Probiotic Properties of Lactic Acid Bacteria from Dairy Milk

Abstract

Lactic Acid Bacteria (LAB) are indigenous habitants of the human Gastro Intestinal Tract and have a long history of use in foods and fermented products as starter cultures. A variety of microorganisms, typically Lactic acid bacteria (LAB), have been evaluated for their probiotic potential and are explored as adjunct cultures in various types of food products or in therapeutic preparations.

Therefore, the present study was aimed at isolation of lactic acid bacteria from buffalo and goat milk and their characterization for probiotic potential.

Three different LAB Species were isolated from buffalo milk and two different species were isolated from goat milk sample. The isolates were all found to be tolerant to acidic pH=3, tolerant to bile and simulated pancreatic fluid. Antimicrobial activity testing showed that most of the isolates were able to produce antimicrobial compounds. These isolates may be further used as probiotics to promote health of hosts, protect hosts from intestinal pathogens and maintain the natural balance of intestinal microflora during antibiotic treatments.

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Introduction

Human beings as well as animals are normally born sterile but shortly after birth, colonization begins with different body parts being occupied by the fittest microbes from the environment, thus creating a balanced ecological system. The normal microflora changes dramatically during the lifetime of the host. The Lactic Acid Bacteria (LAB), if present, constitutes the dominant flora population due to their ability to colonize the human and animal intestinal tract. However, exogenous bacteria, either of probiotic or pathogenic origin, influence the intestinal bacterial flora. The acidity of the stomach maintains a low concentration of bacteria in the upper part of the digestive tract and destroys pathogens.

Interactions that occur between various bacterial species are also important in maintaining the equilibrium of the intestinal microflora (Olanrewaju, 2007).

Milk and milk products are usually associated with LAB, which provide supplements in maintaining beneficial intestinal balance (Isolauri, 2001). Generally, the LAB are the most implicated of the probiotic organisms with respect to intestinal bacterial colonization particularly those of Lactobacilli and Bifidobacterium, which attributes antagonistic property by secreting acids to lower the pH, thereby creating an environment unfavorable to disease-causing bacteria. LAB, a probiotic, must tolerate low pH and high bile concentration, which enables selected strains to survive, grow and perform their therapeutic roles in the intestinal tract (Gilliland & Walker, 1989; Usman & Hosono, 1999), and produce substances that inhibit pathogenic, non-pathogenic and spoilage organisms in fermenting foods and beverages.

Lactic Acid Bacteria (LAB) are indigenous habitants of the human Gastro Intestinal Tract and have a long history of use in foods and fermented products as starter cultures. A variety of microorganisms, typically Lactic acid bacteria (LAB), have been evaluated for their probiotic potential and are explored as adjunct cultures in various types of food products or in therapeutic preparations. Numerous health benefits of LAB have made them promising probiotic candidates and being extensively studied to explore their safety and other desirable properties and contribute to add value to products. New product categories, and thus novel and more complex raw materials with regards to probiotics technology, are certainly a key research and development area for the functional foods market.( Wedajo B .,2015)

Milk is a potent source of lactic acid bacteria. Lactic acid bacteria are industrially important organisms recognized for their fermentative ability as well as their health and nutritional benefits. Lactic acid bacteria are a heterogeneous group of bacteria that are gram positive, non-spore forming, cocci or rods. Fastidious, acid tolerant and strictly fermentative as the major end product during sugar fermentation. LAB are most likely to grow in the nutrient rich habitats such as various food products (Milk, meat, beverages and vegetables), whereas some are also a part of natural flora of mammals. They are found to be in mouth, intestine and vagina of mammals. Lactic acid bacteria comprise the following genera: Aerococcus, Allococcus, Lactococcus, Lactosphera, Leuconostoc.

