Abstract: Background and Objective: The word ‘probiotic’ in greek, means ‘for life’. Probiotics are defined as live microbial feed supplement which have several health benefits. The objective of this study was to screen and identify 20 bacterial strains provided by School of Industrial Technology, USM, Malaysia. Materials and Methods: Twenty bacteria culture was screened using morphological and biochemical studies. From the 20 bacteria cultures, 9 of those exhibited viable count of above 9.0 log10 CFU mL–1 are selected for acid and bile tolerance tests. From 9 bacteria, five bacteria culture were able to tolerate acid and bile which exhibited viable counts of more than 9.0 log10. Results: All the 20 bacteria were confirmed as probiotics Lactobacillus using morphological and biochemical studies. The identity of the five lactobacilli, FTDC 0582, FTDC 0785, FTDC 2916, FTDC 4462 and FTDC 4793 were confirmed to be L. brevis, L. plantarum, L. plantarum, L. casei and L. plantarum, respectively. Conclusion: It can be concluded that, a carbon utilization microplate assay system developed by API CHL 50 has the potential to simplify the identification scheme of probiotic bacteria to the genus level.
INTRODUCTION
The concept of probiotics was introduced by Metchnikoff1. The term "probiotics" was coined by Lilly and Stillwell2 to describe substances produced by micro-organisms that stimulate the growth of another. The word "substances" in Parker’s definition of probiotics were resulted in a wide connotation that included antibiotics3. Lactobacillus and Bifidobacterium are two major groups of micro-organisms which are well known for their probiotic properties4-11. Probiotics produce antimicrobial agents such as organic acids, hydrogen peroxide and bacteriocins which are antagonistic to pathogenic micro-organisms12-16.
Probiotics are also defined as “a live microbial feed supplement which beneficially affects the host by improving its intestinal microbial balance”17. The definition of probiotics with respect to host and habitat of the microflora is given as “a viable mono-or mixed culture of micro-organisms which, when applied to animal or man, beneficially affects the host by improving the properties of the indigenous microflora”18. It was further investigated by Schrezenmeir and de Vrese19 who proposed probiotics as “a preparation of a product containing viable, defined micro-organisms in sufficient numbers, which alter the microflora (by implantation or colonization) in a compartment of the host and by that exert beneficial health effects in this host”. According to the World Health Organization (WHO)20, probiotics are living micro-organisms which when administered in adequate numbers confer a health benefit on the host.
The safety of Lactobacillus sp. and Bifidobacterium sp. as probiotics has been extensively reviewed21-26. Probiotics are treated as Generally Recognized As Safe (GRAS) micro-organism by the Federal Food and Drug Administration27-30. The identification of Lactobacillus depends mainly on physiological and biochemical criteria31-33. The prescreening of different Lactobacillus sp. was performed using various methods, such as conventional biochemical and physiological tests34-36. It can also be identified by more complex technique such as carbohydrate fermentation patterns using commercially available kits37-38.
Twenty probiotics culture bacteria of Lactobacillus used in the present study were provided by the School of Industrial Technology, Universiti Sains Malaysia. It was essential to confirm the identity of the probiotics culture bacteria. Therefore, the objective of this part of study was to identify the twenty bacterial strains provided by School of Industrial Technology, USM, Malaysia.
MATERIALS AND METHODS
The study was conducted in the School of Pharmaceutical Sciences, University Sains Malaysia, Penang, Malaysia from July-December, 2017.
Bacteria cultures: Twenty probiotics bacteria cultures given by the School of Industrial Technology, Universiti Sains Malaysia. The cultures were FTDC 9393, FTDC 0582, FTDC 0785, FTDC 4462, FTDC 5127, FTDC 5030, FTDC 8891, FTDC 4793, FTDC 1295, FTDC 8133, FTDC 8264, FTDC 2916, FTDC 2804, FTDC 1960, FTDC 8592, FTDC 3666, FTDC 3871, FTDC 1903, FTDC 1861 and FTDC 8033.
Enumeration of lactobacilli: For the enumeration of lactobacilli, 10.0 mL of bacterial culture was inoculated into 90.0 mL of MRS broth and incubated at 37°C for 24 h (Memmert, Germany). The enumeration of viable cells was conducted after ten-fold serial dilution of fresh bacterial culture. About 1.0 mL of each dilution was cultivated in MRS agar using pour plate method. The plates were incubated at 37°C for 48 h.
Morphological and biochemical evaluation: The morphological and biochemical characteristics of the bacteria were determined after 48 h of incubation on MRS agar. The evaluation included shape, size, margin, colour and Gram-staining test.
Analytical profile index (API 50 CHL) identification kit: API 50 CHL carbohydrate identification kit is comprised of 50 biochemical tests. The API 50 CHL strip is made up of 5 smaller strips. Each smaller strip consists of 10 wells of different carbohydrate substrates. Briefly, all the strips were removed from their packaging and placed in the tray.
