Production Of Soybean Bioactive Peptides By Fermentation Of Defatted Soybean Flour With Bacillus Subtilis

Soybean peptides, which derived from hydrolysis of soybean protein by acid, alkali or enzyme,were the general name of a group of peptides chain with different amino acid composition. Themolecular weight rang of them was defined from 300 to 1000Da with 3～10 amino acid now. They werealso defined as soybean bioactivity peptide or soybean functional peptide due to their good processfunction and bioactivity beneficial to human body and focused by researchers for their potential offunctional food.There are lots of studies about the production of soybean peptide in China but the industry practicein large scale still under the way due to two reasons: 1 the cost of production is high.2 the bitterness ofhydrolyzate harm to the taste of peptide produce badly.We established a production technology of soybean peptide with biotechnique in order to overcomethe shortage of the studies and industry practices on soybean peptide in our country. The soybeanpeptide with bioactivity was produced by fermentation defatted soybean flour with microorganism. Asuitable technology of fermentation was established by optimization with response surface methodology.The purification of soybean bioactivity peptide by membrane technique was studied. The enzymeproduction and the mechanism of debitterness of fermentation strain were discussed. The bioactivity andprocess character of soybean peptides produced by fermentation was studied and a soybean peptidebeverage with antifatigue function was developed. The main results were described as following:1. A new method which was suitable for fermentation technology and used for detect the degree ofhydrolysis (DH) was established at the base of traditional method. The DH was measured by calculatingthe number of peptide-bond split in new method. It accord to the rule of protein hydrolysis at the theoryand reflect the defining of DH. This method proposed in this paper was shown to be direct, sensitive,accurate and reproducible. 2. The hydrolysis dynamic of soybean protein in defatted soybean peptide under the effect of enzymeproduced by Bacillus subtiliss SHZ was studied. Results showed that B. subtiliss SHZ can yieldedproteinase and carboxypeptidase, at the beginning of fermentation (0～8h), the strain mainly yieldedproteinase which can hydrolyzed the soybean protein into long chain peptide with slight bitterness;during 8～16h of fermentation, long chain peptide were further hydrolyzed into middle and short chainpeptide. The amino acids were exposed at the effect of carboxypeptidase which resulted to bitternessincreasing. During 24～32h, strain yielded Carboxypeptidase in larger number which can cut down theamino acids from the C-terminal of peptide and at the end of fermentation, there are mainly short chainpeptidase and free amino acid with low bitterness. When fermentation for 36h, the DH was 25% andmolecular weight range of soybean peptide was from 300 to 1000Da.3. The bitterness value and amino acid composition of enzyme hydrolyzate of soybean protein andsoybean peptides from the fermented liquid of defatted soybean flour were compared each other. It wasfound that there are 37.69% of hydrophobe amino acid occured in the form of peptide in enzymehydrolyzate which exhibit strong bitterness. While there is 11.75% of hydrophobe amino acid infermented liquid of defatted soybean flour and they are mainly in the form of free amino acid. Thebitterness values of enzyme hydrolyzate were reduced from 4 to 0 when fermented with B. subtilis SHZfor 20h. It proved the debitter ability of B. subtilis SHZ and it may be the effect of carboxypeptidaseproduced from B. subtilis SHZ. The enzyme analysis of strain showed that the strain can excretecarboxypeptidase during growth. After purified, the special activity of the carboxypeptidase from B.subtilis SHZ were increase from 4.5 to 24.5, but the loss of enzyme protein and total carboxypeptidaseactivity is huge, only 12.7% were reclaimed. The amino acid such as Phe, Ala, Tyr and Leu at theC-terminal of peptide of enzyme hydrolyzate can be cut down by the purified carboxypeptidase from B.subtiliss SHZ and result to nonbitternss. This is the main mechanism of debitterness of B. subtiliss SHZ.4. In order to obtain the highest yield of soybean peptides, the medium composition and fermentationconditions of B. subtiliss SHZ were optimization by the response surface methodology. First, themedium composition were optimized used Box-Behnken design and the optimal medium compositionwere defatted soybean flour 52～58 g/l, glucose 7～9g/l, KH₂PO₄ 3g/l. It