Contents Of Beta-defensins In Milk And Their Function

Objective: To evaluate hBD-1and hBD-2concentrations inhuman colostrum and mature milk of Han Chinese, identify factorsregulating milk hBD-1and hBD-2production, and explore thepotential protective effects of milk hBD-1and hBD-2for infants. Toevaluate the concentrations of BNBD-1and BNBD-2in formula milkand fresh bovine milk, and clarify the difference. To test theantimicrobial activity of recombinant hBD-1, hBD-2and hBD-3against3international common probiotic strains, B. longum JDM301,B. lactis HN019and LGG.Methods: A total of100pairs of mothers and their babies wererecruited into the study. Colostrum samples (n=100) and mature milksamples (n=82) on day42postpartum were collected. At the time ofenrollment, a questionnaire was used to acquire information ofmother and neonate characteristics, including potential factors whichmight influence defensin levels. The babies were followed-up for a period of6months. Breast milk concentration of hBD-1and hBD-2were measured using commercially available ELISA kits. Influencingfactors was analyzed by multivariate linear regression statisticalmethods. Association of colostrum hBD-1/hBD-2and infantsinfectious diseases was analyzed. Fresh bovine colostrum samples(n=100) and mature milk samples (n=100) were collected.Concentration of BNBD-1and BNBD-2in fresh bovine milk andformula were measured using ELISA kits. The difference betweenformula, fresh milk and human milk was compared. For antimicrobialactivity assay of hBDs, the microbroth dilution method wasperformed using sterile96-well microtiter plate, slightly modifiedfrom the Clinical Laboratory Standards Institute document. Therecombinant peptides hBD-1,2and3were tested on above strains ofprobiotics to determine their antimicrobial activity in vitro, using E.coli MG1655as the reference strain.Results:(1) Concentration ranges were1.04-12.81μg/ml forhBD-1in human colostrums,0.31-19.12ng/ml for hBD-2in humancolostrum,1.03-31.76ng/ml for hBD-1in mature milk and52.65-182.29pg/ml for hBD-2in mature milk. In multivariableanalysis, pre-pregnancy BMI was significantly associated with hBD-1levels in human colostrum (P=0.001), mode of delivery wassignificantly associated with hBD-2levels in human colostrum (P=0.006) and gestation age was significantly associated with hBD-1levels in mature milk (P=0.010). The rate of upper respiratoryinfection during the first6months of life in high colostrum hBD-1group was less than low colostrum hBD-1group (χ2=4.995, P=0.025).(2) The results showed the different concentrations of BNBD-1andBNBD-2in fresh bovine milk and formula. Formula milk BNBD-1andBNBD-2concentrations were significantly lower than fresh bovinecolostrum and mature milk. Formula milk BNBD-1and BNBD-2concentrations were significantly lower than hBD-1in humancolostrum, lower than hBD-2in human colostrum, and slightly lowerthan hBD-1in human mature milk. Fresh bovine colostrum andmature milk BNBD-1and BNBD-2levels showed dynamiccharacteristics.(3) All probiotic strains tested in this study showedresistance to hBD-1,-2and-3. Then, by carbonyl cyanide3-chlorophenylhydrazone (CCCP) inhibiting efflux pump test, weinvestigated the role of efflux pump in resistance of these3probioticstrains to hBD-1, hBD-2, and hBD-3. In the presence of CCCP, hBD-1,-2and-3exhibited enhanced antibacterial effect against B. longumJDM301and B. lactis HN019, but not against LGG.Conculsions: There were high concentrations of hBD-1andhBD-2in breast milk from healthy mothers. The concentrations ofboth hBD-1and hBD-2in human milk were demonstrated to decrease over the course of lactation. Several factors that can regulatethe levels of hBD-1and hBD-2in breast milk were identified. Thestudy suggested that abundance of hBD-1in colostrum may have aprotective function for infants younger than6months. AbundantBNBD-1and BNBD-2existed in fresh bovine colostrum and maturemilk. However, BNBD-1and BNBD-2concentrations were rather lowin formula milk, which may be caused by the complex productprocess. Recombinant hBD-1,-2and-3might be safe antimicrobialagents without disturbing LAB species. Efflux pump may be one ofthe potential factors contributing to B. longum JDM301and B. lactisHN019’s resistance to hBD-1,-2and-3.