is the peptide content of the supernatant, and B is the total protein content of the sample before digestion. 2.7. Soluble protein fractions and distribution by electrophoretic analysis The protein content of supernatants collected at different stages of 641
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the simulated digestion was determined by the biuret method (Gornall, Bardawill, & David, 1949) using bovine serum albumin (BSA) as a standard. The samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to determine the protein patterns by using NuPAGE® Bis-Tris gradient precast gel (4–12% gradient, 10 × 10 cm2) in a Novex XCell Mini-Cell (Invitrogen, Thermo Scientific, Rockford, IL, USA). NuPAGE® MES SDS Running Buffer was used as the electrophoresis running buffer. According to the difference in protein content of a sample solution, the protein solution was diluted with deionized water to be the same concentration, and then mixed with 12.5 µL NuPAGE® LDS Sample Buffer and 5 µL NuPAGE® Reducing Agent. The mixture was heated at 70 °C for 10 min. The samples (all containing 20 μg of protein per well) and the protein standard markers (Thermo Scientific, Rockford, IL, USA) were loaded onto the gel. Electrophoresis was performed at 200 V for 35 min. Proteins were visualized by staining with SimplyBlue™ SafeStain (Thermo Scientific, Rockford, IL, USA) overnight then de-staining with distilled water for 1 h. The water used for washing was changed every hour until the background was clear, at which point gels were then dried.
method of Ketnawa et al. (2017), with slight modification. ABTS radicals (ABTS%+) were produced in a reaction of 7 mM ABTS dissolved in 2.45 mM potassium persulfate, allowing the mixture to react in the dark at room temperature for 12 h before use. The ABTS+ solution was diluted with distilled water to obtain an absorbance of 0.7xx at 734 nm. To initiate the reaction, 0.02 mL of the sample was mixed with 0.98 mL of diluted ABTS%+ solution. The extent to which the ABTS%+ was quenched was measured at 734 nm after 10 min incubation at 30 °C in the dark. An ascorbic acid standard curve (0–0.1 mg/mL) was prepared. Distilled water was used instead of the sample and prepared in the same manner to obtain the control. ABTS+ radical scavenging activity was expressed as milligrams of ascorbic acid equivalent (AA) per gram of protein (dry matter). 2.12. Ferric reducing antioxidant power (FRAP) activity The capacity of digested fractions to reduce ferric–tripyridyltriazine complex was evaluated by the FRAP assay, as described by Ketnawa et al. (2017), with a slight modification. Freshly prepared FRAP reagent (10 mM TPTZ solution in 40 mM HCl plus 20 mM FeCl3·6H2O solution and 300 mM acetate buffer, pH 3.6, at a ratio of 1:1:10 (v/v/v)) (1.30 mL) was incubated at 37 °C before being mixed with 0.2 mL of the sample. The mixture was incubated at the same temperature for 30 min in the dark. Absorbance at 595 nm was recorded after 30 min of reaction. The control was prepared in the same manner as previously described, with distilled water instead of the sample. FRAP was calculated from the FeSO4 standard curve (0–100 μmol/L) and expressed as micromoles of FeSO4 equivalent per gram of protein (dry matter).
2.8. Free amino acid analysis Free amino acid content was analysed using an automatic amino acid analyser (JLC-500/V2 equipped with an ion exchange column; Jeol, Tokyo, Japan). In summary, freeze-dried powder of each sample was dissolved in distilled water (20 mg/mL protein content), then a 0.2 mL aliquot was added to 1.8 mL of 5% (w/v) TCA solution, mixed and centrifuged at 10000g for 10 min at 10 °C. The supernatant was diluted to an appropriate range, adjusted to pH 2 to 3 by 0.02 N HCl and filtrated through a 0.45-µM filter (polytetrafluoroethylene (PTFE), Advantec® Dismic®-13HP; Toyo Roshi Kaisha, Ltd, Chiba, Japan). Finally, the collected supernatant was subjected to an amino acid analyser, and open Type AN-II and Type B (Wako Pure Chemical, Ltd, Osaka, Japan) were used as reference amino acid standards. Free amino acids were separated in high separation mode (110 min) with a lithium citrate buffer system and measured by photometric detection after derivatization with ninhydrin.
2.13. Metal chelating activity (MIC) The chelating activity on Fe2+ was measured using the method of Ketnawa et al. (2017), with a slight modification. Diluted sample (0.8 mL) was mixed with 2 mM FeSO4 (0.01 mL) and 5 mM FerroZine® (0.02 mL). The reaction mixture was allowed to stand for 10 min at room temperature. The absorbance was then measured at 562 nm. The blank was prepared in the same manner but distilled water was used instead of the sample. A standard curve for EDTA (0–10 mmol/L) was prepared and used as the positive control. Ferrous chelating activity was expressed as micromoles of EDTA equivalent per gram of protein (dry matter).
