Occurrence and probiotic properties of Lactobacillus spp. of maternal breast milk in the Uyghur, Xinjiang China, at different lactation stages

As a secure vehicle of probiotics, breast milk can provide babies with the Lactobacillus which can not only colonize and inhibit the pathogenic bacterial infection in infant's intestines. The purpose of this study was to assess the occurrence and probiotic properties of Lactobacillus spp. of maternal breast milk in the Uyghur population, Xinjiang China, by using culture method. Based on repetitive genomic fingerprinting PCR (rep-PCR), a total of 198 isolates of L actobacill us from 31 different lactation of breast milk samples of Uighur in kashi region of Xinjiang were classified into 11 genotypes, which were identified as Lactobacillus fermentum (82 isolates, prevalence: 61.3%, mean relative abundance: 45.4%, genotype: 3), Lactobacillus brevis (75, 41.9%, 38.1%, 4), Lactobacillus oris (37, 32.3%, 15.1%, 3) and rare Lactobacillus vaginalis (4, 9.7%, 1.4%, 1). From colostrum to mature milk, the number and species of Lactobacillus showed an uptrend, from mature milk to late milk, a downside was found in Lactobacillus . The relative abundance of L. fermentum decreased throughout lactation, while L . brevis showed an opposite trend. Three isolates were randomly selected for each genotype to estimate antimicrobial activity (33 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 KM66 ( L . brevis ) was resistant up to 5/8 antibiotics. KM147 ( L. brevis ) and Y3 ( L. fermentum ) strains were selected as potential probiotics by combining lysozyme, acid and bile salt tolerance. The optical density (OD 600nm 37 and The % resistance = the residual quantity of OD 600nm in MRS broth with different concentrations of lysozyme / increment of OD600 nm in MRS broth without lysozyme.


Background
As a secure vehicle of probiotics, breast milk can provide babies with the Lactobacillus which can not only colonize and inhibit the pathogenic bacterial infection in infant's intestines. The purpose of this study was to assess the occurrence and probiotic properties of Lactobacillus spp. of maternal breast milk in the Uyghur population, Xinjiang China, by using culture method.

Results
Based on repetitive genomic fingerprinting PCR (rep-PCR), a total of 198 isolates of L actobacill us from 31 different lactation of breast milk samples of Uighur in kashi region of Xinjiang were classified into 11 genotypes, which were identified as Lactobacillus fermentum (82 isolates, prevalence: 61.3%, mean relative abundance: 45.4%, genotype: 3), Lactobacillus brevis (75, 41.9%, 38.1%, 4), Lactobacillus oris (37,32.3%, 15.1%, 3) and rare Lactobacillus vaginalis (4, 9.7%, 1.4%, 1). From colostrum to mature milk, the number and species of Lactobacillus showed an uptrend, from mature milk to late milk, a downside was found in Lactobacillus. The relative abundance of L. fermentum decreased throughout lactation, while L . brevis showed an opposite trend. Three isolates were randomly selected for each genotype to estimate antimicrobial activity (33 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 KM66 ( L . brevis ) was resistant up to 5/8 antibiotics. KM147 ( L. brevis ) and Y3 ( L. fermentum ) strains were selected as potential probiotics by combining lysozyme, acid and bile salt tolerance.

Conclusion
Species composition and prevalence of Lactobacillus varied with the study subjects throughout lactation phase. The deliberately selected Lactobacillus strains from breast milk may have a great potential as probiotics to inhibit pathogen infection in infants.

