Identification, characterization, and probiotic potential

LWT - Food Science and Technology 84 (2017) 271e280

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Identification, characterization, and probiotic potential of Lactobacillus rhamnosus isolated from human milk Muhammad Shahid Riaz Rajoka a, Hafiza Mahreen Mehwish c, Muhammad Siddiq b, Zhao Haobin a, Jing Zhu a, Li Yan a, Dongyan Shao a, Xiaoguang Xu a, Junling Shi a, * a Key Laboratory for Space Bioscience and Space Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi'an 710072, Shaanxi, People's Republic of China b Department of Neonatology, The First Affiliated Hospital, College of Medicine, Xi'an 710072, Shaanxi, People's Republic of China c Department of Biotechnology, University of Agriculture Faisalabad, Pakistan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 December 2016 Received in revised form 20 April 2017 Accepted 23 May 2017 Available online 29 May 2017

Breast milk is a source of lactic acid bacteria with remarkable functional and Technological properties; it is also a potential source of probiotics. In the present study, seven strains of Lactobacillus rhamnosus were isolated from breast milk and identified according to their 16S rDNA sequences. Furthermore, their probiotic potential was evaluated. The probiotic properties were tested for aspects of antibiotic susceptibility, antimicrobial activity, lysozyme tolerance, gut condition tolerance (low pH, bile salt tolerance, and 0.4% phenol resistance), hydrophobicity, antioxidant ability, aggregation ability, and adhesion to Caco-2. Most isolates were resistant to Streptomycin, Ampicillin, Gentamicin, Kanamycin, Penicillin, Cephalotoxin, and Ciprofloxacin. The isolate shows a strong ability to adhere to Caco-2 cells as well as DPPH radical scavenging activity in the range of 76%e85%. Isolates SHA113 and SHA117 showed a high survival rate under gastrointestinal tract conditions (>80%), indicating excellent potential for application as probiotics. The results of these tests indicate that the lactic acid bacteria isolated from human milk have excellent potential for use as probiotics in various products. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Lactobacillus rhamnosus Probiotic potential Breast milk Antioxidant

1. Introduction According to definition (Food & Organization, 2006), probiotics are living microbes within hosts that exert health benefits when ingested in sufficient amounts, regulating the immune system, maintaining healthy gut microbiota, reducing hypertension, lowering cholesterol levels, and preventing diarrhea (García-Ruiz et al., 2014; de Vrese & Schrezenmeir, 2008). The genera of Lactobacillus, Bifidobacteria, Lactococcus, Streptococcus, and several strains of yeast have been used as probiotics (Ohland & MacNaughton, 2010; Vidhyasagar & Jeevaratnam, 2013). Numerous species of these genera are “Generally recognized As Safe” by the FDA (US food and drug administration) or received the “Qualified Presumption of Safety” status by the ESFA (European food authority). To be utilized as probiotic, bacteria must have the ability to survive in the acidic condition of the stomach and within

* Corresponding author. E-mail address: [email protected] (J. Shi). http://dx.doi.org/10.1016/j.lwt.2017.05.055 0023-6438/© 2017 Elsevier Ltd. All rights reserved.

€ & Peta €ja €, bile acid at the start of the gastrointestinal tract (Erkkila , Martín, Aymerich, & Garriga, 2014). 2000; Rubio, Jofre To date, numerous probiotics have been isolated from the human gastrointestinal tract and from dairy products. A recent investigation reported that the infectious diseases incidence reduced in breast fed infants compared to infants who received other milk products. This indicated that specific factors are present in breast milk that seem to provide protection against infectious diseases. Breast feeding is an important source of lactic acid bacteria for the infant and the lactic acid bacteria that are present in mother milk can also protect the infant from pathogenic microbes (Kozak, Charbonneau, Sanozky-Dawes, & Klaenhammer, 2015; Martín et al., 2003; Rodríguez et al., 2012). The isolation of probiotic bacteria from breast milk (exerting its protective effect via production of hydrogen peroxide and organic acid) is an attractive approach for the selection of probiotic strains, as their in vivo production might play an important role in the prevention of infectious disease in the gastrointestinal tract of the host (Martín et al., 2005). The aggregation and adhesion ability of

