Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e7, 2014 www.elsevier.com/locate/jbiosc

Isolation of potential probiotic Lactobacillus oris HMI68 from mother’s milk with cholesterol-reducing property Marimuthu Anandharaj and Balayogan Sivasankari* Department of Biology, Gandhigram Rural Institute e Deemed University, Gandhigram 624302, Dindigul, Tamilnadu, India Received 12 December 2013; accepted 30 January 2014 Available online xxx

The objective of this study was to evaluate the probiotic properties of Lactobacillus strains isolated from mother’s milk and their effects on cholesterol assimilation. In this study 120 isolates from mother’s milk were phenotypically and genotypically characterized. Among these, only 6 predominant strains were identified as Lactobacillus spp. The following parameters were selected as important test variables in model stomach passage survival trials: acid and bile tolerance, antimicrobial activity, antibiotic susceptibility and cholesterol reduction. Results showed that the considerable variation existed among six strains. Moreover, the strain HMI68 is the most acid-tolerant and the HMI28 and HMI74 is the most acid-sensitive of all strains tested. HMI118 did not grow at 0.5% and 1% bile concentration after 5 h but the HMI68 and HMI43 showed some tolerance to such bile concentration. The differences found in the growth rate were not significant (P > 0.05). HMI68 showed resistance to most of the antibiotics as well as antagonistic activity against the tested pathogens. The amount of cholesterol reduction is increased when the media supplemented with bile salts. HMI68 assimilate 61.05 ± 0.05 mg/ml cholesterol with the presence of 0.3% bile salt this could be significantly decreased by 25.41 ± 1.09 mg/ ml without bile salt. HMI68 was identified to be Lactobacillus oris HMI68 and 16S rRNA sequence was deposited in the National Center for Biotechnological Information (GenBank). For the first time the cholesterol-reducing property of L. oris isolated from mother’s milk were investigated in this study. Therefore the effective L. oris HMI68 strain was regarded as a candidate probiotic. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: Probiotics; Mother’s milk; Lactobacillus oris HMI68; Cholesterol assimilation; Deconjugation]

Cholesterol is needed in the body to insulate nerves, make cell membranes and produce certain hormones. However, the body makes enough cholesterol, so dietary cholesterol intake is not essential for normal adult metabolism (1). Cholesterol plays a major role in human cardiovascular pathogenesis. The consumption of food rich in lipids, especially saturated fatty acids and cholesterol, plays a predominant role in the rise of heart diseases. At present, cardiovascular diseases are the major causes of death in the world. Elevated serum cholesterol level is widely recognized as a contributory risk factor for the development of cardiovascular diseases (CVD) such as atherosclerosis, coronary heart disease and stroke. The World Health Organization (WHO) has predicted that by 2030, CVD will remain the leading causes of death and affect approximately 23.6 million people globally (2). It has been reported that even a 1% reduction in serum cholesterol could reduce the risk of coronary heart disease by 2e3% (3). Numerous drugs that lower serum cholesterol have been developed to treat hypercholesterolemic subjects, the best example being the statins (Atorvastatin, Simvastatin, Rosuvastatin and Lovastatin). However, the undesirable side effects of these compounds were observed and have caused concerns about their long

* Corresponding author. Tel.: þ91 451 2452371, þ91 9688893123 (mobile); fax: þ91 451 2454466. E-mail addresses: [email protected] (M. Anandharaj), [email protected] (B. Sivasankari).

term therapeutic use. For this reason, a number of non-pharmacological approaches (including dietary ones) resulting in serum cholesterol reduction were tested. Various approaches have been used to alleviate this issue, including the use of probiotics. Probiotic is defined as a “live microbial supplement that beneficially helps the host by improving its intestinal microbial balance” (4). Probiotics are non-pathogenic microorganisms which confer benefits to their host when consumed in adequate amounts. The main probiotics are some lactic acid bacteria (LAB), such as Lactobacillus and Bifidobacterium, which are members of the commensal bacteria in the gastrointestinal tract of human and animals. Over the years, Lactobacilli have been associated with improvements in lactose intolerance, increases natural resistance to infectious disease in the gastrointestinal tract, suppression of cancer, improved digestion and reductions in cholesterol levels in the serum (5). A great number of lactic acid bacteria were isolated from various traditional naturally fermented foods by various researchers (6,7). Mother’s milk is a potential source of effective Lactobacillus strains but surprisingly not so much studies were done on isolation of probiotics from human breast milk. Martin et al. (8) isolated Lactobacillus gasseri, Lactobacillus rhamnosus, and Lactobacillus fermentum from the mother’s milk. It is estimated that an infant ingests 1  105 to 1  107 commensal bacteria, when the infant consumes approximately 800 ml breast milk per day (8e10). Traditionally human milk was believed to be sterile, however recent studies revealed that colostrum and breast milk are continuous sources of commensal, mutualistic and potentially

