PROBIOTIC POTENTIAL AND STRESS TOLERANCE IN LACTOBACILLUS MU1008 ISOLATED FROM CHILLED YOGURT SAMPLES OF MAJMAAH

PROBIOTIC POTENTIAL AND STRESS TOLERANCE IN LACTOBACILLUS MU1008 ISOLATED FROM CHILLED YOGURT SAMPLES OF MAJMAAH

Johra Khan ^{* 1}, Bader Alshehri ¹ and Saeed Banawas ^{1, 2}

Department of Medical Laboratory Sciences ¹, College of Applied Medical Sciences, Majmaah University, Majmaah 11952, Saudi Arabia.

Department of Biomedical Sciences ², Oregon State University, Corvallis, OR 97331, USA.

ABSTRACT: In recent years, scientific research has confirmed the presence of large amounts of probiotic Lactobacillus sp. in fermented dairy products, which shows a positive impact on human health. This study was designed to isolate Lactobacillus with probiotic potential from local and commercial samples. 40 Lactobacilli strained were isolated from commercial and local yogurt samples by tenfold dilution plating method and plated on Man-Rogosa-Sharp agar medium and were screened for their use as potential probiotics. The isolates were tested for their ability to survive at pH 2.0, pH 3.0, in the presence of 0.3% bile salt and antimicrobial activity; sensitivity against 10 specific antibiotics (ciprofloxacin, erythromycin, tetracycline, penicillin G, ampicillin, streptomycin, polymyxin B, vancomycin, chloramphenicol and rifampicin) using filter paper disc diffusion method and two-fold serial dilution methods. Out of the 40 strains, 24 strains (42%) had survival rates above 90% after 2 h of incubation at pH values of 2.0 or 3.0 pH. Further screening performed on the above 40 isolates indicated that 24 strains show to 0.3% bile salt. Lactobacilli strains exhibited inhibitory activity against Salmonella strains and Candida albicans. Moreover, all of the strains were resistant to vancomycin and streptomycin. The 24 strains that were found suitable for Potential probiotic activity including strains Lactobacillus casei (95%), Lactobacillus salivarius (25%), Lactobacillus plantarum (75%), and Lactobacillus buchneri (72%).

Lactobacilli, Probiotic Potential, In-vivo, Antimicrobial Activity, Genotypic identification

INTRODUCTION: From the group of lactic acid-producing bacteria also known as LAB, are among the largest group whose members are found in intestinal tract of vertebrates, including humans, that are involved inz process of fermentation of different food products, resulting in improving the quality of food and safety, including the health status and comfortableness of the consumers.

These characteristic in a microorganism make them safe for use as food and as medication in the form of probiotics ¹. The other probiotic strains also include Pediococcus, Bifidobacterium, Lactobacillus and Enterococcus ².

Probiotics are characterized as living micro-organisms which provide good effects on the organism, and can change as per the host body’s micro-ecological scale, modify intestinal functions, also affect digestion and immune response. Lactobacillus sp. was among the earlier discovered probiotics out of the three types of probiotics, which include Bifidobacterium and Gram-positive cocci ³. A lot of species of Lactobacillus are accepted as safe for intake, so, are often used in food products ⁴. Members of species of Lactobacillus, which include Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus johnsonii, Lactobacillus reuteri, and Lactobacillus rhamnosus, are used as probiotics ⁵. A lot of recent years research has confirmed the existence of large number of probiotic Lactobacillus sp. in fermented food and dairy production which shows positive effect on human health. More emphasis has been given to the probiotic potential of Lactobacillus.

Different elements should be considered while screening the potential probiotic lactic acid bacterial strains in chilled samples which has to be used for consumption. The more advanced studies show that when probiotics are to be used for gut health, the bacterial strains shall survive the passage through the gastrointestinal tract ^{5, 6}. Taking probiotic as food or with food are good way to get its benefits and yogurt and ice cream are commonly consumed by human which are easy transporting vehicles for these beneficial lactic acid bacteria ⁷.

This study was designed to identify the best potential probiotic Lactobacillus isolates from household and commercially available 40 chilled yogurt samples collected from Majmaah. The in-vitro probiotic properties like Bile tolerance, pH, acid tolerance, antimicrobial activity, antibiotic susceptibility, auto and co-aggregation property and cell surface hydrophobicity of the selected isolates were studied.

MATERIALS AND METHODS:

Sampling: Forty samples of yogurt were collected of different commercial brands. Isolation was based on methods Ananthanarayan, R ⁸. Lactobacillus sp were isolated on Man-Rogosa-Sharp (MRS) agar medium and stored at 37 ºC for 24 to 48 h for further phenotypic and genotypic identification ⁹.

