The Occurrence And Probiotic Properties Of Lactobacillus In Breast Milk Of Kashi, Xinjiang During Different Lactation Stages

Object: Breast milk is the most natural and safe food for the growth of infants.It contains all the nutrients and antibodies needed for the growth of infants,which can not only provide the most adequate nutritional needs for infants,but also ensure the development of their immune system.Breast milk,as a safe carrier of probiotics,can provide infants with Lactobacillus that can be colonized and inhibit pathogenic bacterial infections in their intestines.However,whether the species occurrence,and changes of Lactobacillus in breast milk are related to lactation are rarely reported.The experiment was conducted to investigate the occurrence of Lactobacillus population in breast milk and to isolate and screen potential probiotics from breast milk for use in the health care industry by assessing their probiotic properties.Methods: The modified culture method was used to isolate and screen the strain with MRS medium.Fingerprint(Rep-PCR Repetitive genomic fingerprinting)and homology analysis of 16 S r RNA were combined to identify the strains.The evolutionary tree was established by means of MEGA V5.0 to study the occurrence regularity of Lactobacillus populations in different stages of breast milk of uygur in kashi region of xinjiang.After genotyping representative strains of different genotypes were selected for evaluation of the probiotics characteristics such as growth curve,acid production,antimicrobial activity,antibiotic resistance,lysozyme tolerance,simulated gastric and intestinal fluid,acid and bile salt resistance hrough in vitro simulation.Results: 45 samples of Uighur breast milk from kashi region of xinjiang were divided into five stages:colostrum stage A(1-5 days after delivery),mature milk stage B(15-150 d),late mature milk stage C(151-300 d),late milk stage D(301-450 d)and late milk stage E(451-570 d).Results are as follows:(1)242 strains of suspected Lactobacillus were isolated and identified as Lactobacillus,Enterococcus and Streptococcus by homology analysis of 16 S r RNA.Based on rep-PCR,a total of 220 isolates of Lactobacillus belonging to five species,which were classified into 12 genotypes,namely,L.fermentum(82isolates,prevalence: 61.3 %,mean relative abundance: 45.4 %,genotype: 3),L.brevis(75,41.9 %,38.1 %,4),L.oris(37,32.3 %,15.1 %,3),L.gasseri(22,15.4 %,9.24 %,1)and rare L.vaginalis(4,9.7 %,1.4 %,1).(2)The relative abundance of L.fermentum decreased throughout postpartum lactation,only L.fermentum was isolated from colostrum A,while L.brevis showed an opposite trend from mature milk of stage B to late milk of stage E.L.oris was isolated from breast milk of lactation stages B,C and D,and there was no significant difference in the number of separated strains in each stage.However,L.gasseri showed a remarkable increasing trend in breast milk of stage B~C,but the strain was not isolated in breast milk of stage C,and the strain was isolated in stage E of late milk,but its quantity was greatly reduced compared with that in stage C.L.vaginalis were isolated from milk of lactation B and C.The common species of the five stages of breast milk is L.fermentum,and there are no endemic species.On the whole,from colostrum A to mature milk B,the number and species of Lactobacillus showed an uptrend,from mature milk B to late milk E,a downside was found in Lactobacillus,and the population structure of Lactobacillus in mature milk B reached the most complex level.The population variation trend of Lactobacillus was obvious in different lactation stages.(3)From all isolates,each of the three isolates represented one genotype randomly selected to estimate antimicrobial activity(36 isolates in total).Among the isolates,20 isolates exhibited broad antibacterial spectrum with inhibition halos >10 mm against most indicator pathogens,which were selected for assessing probiotic properties.Nineteen isolates showed the resistance to vancomycin and co-trimoxazole,and L.brevis KM66 was resistant up to 5/8 antibiotics.L.brevis KM147 and L.fermentum Y3 strains were selected as potential probiotics by combining growth curve,acid production,antimicrobial activity,antibiotic resistance,lysozyme tolerance,simulated gastric and intestinal fluid,acid and bile salt tolerance.Conclusion: The change of Lactobacillus population is observable throughout lactation.The results of in vitro probiotic properties test showed that among isolates isolated from breast milk with the potential to inhibit pathogen infection two strains could be used as potential probiotics.