1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.01.015

Please cite this article in press as: Anandharaj, M., and Sivasankari, B., Isolation of potential probiotic Lactobacillus oris HMI68 from mother’s milk with cholesterol-reducing property, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.01.015

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ANANDHARAJ AND SIVASANKARI

probiotic microbes to the infant-gut (11). Probiotic bacteria reach the mammary gland through the enteromammary pathway. It has been demonstrated that dendritic cells (DCs) can penetrate the gut epithelium to take up non-pathogenic bacteria from the gut lumen (12). Once the bacteria inside the DCs or macrophages, could spread to other locations via mucosal associated lymphoid system or blood circulation to colonize distant mucosal surfaces such as respiratory tract and genitourinary tracts, salivary and lachrymal glands and most significantly to the mammary gland (13). There are number of cholesterol lowering mechanism by Lactobacillus strains have been proposed, one of the most important mechanisms is through the bile salt hydrolase (BSH) activity, the enzyme responsible for bile salt deconjugation in the enterohepatic circulation. The BSH activity was able to hydrolyze conjugated glycodeoxycholic acid and taurodeoxycholic acid, leading to the deconjugation of glyco- and tauro-bile acids. It has been detected in several LAB species indigenous to the gastrointestinal tract (14e16). It has also been suggested that BSH activity should be a requirement in the selection of probiotic organisms with cholesterol lowering properties, as non deconjugating organisms do not appear to be able to remove cholesterol from the culture medium to any significant extent (17). In recent years, the possibility of using bile salt deconjugation by lactic acid bacteria to treat hypercholesterolemia in humans has received increased attention (18,19). However, if the bacteria with the ability to provide health benefits such as protection of host from pathogenic bacteria were isolated from human milk, they would be considered attractive probiotic organisms (8). Human milk is an important food for neonates for few months to grow them up and protect the infants against some infections. The high concentration of LAB in milk from healthy mother may play an important biological role during the first months of life. Studies on this biological fluid indicate that human milk is a challenging source for potential probiotic bacteria. Hence the present study was aimed to isolate, identify and determine the cholesterol assimilation by Lactobacillus spp. from mother’s milk. MATERIALS AND METHODS Collection of mother’s milk Mother’s milk sample was obtained from a healthy two month lactating mother volunteer at Gandhigram, Dindigul District, Tamilnadu, India. Before collecting the samples the nipple and mammary areola of left breast were cleaned with sterile water and the chlorhexidine (Himedia) were applied to prevent other unwanted normal floral microbes. The initial outcomes from the breast were discarded to avoid chlorhexidine contamination. The samples were collected on sterile carriers and stored on ice until delivery to the laboratory. Isolation and identification of Lactobacillus spp. For isolation of Lactobacillus spp. Mother’s milk (1 ml) was transferred to 9 ml of sterile saline (0.85% sodium chloride) and mixed well. The serial dilutions were subsequently prepared in sterile saline. Appropriate dilutions of the samples were plated on Lactobacillus de Man, Ragosa Sharpe (MRS) medium (HiMedia, Mumbai, India). Plates were incubated at 37 C for 24e48 h under anaerobic condition. Totally 120 strains were isolated and only six predominant strains were identified as Lactobacillus spp. based on colony morphology, catalase reaction, Gram staining, fermentation test using eleven different carbohydrates (glucose, fructose, lactose, mannose, maltose, arabinose, sucrose, galactose, mannitol, sorbitol and xylose) and growth on MRS medium. The pure cultures of the isolates were preserved in MRS broth medium containing 20% (v/v) glycerol as frozen stocks at 80 C. The cultures were grown prior to use by sub culture twice in MRS broth. PCR amplification of the 16S rDNA and sequence determination of Lactobacillus spp. Genomic DNA from the isolate was isolated by a modified method of Smoker and Barnum (20). The 16S rDNA were amplified from the isolated genomic DNA by using EGE1 (50 -AGAGTTTGATCCTGGCTCAG-30 ) as a forward primer and EGE2 (50 -CTACGGCTACCTTGTTACGA-30 ) as a reverse primer, Taq DNA polymerase and buffers in the thermocycler from 30 cycles comprising 94 C denaturation for 30 s, 56 C annealing for 30 s and 72 C for elongation. The PCR amplified rDNA were purified by using the Quick PCR purification kit (Bangalore Genie, India) and sequencing of the amplicons were done. Databases (GenBank) were searched for sequences similar to the 16S rRNA. The analysis of alignment and homology of the partial nucleotide sequence of Lactobacillus spp. was carried out by the basic local alignment search tool (BLAST).