Background
With the improvement of people's living quality and the constant pursuit of healthy food, there is a growing interest in probiotics, especially the lactic acid bacteria (LAB) viz. Lactobacillus spp., Bifidobacterium spp., etc (1). It is well known that Lactobacillus is a fabulous probiotic LAB, a grampositive, non-spore forming, facultative anaerobic bacteria which plays a significant role not only in fermented dairy products, but also in beverages and silages (2). Particularly, the Lactobacillus has been demonstrated to provide many health benefits including inhibition against the infection of pathogenic bacteria, enhancing the body's immunity as well as preventing the occurrence of bacterial flora imbalance and bacterial displacement (3)(4)(5).
It has been found that probiotics in infants gut primarily come from breast milk and the mother's gut and vagina. Given the risk of antibiotic resistance, breast milk is deemed to be a more secure source of probiotics relative to other environments (6). As a vehicle of probiotics, breast milk has the motherto-infant transfer of some bacteria that can colonize in the infant's intestines (7). Probiotics such as Lactobacillus and Bifidobacterium in breast milk have profound effects on the development of intestinal flora and immune system of infants (8). Additionally, the microbiota of breast milk is generally complex and variable: it reaches the most complex level at the mature milk, and then it stays constant and drops sharply at late milk (9). Lactobacillus is abundant in breast milk, among which the most commonly reported are L. fermentum, L. rhamnosus, L. salivarius, etc (7). To date, the occurrence of Lactobacillus in different lactation stages of breast milk is not clear. Whether the prevalence of Lactobacillus in breast milk is related to different lactation periods is of great significance for understanding the occurrence of Lactobacillus in breast milk and the intestinal metastasis of these bacteria in infants. Since most babies are breastfed after birth, and it's confirmed that it can prevent neonatal allergy, malnutrition and infections from breastfeeding (10). Based on it's potential probiotic properties, the antimicrobial activity of the Lactobacillus isolated from breast milk has become a hotter topic to scientists around the world. Some studies have found that Lactobacillus isolated from breast milk has probiotic properties, such as improving intestinal tract, therapeutic effect of diarrhea and adjuvant therapy of some diseases (11). For example, the L. gasseri CECT5714 and L. fermentum CECT5716, originated from breast milk have been shown to play a crucial role in inhibiting pathogen infection based on the mechanism of producing antimicrobial bacteriocins to compete with pathogens in intestinal tract (12,13).
In addition, breast milk microflora is closely related to human lifestyle, dietary habits, regional differences, delivery mode (14,15). Xinjiang is a large and multi-ethnic residential area with unique Uighur living habits and food culture. Thus, the breast milk of Uighur in Xinjiang provide a rich source of unique host for the exploiture and utilization of Lactobacillus. In this study, the aim was to assess the occurrence of Lactobacillus spp. of maternal breast milk in the Uyghur population, northwestern China, by using culture method, and then evaluate their key probiotic characteristics including antimicrobial activity, antibiotic resistance, lysozyme, acid and bile salt resistance. We hope to screen out lactobacilli probiotics with significant antibacterial properties, and finally apply them to health food after in vivo experiments. According to different lactation period, breast milk samples were divided into five stages, they are colostrum stage A (1-5 postpartum days), mature milk B (15-90 days), late mature milk C (91-300 days), late milk D (301-450 days) and late milk E (451-570 days) (16). As shown in Table 1, during the whole lactation period, the number of lactobacilli in breast milk samples showed a trend of mature milk B > late milk E > late milk C > colostrum A > late mature milk D, and the diversity showed mature milk B = late mature milk C > late milk D > late milk E > colostrum A. At the level of species, the mean relative abundance of L. fermentum, L. brevis, L. oris and L. vaginalis in five stages was 45.4%, 38.1%,15.1% and 1.4% respectively. L. fermentum was the dominant species, and only L. fermentum was isolated from colostrum A. L. fermentum, L. oris and L. vaginalis had the highest isolation rate (22.2%, 13.6% and 1.5%) in mature milk B, and L. brevis had the highest isolation rate (16.7%) in late milk E. The 4 isolates of L. vaginalis were isolated from mature breast milk B and late mature milk C.

