Fermentation Of Citrus Pulp Residue With Dominant Strains To Improve Its Nutritional Components And In Vitro Digestion Characteristics

Citrus pulp residue is the main residue of citrus processing industry,which is characterized by seasonal production and high water content.Due to economic and environmental factors,the disposal of citrus pulp residue has been considerably limited.However,citrus pulp residue itself is rich in nutritional value,containing fiber,monosaccharide,organic acids and antioxidant substances,etc.,which is a very potential fermentation substrate.In this study,we screened the fermentable strains which can utilise citrus pulp residue,and preliminarily explored the gene function of this dominant strain.Secondly,combined with the commonly used yeasts in fermentation,the optimal fermentation species combination were determined,and the optimal fermentation conditions to improve the protein content of citrus pulp residue were explored to construct an efficient co-fermentation system of citrus pulp residue.In addition,the relationship between the utilization of organic matter from substrates by microorganisms during fermentation and the changes in the activities of key hydrolases were also analyzed,and the biotransformation mechanism that increased the protein content of citrus pulp residue was preliminarily elucidated.Finally,the effects of fermentation on protein degradation,phenolic release and antioxidant activity of citrus pulp residue during digestion were investigated by simulating gastrointestinal digestion model in vitro.The main conclusions are as follows:(1)The naturally fermented citrus pulp residue was inoculated on pectin medium for selective culture.After isolation and purification,four pectin degrading bacteria were obtained,which were identified as Bacillus subtilis by 16 S r DNA method.Fermentation strains which could better utilize citrus pulp residue were selected by mono-fermentation,and the maximum crude protein content of fermentation products was 199.40 ± 1.60 mg/g when fermented with B.subtilis L7 alone,which increased by 27.33% compared with the unfermented group.Among the 4 216 protein coding genes on the genome of B.subtilis L7,3 191,3 056,2 088 and 144 were annotated on the COG,GO,KEGG and CAZy databases,respectively.The results showed that the genes of B.subtilis L7 were mainly focused on participating in molecular functions and biological processes,among which genes related to amino acid and carbohydrate metabolism were the majority,and those annotated as glycoside hydrolases were the most.(2)Combined with the commonly used yeasts in fermentation,the optimal fermentation species combination was determined to be Bacillus subtilis L7 and Candida utilis.Optimization of solid-state fermentation conditions showed that when the initial water content was 70%,the strain inoculum was 20%,the inoculum ratio was 1:1,and the fermentation time was 96 h,the crude protein content of citrus pulp residue reached the maximum value at242.50 ± 6.50 mg/g;the contents of essential and non-essential amino acids were increased by2.30 and 4.02 times,respectively;and the contents of leucine and phenylalanine were increased to 8.3% and 5.1%,respectively;anti nutritional factors such as pectin and crude fiber decreased by 43.10% and 20.21%,respectively.Correlation analysis showed that the activities of acid protease,neutral protease,pectinase,xylanase and cellulase were significantly correlated(p<0.05)with the changes in soluble protein,total sugar and reducing sugar contents,and the results indicated that the enrichment process of proteins was closely related to the changes in organic matter and enzyme activities.(3)After treatment with simulated in vitro gastrointestinal digestion,the crude protein digestibility of fermented citrus pulp residue was 61.46%,while that of unfermented citrus pulp residue was only 37.06%.Although the total amount of free amino acids in the digestion products(residue)of both unfermented and fermented citrus pulp residue decreased significantly,Thr and Val in fermented citrus pulp residue were completely digested only at the gastric stage.In addition,the soluble protein content,total phenolic content and antioxidant activity of fermented citrus pulp residue were also significantly higher than those of the unfermented treated group(p<0.05).This result confirmed that citrus pulp residue after fermentation has a high digestion performance,and provided a basic theoretical basis for developing and producing new protein feed with high added value of citrus pulp residue.