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M.S. Riaz Rajoka et al. / LWT - Food Science and Technology 84 (2017) 271e280

probiotic microbes is also an important feature as they can form barriers, thus preventing colonization by pathogenic microbes (Ciandrini et al., 2016; Fontana, Cocconcelli, Vignolo, & Saavedra, 2015). Numerous studies furthermore evaluated the probiotic potential of lactic acid bacteria isolated from adult and infant feces, for aspects of their resistance to bile salt, low pH, lysozyme, adhesion to intestinal cells, and immunomodulatory properties (Davoodabadi, Dallal, Lashani, & Ebrahimi, 2015; Fernandez et al., 2014; Halimi & Mirsalehian, 2016; Rubio et al., 2014). Several reports indicated that lactobacilli strains carry antibiotic resistance genes that can be transferred to other bacteria in the host gut. Therefore, the evaluation of the antibiotic resistance properties of probiotics is very important. Furthermore, characteristics related to safety, survival in host gut, and colonization abilities are important for evaluating the proposed probiotic bacteria (Lavillarez-Pulido, Maqueda, & Valdivia, 2013; Tejero-Sarin ~ ena, Lerma, Pe Costabile, Gibson, & Rowland, 2012). The objective of the present study was to perform a thorough screening of Lactobacillus rhamnosus strains from human breast milk and to evaluate their potential use in probiotic products. 2. Material and methods 2.1. Subjects and sample cultivation Ten (10) breast milk samples of healthy women (women's age was in the range of 25e34 years and their infant age was in the range of 8 dayse45 days) were collected from the first affiliated hospital of the Xi'an Jiaotong University, Xi'an, Shaanxi, China. The milk samples were transported within sterile containers and processed immediately upon receipt. 2.2. Isolation and screening of lactic acid bacteria isolates The samples were diluted in PBS and spread on de Man-RogosaSharpe, supplemented with 0.05% L-cysteine (MRSc) agar plates. 1% CaCO3 was added to MRSc agar medium to indicate acid producing bacteria (Chen et al., 2016). The plates were incubated aerobically at 37 C for 72 h. Acid producing colonies were identified by a clear zone around each colony and the ones with different morphologies were selected for further purification. Single, pure colonies were isolated and subcultured for further experiments. Each selected LAB isolate was subjected to tests of morphology, catalase, and Gram reaction. The isolates of gram positive, catalase negative, and rods 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 utilized to identify the obtained strains. The total Genomic DNA of isolates was extracted using the Genomic DNA purification kit (TransGen Biotech Co., Ltd., Beijing, China), following the manufacturer's instructions. The primers used for amplifying the 16S rDNA sequences are forward 500 -AGAGTT TGATCC TGG CTC AG-300 and reverse 500 -CCGTCA ATT CCT TTGAGT TT-3” (Beasley & Saris, 2004). The fragments were amplified in a Techne-TC 512 Thermocycler (England) under the following conditions: 94 C, 30 cycles of 94 C for 45 s, 55 C for 30 s and finally 72 C for 10 min. The amplified fragment was screened on agrose gel and sequenced by the GenScript Company, Nanjing, China. All obtained sequences were screened via the BLAST program (https:// blast.ncbi.nlm.nih.gov/Blast.cgi). The sequences were deposited into Gene Bank. Sequence alignment was performed via ClustalW2 (http://www.ebi.ac.uk/Tool/mas/clustalw2/) and a phylogenetic tree was constructed via neighbor-joining (Saitou & Nei, 1987) and