J. BIOSCI. BIOENG., Phylogenetic analysis The 16S rRNA gene sequences obtained in present study and the taxonomically related Lactobacillus spp. retrieved from the National Center from Biotechnology Information (NCBI) database (www.ncbi.nlm.nih.gov). The GenBank accession numbers for 16S rRNA gene sequences from following bacteria were used for our phylogenetic analysis (accession numbers in parentheses): Lactobacillus panis (NR_026310.1), Lactobacillus vaginalis (KF418829.1), Lactobacillus antri (NR_027206.1), Lactobacillus reuteri (KF149748.1), Lactobacillus brevis RO97 (AF515219.1), L. fermentum MTCC 8711 (GU213430.1), Lactobacillus intestinalis (EF187259.2), Lactobacillus plantarum (EU825657.1), L. rhamnosus IDCC 3201 (EF533991.1), Lactobacillus sakei PSH-313 (DQ989236.2), Lactobacillus crispatus ZDY35b (EU559595.1), Lactobacillus acidophilus IDCC 3301 (EF533992.1), Lactobacillus delbrueckii (EU722745.1), L. gasseri ATCC 33323 (AF519171.1), Lactobacillus paracasei 6W (FJ476126.1), L. reuteri (EU722746.1), Lactobacillus ruminis LBARR16SZ (M58828.1), Lactobacillus salivarius I24 (EF412986.1), and Lactobacillus johnsonii NCC2822 (FJ557011.1). The multiple distance matrix obtained were then used to construct phylogenetic trees using Neighbor-Joining method and the phylogenetic tree were constructed by using Mega 5 software. Resistance to low pH Acid tolerance of the cultures was studied by incubating the organisms in MRS broth (in some experiments 1% skim milk powder was added to MRS broth). The pH was adjusted to 1.0, 2.0 and 3.0 with 1 N HCl and 1 N NaOH the cultures were incubated at 37 C for 3 h. Each of the bacterial strains were sub cultured at least 3 times before experimental use MRS broth was inoculated (10% v/v) with bacterial strain, and growth was monitored using the plate count method 1 ml of sample was taken at 0, 3 and 5 h, and serial dilutions were made using peptone water diluents. Samples were plated onto MRS agar, and the plates were incubated at 37 C for 48 h in an anaerobic jar. Resistance to bile salts Growth rate of bacterial cultures in MRS broth containing different levels (0%, 0.1%, 0.3%, 0.5% and 1%) of bile salts (oxgall) were determined. Freshly prepared cultures were inoculated (1%) into medium and incubated at 37 C for 24 h under anaerobic condition. 1 ml of sample was taken at 0, 3 and 5 h, and serial dilutions were made using peptone water diluents. Samples were plated onto MRS agar, and the plates were incubated at 37 C for 48 h in an anaerobic jar. Antimicrobial activity by agar diffusion assay Modified method of Sirilun et al. (21) was used for screening for antimicrobial activity of the selected strains against variety of indicator organisms: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 15442, Candida albicans ATCC 90028, Staphylococcus aureus ATCC 25923 and Klebsiella pneumonia ATCC 13883 obtained from American Type Culture Collection (ATCC). Briefly, cell free neutralized supernatants were obtained from overnight producer cultures grown in MRS broth. After centrifuging at 12,000 g for 10 min at 4 C, the supernatant was filter sterilized through 0.22 mm millipore membrane filter (Millipore, Billerica, MA, USA). Cell free extracts of bacterial isolates (50 ml) were poured into 2 mm wells previously made on trypticase soy agar (TSA) plates coated with the test pathogens. The plates were incubated at optimal growth conditions required for the pathogenic bacteria and the zone of inhibition was measured. Antibiotic susceptibility test The selected strains were investigated for their antibiotic resistance profile as recommended by the Clinical and Laboratory Standards Institute (CLSI; Wayne, PA, USA). In this study 11 different types of antibiotic disks (HiMedia, Mumbai, India), ampicillin (10 mg), chloramphenicol (30 mg), erythromycin (15 mg), gentamycin (10 mg), cephalexin (30 mg), novobiocin (30 mg), bacitracin (10 mg), cefuroxime (30 mg), rifampicin (30 mg), kanamycin (30 mg), ofloxacin (5 mg), were used. These were subsequently overlaid with 4 ml of MuellereHinton soft agar at 45 C and 100 ml of bacterial isolates were spread over the plates. The HiMedia antibiotic disks were placed onto the agar surface. The agar plates were incubated at 37 C anaerobically for 24 h. Diameter (in mm) of the inhibition zone was measured using an antibiotic zone scale, and results were expressed in terms of resistance, moderate susceptibility or susceptibility by comparing with the interpretative zone diameters provided in the performance standards for antimicrobial disk susceptibility tests (22). Deconjugation of sodium glycocholate and sodium taurocholate Ten milliliters of MRS broth was supplemented with 0.5% (w/v) sodium glycocholate (Sigma) and 0.5% of sodium taurocholate (Sigma). The isolated Lactobacillus spp. was inoculated at 2% (v/v) and incubated anaerobically at 37 C for 24 h (23). Free cholic acid analysis was carried out as prescribed by Walker and Gilliland (15). Cholesterol reduction assay In vitro cholesterol reduction was determined by growing cells at 37 C in MRS broth supplemented with 0.30% oxgall (Himedia) as bile salt. Water soluble cholesterol (polyoxyethanyl-cholesteryl sebacate, Sigma) was filter sterilized and added to the broth at a final concentration of 50e200 mg/ml, inoculated with each strain at 1% level and incubated anaerobically at 37 C for 24 h. Following incubation the cells were harvested by centrifuging (10,000 g, 4 C, 10 min) and the supernatant was collected. The cholesterol content was determined using a modified colorimetric method as described by Rudel and Morris (24) and Gilliland et al. (25). FeCl3 work solution (1.5 ml) was added to the tube containing sample, after through mixing the solution was allowed to stand for 10 min. Then 1 ml of concentrated sulphuric acid (H2SO4) was added. The solutions were placed in the dark for 45 min and absorbance was read at 560 nm (Spectronic 20D, Thermo Scientific, USA) after 24 h. The activity of cholesterol lowering (mg/ml culture broth) was calculated as follows:

Please cite this article in press as: Anandharaj, M., and Sivasankari, B., Isolation of potential probiotic Lactobacillus oris HMI68 from mother’s milk with cholesterol-reducing property, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.01.015

c

þ þ þ þ þ þ      

The bacterial growth at 4% NaCl and 6.5% NaCl is indicated as (þ) positive and (P) partial growth. The bacterial growth at different temperatures is indicated as (þ) positive and () negative. For carbohydrate fermentation number of þ indicates the fermentation of carbohydrates by the human milk isolates: þ, poorly; þþ, moderately; þþþ, fully. a

b

Galactose Sucrose

þþ þþþ þþþ þþ þþþ þþþ      

Mannitol Sorbitol

þþ þþþ þþþ þþ þþ þ      þþþ

Arabinose Maltose

þ þþþ þþþ þþþ þþþ þþ  þ  þ  þþ

Mannose Xylose

      þ  þ þ þ þþ

Lactose Fructose Glucose Catalase test Growth at 6.5% NaClb Growth at 4% NaClb Growth at 45 Ca Growth at 15 Ca

Resistance to bile concentration The strains resistant to low pH were further screened for their ability to tolerate the bile salt (27). Table 2 shows the effect of bile salts on the growth of the strains. Significant variations existed among the cultures with regard to their ability to grow in MRS broth supplemented with bile salts (P > 0.01). HMI118 was found to be the least bile tolerant in comparison while HMI68 was found to be the most resistant. In a comparison of all the other strains, HMI118 was the most sensitive to bile salts, with significantly lower growth rates in the medium with 0.5% (w/v) bile acids than in the absence of bile (P < 0.05). Though HMI68 and HMI43 showed some sensitivity no significant difference in the growth rate was observed (P > 0.05). The HMI74 and HMI21 show no growth at

Biochemical tests

Resistance to low pH condition The effects of acidity on the viability of strains are presented in Fig. 1. Compared to all the other strains HMI68 showed resistant to pH 3 even after 5 h. HMI28 and HMI74 were the most acid-sensitive of all strains tested, losing viability in less than 5 h at pH 2.0 and pH 1.0. The total colony forming unit (CFU) of HMI21 and HMI43 were significantly decreased (P < 0.01) after 5 h at pH 1.0. The viability of HMI21 at pH 2.0 significantly decreased (P < 0.01) after 3 h. HMI68 is the most acid-tolerant strain, retaining around 80% viability for up to 5 h at pH 1.0 and pH 2.0. No significant differences were found between the viability after 3 h for HMI21 and HMI68 (P > 0.05). No growth was observed in HMI28, HMI74 and HMI118 at pH 1.0 and pH 2.0 after 5 h, which showed that these strains are sensitive to intestinal pH conditions. When compared with all other strains HMI68 shows more resistant to the all pH level after 5 h.

TABLE 1. Biochemical characteristics of bacterial isolates.

Identification by 16S rDNA sequencing The amplified 16S rRNA gene from the DNA of Lactobacillus spp. was determined using 2% agarose gel. The size of the amplified fragments was determined by using size standard (Gene ruler 10 bpe330 bp DNA ladder). The PCR products were visualized under UV light and photographed using gel documentation system. Approximately 380 bp of 16S rRNA gene was amplified (Fig. S1). The PCR products were purified and sequenced using 16S forward primer. The BLAST algorithm was used to retrieve for homologous sequences in NCBI GenBank. The Lactobacillus spp. revealed 99% identity to Lactobacillus oris and 98% identity to L. antri (Fig. S2). Based on the morphological, biochemical, and molecular characteristics, the Lactobacillus spp. was designated as L. oris HMI68 and partial 16S rRNA sequence were deposited at NCBI GenBank under the accession number KF718268.

