Breast milk is the best source of nutrition for the baby. But in some cases, the use of milk powder and complementary foods is unavoidable. A variety of these foods are available in different formulations. The formulation of these products is designed to provide the nutrients needed for the optimal growth and development of the baby. One of the most important components in breast milk is its microbial flora, which contains different types of probiotic bacteria and plays a vital role in creating the baby's natural flora and strengthening the immune system. Therefore, some types of baby food formulations fall into the category of probiotic products (1, 2). Initial colonization of the baby's gut occurs during the transmission of germs from the mother or hospital. After this inoculation, the baby's gut flora changes rapidly under the influence of diet and other environmental factors. In the first study, a comparison between breastfed infants and formula-fed infants revealed that the predominant microbial flora of infants breastfed was Bifidobacterium (3). Subsequent studies have shown that the important factors initiating the growth of these bacteria are oligosaccharides in breast milk, which have a complex structure and exist in the form of free oligosaccharides attached to glycolipids or glycoproteins and are known as prebiotics. In the past malnutrition, respiratory infection, and diarrhea were the main causes of mortality and morbidity. Feeding with breast milk had reduced this disease (4). It has been proved that the normal flora of mother milk is able to reduce diseases (5). Approximately 4 million infants die each year, mostly in developing countries (6). One study reported that breastfeeding in the first 6 months and continuing until one year of age may reduce the deaths of 1.3 million infants annually (7). In the first 6 months, nutrition with breast milk reduces the incidence and severity of diarrhea, and the risk of celiac and Helicobacter pylori infection (8). Some research on pathogenic microorganisms  in breast milk were performed, but in 2000 scientists  began to evaluate the potential benefit  of bacteria isolated from breast milk, mainly lactic acid bacteria (9). Lots of research has been carried out on the benefit of Lactobacillus and Bifidobacterium in breast milk (10). Lactobacillus are gram-positive, nonmotile, without spores. Mostly non-pathogen and antagonistic of the bacterial pathogen (11). Interference between normal flora and pathogens were taught to be considered in the treatment of infections followed by the use of non-pathogenic bacteria to control infections. Research shows that probiotics such as lactobacilli inhibit the growth of a wide range of human and animal pathogens. One of the beneficial potentials of probiotics is their antagonistic effect against harmful microorganisms so that these organisms can be useful in preventing and improving the treatment of gastrointestinal infections (12). In recent years the antibiotic resistance of bacteria led to alternative ways of treatment by using useful bacteria against harmful microorganisms, which is called bacteriotherapy (5, 10). Gram-negative intestinal bacteria, especially Salmonella, Shigella, and Escherichia coli, are the most important causes of foodborne illness and diarrhea in developing countries. On the other hand, drug resistance in these bacteria is increasing day by day. Therefore for the inhibition of pathogenic bacteria probiotics, including lactic acid bacteria and especially lactobacilli are used to prevent the growth of pathogenic bacteria (13). Considering the positive effects of breastfeeding and its compounds, including beneficial microbial flora, breast milk can be considered as a candidate to prove the probiotic properties.   The aim of this study was to investigate the probiotic properties of lactobacilli isolated from breast milk and to determine their inhibitory effect on gastrointestinal bacterial pathogens.
 

A total of 122 Lactobacillus strains were isolated from breast milk samples. To identify Lactobacillus species, carbohydrate fermentation, and growth test at 15 and 45 ° C, arginine hydrolysis, and glucose gas production were used. Among 122 isolates, 111 Lactobacillus species (90.98%) were identified by biochemical tests, and 11 isolates (9.02%) were not identified.
A total of 12 different species of Lactobacillus were identified in breast milk samples that had probiotic properties. These species included: Lactobacillus casei, Fermentum, Plantarum, Gasseri, Corvatus, Paracasia, Acidophilus, Ruteri, Bruis, Pentosus, Rhamnosus and Johnsonium. The most prevalent species of Lactobacillus casei was 43 isolates with a frequency of 35.24%. Lactobacillus fermentum, Lactobacillus plantarum and Gasseri were identified as the most abundant species with 14.75, 9.83 and 8.19%, respectively.
Evaluation of probiotic potency of isolates
Resistance acid and bile salt
Out of 122 Lactobacillus isolates, 22 Lactobacillus were resistant to acidic conditions with a pH of 2. In the next step, these 22 acid-resistant strains were examined for resistance to 0.3% oxgall bile salt. Of these isolates, 19 strains were finally resistant to both acidic and bile salt conditions, and these strains were confirmed as strains with probiotic potential.
Evaluation of antimicrobial effects of probiotic lactobacilli against gastrointestinal pathogens
The antimicrobial activity of a supernatant culture of 19 acid and bile-resistant lactobacilli isolates against a number of gastrointestinal pathogens was investigated. Antimicrobial activity of a supernatant culture of all 19 lactobacilli isolates was performed without applying temperature and enzymatic treatments. Among 19 lactobacilli isolates resistant to acid and bile, 10 strains (52.63%) were able to inhibit the growth of E. coli O157 H7, of which 6 strains had a strong inhibitory effect and 4 strains had a moderate inhibitory effect and 9 isolates had no inhibitory effect. The results showed that 12 isolates of probiotic lactobacilli (65.15%) were able to inhibit the growth of Salmonella, of which 9 isolates strongly or strongly inhibited Salmonella. 3 isolates had a moderate inhibitory effect and 7 isolates had no inhibitory effect. All 19 probiotic isolates (100%) were able to inhibit Shigella growth, of which 9 strains had a strong effect, 6 strains had a very strong effect and 4 strains had a moderate inhibitory effect.7 probiotic strains (36.84%) had an inhibitory effect on Staphylococcus aureus, of which 5 strains had a strong inhibitory effect and 2 strains had a moderate inhibitory effect. The other 12 strains of lactobacilli had no inhibitory effect on staphylococcal growth. Eleven strains of probiotic lactobacilli isolates (57.89%) had a strong inhibitory effect on Yersinia growth and 8 of them had no inhibitory effect on growth. Among 19 probiotic lactobacilli strains, 3 L4, L14, and L16 strains were able to strongly or very strongly inhibit all gastrointestinal pathogens tested (Table 1).In this study isolates with growth inhibition zone diameter <11 mm considered as negative (no inhibitory effect), 16-16 mm as a moderate inhibitor (+), 22-17 mm as strong inhibitor (++) and > 23 mm were classified as a very strong inhibitor (+++). Sterile broth MRS was used as negative control and Lactobacillus GG as a positive control.