maximum-composite likelihood methods (Tamura, Nei, & Kumar, 2004) using Mega 6.0 software (http://megasoftware.net/). 2.4. Survival under GIT conditions 2.4.1. Acid tolerance The ability of isolates to survive at pH 2.0, pH 3.0, and pH 6.2 (control) was evaluated following the method described by (Oh & Jung, 2015); acid resistance was evaluated via plate count on MRSc agar. 2.4.2. Bile salt tolerance The bile salt tolerance of isolated strains that survived for 3 h in acidic conditions were determined using MRSc broth containing 0.3%, 0.5%, and 1.0% of bile (w/v), following the method described by (Oh & Jung, 2015). Resistance was evaluated via plate count on MRSc agar. 2.4.3. Lysozyme resistance Tolerance to 100 mg/l lysozyme was assessed following the method reported by (Dias, Vilas-Boas, Hogg, & Couto, 2015). 2.4.4. Bile salt hydrolase activity The fresh bacterial cultures were streaked in triplicate on MRSc agar containing 0.37 g/l CaCl2 and 5 g/l sodium salt of taurodeoxycholic acid. The plates were incubated aerobically at 37 C for 3 days. The presence of precipitated bile salt around the colonies was considered to be a positive result. 2.4.5. Resistance to 0.4% phenol The ability to grow in the presence of phenol was evaluated using MRSc broth, supplemented with 0.4% phenol. The enumeration of bacteria was performed at 0 h and after 24 h of incubation at 37 C. 2.5. Autoaggregation The autoaggregation abilities of the selected isolates were analyzed according to (Collado, Meriluoto, & Salminen, 2008). LAB cells were washed twice with PBS, resuspended in the same buffer, and adjusted OD630 nm ¼ 0.25 ± 0.05. Bacterial cell suspensions were incubated at 37 C for 24 h with sampling at 2 h intervals and the percentage was expressed as autoaggregation % ¼ 1-(At/A0); where A0 represents the absorbance at 0 h and At represent absorbance at 2, 4, 6, 12, and 24 h. 2.6. Coaggregation In the coaggregation test, bacterial suspensions were prepared identical to the autoaggregation analysis above. The same volume of each LAB isolate and pathogen strain were mixed and incubated at room temperature for 24 h and the coaggregation percentages were monitored after incubation according to methods described by (Del Re, Sgorbati, Miglioli, & Palenzona, 2000). 2.7. Cell surface hydrophobicity A cell surface hydrophobicity assay using xylene was conducted following the method reported by (Kotzamanidis, Kourelis, Litopoulou-Tzanetaki, Tzanetakis, & Yiangou, 2010; Mishra & Prasad, 2005) and xylene was chosen as apolar solvent since it reflects both hydrophobicity and hydrophilicity (Kos et al., 2003).


2.8. Exopolysaccharide production LAB isolates were plated on a MRSc agar plate with glucose as the only carbon source and plates were incubated at 37 C for three days. Triplicate plates containing 25 to 250 colonies were scored for their mucoid property, following the method reported by (Manini et al., 2016).

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X-100 solution. The cell lysate was serially diluted and spread on MRSc agar plates. The plates were incubated at 37 C for three days. The percentage of bacterial adhesion was calculated according to the following Equation: Adhesion% ¼ (adhered bacteria/total added bacteria) 100

2.9. Biofilm quantification 2.14. Statistical analysis This assay was performed in a 96 well microtitter plate according to the method described by (Musk, Banko, & Hergenrother, 2005). 2.10. Antibiotic resistance test LAB isolates were screened for resistance against Streptomycin (10 mg/ml), Ampicillin (10 mg/ml), Erythromycin (15 mg/ml), Tetracycline (30 mg/ml), Gentamicin (10 mg/ml), Kanamycin (30 mg/ml), Penicillin (10 mg/ml), Cephalotoxin (15 mg/ml), Ciprofloxacin (5 mg/ ml), and Amoxicillin (30 mg/ml), using the disc diffusion method described by (Angmo, Kumari, & Bhalla, 2016; Jorgensen & Turnidge, 2015). After incubation for 24 h at 37 C, the diameter (mm) of the inhibition zone was measured. 2.11. Antimicrobial activity Agar well diffusion assays were utilized to test the antimicrobial activity as described by (Fontana et al., 2015). The 18 h old LAB cultures were tested against the indicator microorganism E. coli ATCC 25922, Salmonella enterica Serovar Typhimurium CMCC (B) 50115, and Staphylococcus petrasiisubsp. PragensisKY196531, which is a local isolate from human breast milk. 2.12. DPPH free radical scavenging ability The DPPH free radical scavenging ability of isolates was determined for the supernatants from 24 h MRSc medium cultures. 100 ml of supernatant cultures were mixed with 100 ml of ethanolic DPPH solution in 96 well plates. The plates were incubated in the dark for 30 min at room temperature. The absorbance was measured at 517 nm and the scavenging ability was calculated according to the following Equation: Scavenging ability (%) ¼ [1-(A517 of sample/A517 of blank)] 100