Carbohydrate fermentationc

Identification of the bacterial culture The isolated colonies were confirmed as Lactobacillus spp. using morphological and physiological characteristics as previously described (26). The strains were gram-positive, catalase negative, nonsporeforming, rods, grew well at 45 C but not at 15 C and tolerate 4% NaCl and partially tolerate 6.5% NaCl concentrations. They fermented glucose, fructose, lactose, mannose, maltose, arabinose, sucrose and galactose and they have not fermented mannitol, sorbitol and xylose. Results of biochemical tests are represented in Table 1.

þ þ þþ þþ þþ þþ

RESULTS

þþþ þþ þþ þþþ þ þþþ

Statistical analysis All experiments were done in triplicate, and the results were expressed as mean  standard deviations. Data analysis was carried out with MS-Excel and SPSS 13.0 statistical software (SPSS Inc., Chicago, IL, USA). One-way ANOVA was used to study a significant difference between means with a significance level of P < 0.05 when needed.

     

Where C1 and C2 represent the cholesterol concentration of the uninoculated and inoculated medium, respectively, and W1 and W2 represent the weight of culture per milliliter of medium before and after the incubation period.

þ þ P þ P P

(1)

HMI21 HMI28 HMI43 HMI68 HMI74 HMI118

Cholesterol assimilation (mg/ml) ¼ (C1  C2) or (C1  C2)/(W2  W1)

3

þþþ þþ þþþ þþ þþþ þþ

ISOLATION OF L. ORIS HMI68

þ þ þ þ þ þ

VOL. xx, 2014

Please cite this article in press as: Anandharaj, M., and Sivasankari, B., Isolation of potential probiotic Lactobacillus oris HMI68 from mother’s milk with cholesterol-reducing property, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.01.015

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ANANDHARAJ AND SIVASANKARI

J. BIOSCI. BIOENG., organisms was determined by the agar diffusion method. Results are available in Table S1. All the isolates were able to inhibit the growth of pathogens with significant variations. HMI68 strain produced an inhibition zone (mm) of 28.00  1.00 and 11.40  1.72 against C. albicans ATCC 90028 and P. aeruginosa ATCC 15442 strains respectively (Table S1). All strains exhibited marked inhibitory effect against E. coli ATCC 25922 but showed little inhibitory effect against S. aureus ATCC 25923 except HMI68 strain which potentially minimized its growth rate (9.86  0.41). HMI68 and HMI28 strains are resistant to most of the enteric pathogens and prevent their growth. This inhibitory effect may be due to the production of organic acids and other antimicrobial substances. Antibiotic resistance profile The selected six strains were screened for their susceptibility to eleven antimicrobial agents. All of them were sensitive to novobiocin and kanamycin. Ofloxacin (except HMI21, HMI43 and HMI74), bacitracin (except HMI68), chloramphenicol (except HMI68 and HMI118), ampicillin (except HMI21 and HMI68), but they were resistant to gentamycin (except HMI28) and cefuroxime (except HMI21 and HMI43). The L. oris HMI68 shows resistant to most of the antibiotics except novobiocin and kanamycin (Table S2). Deconjugation of sodium glycocholate and sodium taurocholate The amount of free cholic acid released determines the deconjugation ability. All the strains demonstrated deconjugation ability but possess different substrate affinity. Only strains HMI28 and HMI68 were able to deconjugate both sodium taurocholate and sodium glycocholate. The deconjugation ability was observed to higher in L. oris HMI68. Lactobacillus strains HMI74 and HMI118 were able to deconjugate sodium glycocholate while Lactobacillus strains HMI21 and HMI43 deconjugated sodium taurocholate. Higher amount of deconjugation ability with both sodium taurocholate and sodium glycocholate was observed in L. oris HMI68 1.40 mM and 1.91 mM respectively similarly Lactobacillus HMI28 strain shows 0.98 mM and 0.89 mM, respectively. The Lactobacillus strains HMI74 and HMI118 able to deconjugate only sodium glycocholate, on the other hand Lactobacillus strains HMI21 and HMI43 deconjugated only with sodium taurocholate (Table 3). Cholesterol reduction assay The selected strains were found to reduce cholesterol by 23.28e35.41 mg/ml in the in vitro test. The amount of cholesterol assimilated by the different strains showed wide variation after 24 h of anaerobic growth. The amount of cholesterol assimilation is increased tremendously with the addition of bile salts (0.3% w/v). The cholesterol assimilation is increased upto 61.05 mg/ml after addition of bile salt. In general, HMI68 strain exhibited high cholesterol assimilation with (61.05  0.05 mg/ml) or without (35.41  1.09 mg/ml) bile salts compared to other strain. Media supplemented with 50 mg/ml cholesterol the HMI68 assimilate 22.49  1.48 mg/ml but it will decrease at 200 mg/ml concentration (20.61  0.61). Cholesterol assimilation (mg/ml) from MRS broth was significantly high (P  0.05) for strains HMI68 (25.41  1.09) and HMI43 (22.09  1.14)

FIG. 1. Effects of different pH on the viability of Lactobacillus strains isolated from the mother’s milk after 0 h (A), after 3 h (B) and after 5 h (C) represented as log10 CFU/ml. Values are expressed as mean  standard deviations of triplicates. Cultures were incubated anaerobically at 37 C.

