Study On The Enzymatic Hydrolysis Of Glucomannan From Konjac And Its Probiotic Activity In Broilers

Glucomannan oligosaccharide(GMOS)is a functional oligosaccharide composed of glucose and mannose withβ-1,4-glycosidic bonds.It has broad application prospects in the fields of feed,food,and health care products.But there are few reports on the application of mannooligosaccharides in broiler breeding.In view of the low level of intensive processing of konjac rich glucomannan resources in China,the study on the enzymatic preparation of high value-added glucomannan oligosaccharides by konjac glucomannan and its application in broiler breeding was carried out in this paper,which provided the basis for the industrial production and application in breeding industry of glucomannan oligosaccharides by konjac enzymatic preparation.The main findings of this study are as follows:(1)Aiming at the technical bottleneck that theβ-mannanase synthesized by natural microbial strains contains mannosidase,which is not suitable for the directional degradation of glucomannan to prepare glucomannan oligosaccharides,molecular biology methods were used to convert theβ-mannan derived from Aspergillus niger.The glycanase gene was successfully introduced into th e Pichia pastoris for expression.A recombinant Pichia pastoris strain KM71H-MANNAN that can efficiently expressβ-mannanase was obtained by screening.By adjusting high-density fermentation parameters,the concentration of dissolved oxygen was controlled above 20%in 3L fermenter with 10%inoculation amount,p H value 5.0,and the activity ofβ-mannanase reached 1986.31 U/m L in high density fermentation for 96 h,which provided important technical support for the preparation of glucomannan oligosaccharides by enzymatic directed hydrolysis.(2)High purity glucomannan obtained from konjac powder was used as substrate to prepare glucomannan oligosaccharides by directional hydrolysis ofβ-mannanase.Under the optimal enzymatic hydrolysis conditions of 20 g/L glucomannan,30 U/g glucomannan,temperature 50 ~oC,p H 5.0,enzymatic hydrolysis for 2 h,the yield of glucomannan oligosaccharide reached 51.80%,and The selectivity of the enzyme to glucomannan oligosaccharides was 98.0%,and the glucomannan oligosaccharides were mainly disaccharides and trisaccharides.On this basis,the industrially scalable ultrafiltration technology(molecular weight cut-off is3500 Da)was used to prepare high-purity glucomannan oligosaccharide products.The purity of glucomannan oli gosaccharides in the obtained products was as high as96.89%and the weight-average molecular weight was 2200 Da.(3)The application experiment of glucomannan oligosaccharide as a feed additive in Partridge Shank Chicken breeding showed that adding glucoma nnan oligosaccharide in the diet had no significant effect on the growth performance and intestinal flora distribution of broilers,but it could significantly i ncrease the relative weight of the immune organs of Partridge Shank Chicken and it also significantly improved the intestinal morphology and the number of goblet cells,improved the integrity of the intestinal barrier of Partridge Shank Ch ickens,and improved the antioxidant capacity and the secretion level of immunoglobulin of Partridge Shank Chickens.When the amount of glucomannan oligosaccharide added in the feed was0.5 g/kg,the relative weight of bursa of Partridge Shank Chickens was increased by72.45%,which significantly increased the activity of superoxide dismutase(SOD)in the duodenal mucosa of Partridge Shank Chickens,and decreased malondialdehyde(MDA)content,enhanced total antioxidant(T-AOC)capacity,significantly enhanced T-AOC in jejunal mucosa,and decreased MDA content in ileal mucosa,and T-AOC in serum of Partridge Shank Chckens.The contents of s Ig A,Ig G and Ig M in the serum of 42-day chickens increased by 25%,34%and 34%,respectively.(4)The effect of glucomannan oligosaccharide on the drug resistance of Escherichia coli showed that Escherichia coli in this experiment had the most serious resistance to tetracycline and enrofloxacin,and glucomannan oligosaccharide could effectively improve the drug susceptibility of drug-resistant Escherichia coli.When the glucomannan oligosaccharide was 5g/L,the minimum inhibitory concentration of enrofloxacin against Escherichia coli decreased from8μg/m L in the control group to 0.25μg/m L.Glucomannan oligosaccharide had inhibitory effect on the expression of resistance gene of drug-resistant Escherichia coli,and the inhibitory effec t on the expression of enrofloxacin resistance gene was higher than that of tetracycline resistance gene.When the concentration of glucomannan oligosaccharide was 10g/L,the expression level of enrofloxacin resistance gene oqx AB was only 67%of the contro l group,indicating that glucomannan oligosaccharide could interfere with the efflux pump system of Escherichia coli and prevent bacteria from expelling antibiotics,so as to reduce the drug resistance of Escherichia coli.In conclusion,this paper established the preparation technology of recombinantβ-mannanase,and on this basis,it established the technology of enzymatically directed hydrolysis of konjac glucomannan to prepare high-purity glucomannan oligosaccharide products.In addition,the Partridge Shank Chickens breeding experiment and the Escherichia coli drug resistance experiment showed that glucomannan oligosaccharide has the effect of improving the antioxidant and immune capacity of animals,and had potential application value in reducing the drug resistance of microoganism in animals.