2.9. Total phenolic content (TPC) TPC was determined according to the method of Donlao and Ogawa (2018). Briefly, 0.2 mL of the test solution was mixed with 1 mL of 10% (v/v) Folin–Ciocalteu solution and 0.8 mL of 7.5% (w/v) sodium carbonate solution. The mixture was incubated for 1 h at room temperature, and absorbance was then measured using a UV–Vis spectrophotometer (V-630; Jasco, Tokyo, Japan) at 765 nm; distilled water was used as a blank. TPC was reported as the milligrams of gallic acid equivalent (GAE) per gram of protein (dry matter).
2.14. Determination of nitric oxide (NO) inhibition activity The compound sodium nitroprusside (SNP) is known to decompose in aqueous solution at physiological pH (7.2), producing NO radicals (NO%). Under aerobic conditions, NO% reacts with oxygen to produce stable products (nitrate and nitrite), the quantities of which can be determined using Griess reagent. The scavenging effect of the plant extract on NO was measured according to the modified method of Marcocci, Maguire, Droylefaix, and Packer (1994). One millilitre of sample at different concentrations (0–1000 µg/mL protein) was added to test tubes containing 1 mL of SNP solution (100 mM), and the tubes were incubated at 29 °C for 2.5 h. An aliquot of 1 mL of the incubation solution was removed and diluted with 1 mL of Griess reagent by mixing equal amounts of 1% sulfanilamide and 0.1% N-1-naphthylethylenediamine dihydrochloride in 2% H3PO4. The absorbance of the chromophore (purple azo dye) that formed during the diazotization of nitrite ions with sulfanilamide and subsequent coupling with naphthylethylenediamine dihydrochloride was immediately read at 540 nm. The percentage nitrite radical scavenging activity of the sample and gallic acid (0–1000 µg/mL) was calculated using the following formula:
2.10. DPPH radical scavenging activity The DPPH radical scavenging activity of the sample was determined according to the method described by Ketnawa, Benjakul, MartínezAlvarez, and Rawdkuen (2017). Sample solution (0.5 mL) was added to 0.1 mM DPPH in 95% ethanol (0.5 mL). The mixture was vigorously mixed and allowed to stand for 30 min in the dark at room temperature. The absorbance of the resulting solution was measured at 517 nm. The DPPH blank is the value for 0.5 mL of 95% ethanol mixed with 0.5 mL of 0.1 mM DPPH in 95% ethanol. The DPPH radical scavenging activity was calculated by a Trolox standard curve (0–1000 μmol/L) and expressed as micromoles of Trolox equivalent (TE) per gram of protein (dry matter). 2.11. ABTS radical scavenging activity
NO inhibition activity (%) =
ABTS radical scavenging activity was determined according to the 642
Acontrol
Asample
Acontrol
× 100
(4)
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where Acontrol is the absorbance of the control without the tested samples, and Asample is absorbance in the presence of the samples or standards. 2.15. Inhibition of egg albumin denaturation In vitro anti-inflammatory activity of digested proteins during simulated digestion was evaluated with the protein denaturation method of Mizushima and Kobayashi (1968) as modified by Osman, Sidik, Awal, Adam, and Rezali (2016). Diclofenac sodium, a powerful nonsteroidal anti-inflammatory drug, was used as a standard. The reaction mixture consisting of 1 mL of different concentrations of protein isolate (125–500 μg/mL) or standard diclofenac sodium at a final concentration of 125–1000 µg/mL and 1.4 mL of phosphate-buffered saline (pH 6.4) was mixed with 0.2 mL of egg albumin (from fresh hen egg) and incubated at 37 °C for 15 min. Denaturation was induced by keeping the reaction mixture at 57 °C in a water bath for 20 min. After cooling, the absorbance was measured at 660 nm using distilled water as a blank. Increments in the absorbance of test samples with respect to the control indicated stabilization of the protein, i.e. inhibition of heat-induced protein (albumin) denaturation by the test sample and reference drug diclofenac sodium. Each experiment was done in triplicate, and the average was taken. The percentage inhibition of protein denaturation was calculated using following formula:
Inhibition(%) = 1
Asample A control
× 100
Fig. 1. Changes in protein content, nitrogen content, trichloroacetic acid (TCA)-soluble peptide yield and protein digestibility of the fermented soybeans during stimulated in vitro digestion, calculated and reported as percentages (%). Bars represent the standard deviation from triplicate determinations. Significant differences were found in all digestion stage compared to before digestion (F). Non-significant differences are highlighted with an asterisk as a function of digestion stage (P > 0.05).