2.13. Adhesion to the Caco-2 cell line Caco-2 cells were grown in cell culture bottles using DMEM medium supplemented with 2 mM L-glutamine, 10% heat inactivated fetal bovine serum, 100 mg streptomycin/ml, 1% non-essential amino acid and 100 Ul penicillin/ml. Caco-2 cells were subsequently seeded into 24 wells culture plates at a concentration of 2.5 105 cells per well and allowed to differentiate for three days, while the medium was changed daily. The cells were incubated at 37 C in 5% CO2 atmosphere. Overnight cultures of the isolates were centrifuged, washed twice via PBS and resuspended in the same buffer to an appropriate dilution. After that, the bacterial cells were added to each well and plates were incubated at 37 C for 4 h. After incubation, cells were washed with PBS and lysed with 0.1% Triton

Values are presented as mean values and standard deviations of triplicate experiments. Significant ANOVA results were followed up with Tukey's Multiple Comparison Test in all assays and differences were considered statically significant if p < 0.05. 3. Results 3.1. Isolation and identification of Lactobacilli A total of 40 LAB isolates were isolated from human breast milk. Out of these, seven isolates showed an appearance of lactic acid bacteria on MRSc medium supplemented with 1% CaCO3 and were randomly selected for further experiments. All isolates were gram positive, catalase negative, rod shaped, and showed a clear zone around the colonies on MRSc medium supplemented with 1% CaCO3. All isolates were mesophilic and able to grow at 30 C and 37 C in the presence of 2.5%e5% NaCl concentration within medium. A phylogenetic tree was constructed based on their 16S rDNA sequences utilizing the neighboring method. The results showed that the seven strains belonged to Lactobacillus rhamnosus (Fig 1). 3.2. Survival under GIT condition 3.2.1. Acid tolerance Fig. 2A shows survival rates of the isolated strains at pH 2.0 and pH 3.0 after 3 h of incubation. At pH 3.0, the survival rate was higher compared to at pH 2.0 for all tested isolates. The survival rates of SHA111, SHA112, SHA113, SHA114, SHA115, SHA116, and SHA117 remained above 81% and 90% after exposure to pH 2.0 to pH 3.0 for 3 h. The results indicate that all tested strains showed high resistance to acidic conditions. More importantly, the isolates SHA113 and SHA117 showed high resistance to very low pH values. 3.2.2. Bile tolerance The results revealed that all isolates tolerated various concentrations of bile salt (0.3%, 0.5%, and 1%) during 3 h incubation (Fig. 2B). However, with increasing bile salt concentration, their growth rate decreased. The isolate SHA116 showed better tolerance to 0.3%, 0.5%, and 1% bile salt than any other isolates. 3.2.3. Lysozyme tolerance The overall resistance of the isolates to lysozyme (100 mg/l) was expressed as the percentage of survival rate, ranging from a minimum of 49.6% to a maximum of 100%. Isolates SHA113, SHA115, SHA116, and SHA117 showed high resistance (>84%) after 120 min of incubation, which was considered as a severe treatment compared to control (Fig. 2C). 3.2.4. Bile salt hydrolase activity All isolates showed the ability to hydrolyze the sodium salt of taurodeoxycholate. This became apparent due to the presence of a