1% bile after 5 h. L. oris HMI68 is the most bile resistant strain, it shows viability at 1% bile concentration after 5 h. Antimicrobial activity The inhibition ability of supernatants from the different Lactobacillus strains against the indicator

TABLE 2. Effect of bile salt concentration on the viability of Lactobacillus spp. expressed as log10 CFU/ml. Bacterial isolates

0h

0% HMI21 HMI28 HMI43 HMI68 HMI74 HMI118

3h

5h

Bile salt concentration

25.8 24.3 24.8 26.5 23.7 21.8

     

0.3% 0.50 0.20 0.31 0.22 0.15 0.40

17.2 18.6 14.9 22.3 16.4 17.1

     

0.20 0.70 0.50 0.20 0.10 0.50

0.5% 16.3 17.8 16.8 18.7 15.3 16.0

     

0.30 0.20 0.20 0.30 0.50 0.10

1% 18.9 17.3 18.0 19.4 14.2 16.8

     

0% 0.50 0.20 0.50 0.10 0.10 0.30

29.4 28.1 30.6 31.2 28.8 23.9

     

0.3% 0.15 0.20 0.10 0.70 0.50 0.30

20.1 21.5 28.8 30.4 23.8 26.9

     

0.30 0.20 0.25 0.50 0.20 0.50

0.5% 19.8 20.4 18.1 23.1 19.9 18.2

     

0.20 0.20 0.30 0.40 0.20 0.10

1% 8.1 7.6 14.7 17.0 9.4 8.6

     

0% 0.30 0.10 0.20 0.70 0.60 0.20

36.0 34.7 34.1 36.1 32.8 29.8

     

0.3% 0.50 0.60 0.40 0.50 0.20 0.10

19.3 20.4 17.2 15.4 13.6 16.5

     

0.00 0.30 0.00 0.10 0.80 0.10

0.5%

1%

    

ND 4.2  0.02 6.2  0.04 7.8  0.00 ND ND

9.4 10.4 9.8 12.2 9.1 ND

0.10 0.00 0.20 0.80 0.00

CFU, colony forming unit after 24 h incubation at 37  2 C under anaerobic condition in different time intervals. Values are represented as mean  standard deviation (n ¼ 3). ND, not detectable with the indicators employed in this study.

Please cite this article in press as: Anandharaj, M., and Sivasankari, B., Isolation of potential probiotic Lactobacillus oris HMI68 from mother’s milk with cholesterol-reducing property, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.01.015

VOL. xx, 2014

ISOLATION OF L. ORIS HMI68

TABLE 3. Deconjugation of sodium glycocholate and sodium taurocholate by Lactobacillus spp. Bacterial isolates HMI21 HMI28 HMI43 HMI68 HMI74 HMI118 E9 GD2 NCDO1693 NCDO82

Cholic acid release (mM) Sodium glycocholate e 0.89 e 1.91 1.02 0.53 e 0.53 5.20 5.85

Reference

Sodium taurocholate

 0.02  0.81  0.11  0.03  0.01  0.42  0.18

0.59 0.98 1.05 1.40 e

   

0.13 0.18 0.12 0.10

0.61 0.50 0.85 0.54

   

0.02 0.02 0.03 0.04

In this In this In this In this In this In this 48 48 49 49

study study study study study study

without bile and HMI68 (61.05  0.05) and HMI74 (56.18  0.02) with bile salts, whilst the lowest cholesterol removal values (mg/ml), i.e., 18.63  1.15 and 19.00  0.83 were recorded for HMI21 and HMI28, respectively. Results are shown in Tables 4 and 5. DISCUSSION Mother’s milk constitutes an interesting source to obtain new and specific probiotic strains for neonates aiming at assisting a proper development of the gut microbiota and the immune development in infants who, for different reasons, cannot be breast-fed (28). The results of this study indicate that mother’s milk may be used as a potential natural source to isolate the effective strains of Lactobacillus spp. The understanding of communal and potential probiotic bacteria that survive in milk of healthy women is very limited. It has been reported that isolated bacteria from breast milk commonly include Staphylococci, Streptococci, Micrococci, Enterococci and Lactobacilli such as L. gasseri, L. rhamnosus, L. acidophilus, L. plantarum and L. fermentum (10,12,29,30). Resistance to bile salt concentration is considered to be an important property in strains envisage as probiotics. Since, to reach the small intestine they have to pass through from the stressful conditions of stomach. Although in the stomach, pH can be as low as 1.0, in most in vitro assays pH 3.0 has been preferred. Due to the fact that a significant decrease in the viability of strains is often observed at pH 2.0 and below. The time that takes during the digestion in the stomach is 3 h. This study revealed that the six isolated Lactobacillus strains were able to survive in the acidic conditions of the intestine and also tolerate the high bile concentrations. The strains were able to grow around pH 1 for 3 h and also tolerate 1% of bile concentrations. This observation indicates that these strains will not only survive the low pH of the stomach but may be able to grow and colonize in the high bile environment of the intestine.