compounds contained in the soybean samples are also known to inhibit protein absorption. This has been mainly described for high concentrations of tannins that nonspecifically bind and precipitate proteins and therefore, are considered as anti-nutritional compounds (MartinezGonzalez et al., 2017). Previous studies have shown that fermentation of soybeans using several Bacillus spp. results in major biochemical changes in the substrate, leading to an increase in soluble material and material dialysable into water-soluble low molecular weight peptides, oligosaccharides and monosaccharides (Sanjukta, Rai, Muhammed, Jeyaram, & Talukdar, 2015; Weng & Chen, 2010, 2011). It was shown that Bacillus spp. are able to degrade soybean macromolecules to a large extent, resulting in water-soluble low molecular weight compounds as observed by the difference between protein patterns in boiled soybeans (B, lane 3) and fermented soybeans (F, lane 4), as shown in Fig. 2 corresponding to the higher protein digestibility number in Fig. 1. In addition, simulated digestion of fermented soybeans using digestive enzymes also increased the amount of dialysable matter, which clearly demonstrated the beneficial effect of Bacillus fermentation on food nutrient availability in stimulated digestion (Sanjukta et al., 2015). The increase in protein digestion improved the nutritional quality of the soybean proteins by favouring hydrolysis and producing short-chain peptides, which is essential to human metabolism as can be seen from the amino acid composition in Table 1. The progress of hydrolysis was also confirmed by the determination of TCA-soluble peptide content and is shown in Fig. 1. TCA-soluble protein means the amount of digestible soybean protein that dissolves in TCA; it measures the portion of protein soluble in TCA which contains polypeptides of fewer than 10 amino acids and free amino acids whereas nondegraded proteins are precipitated by TCA (Chen, Chiou, & Yu, 2010). TCA-soluble protein content after digestion (59.83%; G2I2) was significantly higher than before digestion (4%; F) (P < 0.05). The higher Ysp is due to the large amount of soluble peptides initially present in the fermented soybeans in cooperation with hydrolysis by digestive enzymes (Chen et al., 2013). The change in TCA-soluble peptides during digestion shows that the protein of fermented soybeans was digested and that protein utilization was significantly improved. Previous research has also reported an increase in TCA-soluble peptides in fermented soybeans compared to raw material or protein flour (Weng & Chen, 2010). The results suggest that digested fermented soybeans can improve the absorption of oligopeptides through simulated digestion.
(5)
where Acontrol is the absorbance of the control without the tested samples, and Asample is absorbance in the presence of the samples or standards. 2.16. Statistical analysis Simulated digestion was done in duplicate for fermented samples. The other experiments were carried out in triplicate. Data were subjected to analysis of variance, and mean comparisons were performed using Duncan’s multiple range test. Statistical analysis was carried out using the Statistical Package for the Social Sciences (SPSS for Windows, SPSS, Inc., Chicago, IL). Differences were considered to be significant at P < 0.05. 3. Results and discussion 3.1. Parameters for in vitro protein digestibility Four parameters were used to evaluate protein digestibility during a two-stage (1–2 h of gastric enzymes, pepsin, pH 1.2, 37 °C: G1 and G2, followed by 1–2 h of intestinal enzymes, a mixture of pancreatin, invertase and α-amyloglucosidase, pH 6.8, 37 °C: G2I1 and G2I2) simulated gastro-small intestinal digestion compared to that in the fermented soybean samples before digestion (F). TCA-soluble peptides, total soluble nitrogen, SDS-PAGE profile and amino acid content were determined. Fig. 1 shows the variability in protein and nitrogen content, in vitro protein digestibility (expressed as the percentage protein content compared to that in F) and Ysp. Protein and nitrogen content increased approximately 1.52-fold during digestion. Protein digestibility for G1 was 30.86%, and increased to 47.29% (G2), 49.19% (G2I1) and 48.99% (G2I2). So, digestibility increased around 1.59-fold (Fig. 1). This increase shows the ability of digestive enzymes in the gastric digestion stage to digest protein in the fermented samples. The digestibility in the intestinal stage was quite stable may be due to trypsin and α-chymotrypsin inhibitors present in the samples (Chen, Zhao, & Sun, 2013). Weng and Chen (2011) and Weng and Chen (2010) reported levels of trypsin and α-chymotrypsin inhibitors of 46 ± 6 and 2.7 ± 1.6 IU/10 g natto, respectively. Moreover, some phenolic 643
