| China is the largest livestock producer in the world.To ensure the animal health,food safety,and green development of animal husbandry,it is the key to develop“antibiotic reduction and replacement”products for industrial transformation.Due to the characteristics of natural ingredients,safe efficacy,and numerous functions,traditional Chinese medicine plays important roles in the prevention of diseases in animal farming.Beneficial microorganisms,namely probiotics,are important feed additives by regulating the intestinal functions,maintaining the ecological balance of gut microbiota,enhancing digestion and immunity,and so on.Probiotics-fermentated traditional Chinese medicine combines their advantages,in which one or more probiotic strains are fermented using the natural Chinese medicine as a substrate.And the fermented products are of great significance for disease prevention and control and food safety in the livestock industry.This study aimed to screen probiotics and traditional Chinese medicine for combination and optimize solid-state fermentation conditions to improve the biomass,enzyme activity,and active components of fermented products.A preliminary animal growth promotion test was carried out to verify the effect of fermentation products.The main research methods and results are as follows:1.Thirteen probiotic strains were screened on plates to test antagonism in co-culture,and Bacillus sp.strains B7-118,ACCC 01957,ACCC 02136,and Candida albicans strain ACCC 21161 had no antagonism against each other.Five traditional Chinese medicines,including Codonopsis pilosula,Astragalus membranaceus,Poria cocos,Atractylodes macrocephala,and Glycyrrhiza uralensis,were selected to prepare the water extract of 1g/m L.Plate antibacterial activity assays showed that except for the water extract of G.uralensis that had mild or moderate inhibitory effects on strains B3-388,B8-116,ACCC01957,and ACCC 02136,other traditional Chinese medicines had no inhibitory effect on the tested strains.2.The cellulase activity of each strain was assayed under pH 3.0-7.0.The preliminary screening on 96-well plates showed that Bacillus sp.B6-10~8at pH 3.0-6.0 and C.albicans ACCC 21161 at pH 7.0 had the highest cellulase activities,while other strains had the highest enzyme activity at pH 5.0-7.0;re-screening in tubes showed that the cellulase activity of ACCC 21161 was the highest,8.796 U/m L,when assayed at pH 7.0 and 50℃for 30 minutes.Under the same assay conditions,the cellulase activities of Bacillus sp.strains were much lower,and the cellulase of strain B3-388 had a wider pH-activity profile and showed the highest activity at pH 7.0.Therefore,a probiotics complex of ACCC21161 and B3-388 was prepared for further studies.3.According to the Chinese Pharmacopoeia,a compound Chinese medicine combination of C.pilosula,A.membranaceus,P.cocos,A.macrocephala,and G.uralensis at the ratio of 3:1.5:1.5:1.5 was prepared,and supplemented to the solid-state fermentation medium at the concentration of 10%to 30%.The results showed that the bacterial biomass and cellulase activity reached maxima of 3.63×10~7CFU/g and 0.314U/g,The polysaccharide content of compound Chinese medicine after fermentation was19.85 mg/m L,which was 57.8%higher than that before fermentation(P<0.05).respectively when fermented with 30%of the compound Chinese medicine for 5 days,and the fungal biomass reached 1.01×10~8CFU/g,significantly lower than the 10%group(P<0.05).4.The conditions of solid fermentation of compound Chinese medicine were optimized by orthogonal test of three factors and three levels.The results showed that 30%water content,35℃fermentation temperature and 10~8CFU/g inoculation amount were the best.The polysaccharide content of compound Chinese medicine after fermentation was 19.85mg/m L,which was 57.8%higher than that before fermentation(P<0.05).The water content and pH value in the fermentation process were monitored.It was found that the water content decreased significantly with the increase of time.The pH value increased and reached the highest value at 24 h to 48 h,and then decreased rapidly.5.Animal experiment:42male Kunming mice were selected,7 in each group,divided into 6 groups,respectively,blank group,model set,unfermented traditional Chinese medicine low dose group,unfermented traditional Chinese medicine high dose group,fermented compound traditional Chinese medicine low dose group,fermented compound traditional Chinese medicine high dose group,blank group was fed with basal diet every day,unfermented traditional Chinese medicine group was added with 2%and 4%unfermented traditional Chinese medicine respectively,fermented traditional Chinese medicine group was added with 2%and 4%fermented traditional Chinese medicine respectively,adaptive feeding for 7 days,and the test period was 9 days,The immunosuppressive mouse model was established by intraperitoneal injection of cyclophosphamide on the 7 th to 9 th day.The serum biochemical indexes and immunoglobulin content were detected 24 h after the last administration.The results showed that the average daily gain of the non-fermented Chinese medicine group was lower than that of the blank group and the fermented high-dose group.The average daily gain of the fermented high-dose group was higher than that of the low-dose group and the non-fermented Chinese medicine group,slightly higher than the normal group.In terms of feed intake,the fermented Chinese medicine group was lower than the other groups.The low-dose group of fermented Chinese medicine had the lowest feed intake,only 43.63g,followed by the high-dose group of fermented Chinese medicine,47.31 g,and the normal group was 55.53g.The high-dose group of fermented Chinese medicine reached the feed intake of the normal group with a lower feed intake and improved the feed utilization rate.In addition,the results also showed that the ALT and AST levels of the group added with traditional Chinese medicine were significantly lower than those of the model group(P<0.05),the CRE level was also significantly decreased,the ALB level was increased,and the IgG and IgM levels in the serum of mice could be effectively recovered,and the protective effect of fermented compound Chinese medicine on immunosuppressive mice was improved compared with unfermented Chinese medicine,but not significant,indicating that fermented compound Chinese medicine can enhance the immune function of immunosuppressive mice.In summary,this study screened probiotics(Bacillus and Candida)and traditional Chinese medicines(C.pilosula,A.membranaceus,P.cocos,A.macrocephala,and G.uralensis)and prepared a probiotics complex,a compound traditional Chinese medicine,and a probiotics-fermented traditional Chinese medicine.After optimizing the fermentation conditions,the fermented product had enhanced biomass and enzyme activity and showed growth promoting and disease preventing activities.It has a certain growth-promoting effect and can effectively protect the liver function of immunosuppressed mice and improve the immunity of the body.It is of great importance in the application of“antibiotic reduction and replacement”products in the animal husbandry. |
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