Phenotypic and Genotypic Identification of Stains: Forty strains were isolated from the from commercial and yogurt samples by tenfold dilution plating method and plated on MRS Medium. All the isolates were first identified using conventional method of Gram staining, catalase activity and gas production in presence of glucose followed by carbohydrate fermentation. 16Sr RNA gene sequence analysis technique was used to identify the species. For this process genomic DNA was isolated using Genomic DNA isolation kit (HiPurA™, HiMedia). The amplification of 16SrRNA was done using thermal cycler and using prokaryotic 16S ribosomal DNA universal primer pair BSF8/27 (5′-AGAGTTTGATCCTGGCTC AG-3′) and BSR1492/20 (5′-GGTTACCTTGTTA CGACTT-3′) ^{10, 11}. All the sequences obtained are then compared using BLAST to the Gene Bank database. Resulting in the identification of 31 Lactobacillus isolates.

Acid Tolerance: Acid tolerance test was done as per the method given by Chung et al., ⁹ with little changes. In in this method, 10 μL of overnight stored bacterial samples in MRS broth were inoculated into 1 mL of broth of MRS with pH 2.0, and pH 3.0, and pH 6.4 as control.

Study of Culture Characters: The enumeration of culture characters and morphology was carried out according to the method described by Banson, H.J ¹⁰.

To study these aspects, streak plate method was performed. Individual colonies grown on MRS agar plates were carefully studied with respect to size, color, opacity, form rise and margin.

Proteolytic Activity: To study proteolytic activity of lactic acid bacteria, MRS agar with additional 10% skimmed milk was introduced, left for solidification and then dry. Whatman paper discs sterilized and kept on the surface of MRS agar. 20μl young culture were given to each paper disk. Incubated at 37 ºC for 24 hour, after which proteolytic activity was measured as diameter of clear zones around the discs ¹¹.

Lipolytic Activity: To measure Lipolytic activity, all strains were introduced on agar having spot in Tween 80 with different concentrations as 1%, 3% and 5% ¹². Incubated for 72 h at 25 ºC. The strains which showed an opaque area around the spot because of esters formation with calcium which produce fatty acids are recorded as positive¹³. Diameter of lytic zone were measured to study the lipolytic activity.

Cell Surface Hydrophobicity: To study the bacterial adherence with hydrocarbons was done as per Mishra and Prasad. Lactobacillus sp. were harvested after incubation for 18 h at 37 °C precede by centrifuging the sample at 5000 rpm for 15 min. The cells collected were then washed two times with Phosphate Urea Magnesium Sulphate (PUM) buffer and then kept on an individual basis in PUM buffer at 10⁸ cfu/ml level. The spectrophotometer reading of the suspension were recorded at 600 nm (A). From each cell suspension an amount of 5 ml was blended in 1 ml of hydrocarbon including ethyl acetate, xylene, toluene and chloroform. The mixture obtained was then vortexed for 1 min and the phases were separate after one hours at 37 °C. The aqueous stage was cautiously separated using a sterilized Pasteur pipette and the absorbency (A0) was read at 600 nm to compute cell surface hydrophobicity ¹⁴.

Medium: LS broth with 0.4%- 1% bile salt (Sodium taurochlate).

Procedure: LS broth was prepared with 0.4%, 0.6%, 0.8% and 1% sodium taurochlate and dispended to test tubes in appropriate proportion. The tubes were inoculated by loop inoculation process and then incubated at 37 ºC for 24 h along with a control. The results were analyzed after 24 hours of incubation spectrophotometrically at 600nm.

Test for Anti-microbial Activity: Probiotic strains have been demonstrated to inhibit the growth of many enteric pathogens. There are several metabolic compounds produced by probiotic bacteria such as organic acids, fatty acids, hydrogen peroxide, diacetyl, bacteriocin and proteinaceous substances, which have antimicrobial effect.

Medium: Soft nutrient agar with 6g/L of agar (pH-7.2).

Procedure: Soft nutrient agar was prepared with 6g/L of agar (pH-7.2), the agar medium was cooled to 45 ºC. The medium was then poured into four sets of sterile petri plates aseptically and allowed to set. After cooling, 100µL of inoculums of three different pathogenic strains were pipetted out separately with the help of micropipettes are poured aseptically to the center of the agar plates of each set. With the help of sterile paper discs (about 8mm thick) were placed aseptically on the center of the inoculation plates. These paper discs were then inoculation at 37 ºC for 24 h. After 24 h of inoculation the plates were observed for zone of inhibition. The four pathogenic strains were Salmonella typhi. E. coli, Staphylococcus aureus and Candida albicans and the test isolate were lactobacillus species.