The inoculum was prepared according to the manufacturer’s instruction by aseptically transferring pure culture of lactobacilli from MRS agar into the API Suspension Medium ampoule (2.0 mL) using sterile swab. The suspension was mixed and 350 μL was transferred to a second API Suspension Medium ampoule (5.0 mL) to reach turbidity equivalent to McFarland standard # 2. The final inoculum was prepared by transferring 700 μL from the initial bacterial suspension (API Suspension Medium ampoule, 2.0 mL) into an API 50 CHL Medium (10.0 mL). The suspension was mixed and 150 μL (inoculated API 50 CHL medium suspensions) was measured into the well using sterilized micropipette and covered with 50 μL mineral oil. The strips were incubated (Memmert, Germany) at 37°C for 48 h.
After the incubation of 48 h (manufacturer instruction), each well was observed for colour changes. The positive result was confirmed by the change of colour of bromocresol purple indicator from purple to yellow, except for well # 26 (for esculin hydrolysis test), by the change in colour from purple to a darker colour or black. The first well on the strip was used as a control. No change in the colour indicated negative result. The result was analyzed using api-webTM identification software database (Biomérieux, France, V 5.1) to identify Lactobacillus species.
RESULTS AND DISCUSSION
Morphological characteristics: The bacteria were creamy white colonies, rod-shaped with sharp margins. The colony diameter was in the range of 2.0-5.0 mm (Fig. 1). All the strains are Gram-positive, non-spore forming and non-motile (Table 1). The results were consistent with the findings of Tabatabaei-Yazdi et al.39and cross reference information provided in Bergey's Manual of Determinative Bacteriology36on Lactobacillus.
Identification using API 50 CHL kit: The results of carbohydrate fermentation test of API 50 CHL identification kit were shown in Table 2. The results obtained coincided with the characteristics of Lactobacillus36,40. The results in Table 3 provided the identity of lactobacilli species based on carbohydrate fermentation profiles using API 50 CHL database. FTDC 0785, 2916 and 4793 showed a 99.9% identity of Lactobacillus plantarum. FTDC 0582 was identified as Lactobacillus brevis with 99.0% identity. On the other hand, FTDC 4462 showed 99.8% identity with Lactobacillus paracasei 3 and Lactobacillus paracasei 1. The result was in good agreement with the findings of other researchers23,31,41,42. API 50 CHL identification kit was reported as an important tool for lactobacilli identification43-45. It can be used for taxonomic identification which is based on phenotypical characteristics to identify the different species of Lactobacillus45,46.
| Fig. 1: | Morphological characteristic of probiotics colonies grown on MRS agar |
| Table 1: | Morphological and biochemical tests for probiotics |
+: Positive result (Gram-positive in case of Gram-staining and capability to motile in case of motility), -: Negative result (Gram-negative in case of Gram-staining and inability to motile in case motility). A: FTDC 9393, B: FTDC 0582, C: FTDC 0785, D: FTDC 4462, E: FTDC 5127, F: FTDC 5030,G: FTDC 8891, H: FTDC 4793, I: FTDC 1295, J: FTDC 8133, K: FTDC 8264, L: FTDC 2916, M: FTDC 2804, N: FTDC 1960, O: FTDC 8592, P: FTDC 3666, Q: FTDC 3871, R: FTDC 1903,S: FTDC 1861, T: FTDC 8033 | |
| Table 2: | Carbohydrate fermentation profiles of Lactobacillus species |
| Pos: Positive reaction: Color changed and Neg: Negative reaction: Color not changed | |
| Table 3: | Identification of Lactobacillus species based on carbohydrate fermentation profiles using API 50 CHL database
|
ID: Identity (%), the percentages following the scientific names of species represent the similarities from the computer-aided database of the API-webTM API 50 CHL V5.1 software | |
CONCLUSION
The identity of 20 probiotic culture was confirmed as Lactobacillus using morphological and biochemical studies prior to the commencement of the study. From the 20 bacteria cultures, five bacteria culture, FTDC 0582, FTDC 0785, FTDC 2916, FTDC4793 and FTDC 4462 were able to tolerate acid and bile which exhibited viable counts of above 9.0 log10 CFU mL–1 were selected for subsequent study. The identity of the five lactobacilli, FTDC 0582, FTDC 0785, FTDC 2916, FTDC 4462 and FTDC 4793 were confirmed as L. brevis, L. plantarum, L. plantarum, L. casei and L. plantarum, respectively using API 50 CHL identification kit.
SIGNIFICANCE STATEMENT
This study discovered the isolation and identification of unknown probiotics strains from Malaysian fermented foods that can be beneficial for use. This study will help the researchers to uncover the critical areas of the identification for research and commercial purposes.
ACKNOWLEDGMENT
The present study was financially supported by the Universiti Sains Malaysia (USM). Grant Number (1001/PFARMASI/843084). Authors are highly appreciate.