A Fermented Soybean Food) Affected by Fermenting Time

Food Sci. Technol. Res., 16 (6), 537–542, 2010

Changes of Protein in Natto (a fermented soybean food) Affected by Fermenting Time

* Tien Man Weng and Ming Tsao Chen

Department of Bioindustry Technology, Da Yeh University, 168 University Rd. Dacun, Changhua, Taiwan

Received May 29, 2010; Accepted August 18, 2010

The purpose of this study was to investigate effect of fermentation time on the proteins of steamed soybean inoculated with Bacillus subtilis (natto). The result was found that the contents of trichloroacetic acid soluble nitrogen (TCA-N) and drgree of hydrolysis (DH) of protein increased with the fermentation time increased, but the protein solubility was declined with increasing the fermentation time in advance, then increased. The most bands of soy proteins were found to disappear from the Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoretogram after 24 h of fermentation. Some of nonessential amino acids were found to remark- edly decline, but some of them were remained unchanged after 24-48 h of fermentation., However the most essential amino acids were found to declined after 36h of fermentation, then all of them increased after 48 h of fermentation except histidine.

Keywords: Natto, soy protein, Bacillus subtilis, SDS-PAGE

Introduction with other fermented soybean foods. However, there seems Natto is a traditional fermented soybean food product to be an upswing in consumption in Taiwan, recently. In with venerable history in Japan. By steaming soybeans and Taiwan, natto is seasoned with soy sauce and mustard like inoculating with Bacillus subtilis, natto spores to ferment it, Japanese. Natto has a short shelf life, partly because it has a and then it would become a characteristic odor, flavor and moisture content of 59.5% (Ohta, 1986) and partly because slimy food. it is usually prepared in small scale plants with poor quality The processing of natto includes soaking, steaming, cool- control. Cold storage has been used to extend its shelf life. ing, inoculating, fermenting and aging (Ohta, 1986; Maruo Because it contains some functional compounds such as iso- and Yoshkawa, 1989). During fermentation the steamed flavones, γ-PGA, nattokinase and oligopeptides, so, besides soybeans will produce a large amount of sticky substances it is consumed as wet and sticky form, some biotechnical or which include glutamic acid, fructan, amino acids (such as functional foods companies try to dry it into powder form glutamic acid, phenylalanine, tyrosine) and volatile fatty ac- or make capsulized form sold as functional foods. Proteases ids (such as butanediol, ethanoic acid, pentanoic acid) (Shih which are produced by B. subtilis, natto can hydrolyze soy and Van, 2001). Natto contains isoflavones, dietary fiber, vi- protein into polypeptides. The higher proteins are hydro- tamins, linoleic acid and some minerals which are originated lyzed the more peptide bonds are broken and the more free from soybeans, in addition, it also contains some functional amino acids and lower molecular weight of oligopeptides are compounds such as enzymes, bioactive peptides, natto ki- produced (Spellman et al., 2003). Shih (2006) considered nase (fibrinolytic agent), gama-polyglutamic acid (γ-PGA) that increase in steaming and fermenting time would ac- etc. which are produced by B. subtilis, natto (El-Safey and celerate the solubility of protein. When the fermenting time Abdul-Raouf, 2004; Ma et al., 2006). and inoculum of B. subtilis, natto number are not enough, Perhaps because of it possesses characteristic musty odor the bacteria are unable to utilize the protein of soybeans to and slimy appearance, natto, even though it is well known raise the solubility of protein. If the fermentation time and in Japan, is not so popular so widely consumed as compared the number of inoculum increased it will be able to raise the solubility of the protein (Chou, 2004). Liu (2005) also found *To whom correspondence should be addressed. that the steamed soybeans were inoculated with B. subtilis, E-mail: [email protected] natto would increase in protein solubility and amino nitrogen 538 T.M. Weng & M.T. Chen content. Wu (2006) reported that B. subtilis, natto inoculated tion, N:Normality of titration solution (0.1N), f :Nitrogen in soy protein added with different concentrations of sucrose factor in soybean and its products = 5.71 and sodium glutamate and incubated for different times and Protein solubility Protein solubility was determined ac- produced different contents and types of amino acids. The cording to the method described by Machado et al. (2008), conditions such as fermentation time and temperature have based on the soluble nitrogen determination in 0.2% sodium been established. Ohta et al. (1986) investigated the effects hydroxide solution and the results were expressed as percent of fermentation time on the organoleptic quality of natto, of soluble nitrogen in relation to total nitrogen in sample. they found that natto made with 8 h fermentation had the Natto (2 g) was stirred with 100 mL of 0.2% sodium hydrox- highest overall scores, whereas natto made with the tradi- ide solution at 150 rpm for 20 min at 25℃ and centrifuged at tional 16 h fermentation received a poor rating. In general, 3840 × g for 15 min. when the ammonium production exceeds a level of 1%, the Supernatant (15 mL) was taken for protein determination product becomes obnoxious. This research is to study the using the Kjedhal method. The soluble nitrogen percentage changes in TCA soluble nitrogen , protein solubility, amino (SN%) was determined according to the formulas: acid composition and electrophoretic patterns of protein, Soluble nitrogen% (SN%) = when the steamed soybeans inoculated with B. subtilis, natto ( (Vs - VB) × 0.05 × f ×1.4 / SW) × 100 and fermented at 43℃ for 0, 24, 36 and 48 h. Protein solubility (%) = (SN% / TN%) × 100