Antibiotic Susceptibility Test: To test the sensitivity of bacteria to different antibiotics disk diffusion method ¹⁴ was performed using dodeca discs (Hi Media, India) of different antibiotics like Ciprofloxian, Erythromycin, Tetracycline, Peni-cillin, Ampicillin, Streptomycin, Vancomycin, Azithromycin, Chloramphenicol, Gentamycin, Neomycin and Oxacillin. The active cultures were poured into the plate with a base layer on Muller Hinton agar plates. The inhibition zone diameters were recorded after 24 h of incubation at 37 °C.

Statistical Analysis: Statistical analyses were done using SPSS 14.0 software (SPSS Inc.; Chicago, IL, USA). Significant differences among treatments were tested by ANOVA developed by Ronald Fisher in 1923 with a level of significance at α = 0.05. Data were expressed as Mean Values ± Standard Deviation (SD). All experiments were performed in duplicate and repeated three times.

RESULTS AND DISCUSSION:

Morphological and Colony Characteristics: Morphological and colony characteristics of the isolated bacterial culture from yogurt samples were observed carried out. All together four types of different bacterial colonies with distinct morphological characters were isolated.

Identification by Molecular Biology Method: Out of 40 stains, 34 were found Gram-positive, rod-shaped, and catalase-negative using the conventional method of identification, whereas by using molecular biology technique, isolated stains were found to be Lactobacillus casei (28 strains), Lactobacillus salivarius (2 strains), Lactobacillus plantarum (3 strains) and Lactobacillus buchneri (1 strain).

Result of Agitation: The growth in the incubator shaker was less than compared to the ordinary incubator; thus, it confirms that the isolates are all facultative anaerobes. The spectrophotometric analysis was shown in graphical representation Table 1.

Acid Tolerance: For a potential probiotic bacteria to work efficiently, it must have the capacity to survive in the gastrointestinal tract. In many studies, the acid resistance for Lactobacillus at pH 2.0 and 3.0 was studied with MRS broth ¹⁴. In our study, 34 strains of Lactobacillus were studied for acid tolerance in this study. Acidic pH affects the growth of Lactobacillus a lot. It was found that 24 strains had good resistance to low pH, as shown in Table 3. It shows that the survival rate of 10 strains after 2 h incubation compared with the control. Lactobacillus 1004, 2014, 3026, 3030, 3035, 4036, 4038, 4039, 1008, and 4037 were found to be ≥ 90% at pH 3.0 and Lactobacillus strains 1004, 1024, 3026, 3030, 3032, 3035, 4036, 5039 and 1008 were found to be ≥ 90% at pH 2.0. Strain 3032 was the most acid-tolerant at pH 2.0, with survival rates of 98%. Out of 24 strains, eleven strains showed a survival rate ≥ 90% at pH, as shown in Table 1.

TABLE 1: RESULT OF VIABILITY (LOG CFU/mL) AT DIFFERENT pH, SURVIVAL PERCENTAGE AND AGITATION AT 600 nm OF DIFFERENT LACTOBACILLI STRAINS

Bile Salt Tolerance: Bile salt concentration 0.4% -1% show different degrees of inhibition on 24 strains. The result obtained was analyzed using Gilliland et al., (1984) ¹⁵.

Two strains Lactobacillus 3032 and 1004 were considered to be resistant (d≤ 15 min); Out of 24 strains, 4 strains were found to be tolerant strains (1008, 1024, 3030 and 3035; 15 < d ≤ 40 min); 11 strains (Lactobacillus 1007, 2011, 2012, 2014, 2022, 3026, 4036, 4038, 4039, 4037 and 5039; 40<d<60) were found to be weakly tolerant strains as shown in Fig. 1.

Antibacterial Activity: The antimicrobial activity against four pathogenic strains were examined on 10 Lactobacilli strains that were found good in screening tests. Lactobacillus strains 1008, 1024, 3030, 3035, 2022, 3026, 4036, 4038, and 5039 showed an inhibitory effect against E. coli GU128 with an inhibition zone 12.60-24.50 mm in diameter. All the 24 strains showed positive results against all the four pathogens (with inhibition zones 12.60-30.50 mm in diameter). Lactobacillus plantarum 3032 shows zone of inhibition from 24.60- 30.50 mm in diameter Table 2.