Result Molecular identification and occurrence of Lactobacillus in breast milk
As shown in Fig. 1, L. fermentum were identified from all 5 stages of lactation (A-E). Meanwhile, the relative abundance of L. fermentum showed a decreasing trend throughout the lactation. On the contrary, the relative abundance of L. brevis increased gradually, and reached the highest value (76.7%) in late milk E. Principal component analysis (PCA) (Fig. 2) showed that all shapes were distributed concentratedly and no significant independent population was formed, indicating that populations of Lactobacillus isolated during five different lactation periods were similar. According to the dispersion degree of the shape in the figure, the population structure of Lactobacillus isolated in later milk D was more similar than that in other stages, while that in mature milk B was vice versa.
Three isolates were randomly selected for each genotype to assess probiotic properties (33 isolates in total). The strain ID, genus names and species names were shown in Fig. 3.

Assessment of the antimicrobial activity
The antibacterial property is one of the significant criteria of Lactobacillus for screening potential 6 probiotics. It was reported that some Lactobacillus strains play a pivotal role in the inhibition of the infection caused by pathogenic bacteria in infants (6,17  The diameter of AGAR wafer are 7 mm, -= no activity, ± = standard deviation (S.D), assays were carried out in quadruplication.

Antibiotic resistance profile
Since the overuse of antibiotics, antibiotic resistance has become one of the important criteria for selecting potential probiotics (18). The antibiotic susceptibility of breast milk Lactobacillus was evaluated by the K-B paper method and results are shown in Table 3. All 20 isolates were found sensitive to ampicillin, rifampicin and tetracycline, and only KM66 was resistant to chloramphenicol.
All isolates exhibited antibiotic resistance to vancomycin and co-trimoxazole except KM4 and KM11 respectively. Fifty-five percent of isolates showed resistance to gentamicin (Fig. 4). In particular, KM66 is the most obvious multidrug-resistant isolate with resistance up to five antibiotics (chloramphenicol, gentamicin, co-trimoxazole, penicillin-G and vancomycin). Table 3 Antibiotic susceptibility of lactobacilli using the K-B paper method after 24 incubation at 37 ℃.

Resistance of lysozyme
Assessing their resistance to extreme conditions in the digestive tract is an important criterion for selecting potential probiotics (19,20). The obstacles to overcome, in turn, are high concentration of salivary lysozyme in the mouth, low pH in the stomach and bile in the intestinal fluid (21). After 30,90 and 120 minutes of lysozyme treatment, bacterial survival activity was shown in Fig. 5. Interestingly, among 20 isolates, only the KM50 and GM3-1 were sensitive (survival: 34.7% and 44.4%) to the lysozyme treatment after 120 min respectively. The rest of isolates exhibited resistance to lysozyme with resistance > 50%, among which 8 isolates showed severe resistance to the treatment of lysozyme (with resistance > 80%), which proved that Lactobacillus had high lysozyme tolerance.
These eight isolates were selected for further research.

Resistance of acid and bile salt
The survival of Lactobacillus under low acid condition and high concentration of bile salt is of great significance to withstand gastrointestinal environment stress (22). Among 8, 5 isolates (KM66, KM86, KM147, Y3 and GM12-2) were resistant at pH 3.0 with resistance > 50%, and the maximum resistance were observed with KM66 (61.078%) and KM147 (61.077%), followed by the Y3 (55.205%) at pH 3.0.