Materials and Methods where VS is the 0.05 M HCl volume (mL) used for sample Natto preparation B. subtilis obtained from Nattomoto titration, VB the 0.05 M HCl volume (mL) used for blank (Yuzo Takahashi Laboratory, Japan) was used as inoculum. titration, f the HCl standardization factor, SW the sample The organisms were grown in brain heart infusion broth weight and TN is the sample total nitrogen. (Difco 0037-17) at 37℃ for 18 h. The culture was diluted in Determination of the Degree of Hydrolysis Sample sterile distilled water containing 0.85% NaCl and 0.1% pep- preparation: 1 g of natto was mixed with 9 mL distilled water 9 tone to a concentration of approximately 10 colony forming and stood for one day at 4℃. The sample was then centri- units (cfu)/mL. fuged at 9,000 × g for 30 min. The supernatant was used for Soybean fermentation Soybeans were cleaned and hydrolysis degree determination. The hydrolysis degree was soaked in fresh water overnight at 4℃. The soaking water determined by quantification of ο-phthaldialdehyde (OPA) was then discarded, and the beans were cooked in fresh tap spectrophotometric assay, as described by Church et al. water for 20 min (ratio of bean:water = 1:3), cooled to 40℃, (1983). The OPA solution was a combination of 25 mL of inoculated with 5 mL of the diluted culture, and fermented at 100 mM in 1 mL methanol and 100 μL of β-mercaptoethanol 43℃ for 48 h. The samples were taken for analysis after 0, to create a total volume of 50 mL. An aliquot of 50 μL of the 24, 36 and 48 h of fermentation. hydrolysates was added to 1 mL OPA solution. The solution TCA soluble nitrogen content TCA soluble nitrogen was was mixed by inversion, incubated for 2 min at room tem- determined according to the method described by Nagai 14) perature, and then measured the absorbance of sample by a and modified. A 5 g dried samples were placed into the volu- spectrophotometer (Hitachi, U-2800 ). metric bottle and added the deionized water to 100mL and The content of peptides was calculated by regression blended with magnetic stirrer. Next 10 mL of blended solu- equation obtained from standard dipeptides (Leu-Gly) as fol- tion was added with 20 mL of 10% trichloroacetic acid (TCA) lows: y = 0.33x + 0.0366, R2 = 0.9979. Degree of hydrolysis solution and stored at 4℃ to extract for 16 h, then filtrated is defined as the percentage of the number of peptide bonds with Whatman #1. A 5mL of the filtrate sample were added in a protein which have been cleaved during hydrolysis 15 mL of the concentrated sulfuric acid and one granule of (Spellmana et al., 2003). DH values were calculated as fol- Kjeldahl tecator (FOSS Analytical, Sweden) and placed into lows: DH% = n × 100 / N, Where n is the average number digestive flask for carried on Kjeldahl method analysis. of peptide bonds hydrolysed, N the total number of peptide TCA soluble nitrogen content was figured out according bonds per protein molecule. to the following equation: Amino acid composition Amino acid composition was determined as the modified procedures from the methods TCA soluble nitrogen content (%) = described by Konosu et al. (1974). A 10 g natto sample (A-B) × N × 14.01 × f / g of sample × 10 was added with 20 mL 12 N HCl and hydrolyzed at 100℃ where A is the 0.05 M HCl volume (mL) used for sample vacuum oven for 24 hr. The hydrolysate 5 mL was added 15 titration, B the 0.05 M HC volume (mL) used for blank titra- mL 7% TCA solution and mixed and centrifuged by 4000 × Changes of Protein in NattoAffected by Fermenting Time 539 g for ten min at 10℃. The supernatant was added 7% TCA be utilized by the starter and resulting in TCA-N decrease. solution to make 50 mL volumetrically. A 20 mL of the so- Thirty six hours of fermentation time although TCA-N con- lution was filtrated through funnel filter and added 20 mL tent being the highest, the product may have ammonia odor. ether to remove TCA solution. This procedure was repeated Thus, this may be the reason why natto fermented for 20 to three times. Finally, the collected solution was freeze dried 24 h. The protein solubility from fermented soybeans ranged or evaporated by vacuum distillation to remove ether resi- from 32.71% to 13.69% after 36 h of fermentation, about 1.5 due. Then the solution was added extra-pure water to make to 3 times lower than the protein solubility of non-fermented 20 mL and stored at -20℃ after filtrated by 0.45 μm filter. To soybean. This could be due to the fact that microorganisms take the sample solution or standard solution 25 μL and then and enzymes involved in the fermentation process can eas- added 25 μL distilled water, 50 μL 100 mM NaHCO3 buffer ily hydrolyze soluble proteins as the inference of Song et (pH 8.3) and 200 μL DABSYL-CL solution and mixed well. al. (2008). After 36 h of fermentation the protein solubility Then the mixture was heated at 70℃ for 15 min., and mixed increased again, which suggests protein hydrolysis occurred with 700 μL 50 mM NaHPO4 and filtrated through 0.45 μm mainly secondary fermentation. filter. Finally, a 20 μL solution was taken for high perfor- Degree of hydrolysis (DH) of soy protein in natto Fig. 1 mance liquid chromatography analysis. showed the degree of hydrolysis of soy protein of natto. The Sodium dodecyl sulfate-polyacrylamide gel electropho- results indicated the peptide content and DH of soy protein resis Sodium dodecyl sulfate-polyacrylamide gel elec- increased with the fermentation time. DH did not increase trophoresis (SDS-PAGE) was performed as the procedures too much during the first 24 h of fermentation. This result described by Mujoo et al. (2003). The total proteins were was in accordance with the report of Gibbs et al. (2004). extracted using the procedures modified from the method de- They pointed out in fermented soy foods, the proteins were scribed by De Mejia et al. (2004). A 20 g milligrams of sam- only partly hydrolyzed because of the inability of most pro- ples were extracted with 0.5 mL 0.03 M Tris [Tris (hydroxy- teases to cleave glycoproteins, phosphoproteins, other post methyl) aminomethane] buffer (pH 8.0) containing 0.01 M β-mercaptoethanol (β-ME) for 1 h with vortexing every 10 Table 1. Effect of fermentation time on TCA-N and Protein min. Samples were then centrifuged at room temperature for solubility of soy protein in natto. 20 min at 11,000 × g. The protein content of the supernatant fermentation time(h) TCA-N(%D.B.) Protein solubility(%) was determined according to the Bradford method (Bradford, 1976). The protein conceration of the supernatant was dilut- 0 2.98 36.96 ed to 4 mg/mL with distilled water, and 20 μL of the distilled 24 2.98 32.71 extract were mixed with 20 μL of SDS-sample buffer (0.15 MTris-HCl, pH 6.8, 4% w/v SDS, 5% v/vβ-ME) and heated 36 3.96 13.69 at 96 for 5 min. A 20 μL of the solution cooled to room ℃ 48 4.93 39.00 temperature (20℃), containing 40 μg of protein, were loaded onto a gel containing 12% polyacrylamide. SDS-PAGE was 1. Data represent the average of triplicate determination. 2. D.B.: Dry basis performed in a vertical electrophoresis unit (Mini-Protein 3 Electrophoresis Cell, Bio-Rad) at 100 V constant voltage for 1 h, followed by 125 V constant voltage until the tracking 17 dye migrated to the bottom edge of the gel (6 h). Gels were stained with Coomassie Brilliant Blue R-250 (0.05%,v/v/v) 16 and destained in the same solution without the dye. 15 Results and Discussion 14 TCA slouble nitrogen and protein solubility Table 1 shows effect of fermentation time on TCA slouble nitrogen