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3.2. Soluble protein fractions and distribution by SDS-PAGE The soluble protein distribution profile of different stages of simulated digestion is presented by Bis-Tris-SDS-PAGE analysis (Fig. 2). The SDS-PAGE technique has originally been used to study soybean protein subfractions and to evaluate the degradation rate of individual digested fractions. The protein subfractions were divided into three parts according to molecular weight. The protein bands of 55 kDa and above were grouped as large-size fractions, 16–55 kDa as medium-size fractions, and smaller than 16 kDa as small-size fractions. Fig. 2 shows a typical protein profile of several polypeptides ranging from 140 to < 10 kDa. Soaked soybeans, S (lane 2, Fig. 2), contained several major storage proteins including α, α′ and β subunits of β-conglycinin and acidic and basic subunits of glycinin, prominent among which were 90, 67, 63, 48, 40, 36, 30 and 15 kDa proteins probably corresponding to α, α′ and β subunits of β-conglycinin (67–48 kDa) and acidic and basic subunits of glycinin (38.5–20.5 kDa). In the electrophoretic patterns for the boiled soybeans, B (lane 3, Fig. 2), a total of seven major protein bands having molecular weights around 120, 75, 70, 55, 40, 36, 25, 17 and 15 kDa were observed. These results agree with previous studies of raw soybean protein profiles (González-Montoya, Hernández-Ledesma, Silván, Mora-Escobedo, & Martínez-Villaluenga, 2018; Liu, Zhou, Tian, & Gai, 2007; Mujoo, Trinh, & Ng, 2003). During pepsin digestion (G1–G2), the intensities of the protein bands corresponding to 7S and 11S fractions were decreased, and many peptide bands appeared at 11–15 kDa, indicating protein hydrolysis. Enzymatic hydrolysis led to gradual breakdown of these two subunits of storage proteins into several subunits, in a hydrolysis time-dependent manner. Enzymatic hydrolysis produced a loss of α′-, α and β-7S subunits, and total disappearance of bands corresponding to these subunits of 7S. Simulated digestion of fermented soybeans caused complete degradation of polypeptides > 20 kDa and increased the abundance of oligopeptides with molecular weight < 10 kDa (G2I2; lane 8, Fig. 2). After simulated digestion, the ratio of small protein fractions in G2I2 was increased. These results show that some peptides with high molecular weight were degraded during simulated digestion, and peptides with low molecular weight were formed. This also indicates that pepsin broke down the intra-chain peptide bonds and mainly produced relatively larger peptide fragments. After subsequent intestinal digestion with pancreatin, the larger peptide fractions were further degraded into oligopeptides and amino acids. Zhang et al. (2018), from their study on simulated digestion of Alcalase–soybean protein hydrolysates, reported that after the first 2 h of gastric digestion with pepsin, the larger fraction with molecular weight > 10 kDa was significantly reduced (P < 0.05), while the smaller fractions with molecular weight of 5–10 kDa and 1–5 kDa were significantly increased (P < 0.05). The evidence from this study showed mainly that proteins are digested to smaller molecular weight sizes by digestive enzymes, which could be an important contributing factor to good bioavailability and benefits to human health.
Fig. 2. Change in sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) electrophoretograms for soybean protein at various process and fermented soybean protein at different digestion stages. Upper left is the original gel image, and right is the image of a portion extracted from the original for easier comparison of each lane. Lane 1 (MK) for standard molecular markers, lane 2 (S) for soaked soybeans, lane 3 (B) for boiled soybeans, lane 4 (F) for fermented soybeans, lane 5 (G1) for the fraction at the gastric digestion stage for 1 h, lane 6 (G2) for the fraction at the gastric digestion stage for 2 h, lane 7 (G2I1) for the fraction at the gastric digestion stage for 2 h and intestinal digestion stage for 1 h, and lane 8 (G2I2) for the fraction at the gastric digestion stage for 2 h and intestinal digestion stage for 2 h.
Table 1 Changes in free amino acid content in fermented soybeans during simulated in vitro digestion. Amino acid
Thr Ser Asp Glu Gly Ala Val Met Cysta*** Ile Leu Tyr Phe His Lys Arg Pro Total
Increment (fold)*
Total increment (fold)**
Before digestion
After digestion
3.32 ± 0.41 3.61 ± 0.49 3.07 ± 0.30 6.10 ± 0.61 8.99 ± 0.80 2.02 ± 0.20 2.61 ± 0.30 10.49 ± 1.19 2.44 ± 1.06 16.14 ± 3.35 39.52 ± 2.84 21.48 ± 2.22 37.96 ± 0.54 6.80 ± 0.78 18.54 ± 1.82 0.30 ± 0.02 2.56 ± 0.00 5.96 ± 0.43
8.02 ± 1.07 7.67 ± 0.75 7.13 ± 1.54 10.15 ± 1.20 15.35 ± 1.65 3.68 ± 0.40 4.79 ± 0.59 24.46 ± 2.62 4.54 ± 0.28 36.79 ± 4.05 144.13 ± 0.15 83.52 ± 0.35 125.48 ± 0.54 10.80 ± 1.14 72.78 ± 8.84 14.84 ± 1.76 2.46 ± 0.00 21.10 ± 2.39
2.49 ± 0.63 2.19 ± 0.51 2.40 ± 0.73 1.70 ± 0.37 1.74 ± 0.34 1.86 ± 0.39 1.88 ± 0.44 2.39 ± 0.52 2.36 ± 1.14 2.44 ± 0.76 3.64 ± 0.13 3.98 ± 0.85 3.30 ± 0.31 1.63 ± 0.36 4.01 ± 0.85 49.43 ± 2.86 0.96 ± 0.00 3.59 ± 0.66
3.3. TPC Changes in TPC for different stages during simulated digestion of fermented soybeans are shown in Fig. 1. A significant increment was found for digested fractions. The level of free phenolics in unfermented soybeans was 21.16 ± 0.31 mg GAE/g protein, and increased dramatically to 10.41–25.65 mg GAE/g protein in the digested fractions (Fig. 3). This may be due to the digestive enzymes playing a role in exposing water-soluble polyphenols from the structure because more water-soluble compounds were free. The effectiveness of polyphenols is related to their antioxidant potential. The increase in free phenolic acids in the fermented soybeans is probably due to β-glucosidase production during fermentation (Sanjukta et al., 2015). An increase in free phenolics is expected to improve their bioavailability in the intestine, which will further improve the beneficial effects associated with them.