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hole around the colonies after growth in MRSc agar plate supplemented with 0.37 g/l CaCl2 with 5 g/l sodium salt of taurodeoxycholic acid (Table 1). 3.2.5. 0.4% phenol tolerance All tested isolates showed impactful tolerance to 0.4% phenol after 24 h incubation (Table 1). 3.3. Autoaggregation and coaggregation All isolates had high autoaggregation and a clear supernatant after 24 h incubation (Fig. 3A). The isolate SHA111 showed the highest autoaggregation rate (59%), while SHA113 showed the lowest (51%). In addition, autoaggregation abilities of all isolates increased after 24 h of incubation. All isolates showed marked coaggregation with E. coli ATCC 25922, S. entericserovar Typhimurium CMCC (B) 50115, and S. petrasiisubsp. PragensisKY196531) (Fig. 3B, C, D). Among the tested isolates, SHA117 showed the highest coaggregation capability with E. coli (49%), Salmonella (52%), and Staphylococcus (29%). 3.4. Cell surface hydrophobicity In general, SHA111, SHA113, SHA114, SHA116, and SHA117 had higher percentages of hydrophobicity (51%, 68%, 50%, 60%, and 69%) compared to other isolates. However, isolate SHA115 had the lowest hydrophobicity (33%) and isolate SHA112 had an intermediate level of hydrophobicity (Fig. 4B).

3.5. Exopolysaccharide production All isolates had the capability to produce exopolysaccharides when they grew on MRSc agar plates that were supplemented with glucose (2% v/v) as a carbon source (Table 1).

3.6. Biofilm quantification A 96-wells micro titter plate assay was carried out to quantify biofilm formation. All isolates were found to form biofilms on the bottom of the well in MRSc broth under aerobic conditions (judging by their capacity to retain crystal violet). However, biofilm-forming capability differed among strains (Fig. 5B). Isolates SHA114, SHA116, and SHA117 showed high biofilm-forming capability (O.D540 > 1.7), followed by SHA111, SHA112, and SHA115 (O.D540 > 1.0).

3.7. Antibiotics resistance The minimum inhibitory concentrations of isolates were measured for ten antibiotics (Table 2). All isolates were resistant to Streptomycin, Ampicillin, Gentamicin, Kanamycin, Penicillin, Cephalotoxin, and Ciprofloxacin and susceptible to Amoxicillin according to the break point introduced by (Panel, 2012). SHA114 and SHA115 were also susceptible to Tetracycline and Erythromycin.

Fig. 1. Position of the selected seven isolates in the neighbor joining phylogenetic tree. The phylogenetic tree was constructed on the basis of 16S rDNA sequences. The scale bar 0.01 indicates the nucleotide substitution rate at each site. Bootstrap probabilities were determined using 1000 replicates and presented as the percentage values. The numbers in parentheses are the accession numbers of selected sequences. The filled circles indicate the strains are from NCBI and the Empty circles are the out groups used for tree construction.


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Fig. 2. Resistance of the selected Lactobacillus rhamnosus isolates against acidic conditions (A), bile salt (B), and lysozyme (C). (A) The treatments were carried out under pH 2.0 or pH 3.0 for 3 h. The results are the value of mean ± SD from three independent runs. The data are significantly different from that of the controls (pH 6.2) at the level of p < 0.05. (B) The broth is supplemented with 0.3%, 0.5% and 1% bile salt. The control is MRSc broth without bile salt. The results are expressed as mean ± SD of three independent replicates. All treatments are significantly different from the control at the level of p < 0.05. (C)The MRS broth was supplemented with 100 mg/l lysozyme. The control is carried out in MRS broth without lysozyme. All treatments are significantly different from the control at the level of p < 0.05.

