Screening And Fermentation Of Probiotic Bacteria From Citrus Pomace To Improve Protein And Digestive Properties Of Citrus Pomace

Citrus pomace is a byproduct of citrus processing with seasonal and high water content.Due to technical,financial,and environmental conditions,the utilization of citrus pomace byproducts is very low.However,it is rich in nutrition,containing a variety of carbohydrates,organic acids and polyphenol flavonoids bioactive substances,etc.In this project,we screened the autotrophic probiotic bacteria in citrus pomace and compared them by their growth and reproduction effects in citrus pomace as well as their acid and enzyme production capacity,and explored the gene functions of some autotrophic probiotics;combined with Pseudomonas aeruginosa,which is commonly used in the production of protein feed,we explored the optimal fermentation conditions for probiotic fermentation of citrus pomace feed to improve its protein content by response surface simulation optimization method;analyzed the fermentation process The effect of microorganisms on the substrate nutrients and the changes of key hydrolytic enzymes activity during the fermentation process were analyzed,and the biotransformation mechanism that increased the protein content of citrus pomace was initially elucidated.The main conclusions are as follows:(1)Naturally fermented citrus pomace was selected and inoculated in LB and MRS media,further selective culture was performed after isolation and purification,and the enzyme and acid production capacity of each strain was detected by pectin medium,cellulose medium,casein medium,and calcium carbonate medium.Thirteen strains of Bacillus and 13 strains of Lactobacillus were identified by the 16 S r DNA method.The treatment was carried out using single bacteria fermentation method,and the changes in physicochemical properties of citrus pomace after fermentation were examined to select the superior probiotic bacteria that could better utilize citrus pomace.(2)Of the 4068 protein-coding genes on the Bacillus subtilis BF2 genome,3045,1619,3648,125 protein-coding genes were annotated on the COG,GO,KEGG,CAZy databases,respectively.Lactobacillus plantarum P10 genome contained 3131 protein-coding genes,and2569,1497,1591,110 protein-coding genes were annotated on COG,GO,KEGG,CAZy databases,respectively.bf2 and P10 functional genes were mainly involved in molecular functions and biological processes,among which genes related to amino acid and carbohydrate metabolism The majority of genes were annotated as glycoside hydrolases,and the most annotated genes were related to amino acid and carbohydrate metabolism.(3)Combining the yeasts commonly used for fermentation,the optimal fermentation strain combination was determined as Bacillus subtilis BF2 with Lactobacillus plantarum P10 and Pseudomonas spp.The single-factor optimization of solid-state fermentation combined with response surface simulation results showed that when the initial water content of the fermentation substrate was 70%,the inoculum of the strain was 20%,the inoculation ratio was at 1:1:1,the fermentation temperature was 30°C and the fermentation time was 5 days,the crude protein content of citrus pomace reached the maximum value of 161.9 mg/g,which increased by 111.91% compared with the unfermented group.Various enzyme activities were also tested during the fermentation process,and the results showed that the protein enrichment process was closely related to the changes in organic matter and enzyme activities.(4)After the simulated in vitro gastrointestinal digestion treatment,the crude protein digestibility of fermented citrus pomace was 72.97%,while that of unfermented citrus pomace was only 34.52%;there was a significant reduction in total crude fiber in the digestion products of fermented citrus pomace.We also further determined the degradation and absorption effects of fermentation and gastrointestinal digestion on citrus pomace feeds by scanning electron microscopy of the structural changes of citrus pomace feeds before and after fermentation.The results confirmed the high digestibility of the fermented citrus pomace and provided a basic theoretical basis for the development of probiotic fermented citrus pomace feed.