Introduction: Lactic acid bacteria have been categorized as probiotics and play a crucial role in human health by stimulating the supply of nutrients, shaping the immune system, and preventing the colonization of pathogenic microbes. Breast milk contains many bioactive compounds such as oligosaccharides, immune cells, and a wide range of different bacteria and their metabolites. Studies have shown that breast milk has 700 different types of bacteria, which include types of probiotics such as Lactobacillus, Streptococcus, and Bifidobacterium.
In this study, a lactic acid bacteria, was isolated from human breast cancer, and was identified by using Basic Local Alignment Search Tool search of its amplified 16S rRNA gene sequences. Several primary tests were conducted on isolated bacterium, including Gram staining, catalase assay, low pH and high bile salt concentration tolerance to prove the probiotic properties of isolates.
The present study characterized the probiotic potential of an isolated stain, originally isolated from human breast milk. The acid tolerance, bile tolerance, antibiotics susceptibility, and antioxidant activity of these strains was evaluated. The MCF-7 and MDA-MB-231 breast cell lines were used to test the anticancer potential of the heat killed and cell free supernatant of this strain.
Methods: 2.1 Subjects and cultivation of samples
Twenty milk samples of healthy women (aged 25 to 34 years old) were collected. All participants signed an informed consent prior to study enrollment. The participants were enrolled according to the following criteria: (1) healthy and without present or past underlying conditions; (2) no antibiotics prescription for at least three months prior to the study; (3) did not taken any probiotics prior to study; (4) normal pregnancy. The milk samples were filled in a sterile container and processed immediately upon receipt.
2.2 Isolation and screening of lactic acid bacteria isolates
The samples were diluted in phosphate buffffer saline (PBS) (0.1 M, pH 7.2) and spread on de Man-Rogosa-Sharpe agar plates. The plates were aerobically incubated at 37 °C for 72 h. Pure colonies were isolated and sub cultured for further experiments. Each selected LAB strains was subjected to tests of morphology, catalase, and Gram’s reaction. The isolates of Gram’s positive, catalase negative and rod shape were suspected to be lactobacilli and then maintained in MRSc supplemented with 20% glycerol at −80 °C.
2.3. Identification of lactic acid bacteria
Molecular identification was used to identify the obtained strain. The total genomic DNA of selected strain was extracted using the Genomic DNA purification kit (Addbio®, Korea) following the manufacturer instructions. The primers used to amplify the 16S rDNA sequences of these strains were forward 5″-AGAGTT TGATCC TGG CTC AG-3″ and reverse 5″-CCGTCA ATT CCT TTGAGT TT-3″. The fragment was amplified using a T100 thermal cycler (Bio-Rad, Hercules, USA) under the following conditions: 94 °C, 30 cycles of 94 °C for 1 min, 58 °C for 45 s, 72 °C for 1 min and finally 72 °C for 10 min. The amplified fragment was screened on an agarose gel (1%) and sequenced by Automated DNA Sequencer (ABI 3500 Genetic Analyzer). The obtained sequence was searched by using the Basic Local Alignment Tool (BLAST) program
Conventional lab techniques for analysis of LAB
2.4.1 Gram Staining
The Gram staining of the isolate was determined by light microscopy using Gram staining reagents. It is known that LABs are gram-positive. This means that these cultures will produce blue-violet color for Grampositive bacteria and vice-versa. The cultures were grown in MRS media at 37 °C for 24 h under micro-aerophilic conditions. Fresh cultures were used for gram staining. After incubation, the cultures were aseptically transferred into 1.5 ml of eppendorf tubes and centrifuged for 3 min at 9000 rpm. The cells were resuspended in sterile water by removing the supernatant.
Results: 3.1 Conventional lab techniques
The isolated strain was subjected to Gram staining and examined under a light microscope (100X magnification). The strain showed blue-purple color staining. Hence the isolated strain was found Gram-positive bacterium.
According to our result the strain was recorded as catalase negative. Absence of hemolytic activity was also observed. The result of Arginine hydrolysis test showed that the isolated stain did not produce ammonia from arginine.
3.2 Identification of Lactobacillus stain
The result showed that the isolated strain belonged to genus Lactobacillus. The 16S rDNA gene sequence result showed that isolate had 99% homology with L. fermentum.
3.3 Low pH and high bile salt concentration tolerance test
The results of acid tolerance test revealed that isolated strain tolerated acid conditions and the residual cells were more than 50% (68%) of the initial cells even after 2 h of incubation at the pH 2.5.
According to the results, Cinh for this strain was bigger than 0.4 (0.9). It means susceptibility of this strain to oxgall.
3.4 Antibiotic resistance test
The antibiotic resistance of the strains was assessed toward six antibiotics via the disk diffusion method. According to the results, the isolated strain was sensitive to tetracycline, cephalexin, ampicillin, penicillin, and trimethoprim/sulfamethoxazole. This strain was only resistant toward gentamicin.
Conclusion: In the present study, the probiotic characteristics and anti-tumor activity of a human breast milk isolated Lactobacillus was investigated. Our results demonstrated that Lactobacillus strain exhibited many typical probiotic characteristics such as Gram staining (Gram-Positive Bacilli), higher survival rate under gastric conditions (lower pH), catalase-negative, L-Arginine test, Lack of hemolytic activity, Lack of Arginase activity.
Despite the fact that this strain had most of the properties of probiotic bacteria, it did not show resistance to bile salts, and among the 6 antibiotics that were examined in this study, this strain was only resistant to gentamicin and sensitive to 5 other antibiotics.
In the investigation of the effect of cell-free supernatant and killed cells of the isolated strain on the viability of two breast cancer cell lines, it was found that the cell-free supernatant of this strain had no effect on the viability of MCF7 cell line but decreased the viability of MDAMB237 cell line in concentration of 100 µg/ml after 72 hours.
The heat killed cells of this strain (in concentration of 25 100 µg/ml after 2 h and 100 µg/ml after 48 hours) significantly reduced the viability of MCF7 cell line.
In investigating the effect of killed cells on MDAMB237cell line in all three concentrations (25, 50, and 100100 µg/ml) after 2h the viability) significantly decreased.
Keywords: cancer-breast cancer -probiotic-brest milk-anti cancer