can obtain 8mg/mL soybeanpeptide at this medium and it was increased about 33% compared with the 6mg/mL before optimization.The fermentation conditions were also optimized used central composition design (CCD) and theoptimal conditions were fermentation time 36h, temperature 37℃, initial pH 7 and rotary speed was140～160rpm. The yield of soybean peptides can attain 14mg/mL at this optimal condition and it was increased about 65% compared with the 8 mg/mL which attained before. The transferring ratio ofsoybean protein to soybean peptides was 60% at optimal medium composition and fermentationconditions which is three times compared with traditional enzyme technique.5. The purification effect of ultrafiltration of fermented liquid was studied. Results showed that, thereare 0.85% of peptide with molecular weight over 30KDa, 2.42% of peptide with molecular weight from30KDa to 10KDa, 20.4% of peptide with molecular weight from 5KDa to 3KDa and 63.42% of peptidewith molecular weight less than 3KDa. The bioactivity analysis showed that the peptide with highmolecular weight (≥10KDa) exhibit low free radical scavenging ability. The peptide with molecularweight≤5KDa exhibited strong free radical scavenging ability. The scavenging ability to O^-2·and·OHof peptide with molecular weight≤3KDa were 49%and 82%, respectively. The optimal flux andisolation effect can be attained at liquid concentration 50mg/mL, liquid temperature 30℃, operatedpressure 0.08MPa. The molecular weight distribution of ultrafiltration fraction showed that the peptidechain was shorter as the cut-off molecular weight decrease and the purity was also increased. The gelfraction of soybean peptides with the highest bioactivity was a single peptide proved by RP-HPLC.6. The study of bioactivity of soybean peptides from fermentation showed that the soybean peptides havesignificant free radical scavenging ability and antioxidant activity in vitro. As the purification developing,the scavenging ability of O2—·was increased from 49% to 62%, the scavenging ability of·OH wasincreased from 78% to 90% and the relative antioxidant value was increased from 2.0 to 2.8. Themolecular weight of the peptide with the most antioxidant activity in vitro is about 1,300Da. The resultsof antioxidant experimental in vivo showed that the activity of GSH-PX and SOD in blood of mouseincreased and the MDA content in heart of mouse decreased after being feed with different doze soybeanpeptide (P＜0.01). The increasing of GSH-PX and SOD in blood serum indicated that the antioxidantactivity of soybean peptide were related to the increase the activity of antioxidant enzyme in vivo. Theantifatigue activity in vivo of soybean peptide produced by fermentation was examined and approved byanimal model. The swimming time of rat feed with different doze of soybean peptide were increasedmarkedly approved the anti-fatigue effect of soybean peptide. The glycogen content of rat increasedmarkedly showed that the anti-fatigue effect was related to the increasing of energy storage. Theincreasing of lactic acid in the blood of rat was deferred and the reduction of lactic acid in the blood ofrat was accelerated after being feed with different doze of soybean peptides. The conclusion can be drawthat the soybean peptides produced by fermentation have the antifatigue function according to theevaluation index of health food published by sanitation ministry. 7. The study of process character of soybean peptides from fermentation showed that the soybeanpeptides have good solubility and can keep the high NSI over 80% at abrooad pH range. Its gelation andhydrophile ability were increased compared with soybean protein. At the same time, soybean peptideshave good emulsification and can keep the good EAI and ESI at pH from 2～8. The soybean peptideswith more short chain peptides produced by fermentation have higher foaming and foaming stabilitycompared with the soybean peptides produced by enzymatic hydrolysis.8. A functional beverage based on the soybean peptides produced by fermentation was developed. Theoptimal prescription was following:β-dextrin 0.4%, citric acid 0.2%, sucrose 9%, OJ essence 0.4%.The optimal sterilization technique was microwave sterilization (80℃, 10min) and the optimal storecondition wascold storage at 4℃. The beverage can keep constant at this condition for 2 monthswithout any additive. The results of animal experiment showed that the swimming time of mice feedwith different doze of beverage increased significantly and the swimming time of high doze group wastwo times compared with check group. It can be conclude that the beverage have some antifatiguefunction.