TABLE 4. In vitro cholesterol assimilation by Lactobacillus sp. without bile salt after 24 h. Cholesterol concentration 50 mg/ml HMI21 HMI28 HMI43 HMI68 HMI74 HMI118

16.01 14.46 13.28 22.49 15.08 15.18

     

1.02 0.09 0.18 1.48 1.70 0.42

100 mg/ml 14.48 16.12 13.64 21.28 14.12 16.19

     

0.62 0.76 1.09 1.00 0.91 1.92

150 mg/ml 20.46 21.12 22.09 25.41 21.49 20.16

     

0.12 0.81 1.14 1.09 1.06 1.51

200 mg/ml 18.63 19.42 19.00 20.61 21.68 24.13

     

TABLE 5. In vitro cholesterol assimilation by Lactobacillus spp. with 0.3% bile salts (oxgall) after 24 h. Bacterial isolates HMI21 HMI28 HMI43 HMI68 HMI74 HMI118

Cholesterol concentration 50 mg/ml 56.02 54.78 54.01 61.05 56.18 43.79

     

1.17 1.01 1.98 0.05 0.02 0.41

100 mg/ml 43.04 48.47 46.91 58.71 48.43 51.01

     

0.06 0.92 0.51 0.62 0.11 0.96

150 mg/ml 51.88 53.09 49.40 56.10 52.00 50.09

     

0.43 1.17 1.71 1.00 1.18 1.16

200 mg/ml 49.61 48.67 43.59 53.10 50.81 42.49

     

0.61 0.42 0.32 1.17 0.87 0.12

Cholesterol assimilation is defined as cholesterol removal per ml of culture broth after 24 h of incubation at 37  2 C under anaerobic condition. Results are expressed as mean  standard deviation; n ¼ 3.

Values are represented as means  standard deviation of triplicates. E9, Lactobacillus rhamnosus E9; GD2, L. plantarum GD2; NCDO1693, L. johnsonii NCDO1693; NCDO82, L. plantarum NCDO82.

Bacterial isolates

5

1.15 0.86 0.83 0.61 1.01 1.49

Cholesterol assimilation is defined as cholesterol removal per ml of culture broth after 24 h of incubation at 37  2 C under anaerobic condition. Results are expressed as mean  standard deviation; n ¼ 3.

As already pointed out by various workers in the field (4, 31e33), in vitro studies can only partially mimic the actual in situ conditions in the gut ecosystem. On the other hand, such in vitro systems remain powerful tools especially for screening numerous samples. Similarly Chou and Weimer (34), tried to isolate acid and bile resistant variants of L. acidophilus. Some of these strains were found resistant to acid at pH 3.5 for 90 min at 37 C. Also these strains were capable of growth in medium at pH 3.5 containing 0.2% mixed bile salts. Prasad et al. (27) used 200 isolates to analyze the series of pH between 1 and 3 also for tolerance against bile at final concentrations of 0%, 0.5% and 1% w/v. They were tolerant for the conditions mentioned above. These strains were identified as L. rhamnosus HN001, L. rhamnosus HN067, L. acidophilus HN017 and Bifidobacterium lactis HN019 (27). In another research (34), 29 Lactobacillus strains of dairy origin were tested in vitro for their probiotic potential. All of the examined strains were resistant to pH 3 during 3 h, but most of them lost their viability in 1 h at pH 1. Also all of them were tolerated 0.3% bile salts concentration for 4 h. They were identified as Lactobacillus casei Shirota ACA-DC 6002, L. plantarum ACA-DC 146, L. paracasei subsp. tolerans ACA-DC 4037 (35). An important selection criterion is that the isolated probiotics must be safe for human consumption. For this purpose the antibiotic resistance profile of the isolated organisms was determined as recommended by CLSI. The organism of interest L. oris HMI68 was found to be resistant to all the tested antibiotics except novobiocin and kanamycin making it palatable. The production of antimicrobial compounds against pathogens by breast milk isolates was determined using agar diffusion test. We observed that most of the isolated strains exhibited antimicrobial activity which could be attributed to the production of organic acids. L. oris showed immense potential to inhibit microbial growth. Savadogo et al. (36) in his work demonstrated the antimicrobial activity of other Lactobacillus strains. L. fermentum (S1) produced the maximum zone of inhibition (12 mm) against Enterococcus faecalis while the minimal was shown by Leuconostoc mesenteroides subsp. mesenteroides (S5) on the same strain. In another research L. salivarius FC113 produced an inhibition zone of 11.5  0.7 mm and 14.3  0.6 mm against S. aureus strains KCCM 11335 and KCCM 11593, respectively (37). This study also indicated that L. oris HMI68 was able to deconjugate both sodium glycocholate and sodium taurocholate. On the other hand, HMI21 and HMI43 were able to deconjugate sodium taurocholate while HMI74 and HMI118 deconjugated sodium glycocholate alone. Glycocholate is the most predominant bile salt in the human intestine. In 1998 Brashears et al., postulated that Bshactive strain that prefers to hydrolyze sodium glycocholate may have far more potential to reduce serum cholesterol levels in vivo (38). In general, deconjugated bile salts (pKa 5.0) possess higher pKa values in comparison with conjugated bile salts (pKa w1.5e3.7) and are less soluble in the aqueous environment of the

