Isolation And Identification Of High-yielding Cellulase Fungi And Application Research On Degradation Of Traditional Chinese Medicine Residues

Microbial transformation of Chinese herb residues is one of the latest research methods for resource reuse.Therefore,this study started from cellulose,the main component of licorice herb residues,isolated and screened fungi with high cellulase activity from vinegar grains,optimized their enzyme production conditions,and carried out solid-state fermentation on licorice herb residues together with probiotics.The activity and function of the fermented product were investigated by measuring the prevention and treatment effect of DSS induced colitis in mice.The results show that:(1)Fourteen cellulase-producing microorganisms were isolated from the distiller’s grains of Shanxi aged vinegar.A fungus with the enzyme production capacity of 25.618 U/m L was obtained through the determination and screening of DNS enzyme activity,and the strain was named as CZ-F.The strain CZ-F was identified as Trichoderma reesei CZ-F based on morphological,microscopic and molecular identification and phylogenetic tree construction.Both the CZ-F bacterial solution and the centrifuge supernatant had obvious degradation effect on the filter paper strip,which further proved that the bacteria had the ability of high production of cellulase.(2)The effect of different fermentation conditions(inoculation amount,fermentation temperature,initial p H value,liquid loading amount,shaking speed,etc.)on cellulase production was determined by single factor test.The results showed that incubation time was 36 h,initial p H value was 5,fermentation temperature was 28 ℃,bran dosage was 0.5 g,rotation speed was 120 r/min,and liquid dosage was 50 m L.Single factor and response surface tests were used to optimize the conditions for enzyme production: incubation time 48 h,initial p H 5.08,fermentation temperature32.2 ℃,bran content 0.47 g.Under the optimized conditions,the measured cellulase activity was 45.14 U/m L,which was close to the predicted value(43.92 U/m L),indicating that the optimized model was effective and reliable.(3)After CZ-F was fermented,the crude enzyme liquid was extracted,ammonium sulfate saturated with different concentrations(30 %,50 %,70 %,80 %,90 %)was selected for precipitation,and after centrifugation,dialysis,concentration and lyophilization,the crude enzyme powder was prepared.The molecular weight and protein content of the enzyme were determined by DNS,SDS-PAGE gel electrophoresis and BCA method.The results showed that the enzyme solution with90% ammonium sulfate precipitation had the best effect,the relative enzyme activity was 100 %,the molecular weight was about 44.3-116 KD,and the protein content was9.3015 ug/u L.The enzymatic properties of the enzyme showed that the optimum temperature was 70 ℃,p H was 3,and Na Cl concentration was 0.2 %.In addition,Fe2+ can promote the production of enzyme activity,and when the enzyme was boiling at 100 ℃ for 10 min and 1h,the relative enzyme activity was 85.30% and80.79 %,respectively,indicating that the cellulase could withstand 100 ℃temperature and had high temperature stability.(4)Through the determination of antioxidant properties,the optimal fermentation agent and fermentation method of glycyrrhiza residue were selected.The results showed that different fermentation agents had certain scavenging ability of DPPH and ABTS free radicals,and showed good antioxidant activity.FB group(Trichoderma Reesei CZ-F and lactic acid bacteria R3)had the best effect,and the solid fermentation effect was better than the liquid fermentation method.(5)Effect of solid fermented glycyrrhiza residue on intestinal inflammation induced by sodium glucosulfate(DSS)in mice.The results showed that the average daily feed intake,average daily water intake and average daily body weight of experimental group and unfermented control group were not significantly different from those of blank control group(P < 0.05),indicating no toxicity to mouse growth.HE staining results showed that glycyrrhiza residue could significantly improve the intestinal mucosal damage induced by DSS and protect the intestinal tissue structure of mice,and the treatment of glycyrrhiza residue could significantly prolong the length of colon,indicating that glycyrrhiza residue had a significant inhibitory effect on colitis in mice.The determination of inflammatory cytokines IL-6and IL-10 in serum,small intestine and large intestine of mice showed that compared with DSS induced group,both fermented and unfermented residues could reduce IL-6and increase IL-10 content,but there was no dose dependence.The oxidative damage indexes of T-SOD,CAT,MDA and GSH-PX in serum,large intestine and small intestine of mice showed that the contents of T-SOD,CAT and GSH-PX in serum,large intestine and small intestine of mice in DSS model group were significantly decreased,while the content of MDA was significantly increased.After treatment with different licorice residues,the content of MDA decreased significantly,the content of T-SOD in serum increased significantly,and the content of GSH-Px and CAT increased significantly in low-dose of fermented TLF group and low-dose of unfermented residue TL group.The contents of GSH-Px,CAT and GSH-Px in small intestine were significantly increased under four treatments.For T-SOD,the content of T-SOD in high-dose fermented residue THF group,unfermented residue TL group and TH group was significantly increased.The contents of GSH-Px,T-SOD and GSH-Px in the large intestine were significantly increased,and the CAT activity in the unfermented residue TL group was significantly increased.It can be concluded that glycyrrhiza residue can play a protective role in mice with acute inflammation by increasing the level of oxidative stress in serum and tissues and regulating the level of inflammatory cytokines.(6)Through high-throughput sequencing,the effects of different treatments of glycyrrhiza residue on intestinal microbial community composition and relative abundance in mice with DSS induced colitis were studied.The results showed that compared with the control group,Bacteroidota and Firmicutes were the dominant bacteria in the intestinal tract of mice at the phylum level.At the family level,Lachnospiraceae was the dominant bacteria in the intestinal tract of mice.At the genus level,Lachnospiraceae was the dominant bacteria in the intestinal tract of mice.Shannon and Rarefaction index indicated that the number of samples was reasonable,and the depth of sequencing was sufficient to reflect most microbial diversity information in the intestinal tract of mice.The results of Beta diversity analysis showed that DSS could disrupt the balance of intestinal microflora and change the difference of intestinal microflora composition between different groups.The treatment of licorice residue with different structure can reduce the difference of microflora among individuals and improve the similarity and stability of intestinal microflora.