Screening Of Probiotic Lactic Acid Bacteria Derived From Kunming Dog And Study On Fermentation Technology Of Completed Formula Feed For Puppies

Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host.Lactic acid bacteria represent a main type of probiotics,which can enhance immune regulation,produce a variety of digestive enzymes to promote animal growth,regulate gastrointestinal balance and other probiotic functions Feed fermented with lactic acid bacteria can enhance the immune function and antistress ability of livestock and poultry,and reduce the use of antibiotic prophylactic drugs.As the only independently developed working dog breed in China,Kunming dog has been widely used by the Chinese army and police.It exhibits the police performance including sensitive sense of smell,persistent galloping,alertness and flexibility,and its intestinal lactic acid bacteria resources need to be developed urgently.Rational development and utilization of fermented feed with lactic acid bacteria would be applied in the future.In this study,Kunming dog was taken as the research object,and lactic acid bacteria strains in Kunming dog feces were isolated and identified.Then,the probiotic characteristics of these strains were studied.The strains with excellent probiotic characteristics were used to ferment the completed formula puppy feed and the fermentation technology was optimized followed by analysis of nutrient level in feed.The main study contents and results are as follows:(1)Lactic acid bacteria from fresh feces of Kunming dog were isolated and identified and then their probiotic properties were determined.Using MRS supplemented with calcium carbonate selective medium,21 suspected lactic acid bacteria strains with strong acid–producing ability were isolated from the fresh feces of3807 dogs of Langqing breed and 4704 dogs and 1884 dogs of Caohuang breed.MKLQ3807-13,MKLQ3807-35,MKLQ4704-19,MKLQ4704-27,MKLQ4704-49,MKCH1884-30 and MKCH1884-43 showed good antibiotic sensitivity,antibacterial activity and tolerance to gastrointestinal fluids.Furthermore,MKLQ3807-13,MKLQ3807-35,MKLQ4704-19,MKLQ4704-27 and MKCH1884-43 had greater adhesion ability to intestinal epithelial cells HT-29.Based on physiological and biochemical characteristics and molecular biological analysis,MKLQ3807-13,MKLQ3807-35,MKLQ4704-19,MKLQ4704-27,MKLQ4704-49 and MKCH1884-30were identified as Limosilactobacillus reuteri,and MKCH1884-43 was Lactobacillus gasseri.(2)Strains of L.reuteri MKLQ3807-13,MKLQ3807-35,MKLQ4704-19,MKLQ4704-27 and L.gasseri MKCH1884-43(10~8 CFU/m L)were preincubated with HT-29 human intestinal epithelial cells for 16 h,and then pro-inflammatory agent TNF-α(50 ng/m L)was added for 3 h,total RNA was extracted,and the relative expression level of pro-inflammatory gene interleukin-8(IL-8)was detected with RT-q PCR.These results suggest that both L.reuteri MKLQ3807-13 and L.gasseri MKCH1884-43 could significantly inhibit the m RNA expression of IL-8 gene in HT-29 intestinal epithelial cells induced with TNF-α.(3)Processing technology of fermented dog feed with lactic acid bacteria was optimized.Based on the study on the probiotic characteristics of lactic acid bacteria strains and the influence on the relative expression level of IL-8 inflammatory gene,L.reuteri MKLQ3807-13 and L.gasseri MKCH1884-43 were chosen to completed formula feed for puppies.The inoculum amount of complex bacteria,the inoculum ratio of complex bacteria,the additive amount of single ester,the ratio of solid to liquid,fermentation temperature and fermentation time were selected as the factors.The response surface was used to optimize the fermentation processing of ferment the completed formula puppy feed based on single factor test with the number of lactic acid bacteria colonies as the index.The results showed that the optimal conditions of fermented feed with lactic acid bacteria were as follows:the inoculum amount of complex bacteria was 1.8×10~7 CFU/g,the inoculum ratio of complex bacteria was 1:1,the inoculum amount of single ester was 200 mg/kg,the ratio of solid to liquid was1:1.2 g/m L,the fermentation temperature was 35℃,and the fermentation time was 36h,The viable lactic acid bacteria count in final product prepared under the optimal technology was 2.32×10~9CFU/g.(4)Changes in nutrient content and sensory quality in fermented feed were determined.Compared with unfermented dog feed,the contents of crude protein and crude fat were significantly increased in fermented feed(P<0.05),while the amounts of ash,calcium and phosphorus were not significantly changed(P>0.05).After fermentation,Escherichia coli,Shigella flexneri,Salmonella typhimurium and Mold were not detected.There was obvious sour flavor,no pollution or rot,and the drying color was slightly discolored and brown.The comprehensive score according to odor,texture and color was16.In conclusion,L.reuteri MKLQ3807-13 and L.gasseri MKCH1884-43 were isolated from fresh feces of Kunming dogs,which had antibiotic susceptibility,good antagonistic effect,against pathogens and resistance to simulated gastrointestinal fluids as well as good adhesion to HT-29 intestinal epithelial cells.Completed formula puppy feed with these two strains contained greater crude fat and crude protein and abundant viable lactic acid bacteria.The results provide a theoretical basis for further research on intestinal lactic acid bacteria and fermentation of completed formula feed for puppies.