3.8. Antimicrobial activity All isolates expressed a clear inhibition zone (with a diameter above 15 mm) against the three indicator strains. However, the

results obtained from the agar well diffusion method varied from 6 mm to 14 mm among different strains. Comparatively, the antimicrobial activity of all isolates against E. coli and Staphylococcus (8 mme13 mm) was higher than those against Salmonella

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Table 1 Tolerance to 0.4% Phenol, BSH activity and exopolysaccharide production of the selected isolates. Isolates

Tolerance to 0.4% phenol (indicated as the log value of CFU/ml)a 0h

SHA111 SHA112 SHA113 SHA114 SHA115 SHA116 SHA117

8.78 8.65 8.20 8.86 7.95 8.19 7.85

0:05 0:2 0:1 0:1 0:05 0:1 0:1

8.25 8.89 7.89 9.89 8.13 8.65 8.54

Exopolysaccharide productionc

þ þ þ þ þ þ þ

þ þ þ þ þ þ þ

△(24 h-0 h)

24 h ± ± ± ± ± ± ±

BSH activityb

± ± ± ± ± ± ±

0:02 0:1 0:1 0:05 0:05 0:05 0:05

0.53 0.24 0.31 1.03 0.18 0.46 0.69

a

Values are represented as mean± SD (n ¼ 3). þ, positive in BSH activity, identified as the occurrence of precipitated zone (mm) around bacterial colonies on MRSc agar plates supplemented with 0.37 g/l CaCl2 and 5 g/l sodium salt of taurodeoxycholic acid.a. c þ, positive in exopolysaccharide production, identified as the occurrence of sticky bacterial colonies. b

typhimurium (Table 3). 3.9. DPPH scavenging ability Fig. 5A shows that the isolate SHA112 had the highest DPPH scavenging ability (88%), followed by SHA115 (85%), SHA116 (85%), SHA114 (85%), SHA113 (84%), SHA111 (76%), and SHA117 (79%). 3.10. Adhesion to Caco-2 cells No significant differences were found among different isolates regarding their adhesion abilities (Fig. 4A). Comparatively, the isolate SHA113 showed the strongest adhesion ability (82%) to Caco-2 cells. However, it should be mentioned that other isolates also showed high adhesion abilities to Caco-2 cells in a range of 63%e76%. 4. Discussion In infants, the establishment of gastrointestinal tract microbiota is assumed critical for maintaining health and homeostasis of animals, including humans. Breast milk is the most important factor for immunological programming, metabolome, and microbiome (Aaltonen et al., 2011). It is also an important source of gut microflora for new born babies, since it is the only food babies receive (Arici, Sagdic, & Ozdemir, 2004). Numerous studies indicate that the decrease of the abundance of lactic acid bacteria in the gut is one of the major factors related to the reduction of immunological capability and metabolic disturbance in adults and aging persons (Tsai, Cheng, & Pan, 2012). Therefore, microorganisms from breast milk samples are suggested to aid the maintenance of human health. To verify this hypothesis and to retain lactic acid bacteria with high potential for human health, we tested isolates from different breast milk samples and their potential for health-keeping in aspects of different conditions similar to that in the stomach and initial intestines. Many lactic acid bacteria have been developed as probiotic microbes to improve the health of animals and humans (Rajoka, Shi, Zhu, Shao, Huang & Yang, 2017). Lactobacillus rhamnosus was reported to have multiple functions in the maintenance of human health, such as modulation of immune system and protection against invading pathogenic microbes (Kaewiad, Kaewnopparat, & Kaewnopparat, 2015). However, many studies are still being carried out to isolate new strains with higher potential, since the currently obtained strains still have weaknesses in practical application, especially low survival rate and poor proliferation ability through stomach and guts. The ability to survive under high bile salt concentrations and low pH are important features for the successful passage through the gastrointestinal tract (Mandal, Jariwala, &