* The increment of amino acids was calculated by dividing the quantity (nmol/mL) of each individual amino acid by the concentration of each amino acid in soaked soybeans (data not shown). Values are expressed as increment folds and are means of duplicate measurements. ** The increment of amino acids was calculated by dividing the quantity (nmol/ml) of each individual amino acid by the concentration of each amino acid in fermented soybeans before and after digestion. Values are expressed as increment folds and are means of duplicate measurements. *** Cysteine (Cys) was determined in the form of cystathionine (Cysta). Cysta is a dipeptide formed by serine and homocysteine; the trans-sulfuration of methionine yields homocysteine which combines with serine to form cystathionine. Tryptophan (Trp) cannot be reported because it is unstable and produces ammonia, so it was not contained in the amino acid standard solution. 644
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(1.72 ± 0.02 mg AA/g protein) compared to F (0.84 ± 0.03 mg AA/g protein). When the pepsin digest was hydrolysed with pancreatin, additional peptide-bond cleavage led to the accumulation of shorter peptides (tri- and di-peptides) and free amino acids, thus becoming more hydrophilic. Digests with increased polarity (amino acids, small peptides) can readily react with water-soluble ABTS%+ (Zhu, Chen, Tang, & Xiong, 2008). The conceivable structural changes resulting from pepsin digestion may also favour trapping of free radicals like DPPH and ABTS, thus further enhancing quenching by the sample digest. Furthermore, reducing power (FRAP) continuously increased during simulated digestion, up to 1.72-fold for the gastric stage (18.80 ± 0.18 µmol FeSO4/g protein) and 1.93-fold for G2I1 and G2I2 (21.10 ± 0.05 µmol FeSO4/g protein) (P < 0.05). This increase in the reducing power of digested samples shows that the proteins of fermented soybeans can be more effective hydrogen or electron donors after simulated digestion (Fig. 3). The reducing power of fermented soybeans increased with increasing digestion time (0–4 hrs). Besides that, increasing MIC activity during the simulated digestion was also observed; the value was increased approximately 1.98-fold for the gastric stage (5.47 ± 0.02 µmol EDTA)/g protein) and 2.21-fold for the intestinal stage (6.10 ± 0.03 µmol EDTA)/g protein) (P < 0.05). The activity changes for DPPH, FRAP, Fe2+ chelation and ABTS%+ scavenging followed a similar trend during simulated digestion, suggesting that the two might be related. It is possible that treatment with pepsin disrupted the spatial structure of fermented soybean peptides, conducive to binding and trapping of Fe2+ and ABTS%+ and resulting in reduced chelation capability. On the other hand, as high-affinity metal-binding groups became fully exposed or newly formed by pancreatic digestion, for example, the imidazole and carboxylic groups and electrostatic and ionic interactions with Fe2+ were probably enforced. So, digested protein of fermented soybeans showed capability to bind metal ions. Sanjukta et al. (2015) reported that soybeans fermented with B. subtilis MTCC5480 showed a decrease in antioxidant activity on pepsin digestion, followed by an increase in activity on pancreatin digestion. The results clearly indicate that the protein of fermented soybeans should be resistant to gastrointestinal digestion to reach the target tissue in an active form. In addition, the digested fraction of fermented soybeans after the digestion stage (G2I2) showed the highest activity because the resulting short-chain peptides and smaller sizes led to higher activity than for the parental proteins, containing peptides, and are electron donors and can react with free radicals to convert them into more stable products and terminate the radical chain reaction (Aluko, 2015). From Fig. 2, it can be seen that the smaller sizes presented in SDS-PAGE may support the antioxidant activity. In the present study, in the final digest of fermented soybeans (G2I2), which exhibited the highest overall antioxidant potential, larger molecular weight fractions disappeared.
