Abstract:
Background and Objective: Lactic acid bacteria (LAB) have been used for centuries as food preservative in food technology and food production. The objective of the study was to isolate and identify naturally occurring lactic acid bacteria from Gibna Bayda produced in local markets in El-Dueium City, Sudan. Materials and Methods: A total of 25 white cheese (Gibna Bayda) samples were collected from individual local cheese producers in El Dueim City in White Nile state. Microbiological analysis of the cheese samples was performed. Results: Among 92 LAB strains isolated from Gibna Bayda, bacilli were accounted for 70 isolates (76.09%), while cocci were 22 isolates (23.91%). The isolated LABs were dominated by Lactobacillus bulgaricus (22.8%), followed by L. fermentum (18.48%), Streptococcus lactis (17.4%) and L-casei (13.04%). The least isolated LABs were L. helveticus (3.26%). Conclusion: Dominant LAB (Lactobacilli and Streptococci) were successfully isolated from Gibna Bayda. The wide diversity in microbial community could be attributed to variations in the specific environmental conditions found in Sudan as well as the manufacturing processes for indigenous Gibna Bayda.
INTRODUCTION
Gibna Bayda produced at different sites of many cities in White Nile states like El Dueim and Rabak1. The highest production of Gibna Bayda is obtained during the rainy season2. Gibna Bayda is a traditional fermented milk containing high concentrations of salt (Sodium Chloride) which is added to the milk before processing. Also the processing includes heating of the fresh milk at 35°C followed by addition of salt to give 6-10% solution in milk.
Traditional dairy products differ from commercial dairy products in taste, flavour and the consistency due to the natural flora of the lactic acid bacteria present in it3. Cheese is a very involved microbial ecosystem and a very complex microflora develops in most cheese varieties. The microflora plays a major role in cheese ripening and selection of suitable strains would enable the cheese maker to control or modify flavor development. However, due to the complexity of the flora and the interactions which occur between individual components of it and the cheese environment, strain selection for flavor improvement is not always very obvious4. Lactic acid bacteria (LAB) are naturally present in milk and milk products. Many LAB species play an important role in the ripening process of cheese, especially to improve the consistency, aroma and flavor. Certain LAB strains are characterized by their ability to transform lactose and improves the digestibility of fermented dairy products5 as well as their preservation6. They also employed for improvement of the taste, texture and viscosity in the manufacture of dairy products7. Lactic acid bacteria can be recovered from fermented foods and beverages, vegetables, milk and milk products.
This is in addition to its ability to withstand cheese manufacturing processes (whether fresh or ripe), it can grow in a moisture content of 39% and a salinity between 4 and 6% of sodium chloride (NaCl) and the pH ranges from 9.4-3.5.
Today, lactic acid bacteria (LAB) are a focus of intensive international research for their essential role in most fermented food. Lactic acid bacteria have the ability to produce various antimicrobial compounds promoting probiotic properties8 including anti-tumoral activity9,10. They can reduce serum cholesterol11,12, alleviate lactose intolerance13, stimulate the immune system14 and stabilize gut microflora15. The objective of the study was to isolate and identify naturally occurring lactic acid bacteria from Gibna Bayda produced in local markets in El-Dueim City, Sudan.
MATERIALS AND METHODS
Sample collection: The study was conducted from February to April 2020 to isolate and identify lactic acid bacteria in Gibna Bayda (Fig. 1). A total of Twenty-Five Gibna samples were collected from different local markets in the city of El Dueim, White Nile State, Sudan. Samples were kept in a refrigerator (around 4°C) until the analysis was started.
Isolation of lactic acid bacteria: A total of 10 gm of each item (Gibna Sample) was obtained from five different local markets in El Dueium city, aseptically and transferred to the separate sterile container, containing 90 mL of sterile saline solution. The latter was shaken well until a homogeneous dispersion of 1:10 dilution obtained, then serially diluted and inoculated on plates16, where Lactococci were grown on M17 agar (Oxoid) and enumerated after 48h of incubation at 32°C17. Lactobacilli were grown on MRSA; Merck, carried out anaerobically using the gas pack system at 30°C for 48h18.
