Project Abstract

                      ABSTRACT

The commonly used probiotics bacteria are lactic acid bacteria (LAB) from gastro intestinal tract. However, other LAB from exogenous origin having similar functional properties may also confer health benefit to the host. Palm wine has been described as a rich source of LAB. But very few studies have investigated their probiotic potential. Twenty LAB were isolated from palm wine collected in the South West Region of Cameroon by pour plate method on MRS agar. These isolates were assessed in vitro for their potential to inhibit the growth of some foodborne pathogens, mainly Salmonella sp. and Escherichia coli using disc diffusion method. Acid and bile tolerance were evaluated by measuring the survival rate of LAB after incubation at pH range from 1.0 to 3.0 and various bile salt concentrations (0.15-0.30%). Only five isolates were selected based on their potential to inhibit food borne pathogens tested and their tolerance in acid and bile. They were identified using API kit 50 CHL BioMerieux as strains of Lactobacillus pentosus, Lactobacillus plantarum and Lactobacillus brevis. All these strains showed antimicrobial activity against strains of Salmonella sp. and E. coli with diameters of inhibition varying from 12 to 20 mm. Only L. pentosus and L. brevis1 tolerated pH 3.0 (acidic condition of interest) with survival rates of 55 and 69% respectively, while all survived in bile with survival rates above 60%. Key words Probiotics, antimicrobial activity, acid tolerance, bile tolerance.

Project Overview

1.0 INTRODUCTION 

1.1 BACKGROUND STUDY

The concept of food having medicinal value has been reborn as 'functional foods'. The list of health benefits accredited to functional foods continue to increase and the gut is an obvious target for the development of functional foods, because it acts as an interface between the diet and all other body functions. One of the most promising areas for the development of functional food components lies in the use of probiotics. Probiotics, are live microorganisms that, when administered in adequate amounts, confer health benefits on the host (FAO/WHO, 2002). One of the most accepted approaches through which the gut microbiota composition can be influenced is through the use of probiotics; which are life microbial dietary additives. Besides the nutritional values, ingestion of lactic acid bacteria (LAB) and their fermented foods has been suggested to confer a range of health benefits including immune system modulation, increased resistance to malignancy and infectious illness (Soccol et al., 2010). Vergin in 1954 suggested that the microbial imbalance in the body caused by antibiotic treatment could have been restored by a probiotic rich diet; a suggestion cited by many as the first reference to probiotics as they are defined nowadays. 

Similarly, Vasiljevic and Shah (2008) recognized detrimental effects of antibiotic therapy and proposed the prevention by probiotics. The idea of health-promoting effects of LAB is by no means new, as Metchnikoff proposed that lactobacilli may fight against intestinal putrefaction and contribute to long life (Brant and Todd, 2014). Such microorganisms may not necessarily be constant inhabitants of the gut, but they should have a “beneficial effect on the health status of man and animal” (Belhadj et al., 2010). For the gastrointestinal ecosystem, the most important microbial species that are used as probiotics are LAB. Lactic acid bacteria (LAB) are the most prominent nonpathogenic bacteria that play a vital role in our everyday life, from fermentation to preservation, food and vitamin production, and to prevention of certain diseases and cancer due to their probiotics properties. These microorganisms are one of the prominent groups of bacteria which inhabit the gastrointestinal tract, and the importance of these non-pathogenic bacteria has been more noticed (Krishnendra et al., 2013). Several lactobacilli have been noted to have nutritional benefits, improved lactose utilization, have anti-cholesterol and anti-carcinogenic, and protection against other diseases (Krishnendra et al., 2013). 

Especially, Lactobacillus spp. are well known producers of antimicrobial compounds especially bacteriocins which have high antimicrobial activity (Aween et al., 2012). The production of these compounds by intestinal microflora is one of the most important mechanisms responsible for the antagonistic activity against intestinal pathogens and therefore it is essential to examine this property in isolates that are candidates for probiotics (Bilkova et al., 2011). Effective probiotics should possess antimicrobial activity particularly to the pathogens of the gastrointestinal tract (Klayraung et al., 2008). Palm wine is an alcoholic beverage produced from the sap of various palm tree species. The drink is particularly common in parts of Africa, South India and the Philippines. In Africa, the sap is most often taken from oil palms such as Elaeis guineensis, or from Raffia, kithul or Nipa palms (Ukhum et al., 2005). Besides fermenting yeast belonging to various genera e.g Saccharomyces, Candida, Endomycopsis, Hausenula, Pichia, Saccharomy Fossi et al. 43 codes and Schizosaccharomyces (Ezeronye and Legras, 2009; Chandrasekhar et al., 2012), the dominant bacterial population of palm wine was previously described as lactic acid bacteria-strains of Lactobacillus plantarum, Leuconostoc mesenteriodes and L. mesenteroides subsp. dextranicum.. 