Fig. 3. Total phenolic content (TPC) expressed as “mg of gallic acid equivalents/g protein”, DPPH radical scavenging capacity (DPPH) expressed as “μmol Trolox equivalents/g protein”, ABTS radical scavenging capacity (ABTS) expressed as “mg of ascorbic acid equivalents/g protein”, ferric reducing antioxidant power activity (FRAP) expressed as “mmol FeSO4·7H2O equivalents/g protein”, and metal ion chelating activity (MIC) expressed as “μmol EDTA equivalents/g protein” of the fermented soybeans at each digestion stage. F represents fermented soybeans before digestion, G1 represents the gastric digestion stage for 1 h, G2 represents the gastric digestion stage for 2 h, G2I1 represents the gastric digestion stage for 2 h and intestinal digestion stage for 1 h, and G2I2 represents the gastric digestion stage for 2 h and intestinal digestion stage for 2 h. Bars represent the standard deviation from triplicate determinations. Significant differences were found in all digestion stage compared to before digestion (F). Non-significant differences are highlighted with an asterisk as a function of digestion stage (P > 0.05).
Many researchers have suggested that the increase in antioxidant activity is due to an increase in free polyphenols. Watanabe, Fujimoto, and Aoki (2007) suggested that antioxidant activity in the water-soluble fraction of Rhizopus-fermented tempeh may be due to the amino acids and peptides formed during fermentation. 3.4. Antioxidant activity The antioxidant activity of protein foods or hydrolysed proteins and peptides has been well documented in the literature, but relatively few studies have been conducted to evaluate their activity and fate in the human digestive system. Because of the unique peptide-bond specificity of digestive proteases, the products from pepsin and pancreatin (trypsin and chymotrypsin) digestion will depend on the characteristics of the peptides ingested. Antioxidant activity is enhanced, contributed by the increase of short-chain peptides and phenolic compounds (Möller, Scholz-Ahrens, Roos, & Schrezenmeir, 2008). The antioxidant activity of fermented soybeans came from both phenolic compounds and bioactive peptides. From preliminary tests for the digestion of raw soybeans and boiled soybeans, TPC not only plays a part in antioxidant properties but peptides released during digestion also improve antioxidant activity (Fig. 3). In addition, Sanjukta et al. (2015) suggested that antioxidant activity in the water-soluble fraction of B. subtilis MTCC5480-fermented soybeans is due to the amino acids and peptides formed during fermentation as well as antioxidant peptides that are released from gastrointestinal digestion. The increment in DPPH, ABTS, FRAP and MIC was 2.85-, 4.32-, 1.93- and 2.21-fold, respectively. The biggest change (4.32-fold) was observed for ABTS. Strong ABTS%+ scavenging activity for the watersoluble ABTS%+ free radicals, expressed as L-ascorbic acid equivalent antioxidant capacity (AA), was demonstrated by digested samples (Fig. 3). Following gastric (or pepsin) digestion (G1 and G2), ABTS increased (P < 0.05) around 2.05-fold in the gastric stage
3.5. Changes in free amino acid composition Pepsin plays an important role in the primary digestion of food proteins, resulting in long-chain peptides, followed by secondary digestion by intestinal enzymes, resulting in the formation of short-chain peptides (Goodman, 2010). The nutritional quality of a protein is primarily related to its amino acid composition. In addition, amino acid availability is also a key factor in protein quality. Bioavailability depends on the process of digestion, which may be affected by many factors such as the characteristics of the protein itself. Fermented soybeans and their digested fractions were analysed for their free amino acid profile, responsible for nutritional aspects and bioactive properties, divided by groups of amino acids. The change in free amino acid profile was reported in terms of the free amino acid increment between F and G2I2 and is shown in Tables 1 and 2. When compared to that for the raw material (raw soaked soybeans, S), the total free amino acid composition of fermented soybeans was 645
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Table 2 Increment of selected amino acid groups of fermented soybeans during simulated in vitro gastrointestinal digestion. Amino acid group
Increment (fold)*
EAA HAA AAA AXA
4.13 3.01 3.20 2.99
± ± ± ±
Table 3 In vitro anti-inflammatory activity of fermented soybeans at different stages of simulated in vitro digestion.
0.47a 0.33b 0.35b 0.32b
EAA = essential amino acids: Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Try and Val; HAA = hydrophobic amino acids: Ala, Val, Ile, Leu, Tyr, Phe, Trp, Pro, Met and Cys; AAA = aromatic amino acids: Phe, Trp, Tyr and His; AXA = antioxidant amino acids: Trp, Tyr, Met, Cys, His, Phe and Pro. * The increment of amino acids was calculated by dividing the quantity (nmol/ml) of each individual amino acid by the concentration of each amino acid in fermented soybeans before and after simulated digestion. Values are expressed as increment folds and are means of duplicate measurements. Different letters in the same column indicate a significant difference (P < 0.05) among different amino acid groups.
Digestion stage
Inhibition of nitric oxide production (%)
Inhibition of egg albumin denaturation (%)
F G1 G2 G2I1 G2I2
9.01 ± 0.29e 13.66 ± 0.62d 17.41 ± 0.66c 23.58 ± 0.87a 28.54 ± 0.38a
73.09 73.53 67.47 57.81 48.08
± ± ± ± ±
1.69a 1.39a 0.00b 1.28c 1.15d
Different small letters in the same column indicate a significant difference (P < 0.05) among different digestion stages. Digestion stage captions are described in Fig. 3.
