Bacillus Natto Liquid Fermentation With Buckwheat To Produce Nattokinase And Identification Of Antioxidants In Fermentation Products

Nattokinase(NK)is a serine protease produced by Bacillus natto during natto fermentation.The development of products with high nattokinase activity had become a research focus in the field of functional food with thrombolytic efficacy.It was an important method for reducing the cost of nattokinase production by selecting the characteristic fermentation substrate and optimizing the fermentation medium and fermentation conditions to achieve efficient use of the fermentation substrate.The enzymatic preparation of soybean protein hydrolysate from soy protein isolate was environmentally friendly and safe,which could replace the expensive peptone products used in the domestic fermentation industry to reduce the cost and improve the safety of food.In this paper,buckwheat was used as a substrate to obtain high-activity nattokinase and fermentation products rich in phenolics and with strong antioxidant activity through liquid fermentation.This provided a theoretical basis for the development of functional foods with significant thrombolytic and antioxidant activity using Bacillus natto.The main research results are as follows:(1)The protease activity was increased from 423.38 U/mL to 1340 U/mL,and nattokinase activity was increased from 340.45 U/mL to 630 U/mL by single-factor optimization.Brown rice and buckwheat could significantly promote the production of nattokinase and protease by Bacillus natto fermentation.The enzyme activities of protease and nattokinase of the fermentation product could be significantly improved by soaking and cooking four grains and extending fermentation time.Addition of cereals(especially buckwheat)could significantly increase the antioxidant activity of Bacillus natto liquid fermentation products.Cereal phenolics,peptides and microbial metabolites contributed largely to the antioxidant activity of the fermentation product.(2)The fermentation medium with buckwheat as a substrate was obtained by soaking at room temperature,pulping,and pre-treating with heat-resistant ?-amylase.The fermentation product rich in nattokinase(152.5 FU/mL),phenolics(0.109mg/mL),antioxidants(27.43 ? mol trolox equiv/mL)was obtained by the optimal liquid-state fermentation of buckwheat in shakeflask and 2.5 L fermentor using Bacillus natto when soy protein isolate hydrolysate was used as supplementary nitrogen during fermentation.The changes in the growth of Bacillus natto and metabolism of nutrients(nattokinase,soluble protein,reducing sugar,phenolics and antioxidants)during fermentation using soy protein isolate hydrolysate and commercial soybean peptone as supplementary nitrogen were comparatively studied.The enzyme activity and stability of natto freeze-dried powder with the supplyment of skim milk powder could be comparable to famous import goods,and was significantly higher than domestic natto freeze-dried powder in acceleration experiment of stability.(3)Eight phenolic compounds including caffeic acid,protocatechuic acid,syringic acid,chlorogenic acid,catechin,rutin,vitexin and hyperin were identified.The elution fraction rich in antioxidant peptides was obtained(protein content:66.51%;ORAC:1731.52 ?mol Trolox equiv/g)by isolation and purification using ultrafiltration,ethanol fractionation and XAD-16 macroporous resin from the fermentation product.Twenty four peptides(>heptapeptides)were obtained using MASCOT database.The peptides had not been reported in BIOPEP database.All peptides contained hydrophobic amino acids.Thirteen peptides contained at least one strong antioxidant amino acid.The IY dipeptide fragment in GDKIYNVF,the LY dipeptide fragment in CRNLLLYPAGA,the PW dipeptide fragment in PWFFTST,and the GPP tripeptide fragment in QGSGGPPIV had been reported to be antioxidants in BIOPEP database.The identified phenolics and peptides contributed largely to the antioxidant activity of the fermentation product.