Please cite this article in press as: Anandharaj, M., and Sivasankari, B., Isolation of potential probiotic Lactobacillus oris HMI68 from mother’s milk with cholesterol-reducing property, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.01.015

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ANANDHARAJ AND SIVASANKARI

lumen (39). Bile salt hydrolysis and deconjugation ability could not be correlated well amongst majority of the Lactobacilli as noticed by Liong and Shah (40). Such explanation can be supported with the findings of Kurdi et al. (41,42) who proposed the cholic acid accumulation by Lactobacilli and Bifidobacteria in an ATP-dependent manner. Furthermore, besides the contribution of bile salt hydrolysis in lowering serum cholesterol, incorporation of cholesterol micelles by probiotic bacterial cells in the gut could be an additional attribute for exerting its hypocholesterolemic effect (25,43). In this study the Lactobacillus strains effectively lowered cholesterol in the media at varying degree. The amount of cholesterol assimilated by the strains after 24 h of incubation ranged from 23.28 to 35.41 mg/ml at varying concentration of cholesterol in the broth. The amount of cholesterol assimilation is increased tremendously to 61.05 mg/ml with the addition of bile salts (0.3% w/ v). This observation confers that though cholesterol can be assimilated from media even in the absence of bile salts its reduction increases tremendously on its addition. Furthermore cholesterol reduction ability of Lactobacillus is influenced by its individual BSH activity (44). Liong and Shah (40) and Gilliland and Walker (45) reported that B. longum and L. acidophilus posses the ability to uptake cholesterol into their cellular membrane. Pereira and Gibson (46) observed that the uptake of cholesterol by lactic acid bacteria and Bifidobacteria was higher in the medium containing 0.4% oxgall. Brashears et al. (38) reported that L. casei assimilate about 16.9e73.3 mg/ml cholesterol. One of the most promising strategies for decreasing serum cholesterol level is supplementing diets with probiotic strains. However, for promoting probiotics effects, it is important for the bacterial strain to survive gastrointestinal tract environment of the human such as the acidic conditions, bile acids, and digestive enzymes in intestine (25). Studies about the cholesterol-reducing abilities of probiotics do not sufficiently explain the mechanisms of hypocholesterolemic action, optimum dose, and the frequency of the probiotic strains (47). Further studies are currently underway to verify the cholesterol removing effects in vitro. The hypocholesterolemic effects of some probiotics, which showed high BSH activity from in vitro assays, have been confirmed in human as well as in animals (25). However, the hypocholesterolemic mechanisms of probiotics based on the BSH hypothesis have not yet been clearly defined. It is essential to determine the mechanisms underlying the cholesterol lowering effect in the future. This can be supported by many researchers. In conclusion, the data revealed that L. oris HMI68 isolated from mother’s milk is a commensal bacterium with cholesterol reduction properties, ability to survive the human gastrointestinal conditions. L. oris HMI68 strain needs to be further investigation to improve their health benefits and molecular approaches can be made to increase their cholesterol reduction potential and used it can be incorporated into food supplement. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.jbiosc.2014.01.015. ACKNOWLEDGMENT The authors are thankful to the Department of Biology, Gandhigram Rural Institute e Deemed University, Gandhigram, Tamilnadu, India for providing laboratory facility to carry out the entire research. This research was partially funded by the University Grants Commission (UGC), Research Fellowship in Science for Meritorious Students (BSR) (Grant no. F.4-1/2008). References 1. Tabas, I.: Cholesterol in health and disease, J. Clin. Invest., 110, 583e590 (2002).

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Isolation of potential probiotic Lactobacillus oris HMI68 from mother's milk with cholesterol-reducing property.

The objective of this study was to evaluate the probiotic properties of Lactobacillus strains isolated from mother's milk and their effects on cholest...
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