Bagchi, 2015; Oh & Jung, 2015). In this study, seven Lactobacilli isolates were obtained from the breast milk of healthy mothers and were selected according to their survival capability under artificially simulated conditions in digestive systems. Among these seven, four isolates showed more than 80% survival rate at a bile concentration of 1.0% (w/v), a level much higher than that typically used in other studies in which Lactobacillus rhamnosus IMC501 and Lactobacillus paracasei IMC502 were tested at a bile concentration of 0.3% (m/v) (Verdenelli et al., 2009). In addition, all seven isolates selected in the study showed more than 80% survival rate at pH 2.0 and more than 90% at pH 3.0 after 3 h exposure, indicating that they can survive passage through the digestive systems. Their survival rate was higher than that of previously reported strains such as Lactobacillus rhamnosus and Lactobacillus casei under such low pH values (Manini et al., 2016; Tulumoglu et al., 2013). The response of Lactobacilli to lysozyme was previously found to be species and strain dependent (Dias et al, 2015). Our results revealed that the isolate SHA115 was highly resistant to 100 mg/l lysozyme (resulting in a survival rate above 91%, which is much higher than previously reported for Lactobacillus rhamnosus strains PRA211 and PRA323 (Solieri, Bianchi, Lemmetti, & Giudici, 2014)). Phenol is a common byproduct of the aromatic amino acid metabolism in the intestine. In our study, all isolates tolerated high phenol concentrations (0.4%), indicating that they can resist the bacteriostatic effect of phenol in the intestine (Palaniswamy & Govindaswamy, 2016). The properties of cell surface hydrophobicity, autoaggregation, and coaggregation are related to the adhesion properties of lactobacilli strains and are essential for the protection and colonization of the gastrointestinal tract. A minimum value of 40% hydrophobicity is an essential requirement for a probiotic strain (Del Re et al., 2000). In this study, the isolates SHA113 (68%) and SHA117 (69%) showed high hydrophobicity to xylene. They also showed high autoaggregation (SHA113: 52% and SHA117: 57%) and coaggregation abilities with E. coli (SHA113: 42% and SHA117: 49%) and Salmonella (SHA113: 48% and SHA117: 52%) during a 24 h incubation period, indicating that these two isolates from breast milk are suitable for animal and human use. This is consistent with studies reported by others (Dias, Duarte, & Schwan, 2013; Palaniswamy & Govindaswamy, 2016). Exopolysaccharide is a multifunctional compound that has interesting applications in both pharmaceutical and food industries (Rendueles, Kaplan, & Ghigo, 2013; Russo et al., 2012). All isolates in the study showed a ropy phenotype that is clearly related to exopolysaccharide production on MRSc agar medium. This is in agreement with results reported by others (Degeest, Janssens, & De Vuyst, 2001; Minervini et al., 2010). Bacterial biofilms are an important factor to understand the mechanisms of bacteria to adapt to environmental stress and


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Fig. 3. Aggregation ability (A) and the coaggregation ability of the selected Lactobacillus rhamnosus isolates with E. coli ATCC 25922 (B), Salmonella Typhimurium CMCC (C) and Staphylococcus petrasii subsp. Pragensis KY196531 (D). All the results were obtained after 24 h and the values are represented as mean ± SD of three independent replicates. (A) The aggregation ability significantly different between different isolates at the level of p < 0.05 as measured by Tukey's test. (B) No significant difference was found among different isolates on the data of coaggregation ability at the level of p < 0.05 as measured by Tukey's test.

278


Fig. 4. The adhesion ability of the selected isolates to Caco-2 cells (A) and the hydrophobicity ability of the selected isolates (B). (A) The error bars indicate the SD from three replicate. (B) The data are the mean ± SD of three independent runs of three analyses. The results are significantly different among different strains at the level of p < 0.05.

colonize different niches. L. rhamnosus has the ability to form biofilms in vitro (Jones & Versalovic, 2009). We found three isolates from mother milk (SHA112, SHA116, and SHA117) that had the ability to form biofilms on the bottom of 96-well microtitter plates. This is consistent with the reported results of L. rhamnosus 10863 n, Camino, & Sua rez, 2008). (Rebeca Martín, Sobero Due to safety considerations, the obtained isolates were also tested on their capabilities for antibiotics resistance. As a result, none of the tested isolates were resistant to amoxicillin, while all strains were resistant to streptomycin, ampicillin, gentamicin, kanamycin, penicillin, cephalotoxin, and ciprofloxacin. These results are consistent with previous results that were obtained using L. rhamnosus GG species (Chang, Zhang, Ke, Jian-Ping, & Xiao-Kui, 2009; Maragkoudakis et al., 2006). The production of antimicrobial compounds is one of the centrally important features for probiotics to compete with and exclude pathogens, ultimately to survive in the intestinal tract and to express probiotic effect in their hosts (M Carmen Collado, Gueimonde, Sanz, & Salminen, 2005). In this study, all selected isolates had strong antimicrobial activity against E. coli, which is in agreement with a previous study reported by Tulumoglu et al. (2013). Some recent investigations revealed that L. rhamnosus could act as an antioxidant by releasing some peptide with the ability to quench oxygen radicals (Siragusa et al., 2007). In this study, the cell free supernatant culture of isolates SHA112