 
Table 1. Antimicrobial activity of probiotic lactobacilli against pathogens

Evaluation of antibiotic resistance
All lactobacilli isolates with probiotic potential were tested for resistance to 8 different antibiotics (Table 2). Among these isolates, the highest antibiotic resistance was seen to vancomycin (7.73%), followed by clindamycin (1.42%), gentamicin (3.26%) rifampin (3.5%), chloramphenicol (0%) and penicillin (0%) respectively. The lowest resistance to the studied antibiotics was observed in chloramphenicol (0%) penicillin (0%).In this study, the isolates were divided into resistant (≤ 15 mm), medium sensitivity (20-16 mm), and sensitive (≥ 21 mm) based on the diameter of the growth inhibition zone.

Discussion

The presence of lactic acid bacteria, especially lactobacilli and Bifidobacterium, is very important ,because of the balance of intestinal flora(3).due to their importance in creating a proper balance in intestinal microorganisms as well as beneficial properties on neonatal health and immune system promotion (especially cellular immunity(3). The lactobacilli are the normal intestine flora (20), a heterogeneous group of lactic acid bacteria and are present in the intestinal flora of healthy individuals (20) .The baby's gastrointestinal tract is sterile until birth, and microbial colonization begins gradually after birth.  The feeding influence by different factors such as normal birth and use of antibiotic by mother (4, 9).
Probiotic strains of human origin have a significant advantage over strains that are isolated from other sources and the reason for this is the greater adaptation of these strains to the body and gastrointestinal tract (21).
The breast milk is important in for the development and production of microorganisms in the baby's intestine (22).In this study, the number of lactobacilli isolated from 100 samples of breast milk was reported to be 122, among which Lactobacillus casei with 35.2% was the most abundant species. Out of 122 strains, 11 strains (9%) could not be identified by phenotypic methods of fermentation of carbohydrates. This may be due to genes mutation related to intermediate enzymes in the fermentation of sugars that have mutated or deleted during mutation (23, 24).A study by Vega-Bautista et al, showed that 67% of lactobacilli isolated from neonatal feces belong to the group that is exclusively breastfed. It also showed an increase in the number as well as the predominance of lactobacilli in the gastrointestinal tract of breastfed infants (25). The results of the questionnaire information provided in this study showed that the incidence of diarrhea in breastfed infants was only 3%. It has been proven that the normal flora of breast milk is effective in preventing infants from infectious diseases and diarrhea (5). The highest rate of isolation was Lactobacillus casei, which is consistent with the results of the study carried out by Davoodabadi et al. They identified Lactobacillus casei as the predominant fecal flora of infants who were exclusively breastfed (13). This correlation can clearly confirm the relationship between breast milk flora and fecal flora of Iranian infants who are exclusively breastfed. Similar results were obtained in a study by Martin et al. They showed the probiotic potential of Rhamnosus Lc 705 and Lactobacillus strains, which were isolated from both breast milk and infant feces (26). In the study of Martin et al., the rate of isolation of lactobacilli from eight groups of mothers from their milk and skin flora were  178 strains, compared to our study, the rate of isolation of lactobacilli was 122 strains out of 100 mothers. This difference in the rate of separation can be affected by the type of nutrition, which varies according to geographical conditions (27). Soto et al were examined 160 samples of breast milk for the presence of lactobacilli and bifidobacteria. They isolated lactobacilli from 91.4% of the samples. The predominant species were Lactobacillus salivarius, fermentum and Gasseri, respectively. They noted that lactobacilli and Bifidobacterium was significantly lower in mothers treated with antibiotics compared to healthy mothers. The isolation rate of lactobacilli from breast milk samples was 97%. In order to isolate maximum lactobacilli in breast milk flora, mothers who were on antibiotic treatment for two weeks before sampling were excluded from the study (28). In study carreid out by Martin and Gomez on the   isolation of lactic acid family, only Plantarum species were isolated from lactobacilli, compared to our study, 12 species of Lactobacillus were isolated. This difference might be due to regional such as genetics, health status, maternal nutrition and geographical (29, 30).
The results of the study of antibiotic resistance pattern of lactobacilli isolated from breast milk in this study indicate the sensitivity of this strain to a large number of antibiotics and, conversely, resistance to vancomycin. The vancomycin resistance is an intrinsic feature of lactobacilli (31). Kumar et al. evaluated 80 breast milk samples from four different geographic regions, in Europe, South Africa, and China. Their results showed that breast milk in different regions were completely different for the presence of microbial flora (32). Also, the results of studies performed on 133 breastfeeding mothers from different regions of Taiwan and China, showed that microbiome diversity and the composition of the microbial flora of breast milk was different (33).

 

Conclusion

The results of this study showed the diversity of species of Lactobacillus in the milk of healthy mothers. This might be due to the type of nutrition, diet, lifestyle, age, climatic and geographical conditions.

 

Acknowledgements

This research was supported by TUMS under contract number 29352.

 

Conflicts of Interest

The authors declare that they have no conflict of interest.