FIG. 1: BILE ACID TOLERANCE (0.4% TO 1.0%) OF DIFFERENT STRAINS AT OPTIMUM DENSITY OF 600 nm

TABLE 2: ANTIMICROBIAL ACTIVITY OF DIFFERENT LACTOBACILLI STRAIN AGAINST DIFFERENT E. COLI, STAPHYLOCOCCUS AUREUS, SALMONELLA TYPHI AND CANDIDA ALBICANS

*+, Diameter of inhibition zone: 8-12 mm; ++, 12 to 16mm; +++, 16 to 20mm; ++++, more than 20mm

Antibiotic Susceptibility Test: To study the resistance of different 20 lactobacilli strains against 12 different antibiotics susceptibility test was carried out and the result are shown in Table 3.

TABLE 3: ANTIBIOTIC SUSCEPTIBILITY TEST FOR LACTOBACILLUS ISOLATES AGAINST DIFFERENT ANTIBIOTICS

Cell Surface Hydrophobicity: The cell surface hydrophobicity of 24 different strains of lactobacilli were studied. The hydrophobicity of these strains was found significant (p<0.05; p<0.01), and the percentage of hydrophobicity was found between 10% to 97%.  It was found that the hydrophobicity of five strains was more than 60%: 3031 (96%), 3015(92%), 4038 (87%), 4040 (73%), and 2019 (68%). Result as shown in Table 4.

TABLE 4: PERCENTAGE OF CELL SURFACE HYDROPHOBICITY OF SEVEN STRAINS WITH HIGH HYDROPHOBICITY

The bacterial species isolated from curd samples were subjected to general bacteriological isolation techniques. Among the biochemical test, the important results showed by the isolates were catalase-negative, lactic acid production from glucose, and heavy growth on Tomato juice agar. All the isolates give pigmentation, which ranges from orange to brown color. These results have brought the isolates under Lactobacillus genus. 16Sr RNA gene sequence analysis technique was used to identify the species. The growth of isolates on MRS agar plates at pH 6.5 also confirms the lactobacillus sp. ¹⁶. For probiotic bacteria to work efficiently, it must have the capacity to survive in the gastrointestinal tract. In many studies, the acid resistance for Lactobacillus at pH 2.0 and 3.0 was studied with MRS broth. In a study on ewe’s milk, the strains showed favorable resistance at pH 2.0 of 72% from 50 lactobacillus strains ¹⁷.

In the present study, more than 40% strains showed the survival rate of at pH 3.0, whereas 37.5% of the strains showed the survival rate at pH 2.0 Table 1. In the human gut, NaCl is an inhibitory substance that can inhibit the growth of the microbe so, the isolates were grown in a medium with NaCl concentration ranging between 0.4-1% bile concentration, and it was found that all 24 strains were able to survive at 0.5% bile salt, but only 6 were found to be tolerant to all concentrations between 0.4-1% whereas the rest were found to be weakly tolerant Fig. 1. All the bacterial strains to be used for probiotics must not have any antibiotic-resistant gene present ¹⁸; for this reason, all the strains were assessed for antibiotic resistance test. The result, as shown in Table 4, all the strains were found susceptible to all antibiotics tested except oxacillin and ampicillin, which shows the strains are safe to be used for probiotic potential. The most important use of probiotic bacteria is to protect the organism from pathogenic bacteria within the intestinal eco-system ¹⁹.

In the present study, all 24 strains showed different levels of inhibition against E. coli, Staphylococcus aureus, Salmonella typhi, and Candida albicans. A similar result was reported in many studies against gram-positive and gram-negative bacteria ²⁰. In comparison to a study by Osuntoki et al., ²¹ where the Lactobacillus spp. showed antimicrobial activity only against some clinically important pathogens like E. coli (4.2 mm), Salmonella typhi (4.3 mm), and Listeria monocytogenes (5.0 mm).

Cell surface hydrophobicity is an important character of probiotic bacteria to adhere to the intestinal mucus. The results of the present study Table 4 shows that the lactobacillus strains isolated from the curd sample of Majmaah have very high cell surface hydrophobicity 96%, which is higher than the study by Zhang et al., ²² which was reported on 92.15%.

CONCLUSION: In conclusion, out of 40 strains, 24 strains were selected as appropriate probiotic potential strains which can be used for promoting hosts intestinal health and to maintain healthy natural micro-flora during antibiotic treatment. Also need for in vivo study is there to verify the effectiveness of selected strains.

ACKNOWLEDGEMENT: The author would like to thank the Deanship of Scientific Research at Majmaah University for supporting this work under Project Number No. 64/51942.

CONFLICTS OF INTEREST: None declared.

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    How to cite this article:

    Khan J, Alshehri B and Banawas S: Probiotic potential and stress tolerance in Lactobacillus MU1008 isolated from chilled yogurt samples of majmaah. Int J Pharm Sci & Res 2021; 12(1): 654-60. doi: 10.13040/IJPSR.0975-8232.12(1).654-60.

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