Because of the growing interest in probiotics, LAB are a focus of intensive international research into their essential role in fermented foods and for their ability to produce various antimicrobial compounds promoting probiotic properties like favor¬ably altering the intestinal microflora balance, inhibiting the growth of pathogenic bacteria, promoting digestion, boosting immune function and increasing resistance to infection. Other physiological benefits of probiotics include re¬moval of carcinogens, lowering of cholesterol and im¬munostimulants, lowering the effect of allergies, allevia¬tion of lactose intolerance. But the strains must survive exposure to harsh conditions imposed by gastric acids (pH = 3), bile and digestive enzymes (0.4% bile salts). Hence there is a need for isolation of novel strains of LAB with potential capabilities which would be useful in fermentation and production functional foods.( Mokoena, M. P., Mutanda, T., & Olaniran, A. O.,2016). Therefore, the current study was aimed to isolate and characterize lactic acid bacteria from buffalo and goat milk for their probiotic potential.

Materials and Methods

Collection of Samples

Fifteen raw samples each of Goat milk and Buffalo Milk were collected from dairy farms situated in Goregaon, Naigaon and Dahanu. The samples were transported and stored under refrigeration.

Isolation of Organisms

The Milk samples were serially diluted using Saline and the diluted samples were then spread plated on Lactobacillus MRS Agar plate by ensuring the criteria of PH 6.5, incubation temperature 37°c and incubation time 48 hr. After incubation, the plates were observed for isolated colonies.

Purification of Isolates

Well isolated bacterial colonies were picked and purified by streaking on MRS agar plates. The pure cultures of the isolated LAB were stored in MRS agar slants till further use.

Biochemical Characterisation of the Isolated LAB

The isolated LAB were identified biochemically by using HiLacto Identification Kit (HiMedia). The isolated LAB will be tested for acid tolerance, Bile tolerance and pancreatic fluid (enzymes) tolerance by using appropriate tests.

Acid and bile salt tolerance: Isolated LAB were inoculated into MRS medium of varying pH, i.e. pH 2, 3, 4 and 5; as well as broth with varying concentrations of bile salt (0.5, 1.0, 1.5 and 2.0%), and incubated at 37°C for 48h. Then 0.1mL inoculum was transferred to MRS agar by pour plate method and incubated at 37°C for 48h. The growth of LABs on MRS agar plate was used to designate isolates as acid or bile salt tolerant (Gotcheva et al., 2002).

For the pancreatic fluid tolerance test, 0.35 g of pepsin and 100 mL of a 0.2% sterile NaCl solution were used at pH 2.5 (Charteris et al.,1998). The lactobacilli were incubated in MRS broth at 37°C for 24 h. The cultures were then centrifuged for 5 min at 10,000 g and washed three times with pH 7.0 phosphate-buffered saline (PBS) (108 to 109 CFU/mL). A 10% solution of each sample was transferred into the simulated gastric juices. Viability in the simulated gastric juice was counted at 0 and 3 h on MRS agar. The survival rate was calculated in the same manner as for the determination of the acid resistance.

Evaluation of Antimicrobial Activity of Isolated LAB

The possible probiotic potentials or the antimicrobial activity of the isolate against the selected pathogens was investigated by well diffusion method on a solid medium. In the well diffusion assay, isolated colonies of probiotic cultures were inoculated in 5 ml MRS broth and grown at 30°C on a shaking incubator at 150 rpm for 72h, and cells were removed by centrifugation at 8,000 rpm for 5 min and the culture supernatant were sterilized by passage through 0.45 μm pore size filters. Four embattled pathogenic bacteria Salmonella typhi, S.aureus and E. coli. were precultured in LB broth medium, incubated at 30°C for two days and the culture was bulk seeded into the MH agar plate. Wells (4 mm in diameter) were punched into the Bulk seeded MH agar plates and 100 μl of culture supernatants from the probiotic isolate were added. The plates were then incubated at 37°C for 24 h. Antibacterial activity was estimated as the diameter (mm) of the clear inhibitory zone formed around the wells.

Results and Discussion

Table 1: Isolation Source and Probiotic Characteristics of LAB Strains

Table 2: Biochemical Identification of LAB Strains from Buffalo Milk

Table 3: Biochemical Identification of LAB Strains from Goat Milk

Note: N - Negative, P - Positive, D - Variable

Three different isolates were obtained from buffalo milk sample and two different isolates were obtained from goat milk samples. From the colony characteristics and the biochemical tests it was found that the strains were Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus lactis for buffalo milk sample and Lactobacillus paracasei ,Lactobacillus plantarum for goat milk sample. All the 5 strains exhibited acid tolerance, bile tolerance and pancreatic fluid tolerance which are essential characteristics of bacteria to be used for probiotics. The antimicrobial testing of the strains showed that the strain B1,B4 and G4 had better activity than the other strains.