Fig. 5. DPPH Scavenging ability (A) and the biofilm production of the selected isolates (B). All data are the mean ± SD values of three independent experiments of three analyses. The results are significantly different among different isolates at the level of p < 0.05 as measured by Tukey's test.

(88%), SHA113 (84%), SHA114 (85%), SHA115 (85%), and SHA116 (85%) showed strong antioxidant activity, which was better than results found by others (Oh & Jung, 2015; Palaniswamy & Govindaswamy, 2016). The ability to adhere to Caco-2 cells is also an important criterion for the selection of probiotic lactic acid bacteria. This characteristic provides beneficial effects, such as immune system modulation and exclusion of pathogenic microbes (Lee, Puong, Ouwehand, & Salminen, 2003; Schiffrin, Rochat, Aeschlimann, & Donnet-Hughes, 1995). Various in vitro model systems have been developed for the preliminary selection of adhered strains (Vesterlund, Paltta, Karp, & Ouwehand, 2005). In our study, the adhesion capability to Caco-2 cells varied from 60% to 84% among different isolates, which was much better than in previously found isolates (Caggia, De Angelis, Pitino, Pino, & Randazzo, 2015; M Carmen; Collado, Jalonen, Meriluoto, & Salminen, 2006).

5. Conclusion Breast milk is an excellent source of lactic acid bacteria. In this study, seven L. rhamnosus strains with probiotic potential were isolated from breast milk of healthy women. All L. rhamnosus


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Table 2 Antibiotic resistance of the selected isolates.a Antibiotics

SHA111

SHA112

SHA113

SHA114

SHA115

SHA116

SHA117

Streptomycin (10 mg/ml) Ampicillin (10 mg/ml) Erythromycin (15 mg/ml) Tetracycline (30 mg/ml) Gentamicin (10 mg/ml) Kanamycin (30 mg/ml) Penicillin (10 mg/ml) Cephalotoxin (15 mg/ml) Ciprofloxacin (5 mg/ml) Amoxicillin (30 mg/ml)

þ þ e þ þ þ þ þ þ e

þ þ þ þ þ þ þ þ þ e

þ þ þ e þ þ þ þ þ e

þ þ e e þ þ þ þ þ e

þ þ e e þ þ þ þ þ e

þ þ þ e þ þ þ þ þ e

þ þ þ þ þ þ þ þ þ e

a

Indicated as ‘þ’, resistance and ‘-‘, susceptible.

Table 3 Antimicrobial activity of the selected isolates against different bacteria (mm).a Isolates

E.coli ATCC 25922

Salmonella enteric Serovar Typhimurium CMCC (B) 50115

SHA111 SHA112 SHA113 SHA114 SHA115 SHA116 SHA117

11 ± 0.5 12 ± 0.5 10 ± 0.5 9 ± 0.4 10 ± 0.6 8 ± 0.3 9 ± 0.5

8 7 6 7 7 9 9

± ± ± ± ± ± ±

0.1 0.1 0.5 0.6 0.3 0:4 0.5

Staphylococcus petrasii subsp. PragensisKY196531 11 ± 0.3 11 ± 0.2 9 ± 0.5 10 ± 0.5 10 ± 0.4 11 ± 0.2 13 ± 0.2

a The antimicrobial activity is indicated as the diameter of the inhibiting zone around the isolate colonies. The values are the expressed as mean ± SD (n ¼ 3). The data less than 5 mm indicate weak antimicrobial activity; 6 mme8 mm indicate strong antimicrobial activity, 9 mme14 mm indicate strong antimicrobial activity.

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