Identification of the bacterial strains: Morphological and Biochemical characteristics were considered for identification of lactic acid bacteria genera. Preserved isolated strains were tested for gram staining, catalase production and spore formation using the method of Harrigan and McCance19. The other tests were: anaerobic growth20, Motility test21, oxidase test22 and fermentation/oxidation test23. Hydrolysis of arginine, citrate utilizations, gas formations from glucose in MRS broths containing inverted Durham tubes, dextran productions from sucrose in MRS+ST agar, growth on different temperature (10, 37 and 45°C) for 5 days, resistance to 60°C for 30 min (Sherman test), growth in the presence of 4 and 6.5% (w/v) NaCl and different pH (4.5 and 6.5) and changes in turbidity of MRS broth after 24, 48 and 72 h of incubations were implicated to identify the strains24-26. Arginine MRS medium and Nessler reagent were employed to perform the hydrolysis tests as described by Yavuzdurmaz27. Citrate utilization and colored colonies growth were observed in SL and D agars and results were interpreted according to Reddy et al.28 and Kempler and McKay29. Sugar fermentation tests Membrane (0.45 μm) filtered 1% (w/v) solutions of different sugars (glucose, fructose, lactose, galactose, maltose and mannitol) were deployed to study fermentation characteristics of the isolates. Nutrient broth (0.8%) with 1 mL phenol red was autoclaved at 121+1°C for 15 min then cooled to room temperature. Five ml of broth and 100 μL of sugars were taken into sterilized test tubes. These tubes were checked for contamination by placing at room temperature for 24 h. After 24 h, the purified colonies were inoculated into test tubes with specific sugar containing broth and incubated at 37°C for 48 h. The positive test for sugar fermentation was indicated by color change from red to yellow in the test tubes as mentioned by Mehmood et al.30.
RESULTS AND DISCUSSION
LAB strains isolated, purified and further identified and differential tests were applied including morphological and physiological characteristics which facilitate the opportunity for identification of the LAB. The identification results were confirmed by the carbohydrates fermentation and assimilation profile obtained in correlation with bergey’s manual and also using the manuals of Sharpe25, Holt et al.31, Garrity et al.32 and Hardie33.
The isolated LABs were dominated by the species Lactobacillus bulgaricus (22.8%), followed by L. fermentum (18.48%), Streptococcus lactis (17.4%) and L-casei (13.04%). The least isolates were L. helveticus (3.26%) (Table 1).
Table 2 shows the general properties of the 92 LAB strains isolated from Gibna Bayda. Among these LAB isolates, bacilli were accounted for 70 isolates (76.09%), while cocci were 22 isolates (23.91%). The isolates were classified into two genera according to De Vos et al.34:
Lactobacilli commonly found in the gastrointestinal tract of animals and humans can also be found in fermented food such as milk and milk product. The creamy or whitish appearance of the isolated Lactobacillus species on MRS agar confirms that Lactobacilli have dominance in fermented milk products when compared to other lactic bacteria35.
Lactic acid bacteria (LAB) are widely used as starters in fermented dairy products, mainly Streptococcus thermophilus, Lactococcus lactis, Lactobacillus helveticus and Lactobacillus delbrueckii subsp. bulgaricus, in which the proteolytic system plays a vital role36. The non-starter LAB (NSLAB) are very important in cheese ripening, such as Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Lactobacillus brevis, Lactobacillus casei, Lactobacillus curvatus, Lactobacillus fermentum, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Pediococcus acidilactici, Pediococcus pentosaceus and other LAB37. During the process of milk fermentation, LAB hydrolyze milk proteins, thereby releasing bioactive peptides. The characteristics of this process are important to scale up the fermentation at industrial levels38,39.
These findings agree with some previous studies on the isolation of L. lactis subsp. lactis, L. lactis subsp. cremoris from dahi of Himalayas40 and L. lactis subsp. cremoris from Kazerun’s traditional fermented yoghurt41.
CONCLUSION
This study showed a clear picture of microbial diversity and density in Sudanese white cheese (Gibna Bayda) that might largely contribute to its typical texture and flavor. Dominant LAB comprising of Lactobacilli and Streptococci were successfully isolated from Gibna Bayda which is locally produced in El Dueim City (Sudan). The wide diversity in microbial community could be attributed to variations in the specific environmental conditions found in Sudan as well as the manufacturing processes for indigenous Gibna Bayda. Selection, propagation and preservation of the best performing strain to make starter culture could be done in future through genetic characterization.