| Brewer’s spent grain(BSG)is the main by-product of beer production industry,which is rich in lignocellulose,protein and amino acids.The low efficiency of digestion and utilization of BSG restricts its application in food and feed production.In this study,BSG was used as raw material to isolate and screen for new probiotics,which have the ability to adapt,colonize and ferment on BSG.A multi-strain symbiotic fermentation system was constructed to improve the nutritional value of BSG.According to the investigation of the physiological characteristics and fermentative functions of the microorganisms,combined with comparative genomics analysis,the synergistic growth-promoting mechanism between the fermentation strains was revealed.At the same time,the appropriate coordination material of symbiotic fermentation was explored,and its coordination mechanism on microorganisms during the fermentation was investigated,so as to enhance the efficiency of symbiotic fermentation and further improve the production of nutrients in BSG.The main results are as follows:(1)Lactic acid bacteria with good adaptability and growth ability were isolated and screened from BSG,and a novel Levilactobacillus brevis strain LZB2 was obtained.Different combinations of cellulase,xylanase and protease were used for BSG pretreatment,and explored the fermentation characteristics of LZB2.Cellulase pretreatment could promote the growth of LZB2 during BSG fermentation,and the number of viable cells reached the highest value of 10.2 ± 0.02 log CFU/g.With the protease involved in the pretreatment,the contents of free amino acid and GABA in the fermented BSG reached 754.91 ± 49.32 μm /g and 9.51± 0.81 mg/g,respectively.Cellulase and xylanase worked together to increase lactic acid content.With the xylanase involved in the pretreatment,the soluble protein content in fermented BSG could reach 5.66 ± 0.31 mg/g.With the three enzymes worked together,the yield of total soluble sugar in fermented BSG could reach 195.57 ± 8.84 mg/g.These results indicated that enzyme played an important role in improving the fermentation efficiency of LZB2 on BSG,and LZB2 combined with different enzymes performed different effects on the improvement of fermentation products.(2)A new strain of Bacillus velezensis K8,which can produce abundant extracellular hydrolase,was isolated and screened from BSG.K8 has good capacity of growth and fermentation in BSG.The fermentative characteristics of K8 on degrading lignocellulose and increasing soluble sugar and protein in BSG were investigated.The lignocellulose contents(including cellulose,hemicellulose and lignin)decreased by 36.85 % and 27.68 % in BSG and ultrasonic-pretreated BSG(UBSG),respectively.The reducing sugar contents increased by 226.8 % and 198.1 %,and the soluble protein contents increased by 260.7 % and 258.3 %in BSG and UBSG,respectively.The arabinose,cellobiose,xylose,glucose,fructose,and xylooligosaccharides contents were significantly increased,indicating effective conversion of cellulose and hemicellulose into soluble sugars.High cellulase activity was obtained in BSG fermentation,especially CMCase and filter paper activities.Protease secretion increased but cellulase decreased,and the activities of xylanase and fructofuranosidase remained stable in UBSG fermentation.Genome sequencing and functional gene annotations showed that the proportion of amino acid metabolism related genes in B.velezensis was generally higher than that of carbohydrate metabolism related genes.Combined with the above results,it was speculated that K8 might be active in metabolism and utilization of protein nutrients but left more sugars.Pan-genome analysis of B.velezensis showed that the cellulase-coding genes pertained to the core gene families,and the xylanase-coding genes pertained to the accessory or the unique gene families,indicating that B.velezensis were more conservative in cellulase synthesis and might have more specificity in xylanase synthesis.(3)Construction of the symbiotic fermentation system with Bacillus velezensis K8 and Levilactobacillus brevis LZB2 on BSG was explored.The effects of K8 and LZB2,with different inoculated proportions,on the fermentation products of BSG were investigated,to reveal the role of symbiotic fermentation in improving the nutritional value of BSG.When the inoculation ratio(LZB2:K8 log CFU/g)was 5:5 and 5:5.5,the number of viable cells of LZB2 reached the highest level.At the later stage of fermentation,the LZB2 growth could be over thirty-fold higher than that in the single-strain system.Simultaneously,cellulase and protease activities were substantially increased,and more soluble sugars were produced.Total amino acid,glutamic acid,and γ-aminobutyric acid(GABA)levels increased by 52.2 %,155.1 %,and 144.1 %,respectively.Conversely,lactic acid production decreased.Genetic analysis showed that the glycolysis-related gene pyk A,pentose phosphate pathway gene gnd,and GABA-related gene gad were upregulated,while the heterolactin-related gene xpk A was downregulated,explaining that B.velezensis might inhibit heterolactin fermentation while enhancing growth and GABA biosynthesis in LZB2.Genome sequencing and comparative genomic analysis showed that B.velezensis might be stronger in polysaccharide degradation,while L.brevis might have advantages in carbohydrate absorption and utilization.This study provides a potential strategy for the high-value utilization of BSG in the food or feed industry.