Discussion
The 198 isolates of the isolated Lactobacillus belong to four species: L. fermentum, L. brevis, L. oris, and L. vaginalis. It has been reported that L. fermentum and L. rhamnosus were the most frequent species from breast milk at the level of species (23). In the present study, L. fermentum was the dominant species, which was the same as the results obtained by Soto et al. (24). Moreover, the present study also found that L. fermentum had 100% of relative abundance in colostrum. However, Ozgun et al. revealed that L. brevis was frequently found from colostrum samples from Turkey (25).
This may be related to differences in region, the lifestyle and dietary habits of the mother. For colostrum A to mature milk B, the number and species of Lactobacillus isolated showed an uptrend, mature milk B reached the highest level, and showed a downside from mature milk B to late milk E.
Similar results were reported by Solis et al., who also found that the Lactobacillus spp. in mature milk (10-90 postpartum days) has higher isolation ratio and more species than colostrum (1 day) (26). It has been reported that the breast milk composition influence microbiota (27). Therefore, we can speculate that the growth of some Lactobacillus may be related to changes of nutrient composition in breast milk during different lactation periods. And mature milk is more suitable for Lactobacillus multiplication. In the present study, four isolates of L. vaginalis were rarely isolated from breast milk of stages B and C, which is seldom reported in other studies. Ana et al. detected L. vaginalis in only 2 of the 27 breast milk samples (24). This may due to its harsh survival requirement or the introduction of new technologies, such as metagenomics and sequencing, which allowed the rapid development of Lactobacillus isolation. Lactobacillus are notoriously widespread in the skin, mouth cavity, gastrointestinal and vaginal cavities of humans (28). As previously reported, L. vaginalis was usually isolated from the vaginas of women (29). As demonstrated by Ramsay et al., a degree of reflux into the mammary duct during breast-feeding may facilitate the exchange of bacteria between mammary glands and the oral cavity of infants who acquire bacteria from the vaginal microbiome at birth (27).
This shows that the presence of L. vaginalis in breast milk may be associated with vertical transmission during vaginal birth and breastfeeding.
Since the probiotic characteristics of Lactobacillus are specific to strains, the feasibility of exploring new strains is determined by these probiotic characteristics (30). The authors found that the isolates The risk of Lactobacillus transmitting antibiotic-resistant genes to intestinal pathogens cannot be ignored. With regard to antibiotic resistance, 95% of isolates exhibited antibiotic resistance to vancomycin and co-trimoxazole, though the majority of isolates were found susceptible to other antibiotics. These results are similar with other research (36,37). Mohammadi et al also showed that 94% of the Lactobacillus isolated from human milk were resistant to vancomycin (38). It has been reported that resistance to vancomycin and co-trimoxazole in certain Lactobacillus strains is not a safety issue since it is encoded by chromosomes rather than acquired, and therefore not transmissible (39). For safety assessment, the resistance to vancomycin can be used as a useful criterion for screening safe probiotic strains of Lactobacillus (40).
Ninety percent of candidates were tolerant to the lysozyme, suggesting that Lactobacillus had high lysozyme tolerance, which was confirmed by other authors (16,41). However, only 3 isolates were   Table 4 Rep-PCR amplification system Assessment of antimicrobial activity Antimicrobial activity of Lactobacillus against pathogens was detected by the modified sandwich overlay method as described (46). The 100 µl bacterial suspension of each bacterium (10 7 - was drilled with a hole punch (diameter:7 mm) and the agar blocks were immediately placed onto each indicator pathogen plate. The inhibition zone diameter, which inhibited the growth of pathogens obviously near the colony was measured and the results were divided into < 10, 10-20 and > 20 mm.
MRS agar was taken as the negative control and assays were carried out in quadruplication.

Antibiotic resistance
The antibiotic resistance profiles of isolates were determined by the K-B paper method with the following 8 different types of antibiotics tablets: penicillin-G (10 µg), gentamicin (120 µg), rifampicin

Consent for publication
Not applicable

Availability of data and materials
All data and materials are available on request for academic use. No administrative permission is required but approval from a local hospital is required to obtain breast milk samples from healthy individuals. The individuals mentioned in the method had no associated disease. The 16S r RNA gene sequence can be submitted to the National Center from Biotechnology Information (NCBI) database.

Competing interests
The authors declare that they have no conflict of interests.

Authors' contributions
YQ and FW conceived the study. YQ participated in the experimental design. Manuscript was written by XJ and edited by YQ, XL and YZ. Each author informed the consent to participate of this publication.

Figure 1
Relative abundance of Lactobacillus species in different lactation period Antibiotic resistance rate of Lactobacillus from breast milk Effect of lysozyme treatment on the viability of Lactobacillus Effect of different acid pH values and bile salt concentration on survival of Lactobacillus isolates