In order to have probiotic effects in the intestinal tract, LAB must be capable of surviving passage through the gastrointestinal tract (GIT). Therefore, for probiotic LAB, resistance to the gastric acid environment is a prerequisite for survival and function in the intestinal tract. Depending on the specific individual’s diet, the pH of the human gastric environment varies from 1.5 to 3.0 (Solieri et al., 2014), and is usually around 3. With the intake of foods such as dairy products, the gastric pH value rises to 3.0 or even higher. In most studies, MRS broth with a pH value of 2.0 to 3.0 has been used to determine the acid resistance of Lactobacillus (Jacobsen et al., 1999; Tulumoglu et al., 2013; Solieri et al., 2014). Acid conditions have a large effect on the growth of Lactobacillus. In the present study, all of the 30 isolates survived at conditions of pH 3.0.

Cholate damages the structure of cell membranes, leading to leakage of substances inside the cell, and making it difficult for thallus to survive. Therefore, a strain’s tolerance to cholate is also of vital importance when assessing probiotic ability. The concentration of cholate inside healthy intestinal tracts varies from 0.03% to 0.30%, and generally does not surpass 0.4% (w/v) (Gilliland et al., 1984), which is considered to be the critical concentration when screening for bile-tolerant strains (Gilliland et al., 1984; Jacobsen et al., 1999). Therefore, 0.4% bile was used in this study, and all strains tested showed growth delays in the 0.4% bile. Conversely, Jacobsen et al. (1999) found no growth delay in 0.4% bile for three strains isolated from Ghanaian fermented maize. In the present study, eight strains exhibited high levels of tolerance to bile (with delayed growth ≤40 min).

This result is superior to that of the Lactobacillus strains isolated from cow excrement in a study by Hyronimus et al. (2000), in which the growth delay for all studied strains were >40 min. The strains with the best tolerance in this study had growth delays of The low pH of gastric juices and the gastric protease in gastric juices inhibit the growth of thallus. The small intestine is the major site of probiotic action, and various enzymes, bile acids, and other substances in small intestinal juice also inhibit probiotic growth. Therefore, GIT tolerance is an important criterion for the selection of potential probiotics. In the present study, during the GIT tolerance tests, almost all the strains exhibited better tolerance for simulated intestinal juice than simulated gastric juice.

Further, Bao et al. (2010) reported that pancreatic fluid did not significantly affect LAB survival. In the present study, all the studied strains had survival rates >90% in the simulated gastrointestinal fluid. This result is superior to that of de Almeida Júnior et al. (2015). In a study by Prasad et al. (1998), significantly inferior results were found compared to those in the present study in terms of the simulated GIT tolerance of two commercial fermented strains, with decreases in the viable counts of 7.60 log CFU/mL. The results of the present study are similar to those of studies by Charteris et al. (1998) and Musikasang et al. (2009).

Probiotics can protect organisms via various mechanisms, including bacteriostasis, which plays the most important role in the determination of the dominant bacterial communities within intestinal ecological systems (Tulumoglu et al., 2013). In this study, 30 Lactobacillus strains showed different levels of inhibition against S. aureus, E. coli & S. typhi. The inhibition of these pathogenic bacteria because of Lactobacillus had been reported in previous studies (Ammor et al., 2006; Tulumoglu et al., 2013; Asurmendi et al., 2015). Both Gram-positive and -negative bacteria were tested in the present study. Aymerich et al. (2000) reported that Gram-positive bacteria are more sensitive to Lactobacillus.

In conclusion, five LAB strains were selected as appropriate probiotic candidates in this study. Due to their probiotic properties tested, these strains might help to promote health of hosts, protect hosts from intestinal pathogens and maintain the natural balance of intestinal microflora during antibiotic treatments. However, additional studies are required to verify in vivo the effectiveness of selected strains.