Palm wine is milkywhite and effervescent because of the presence of live bacteria and yeast (Ezeronye, 2009) resulting from natural fermentation. The sap of palm tree has been shown to be a rich medium capable of supporting the growth of various types of microorganisms. In general, the methods of palm wine tapping and collection of palm sap, including air and the environment as a whole, influence the microbial content of the sap (Amoa-Awwa et al., 2007; Naknean et al., 2010). Palm wine plays an important role in many ceremonies in Cameroon, parts of Nigeria such as among the Igbo people, and elsewhere in Central and Western Africa. Guests at weddings, birth celebrations and funeral wakes are served generous quantities. The wine is often infused with medicinal herbs to treat a wide variety of physical complaints. The widely used probiotic bacteria reported in literature were isolated from gastro intestinal tract, but very few are from exogenous origin such as palm wine. This study aimed at investigating the probiotic potential of lactic acid bacteria from palm wine. 

MATERIALS AND METHODS 

Sample collection Thirty samples of fresh palm sap were collected in sterile widemouth bottles directly from the farmers and transported to the laboratory for processing. The samples were kept at room temperature for 48 h for fermentation to take place. After which they were carefully processed under aseptic conditions. Isolation and phenotypic identification of lactic acid bacteria LAB was isolated from palm wine by pour plate method using De Man Rogosa and Sharpe (MRS) agar. For this purpose, 1 ml of each sample was added to 9 ml of saline solution (NaCl, 0.85%). 1 ml aliquot of the 10-4 and 10-5 dilutions was aseptically disposed on sterile plates. About 15 ml of MRS agar was poured onto it and allowed to solidify. All plates were incubated at 30°C for 48 h under anaerobic conditions. After the incubation, a preliminary catalase test was carried out. Catalase negative discrete colonies which appeared on the plates with distinct morphological differences such as color, shape and size were picked and purified 2-3 times by restreaking on fresh MRS agar. The pure colonies were further characterized using Gram staining test and cell morphology examinations. Catalase negative and Gram positive isolates were preserved in 15% glycerol at -80°C until identification. Carbohydrate fermentation patterns of LAB were determined using API 50 CHL kit (bioMerieux, France). 

The APILAB PLUS database software was used to interpret the results. Antimicrobial activity of LAB The antimicrobial activity of LAB was determined by modifying the disc diffusion method of Hamdan and Mikolajcik (1974). Sterile filter discs (diameter; 6 mm) were dipped into the cultured MRS broth of LAB incubated at 30°C for 24 h in a shaker (187 rpm) and placed on solidified Mueller-Hinton agar (LIOFILCHEM DIAGNOSTICI) seeded with 14 h cultures of indicator microorganisms. The plates were kept at 4°C for 3 h to permit diffusion on the assay material, and incubated at 37°C for 16 h. Some of the discs were dipped in un-inoculated MRS broth which served as negative control. Also, antibiotic discs of Ofloxacin and Azithromycine were placed on solidified Muller-Hinton agar (LIOFILCHEM DIAGNOSTICI) seeded with 14 h cultures of indicator microorganisms and incubated under the same conditions. These served as positive control for the tests on Salmonella enteric subsp. enterica and E. coli, respectively. Their zones of inhibition (clear zones around the discs) were evaluated. This was done by using a ruler to measure the diameter of the disk plus the surrounding clear area in millimeters (mm). Tolerance to acidic conditions The lactic acid bacteria isolates were cultured in MRS broth for 18 h. The LAB cells were harvested by centrifugation for 10 min at 5000 rpm and 4°C. Pellets were washed trice in phosphate-saline buffer (PBS at pH 6.2). The pH was adjusted by a pH meter with the use of HCl 1 N to pH 1.0, 2.0, 3.0 and 6.2 (control pH). The cell pellets (107 -108 CFU/ml) were resuspended in 10 ml of PBS (pH 1.0, 2.0, 3.0 and 6.2) and incubated at 30°C for 1, 2, 3 and 4 h. The cells were enumerated by plating 100 µL aliquot of the inoculated PBS solutions at the various tested times, for 24 h. The experiments were performed in duplicates. Bile tolerance These lactic acid bacteria isolates were cultured in MRS broth, for 16-18 h. The LAB cells were harvested by centrifugation for 10 min at 5000 rpm and 4°C. Pellets were washed trice in phosphatesaline buffer (PBS at pH 6.2) and resuspended in PBS (pH 6.2). Two sets of MRS broth were prepared containing 0.15 % (w/v) oxgall-bile and the other 0.30% (w/v) oxgall-bile. Also, one set of MRS broth was prepared without oxgall-bile. This served as the control. These sets of MRS broth were inoculated with 100 µl aliquot of the LAB suspensions (107 -108 CFU/ml) and incubated for 1, 2, 3 and 4 h. Then, viable bacteria counts were obtained after 24 h incubation at 37°C (Barakat et al., 2011). The experiments were performed in duplicates. In both cases, the survival percentage of LAB was calculated by the following formula: