Characterization of Biosurfactant Produced by Probiotic Bacteria Isolated from Human Breast Milk

Introduction: Breast milk is an important nutrient source for rapidly growing neonates since breastfeeding protects newborns against a variety of diseases. This effect may be due to the useful and natural microflora of breast milk. Biosurfactants are unique amphipathic compounds produced by some microorganisms. The present study demonstrates the isolation and characterization of biosurfactant generating bacteria from human breast milk samples. Methods: The human breast milk samples were collected aseptically and then cultured in MRS agar media. The biosurfactant producing ability of the isolated strains was investigated by hemolytic assay, oil spreading method, drop collapse test and emulsification index assay. The screened isolates were identified by 16S rRNA gene sequencing. In vitro antibacterial activities of biosurfactants against a number of common bacterial strains were investigated by the agar disc diffusion method. This biosurfactant was characterized by Fourier-transform infrared spectroscopy (FTIR). Results: In this study, 337 different colonies were isolated from 42 breast milk samples. The best isolates were identified as Pediococcus pentosaceus HM-1, P. pentosaceus HM-2 and P. pentosaceus HM-3 based on microscopic and 16S rRNA gene sequencing analysis. The biosurfactant extracted from screened strains exhibited a broad spectrum of antagonistic activity against some pathogenic bacteria. Conclusion: Bacterial strains isolated here can be valuable sources for novel biosurfactants. The Human breast milk is a safe source for obtaining biosurfactant producing probiotic bacteria and for improving gut microflora of infants.


Introduction
Human breast milk is the best food for infants because it fulfills all the nutritional requirements for them, additionally; it educates the infant immunity functions and confers a certain degree of protection against infectious diseases. 1These effects seem a result of the action of many bioactive molecules, present in breast milk, including different antimicrobial compounds, immunoglobulins, and immune cells. 2,3The breast milk contains prebiotic substances that preserve the beneficiary bacteria in the infant gut. 3,4uman breast milk is constituted from several bacterial species, such as Staphylococci, Streptococci, Micrococci, Lactobacilli, Enterococci, Lactococci and Bifidobacteria. 5,6iosurfactants are heterogeneous amphipathic surface active molecules that belong to a wide range of chemical classes.These are mostly excreted by microorganisms outside the cells, and in some cases attached to the cells. 7Microbial biosurfactants produced by a wide variety of microorganisms, have a lower toxicity and a higher biodegradability than chemical surfactants.They are also effective at extreme conditions regarding temperatures, pH and saline concentration.][9][10] This paper describes the separation and characterization of biosurfactant generating probiotics bacteria from human breast milk.

Sample Collection
Human breast milk samples were obtained from 42 healthy mothers in Kerman province of Iran.The samples were collected in an aseptic condition and stored on ice until delivery to the laboratory.

Isolation of Probiotics Bacteria
Direct isolation of the microorganisms was carried out using serial dilution (up to 10 -7 ) of breast milk samples in 0.85% sterile saline.Serial dilutions were plated onto Man Rogosa Sharp (MRS; Biolife.Italia)) agar using the spreading method.The cultures were incubated anaerobically at 35°C for 5-7 days.Morphologically distinct colonies were isolated and purified by replicating on the MRS agar medium to obtain pure cultures.

Preliminary Characterization of Biosurfactant Producing Bacteria Hemolytic Activity
The bacteria were screened on blood agar (Merck, Germany) plates containing 5% (v/v) sheep blood and incubated at 35°C for 72 h.[13] Oil Spreading Method Pure isolates were cultured in MRS broth at 35°C and 200 rpm for 7 days.The broth cultures were centrifuged at 20000 rpm for 45 minutes. 11,14The supernatant was subsequently subjected to the screening methods using oil spreading and oil collapse methods as below.Distilled water (25 mL) was added followed by addition of crude oil (100 μL).Then, the cell-free culture broth (20 μL) was dropped on to the crude oil surface.[13] Oil Collapse Method Ten microliters of the crude oil were poured into the Petri dishes.After that, 10 μL of cell-free culture broth was added and the picture of the drop on the oil surface was inspected after 1 minute.Biosurfactant production was considered positive when the cultures giving flat drops.Those isolates that gave rounded drops were scored as negative, indicative of the lack of biosurfactant production. 11mplementary Screening of Biosurfactant Producing Bacteria Bacterial isolates positive for at least one primary screening methods were subjected to the complementary assays to verify their ability to produce biosurfactant.

Emulsification Capacity (E24)
The emulsifying capacity of isolated strains was evaluated by an emulsifica tion index (E24).Six milliliters of crude oil and 4 mL of culture supernatant were combined.This mixture was vortexed at high speed for 2 minutes and allowed to stand for 24 hours.The E24 index was calculated as the ratio of the height of emulsified layer (cm) divided respectively to the total height of the liquid column (cm).The results were compared with distilled water as negative control. 15,16entification of Bacterial Isolates Phenotypic Characterization Different phenotypic characteristics were evaluated, as outlined in Bergey's manual of determinative bacteriology, such as motility, Gram reaction, oxi dase and catalase. 17R Amplification of Bacterial 16S rRNA Genes Genomic DNA was extracted from a pure culture of three isolated bacteria using DNA extraction kit (Cinnagen Cat No: DN8115C), according to the manufacturer's instructions.These isolates showed the highest biosurfactant production and were isolated from three independent samples obtained from the separate individual.The purity of the extracted DNA was checked by recording the ab sorbance in 260 nm and 280 nm.The 2 universal oligonucleotide primers used to amplify the 16S rRNA samples were as follows: forward primer 27F (5´-AGAGTTTGATCCTGGCTCAG-3´) and reverse primer 1492R (5´-CGGTTACCTTGTTACGACTT-3´).Polymerase chain reaction (PCR) amplification was performed in a total volume of 50 µL mixture, containing 4 µL templates DNA (50 ng), 5 µL of F primer (10 pmol), 5 µL of R primer (10 pmol), 1 µL of 10 mM 4 dNTP mix, 1.5 µL of 50 mM MgCl 2 , 5 µL of 10X PCR buffer, 0.5 µL of Taq DNA polymerase (5 U µL -1 , Fermentas, Germany), 28 µL of sterile distilled water and microcentrifuged briefly.An initial denaturing step of 94ºC for 3 minutes was followed by 25 cycles of amplification (1 minute 94 o C, 1 minute 56°C, 1.5 minutes 72°C) and a final extension step at 72°C for 5 minutes.PCR products were separated by electrophoresis of 5 μL of PCR product in a 1% agarose gel for 2 hours and by staining with ethidium bromide.Amplification products were stored at -20°C.The clean PCR product was subjected to cycle sequencing in both directions.
Phylogenetic Analysis Phylogenetic analysis was carried out as follows: the sequences were checked using Bioedit V.5.0.9. 18A BLAST was carried out at http://blast.ncbi.nlm.nih.gov/Blast.cgi to identify the nearest neighbors. 19Alignment, phylogenetic and molecular evolutionary analyses were conducted using MEGA version 5. 20 A bootstrap test and reconstruction were done based on 1000 replications to confirm the validity of the phylogenetic tree. 21The nucleotide sequences of 16S rRNA gene of bacterial strains (Pediococcus pentosaceus HM-1, P. pentosaceus HM-2, and P. pentosaceus HM-3) reported in this study have been deposited in GenBank under Accession No: KU527555, KU527556 and KU527557, respectively.

Characterization of Obtained Biosurfactant Biosurfactant Recovery
Ten milliliters of an overnight culture of bacterial strains, in MRS broth, was added to 500 mL of MRS broth and incubated for 7 days at 35°C.The bacteria were removed (10 000 rpm for 15 minutes) to recover the biosurfactant.The pH of the supernatant was adjusted to 2 with 6 N HCl and then the solution was stored at 4°C for 24 hours.The precipitated biosurfactants recovered using a combination of chloroform and methanol (2:1 v/v) and mixed vigorously to obtain the biosurfactant within the organic layer.The biosurfactants move from the hydrophilic phase (MRS broth) into the organic, hydrophobic phase.This layer was separated using a separating funnel and dried at 50 o C for 4-5 hours to obtain dry mass and analyzed by FTIR spectroscopy. 22urier-Transform Infrared Spectroscopy Four milligrams of partially purified biosurfactant was dried (applying a freeze dryer) and then grounded with 100 mg of potassium bromide (KBr) and pressed to obtain translucent pellets.Then analyzed in an FTIR (Bruker : Tensor 27, Germany), device at the range of 400-4000 wavenumbers (cm -1 ).

Separation and Characterization of Biosurfactant Generating Bacteria
The initial isolation yielded a total of 337 morphologically distinct microbial colonies.Among them, 63 isolates (54 gram-positive, catalase-negative cocci and 9 grampositive, catalase-negative bacilli) were isolated.Among the isolated bacteria, 25 isolates gave a positive response to hemolytic activity; of these 10 isolates showed positive oil spreading test (Figure 1) and 9 positive responses were obtained by the oil collapse method.Data presented in Table 1 exhibits the screening methods of biosurfactant producing bacteria.The positive isolates were further evaluated by the secondary assay; i.e.Emulsification activity test (Figure 2).Following complementary screening, three potential biosurfactant producing strains (HM-1, HM-2 and HM-3) were selected.
Phylogenetic Analysis Phylogenetic affiliation of the screened strains was ascertained by 16S rRNA gene sequence analysis.In order to find the most similar available sequences, a BLAST search was done in NCBI database.16S rRNA gene sequence data of most closely related species were retrieved and used in tree construction to demonstrate the taxonomy of these isolates.Figure 3 shows the inferred phylogenetic relationships derived from a  neighbor-joining analysis of 16S rRNA gene sequence of the P. pentosaceus HM-1, P. pentosaceus HM-2 and P. pentosaceus HM-3 with most validly described species of the genus Pediococcus.

Antibacterial Activity of Biosurfactant
The results regarding the antibacterial activity of the different concentrations of biosurfactants are indicated in Table 2. Biosurfactants isolated from screened strains showed a wide activity against the indicator strains (Table 2 and Figure 4).The antibiotic sensitivity pattern analysis of indicator strains was tested against 5 different commonly used antibiotics and the results were presented in Table 3.

Fourier-Transform Infrared Spectroscopy
The Fourier-transform infrared spectroscopy (FTIR) spectrum is illustrated in Figure 5, which shows broad stretching peaks at 3442 cm -1 (as the indicator of N-H stretching vibrations and strong hydrogen bonding).Absorption at 2997 and 2913 cm -1 are assigned to the symmetric stretch of CH 2 and CH 3 groups of aliphatic chains.The appearance of a weak absorbance signal at 2093 cm -1 may be due to C-N stretch.The bands observed at 1659 cm -1 is a definite indicator of linkages between the amides I and II.The absorbance in this region is significant in the presence of the peptide group in the molecule.The band peaks at 1437, 1408 and 1312 cm -1 , are characteristic of aliphatic chains' (-CH 3 , -CH 2 ) bending vibrations.High-intensity peak in the region of 1047 cm -1 is assigned to O-C-O extend vibrations.The following vibrations observed at 500-1000 cm -1 are due to out of plane C-H bending.

Discussion
Human milk is a complex biological fluid adapted to fulfill the nutritional requirements of the rapidly growing infant.The characterization of the breast milk microorganisms contributes to addressing the biological role of milk microbiota in the maintenance of health of the newborn and lactating mother. 2,3The studies microbial features of human breast milk are restricted to those bacteria responsible for infancy infections.However, it is clear that the prevention of infant against pathogens is related to the natural flora of human milk. 3Although there is limited knowledge about the commensal or probiotic bacteria in breast milk, Staphylococci, Streptococci, Micrococci, Lactobacilli, and Enterococci constitute the majority. 2,3,23Biosurfactants are diverse groups of amphiphilic compounds with great diversity, environmental acceptability and a broad spectrum of functions and industrial applications.The biosurfactant producing bacteria were checked using hemolytic activity, oil collapse and oil spreading tests.Selection of these methods was due to their strong advantages including simplicity, low cost, quick implementation and use of relatively common equipment that is accessible in almost every microbiological laboratory. 11The results of our experiments indicated,  39.6% of total isolates were positive for hemolytic activity, 36% were positive for oil collapse and 40% were considered positive based on oil spreading and since these methods have shown differences, the isolates with more than one positive response were exposed to complementary screening including emulsion activity measurements.In the present study, three bacterial isolates (P.pentosaceus HM-1, P. pentosaceus HM-2 and P. pentosaceus HM-3) with biosurfactant-producing ability were isolated and screened from human milk samples.The biosurfactants derived from isolated strains showed significant antimicrobial activities against indicator bacteria at different concentrations.Baraka and Al-Rubayyi reported that the breast milk exerted bactericidal activity against E. coli, P. aeruginosa S. aureus and Salmonella sp. 24The antimicrobial activity results generated from our studies are in agreement with that reported by Ibhanesebhor and Otobo for inhibitory activities of human colostrum against S. aureus and coliform organisms. 25ur findings demonstrated that biosurfactants produced by screened strains, compared with synthetic antibiotics, had acceptable antimicrobial activity.Antimicrobial activity is one of the most important selection criteria for probiotics.Antimicrobial effects of prebiotic bacteria are formed by producing a variety of active biological compounds. 26,27For identifying types of functional groups in the unknown biosurfactants, FTIR analysis was used.The FTIR profile of the biosurfactant showed similarity to surfactin, a lipopeptide biosurfactant, and other lipopeptide biosurfactants like arthrofactin 28 and lichenysin 29 confirming the lipopeptide nature of the produced biosurfactant.According to Khopade et al, lipopeptide surfactants are potent antibiotics and had a wide antimicrobial activity. 30

Conclusion
Biosurfactant producing probiotic bacteria could be safely isolated from human milk.These bacteria are necessary for improving intestinal microflora of infants.The antibacterial properties of the produced biosurfactant against bacterial strains suggest its potential use in the development of new pharmaceutical preparations.

Ethical Approval
Not applicable.

Competing Interests
None.

Figure 1 .
Figure 1.Oil Spreading Zone Exhibited by Isolated Strains.

Figure 3 .
Figure 3. Neighbor-Joining Tree Based on 16S rRNA Gene Sequences, Showing Relationships of Screened Strains With Closely Related Members of the Genus Pediococcus.

Figure 4 .
Figure 4. Antibacterial Activity of the Biosurfactants on the Growth of the Indicator Bacteria by Disc-Diffusion Method.

Figure 5 .
Figure 5. FTIR Spectra of the Biosurfactant Produced by HM-3 Strain

Table 1 .
Detection of Biosurfactant Producing Isolates by Preliminary and Complementary Screening Methods * Diameter of the clear zone in mm.

Table 2 .
Antibacterial Activity of the Different Concentrations of Biosurfactants Against Indicator Strains

Table 3 .
Antibacterial Activity of the Standard Drugs Against Indicator Bacterial Strains S: Susceptible, I: Intermediate, R: Resista.