Preparation And Application Of IgY Against Helicobacter Pylori

Helicobacter pylori(Hp)is a gram-negative bacterium resided on human’s gastric mucosa.Currently,it has been demonstrated that Hp is closely associated with several gastrointestinal diseases including chronic gastritis,gastric ulcer and gastric cancer.Thus,Hp has been confirmed as one of carcinogenic factors by the World Health Organization.To date,the most widely used strategy in clinical to against Hp infection is the combination of multi-antibiotics.However,the disadvantages of antibiotics cannot be evitable such as the irrational antibiotic compatibility,the dysbiosis in gut microbiota and increasing drug-resistance caused by long-term medication,and other side effects.Based on this,we developed a functional instant food to against the infection of Hp.Firstly,the recombinant Escherichia coli BL21(DE3)individually expressing virulence proteins UreB-,BabA2-or FlaA of Hp were initially constructed and their fermentation conditions in 30-liter fermenter were explored.About 28.6g/L of wet bacteria were obtained under the following conditions: TB medium was used as the basal medium,the inoculation dose was 5%,fermentation temperature was 37 °C,the pH was 6.8,the dissolved oxygen content was about 30%.After 2h’s culture,50% glycerol was added at a constant speed,50% yeast extract and 50% tryptone were added at 4 h.Then,IPTG with a final concentration of 0.5 mmol/L was added followed by incubation for 4 h.SDS-PAGE showed the expression level of each protein is over 31.4%.Then,the recombinant UreB,FlaA and BabA2 proteins were purified individually and mixed equally.After emulsifying with freund’s adjuvants and immunizing to laying hens,multivalent yolk antibody(mIgY)were obtained from egg yolks by water dilution and salting out.SDS-PAGE showed the purity of mIgY is over 95%.The recognition of prepared mIgY to recombinant UreB still can be obviously detected under 1:150000 dilution.Particularly,mIgY can effectively inhibit the growth of Hp in vitro.The high-immunity egg powder,which is obtained by the freeze-spray drying technology,was further mixed with other ingredients to produce the instant food whose brewing and liquidity are optimized.The results showed that the instant food is of the best quality when the maltodextrin,xylitol and high-immunity egg powder were added at 20%,10% and 15%,respectively.In vitro,the instant food of 60mg/mL can effectively inhibit the growth of Hp.The safety and efficacy of instant food were evaluated in vivo.After being infected with Hp for 8 weeks,the weights of Hp-infecting mice were largely decreasing and serum titer against Hp was increasing.Hp can be isolated from their atrophied and congested stomachs.However,Hp-infecting mice fed with the instant food for 1 month later showed an increasing weight and a loss of serum titer against Hp compared with placebo-treated mice,and their stomach presents an eggplant-like shape,which was similar to the shape of stomachs from normal mice.Taken together,our present study developed an instant food possessing anti-Hp.