(4)The effects of different carbon sources on the symbiotic fermentation of BSG with Bacillus velezensis K8 and Levilactobacillus brevis LZB2 were investigated.It was found that chitosan(10 % w/w)could promote and maintain the growth of LZB2 and K8,and the maximum viable cells could reach 9.43 ± 0.03 and 8.79 ± 0.02 log CFU/g,respectively.At the same time,the cellulase and protease activities during fermentation were greatly improved.Compared with the fermentation without chitosan,the contents of reducing sugar,total soluble sugar and soluble protein were increased by 29.3 %,105.1 % and 117.1 %,respectively.The contents of lactic acid and citric acid were also significantly increased(p <0.05),while the contents of acetic acid,free amino acid and GABA were decreased.Genomic functional annotation analysis showed that the proportion of carbohydrate metabolism related genes in L.brevis was higher than that of amino acid metabolism related genes.Combined with pan-genome and KEGG annotation analysis,it was found that B.velezensis K8 had more abundant genes related to chitosan hydrolysis,transport and metabolism than L.brevis LZB2,indicating that K8 might have stronger ability in chitosan metabolism and utilization,revealing the mechanism of chitosan promoting the symbiotic fermentation of BSG by LZB2 and K8.(5)The chitosan microparticle(CMP)and chitosan microparticle loaded with carboxymethyl cellulose(CMC-CMP)were prepared by ion cross-linking method,to investigate the coordination of CMP and CMC-CMP in the symbiotic fermentation with Bacillus velezensis K8 and Levilactobacillus brevis LZB2 on BSG.The particle size distribution of CMP and CMC-CMP mainly concentrated in 60-200 μm,and the frequency of particle size distribution near 116 μm was the highest.Compared with CMC-CMP,CMP exhibited stronger adsorption and separation effects on lactic acid,citric acid,extracellular polysaccharides produced by LZB2 and extracellular peptides produced by K8.The addition of CMP(10 % w/w)greatly increased the cellulase and protease activities during symbiotic fermentation,promoted and maintained the growth of LZB2.The maximum viable cells reached 9.32 ± 0.05 log CFU/g,and did not cause the massive growth of K8.The reducing sugar,total soluble sugar,and soluble protein contents were increased.In addition,CMP improved the acetic acid but reduced the citric acid content at the early stage of fermentation,then reduced the acetic acid while improved the citric acid content at the later stage of fermentation.This showed a regulatory effect on the contents of free citric acid and acetic acid during fermentation.The free amino acid and GABA content reached 436.44 ± 25.80μM/g and 11.64 ± 1.07 mg/g,respectively.These results indicated that CMP could be used as an effective coordination substance in symbiotic fermentation,and improved the enzyme activities and nutrient yield during the BSG fermentation.(6)In the in vitro simulated gastric and intestinal digestion experiment,BSG pretreated with cellulase,xylanase and protease released more reducing sugar after digestion,with highest value of 256.99 ± 24.83 mg/g.BSG fermented by Levilactobacillus brevis LZB2 and Bacillus velezensis K8 produced more total soluble sugar after digestion,with highest value of683.26 ± 44.38 mg/g.BSG involved in K8 fermentation released more xylooligosaccharide after digestion,with highest value of 36.37 ± 3.50 mg/g.When protease was involved in the fermentation,the soluble protein content and crude protein degradation rate were at the high level in the digestion stage.When chitosan microparticles(CMP)was involved in the regulation of BSG fermentation,the free amino acid and GABA content in the digestion stage reached 6.27 ± 0.42 m M/g and 14.14 ± 0.96 mg/g,respectively.BSG Fermentation increased the release of lactic acid,acetic acid and citric acid during digestion,and most of the organic acids were released during gastric digestion.These results indicated that the K8 and LZB2 symbiotic fermented BSG,with high growth activity of LZB2,and high enzyme activity while K8 growth is controlled,was conducive to releasing more nutrient products during digestion.Solid-state symbiotic fermentation can improve the nutritional value and digestion efficiency of BSG,promoted its application of in food and feed production. |
PDF Full Text Request