Degree of Hydrolysis (%) 13 (TCA-N), protein solubility of soy protein in natto. The result showed TCA-N increased with fermentation time in- 12 creased at first and then decreased. Generally, natto fermen- 0 24 36 48 tation time takes 24 h, thus we inferred 36 h of fermentation Time (h) may be the time of stationary phase of growth of starter. Af- Fig. 1. Relationship between degree of hydrolysis of fermented ter 36h of fermentation non protein nitrogen compounds may soybean and the fermenting time. 540 T.M. Weng & M.T. Chen translationally modified species, or domains that contained matter), and the next was lysine, phenylalanine, threonine, a higher number of disulfide bridges. Terlabie et al. (2006) tyrosin,valine, histidine, and isoleucine in the decending or- reported that the first 36 h of fermentation, protease activity der. The lowest content of essential amino acid was methio- of the spontaneously fermented soy bean (Contain more than nine plus cystein. The result in sequence of content of amino one species of Bacillus) was lower, however, sharp increase acid was the same trend as the content of amino acids of the in the protease activity occurred at the advanced stages of the fermented soybean meal which was reported by Song et al. fermentation. (2008). And the highest content of nonessential amino acid Contents of amino acids of natto at different fermenta- was glutamic acid (15.57 g/100 g dry matter), and next was tion time In non-fermented cooked soybean, the highest aspartic acid, proline, arginine, Ser, Ala and glycine in the content of essential amino acid was leucine(7.36 g/100 g dry decending order. The lowest content of nonessential amino

Table 2. Effect of fermentation time on amino acid composition of soy protein in natto.

