Research On Antioxidant Peptides Prepared From Fermented Peanut Meal By Using Bacillus Subtilis

Peanut, a sort of one-year-born plant, belongs to Arachis hypogaea L, and it has been grown in everypart of the world. The annual output of peanut in China exceeds14million tons and more than7milliontons of peanut is used to extract edible oil, which produces3.5million peanut meal. The peanut mealcontaining40-55%protein and many kinds of amino acids, possesses high nutritional value. However,because of severe denaturation of peanut proteins, the peanut meal is mainly used as feed or fertilizer afterextraction of peanut oil. China has abundant peanut meal sources, so it is of great practical significance tofully develope and utilise peanut meal, and there will be wide marketing prospect.In this study, the high-temperature-defatted peanut meal was fermented using microorganisms toprepare antioxidant peptides with the low molecular weight. The research covers components analysis ofpeanut meal, screening of strain, analysis of antioxidant activities of fermentation products at differentfermentation time, optimization of medium and culture conditions, isolation and purification of peanutmeal antioxidant petides, characterization of amino acid sequences, chemical synthesis of antioxidantpeptides, verification of antioxidant activities, and structure-acticity relationship. And the results are shownin the following.By comparing peptide content of fermentation liquid, DPPH radical-scavenging effect, superoxideanion radical-scavenging effect and reducing power of fermentation products, we chose target strain fromBacillus subtilis CICC20029, CICC20001, CICC20002and Bacillus licheniformis CICC20033,CICC23631, and CICC20002. The peptide concentration in fermented liquid of Bacillus subtilisCICC20001was higher （4.92mg/mL） than that of the other five strains. And the DPPH radical-scavengingeffect, superoxide anion radical-scavenging effect, and reducing power of fermentation products ofBacillus subtilis CICC20001were54.58%,25.12%, and30.8%, respectively, and all higher than those ofthe other five strains. Therefore, Bacillus subtilis CICC20001was screened as target strain.The antioxidant properties of fermentation products of Bacillus subtilis CICC20001at fermentationtime of48h,72h,96h,120h and144h were evaluated using8antioxidant assays in vitro. The resultsshowed that the fermentation products at96h took possession of better DPPH radical-scavenging effect,superoxide anion radical-scavenging effect, ABTS·＋scavenging effect and reducing power and they were72.18%,28.15%,64.23%,35.77%, respectively. In the meanwhile, the fermentation products couldeffectively inhibit autooxidation of linoleic acid, hemolysis of red blood cell induced by H2O2, andproduction of malonaldehyde （MDA）, and they were60.73%,64.28%, and61.05%, respectively. Thefermentation products at fermentation time of120h showed excellent metal chelating capacity on Fe²⁺,and the metal chelating capacity was70.18%.In order to maximize DPPH radical-scavenging effect, response surface method in Box-Behnkenprogram of Design-Expert Software was used to optimize medium ingredients and fermentationcongditions for Bacillus subtilis CICC20001. And the optimum conditions for Bacillus subtilis CICC20001were51.39g/L of peanut meal,5.27g/L of glucose,3.56g/L of KH₂PO₄,94.14h of fermentation time,initial pH8.06,36.38℃of fermentation temperature, and2.81%of inoculation ratio. Under this condition,the predicted value of DPPH radical-scavenging effect was79.40%, and the mean value of three tests was80.06%. The actual value was very close to the predicted value, and the degree of hydrolysis of thefermentation products at optimum conditions was21.41%.DA201-C macroporous resin, ultrafiltration equipment made by Pall Company, AKTA Purififierequiped with gel column, and reverse phase high-performance liquid chromatography （RP-HPLC） wereused one by one to isolate and purify peanut peptides, and the peanut peptides fraction with higher DPPHradical-scavenging effect was chosen as targer for next purification. After RP-HPLC, two peptides with higher antixodiant activity were obtained, and they were F2c and F2e. The two peptides were subjected toamino acid analysis by amino acid analyzer, and afterwards they were analysed by matrix-assisted laserdesorption/ionization time of flight mass spectrometry （MALDI-TOF/TOF-MS/MS）. The results showedthat the molecular weight of F2c and F2e was930Da and639Da, and their amino acid sequences wereLeu-Ser-Val-Cys-Phe-Cys-Phe-Leu （LSVCFCFL） and Phe-Thr-Pro-Glu-Phe （FTPEF）.In order to verify antioxidant properties of antioxidant peptides, the LSVCFCFL was synthesizedusing soLid-phase peptide synthesis and its antioxidant activities were assayed. The results showed that thescavenging effect on O₂^·-was close to that of Vc ranging from0-0.6mg/mL, and it possessed nice DPPHradical-scavenging effect. The scavenging effect followed this order: DPPH> ABTS^·+> O₂^·-. Besides,LSVCFCFL showed62.4%of reducing power at2mg/mL and68.5%of inhibition of linoleic acidautooxidation at0.8mg/mL.At last, the Hyperchem Software was used to build molecular structures of PTFEP and LSVCFCFL,optimize their geometry conformation and make them turn into preferred conformation. And then theenergies of frontier orbital and the distribution of charges of PTPEF and LSVCFCFL were simulated usingcalculation of quantum chemistry under Gaussian09Software. The results showed that antioxidant activityof peptides was related to the frontier orbital energy gap, and the antioxidant activity of the peptides can bedescribed using the frontier orbital energy gap.