(Udenigwe, Je, Cho, & Yada, 2013). The assay is based on the principle that sodium nitroprusside (SNP) in aqueous solution at physiological pH spontaneously generates NO which interacts with oxygen to produce nitrite ions that can be estimated using Griess reagent. NO released from SNP has a strong NO character which can alter the structure and function of many cellular components. Scavengers of N compete with oxygen, leading to reduced production of nitrite ions (GonzálezMontoya et al., 2018). In this way, the digested fraction of fermented soybeans at different stages showed anti-inflammatory activity by inhibiting NO production, reported as a percentage and shown in Table 3. The inhibition increased with an increase in digestion time. Before digestion, F showed inhibition of NO production of 9.01 ± 0.29%; inhibition activity gradually increased up to 28.54 ± 0.38% in G2I2. From the results, we assume that the peptides released during gastrointestinal digestion of fermented soybean proteins have potential bioactivity against inflammation. Another in vitro anti-inflammatory effect of digested proteins of fermented soybeans was evaluated: denaturation of bovine/egg albumin. Denaturation of proteins is a well-documented cause of inflammation. The present findings were that there was a slight decrease in the inhibition of egg albumin denaturation during digestion and are summarized in Table 3. At the protein concentration used of 1.25 mg/ mL, the inhibition of egg albumin denaturation decreased from 73.09 ± 1.69% to 48.08 ± 1.15% before and after digestion, respectively. Non-steroidal anti-inflammatory drugs have the ability to stabilize (prevent denaturation) heat-treated albumin at physiological pH (6.2–6.5). Meanwhile, the non-steroidal anti-inflammatory drug (NSAID) used, diclofenac sodium, exhibited a relatively higher inhibition capacity of 4.71 ± 0.06% at the same test concentration (1.25 mg/mL). It is suggested that the regular intake of soymilk or fermented soymilk may provide protection against cardiovascular diseases and chronic inflammatory diseases in LPS-induced macrophages (Liao et al., 2010). However, not only proteins can play an anti-inflammatory role: the major phytochemicals (from high to low abundance) in soybeans, phytic acid (1.0–2.2%), sterols (0.23–0.46%), saponins (0.17–6.16%), isoflavones (0.1–0.3%) and lignans (0.02%), also show a strong anti-inflammatory effect and display a wide range of other bioactivities, including anti-cancer, antioxidative, anti-viral, cardiovascular protective and hepatoprotective actions (Sanjukta & Rai, 2016). Another point to consider is that a decrease in in vitro anti-inflammatory ability depends on molecular weight as well; higher antiinflammatory potential of peptides with molecular weight from 5 to 10 kDa was observed in germinated soybean peptides by GonzálezMontoya et al. (2018). Udenigwe et al. (2013) also reported that a high molecular weight almond protein fraction (> 5 kDa) showed the best inhibitory activity for NO production.
higher, 5.96-fold and 21.10-fold before and after simulated digestion, respectively. The content of all other amino acids also increased; the highest increment was observed for the fermented soybeans after simulated digestion (Table 1). When comparing F and G2I2 (after simulated digestion), the total increment was 3.59-fold. Most represented amino acids were increased significantly, around 1.5- to 3.5-fold. Table 2 shows the changes in free amino acid content by different groups. The maximum change was found for Arg (49.43-fold) while Pro was found to slightly decrease (0.95-fold). Considering amino acids divided by group, we found the maximum change (4.59-fold) in the essential amino acid group (EAA: Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Try and Val). As mentioned, Arg was increased the most, while His was increased the least. This could imply that simulated digestion improves the generation of EAA. Apart from EAA, we also found an increment in other groups of amino acids, for example, hydrophobic amino acids (HAA: Ala, Val, Ile, Leu, Tyr, Phe, Trp, Pro, Met and Cys), aromatic amino acids (AAA: Phe, Trp, Tyr and His) and antioxidant amino acids (AXA: Trp, Tyr, Met, Cys, His, Phe and Pro), of around 3-fold. Pepsin is most effective at cleaving peptide bonds between HAA and preferably AAA such as Phe, Trp and Try. Pancreatin exhibits the activity of trypsin (cleavage of peptide bonds at Arg and Lys sites), chymotrypsin (cleavage of peptide bonds at Phe, Trp, Tyr and Leu sites) and elastase (cleavage of peptide bonds at Ala and other aliphatic amino acids) (Udenigwe & Aluko, 2012). Beyond its effect on nutritional quality, the increase in free amino acid content is responsible for the remarkable increase in antioxidant properties shown in Fig. 1. The results for amino acid content showed the same trend as in a study of the amino acid content of soybean meal fermented with B. subtilis; Glu, Val, Tyr, Leu and Phe were higher (Song, Frias, Martínez-Villaluenga, VidalValdeverde, & de Mejia, 2008). From the increase in amino acids in this study, we infer that the digested fraction of fermented soybeans could increase the nutritional value and physiological functions in humans. 3.6. In vitro anti-inflammatory activity In vitro anti-inflammatory activity was evaluated using NO production inhibition and albumin denaturation assays. NO is a signalling molecule that plays a key role in the pathogenesis of inflammation. It gives an anti-inflammatory effect under normal physiological conditions. On the other hand, NO is considered as a pro-inflammatory mediator that induces inflammation due to overproduction in abnormal situations (Sharma, Al-Omran, & Parvathy, 2007). Various inflammatory diseases are related to the overproduction of NO
4. Conclusions Fermented soybeans digested through simulated in vitro gastrointestinal digestion had improved protein digestibility, leading to an 646
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improvement in protein utilization. Besides that, antioxidant properties and NO inhibition improved after simulated digestion, due to leaching out of bioactive peptides during digestion. These activities of fermented soybeans were retained or improved following stepwise enzyme digestion simulating the human digestive tract. The concentration of free amino acids as well as of polyphenols appears to collectively contribute to higher nutritional value by increment of EAA and the strong bioactivity of the protein digests. Therefore, the benefits of fermented soybean, ‘natto’, could be extended beyond its role as a good nutritional and digestible food. Our results suggest that the Bacillus fermentation process could be a potential functional processing method with good bioavailability characteristics due to antioxidant and anti-inflammatory activity.
