| In recent years,the development of new alternative feed in feed industry has attracted wide attention.Microbial fermented feed is a green feed which is easy for animals to feed and absorb,rich in amino acids,short-chain fatty acids and other nutritional components and active probiotics.Mushroom feet is a kind of by-products in mushroom cultivation,which is rich in protein,amino acids,vitamins,polysaccharides,dietary fiber and other nutrients,and has the potential to develop fermented feed.However,there are few research reports on this biotransformation and its resource utilization rate is very low.In this study,Lentinus edodes feet was used as raw material for stepwise fermentation by Bacillus subtilis,Saccharomyces cerevisiae and Lactobacillus.The effects of different conditions on cellulose degradation,organic acid accumulation,growth and metabolism of probiotics in the process of feed fermentation and transformation were investigated.At the same time,analyzed the antagonism between probiotics.The thermograms and metabolic pathways of differential metabolites in the fermentation process were constructed by multivariate statistical analysis according to metabolomics technology.Then,the mechanism of feed transformation of Lentinus edodes feet at different fermentation stages,and the effect of differential metabolites on feed quality in the process of microbial fermentation were investigated,respectively,expected to provide theoretical guidance for the comprehensive utilization of mushroom feet waste resources.The main research work in this paper is as follows :In this study,5 strains of acid-producing bacteria were isolated and screened,and molecular identification and phylogenetic tree analysis were carried out.Finally,a strain with excellent acid resistance,bile salt resistance,in vitro antibacterial and antioxidant ability,Lactobacillus plantarum LP-24,was selected as the target strain.On this basis,the antagonism among the Lactobacillus LP-24,Saccharomyces cerevisiae and Bacillus subtilis was investigated.The results showed that Bacillus subtilis didn’t cause inhibition to the growth of Saccharomyces cerevisiae and Lactobacillus LP-24.Saccharomyces cerevisiae didn’t demonstrate inhibition to the growth of Bacillus subtilis,but could inhibit the growth of Lactobacillus LP-24.LP-24 showed obvious inhibition to the growth of Bacillus subtilis and Saccharomyces cerevisiae,and the antagonism was not conducive to enzymatic saccharification and fermentation of mushroom feet.Therefore,in the subsequent experiments,Bacillus subtilis was first used to aerobically ferment mushroom feet,and then the anaerobic fermentation experiments by Saccharomyces cerevisiae and Lactobacillus LP-24 were carried out step by step using the enzymatic saccharification solution.In this paper,the effects of different fermentation parameters on the saccharification performance of cellulase during aerobic fermentation of mushroom feet by Bacillus subtilis were investigated,and the experimental conditions were further optimized by orthogonal experiment.The results showed that the optimal nitrogen source was corn steep liquor,and the optimal fermentation conditions were as follows :temperature of 40°C,material-water ratio of 1: 4,inoculation amount of 30 %,and fermentation time of 48 h.Under the conditions,the degradation rate of crude fiber was35.48 %,and the degradation rates of hemicellulose,cellulose and lignin were 31.48 %,39.29 % and 22.34 %,respectively,which were 33 %,46 % and 43 % higher than those before optimization.The stepwise anaerobic fermentation experiments of Saccharomyces cerevisiae and Lactobacillus LP-24 were carried out using saccharification solution of mushroom feet as the fermentation substrate,and the fermentation conditions were further optimized by orthogonal experiment.Inoculating Lactobacillus LP-24 at 12 h after inoculation of Saccharomyces cerevisiae was most beneficial to the co-culture of Saccharomyces cerevisiae and Lactobacillus LP-24.As a result,the accumulated ethanol produced by Saccharomyces cerevisiae was less,and the ethanol stress to Lactobacillus LP-24 was slight.At the same time,the growth and metabolism of Lactobacillus LP-24 were slow,and the acid stress to Saccharomyces cerevisiae was small,which was most beneficial to production of free amino acids and organic acids.The optimal fermentation conditions for co-culture included fermentation temperature of 35 °C,the inoculation ratio of Saccharomyces cerevisiae to Lactobacillus of 1: 2,the optimal inoculation amount of20 %,and the optimal fermentation time of 48 h.Under these conditions,the viable counts of Saccharomyces cerevisiae and Lactobacillus LP-24 in the fermentation broth were 8.35 lg CFU / m L and 8.51 lg CFU / m L,respectively.The concentrations of free amino acids,acetic acid,lactic acid and p H were 8.04 μg / m L,9.26 mg / m L,29.68 mg/ m L and 4.37,respectively.The metabolites of fermented mushroom feet were detected using non-targeted metabolomics method at different fermentation stages of mushroom feet such as CK(control),Ta(Bacillus subtilis fermentation 48 h),Tb(Saccharomyces cerevisiae fermentation 12 h),Tc(Lactobacillus LP-24 fermentation 36 h).The results showed that 1262 metabolites were identified in the fermentation solution of mushroom feet,including organic acids,amino acids,carbohydrates,nucleotides,lipids,phenylpropanoids,glycosides,alkaloids,terpenoids,steroids and other secondary metabolites.The aerobic fermentation by Bacillus subtilis promoted the accumulation of small molecules such as sugars,short peptides and protocatechuic acid,stevioside and alginoside E,which provided nutrients and suitable environment for subsequent probiotics.After the inoculation of Saccharomyces cerevisiae,sugars were utilized by Saccharomyces cerevisiae,and the contents of alcohols,esters,fatty acids,amino acids and their derivatives,vitamins and terpenoids were up-regulated.A large number of flavor substances produced by Saccharomyces cerevisiae significantly improved the aroma quality of the fermented feed of mushroom feet.After inoculation of Lactobacillus LP-24,phenyllactic acid,lactic acid,protocatechuic acid,GABA,gulose,ascorbic acid and other substances were obviously riched at 96 h of fermentation,thus,enriching the nutritional and functional components of fermented mushroom feet and improving the digestion and absorption of fermented mushroom feet feed.At the same time,with the inoculation of Lactobacillus LP-24,the vitamin content was significantly reduced,indicating that the vitamin synthesized by Saccharomyces cerevisiae may be an important reason for promoting Lactobacillus LP-24 for promoting the growth and reproduction of cells.Through the metabolomics analysis of CK and Tc,801 differential metabolites in fermentation products were found.With FC > 2 or FC < 0.5 as the screening criteria,201 distinct indigenous differential metabolites were obtained,belonging to 7 primary metabolites(organic acids,amino acids,carbohydrates,esters,nucleotides,vitamins,alcohols)and 5 secondary metabolites(phenylpropanoids,glycosides,alkaloids,terpenoids,steroids).This work shown that using the step-by-step fermentation of mushroom feet by Bacillus subtilis,Saccharomyces cerevisiae and Lactobacillus LP-24,the fermentation products possessed a variety of nutrients and functional components,and have the potential to be used as alternative feeds,... |
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