Fermented Soybean Unfermented Soybean 24 h 36 h 48 h

Non essential amino acids b 2.57 4.38 15.65 Glu 15.57 (-83.5) (-71.9) (+0.5) 1.1 3.06 4.58 Asp 9.0 (-87.8) (-66.0) (-49.1) 3.24 2.41 6.58 Arg 7.17 (-54.8) (-66.4) (-8.2) 4.16 2.38 8.25 Ala 5.65 (-26.4) (-57.9) (+46.0) 3.01 1.93 8.16 Gly 5.44 (-44.0) (-64.5) (+50.0) 2.01 1.70 5.51 Ser 5.88 (-65.8) (-71.1) (-6.3) 7.87 2.59 11.90 Pro 7.62 (+3.3) (-66.0) (+56.2) Essential amino acids b 7.56 3.03 12.13 Leu 7.36 (+2.7) (-58.4) (+64.8) 2.85 2.81 8.25 Lys 5.24 (-45.6) (-46.4) (+64.8) 3.51 1.60 5.43 Ile 3.34 (+5.1) (-52.1) (+64.8) 4.03 1.82 6.91 Val 3.57 (+12.9) (-49.0) (+93.6) 1.95 1.51 4.57 Thr 3.79 (-48.5) (-60.2) (+20.6) 4.33 1.82 6.33 Tyr 3.74 (+15.8) (-51.3) (+69.2) 5.58 2.2 9.18 Phe 5.02 (+11.2) (-56.2) (+82.9) 1.39 1.81 3.29 His 3.40 (-59.1) (-46.8) (-3.2) 0.91 0.70 0.99 Cys 0.9 (+1.1) (-22.2) (+10.0) 2.63 1.26 4.71 Met 2.46 (+6.9) (-48.8) (+91.5)

a: The figures in parentheses represented increase or decrease ratio (%) of the amino acids after fermentation as compare with those of the sample before fermentation. b: grams of amino acids per 100 g of dry matter. Changes of Protein in NattoAffected by Fermenting Time 541 acid in this study was tyrosine which was different from that in the extraction buffer solution overnight at 4℃ before ex- of the fermented soybean meal was proline which was re- traction by ultrasonic treatment. The electrophoretic patterns ported by Song et al. (2008). showed the most of soy protein components in natto disap- After 36 h of fermentation all of the content of amino peared after 24 h fermentation. This result may provide for acids decreased over 46% except for the cystein decreased industry reference. Several studies have reported the degra- 22%, and among which the glutamic acid and serine de- dation of soybean allergens (glycinin and β-conglycinin) dur- creased 71%. When the soybean was fermented for 48 h, ing fermentation by microbial proteolytic enzymes such as in most of the essential amino acids increased significantly, ex- soy sauce, miso, soybean ingredients and feed grade soybean cept for the histidine, and among which the valine increased meals improving their nutritional and functional properties 93.6% and glycine increased 50%. By the increase of amino (Hong et al., 2004; Ito et al., 2005; Kobayashi, 2005; Ogawa acids in this study we inferred that it could increase the nutri- et al., 2000; Yamanihi et al., 1995). In this study, those aller- tion values and functions of the steamed soybean after 48h of gens of soy protein subunits all degraded after 24 h fermen- fermentation. tation. However, what changes of soy protein subunits at the Changes in SDS- PAGE patterns of soya protein Fig. 2 fermenting time between 12 and 24 h may need further more shows the electrophoretic patterns of proteins in the ferment- study in the future. ed soybeans (natto) at different times. Lane 1 presents marker of protein, lane 2 presents soy beans (original sample), References lane 3 presents soaked soy beans, lane 4 presents soaked and Bradford, M. (1976). A rapid and sensitive method for the quantiza- steamed soybeans, Lanes 5-8 presents the steamed soybeans tion of microgram quantities of protein utilizing the principle of fermented for 12, 24, 36 and 48 h. From the fig 2 it can be protein-dye binding. Anal. Biochem., 72, 248-254. seen the soy protein components with molecular weight Chou, C.C. (2004). 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