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5. Ethics statements Our research did not include any human subjects and animal experiments. Acknowledgements The authors are grateful for the JSPS Postdoctoral Fellowship for Research in Japan awarded to Dr. Sunantha Ketnawa. We would like to thank The Tojuro Iijima Foundation for Food Science and Technology. We would also like to thank Assoc. Prof. Dr. Terumasa Takahashi, Graduate School of Horticulture, Chiba University, and Dr. Tamaki Hirose, Research Facility Centre for Science and Technology, University of Tsukuba, for technical support for measurement of crude protein content and free amino acid analysis, respectively. Conflicts of interest The authors declare no conflict of interest. The project was funded by JSPS and The Tojuro Iijima Foundation for Food Science and Technology, but they had no role in study design, data collection or analysis. The authors alone are responsible for the content and writing of the paper. References Almeida, C. C., Monteiro, M. L. G., Costa-Lima, B. R. C. D., Alvares, T. S., & Conte-Junior, C. A. (2015). In vitro digestibility of commercial whey protein supplements. LWT Food Science and Technology, 61(1), 7–11. https://doi.org/10.1016/j.lwt.2014.11.038. Aluko, R. E. (2015). Amino acids, peptides, and proteins as antioxidants for food preservation. In F. Shahidi (Ed.). Handbook of antioxidants for food preservation (pp. 105– 140). Amsterdam: Woodhead Publishing. Chen, C., Chiou, P., & Yu, B. (2010). Evaluating nutritional quality of single stage- and two stage-fermented soybean meal. Asian-Australasian Journal of Animal Sciences, 23(5), 598. Chen, N., Zhao, M., & Sun, W. (2013). Effect of protein oxidation on the in vitro digestibility of soy protein isolate. Food Chemistry, 141(3), 3224–3229. https://doi.org/10. 1016/j.foodchem.2013.05.113. Dartois, A., Singh, J., Kaur, L., & Singh, H. (2010). Influence of Guar Gum on the in vitro starch digestibility-rheological and microstructural characteristics. Food Biophysics, 5(3), 149–160. https://doi.org/10.1007/s11483-010-9155-2. Donlao, N., & Ogawa, Y. (2018). Impacts of processing conditions on digestive recovery of polyphenolic compounds and stability of the antioxidant activity of green tea infusion during in vitro gastrointestinal digestion. LWT Food Science and Technology, 89, 648–656. https://doi.org/10.1016/j.lwt.2017.11.051. González-Montoya, M., Hernández-Ledesma, B., Silván, J. M., Mora-Escobedo, R., & Martínez-Villaluenga, C. (2018). Peptides derived from in vitro gastrointestinal digestion of germinated soybean proteins inhibit human colon cancer cells proliferation and inflammation. Food Chemistry, 242, 75–82. https://doi.org/10.1016/j.foodchem. 2017.09.035. Goodman, B. E. (2010). Insights into digestion and absorption of major nutrients in humans. Advances in Physiology Education, 34(2), 44–53. Gornall, A. G., Bardawill, C. J., & David, M. M. (1949). Determination of serum proteins by means of the biuret reaction. Journal of Biological Chemistry, 177(2), 751–766. Horwitz, W., & Latimer, G. (2000). Official methods of analysis of AOAC International. Gaithersburg, MA, USA: Association of Official Analytical Chemists. Hur, S. J., Lim, B. O., Decker, E. A., & McClements, D. J. (2011). In vitro human digestion models for food applications. Food Chemistry, 125(1), 1–12. https://doi.org/10.1016/
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