Bacteriocin Production using Lactic Acid Bacteria Isolated from Ugba (Pentaclethra macrophylla Benth)
Abstract
Lactic acid bacteria (LAB) play vital roles in our everyday life such as in fermentation, preservation and production of wholesome foods. This work is aimed at studying the bacteriocin production of lactic acid bacteria from Ugba (a local condiment) sold in Anambra state. A total of 5 samples of Ugba each were purchased randomly from three locations in Anambra state. Lactic acid bacteria were isolated and characterized using standard microbiological procedures. The isolates were subjected to antimicrobial activities by disc diffusion method while bacteriocin production was achieved by culture on appropriate medium and partially purified by centrifugation method and then characterized. Antimicrobial activities using cell free supernatant and partially purified bacteriocin of the isolates against Escherichia coli, Staph. aureus and Candida albicans found in Ugba were equally assessed. The isolate that gave the highest zone of inhibition was identified by 16s rDNA sequencing and was selected for further assay. Biopreservative study of the choice isolate against Escherichia coli, Staph. aureus and Candida albicans which were extraneously introduced into the Ugba was conducted. A total of Six LAB isolates designated with codes were obtained based on their catalase spot test reaction. zones of inhibition ranging from 13-20mm, 12-18 mm and 10-19mm were obtained against Escherichia coli, Staphylococcus aureus and Candida albicians respectively. The LAB isolate code A1 with the Best Antimicrobial Activity was identified as Lactobacillus plantarum. The bacteriocin produces by Lactobacillus plantarum (A) was slightly stable at 40 and 60°C with a continuous decrease at 80 and 100°C for 30 mins but totally lost the activity at 121 °C for 15 min. The effect of pH on partially purified bacteriocin showed maximum activity against Escherichia coli, Staphylococcus auerus and Candida albicians. at pH 5.0 (18 mm ,17mm and 15mm respectively) and a continuous decrease as the pH increased from 6 to 9. The antimicrobial activity of bacteriocin was lost after treatment with proteolytic enzyme: trypsin and pepsin. This study show that Lactobacillus plantarum isolated from ugba has great potential for exploitation in food safety and preservation as a result of its bacteriocin content. Thus this locally fermented food plays a dual role of protection from pathogenic agents as well as serving as a functional food. It is therefore recommended that everybody should begin to consume ugba on a regular basis.
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Introduction
Ugba also called ‘Ukpaka’ in Eastern part of Nigeria is an indigenous fermented food, rich in protein and produced by a solid state fermentation of seeds of African oil bean tree (Pentaclethra macrophylla), a perennial legume tree. It is a popular food delicacy in Nigeria especially among the Ibo ethnic group where it serves as snack or as a food condiment. It is an important food item for various traditional celebrations where it is mixed with slices of boiled stock fish, garnished with vegetables and consumed. The unfermented seeds are bitter to taste as a result of anti-nutrition substances inherent in them. The natural fermentation of the seeds still done at the domestic level renders the product nutritious, palatable and non-toxic. Diverse groups of microorganisms including Bacillus, Micrococcus, Leuconostoc, Staphylococcus, Enterobacteriaceae and lactic acid bacteria (LAB) have been reported to play active roles in the process of ugba fermentation (Enujiugha 2009). They produce antimicrobial substances such as organic acids, diacetyl, hydrogen peroxide and bacteriocins (Ogunbanwo et al. 2003) which are alleged to be connected with the preservation of many fermented food condiments in Nigeria. The preservation and safety are currently the two major challenges of the food industry as huge economic losses are sustained yearly due to food spoilage while many consumers have been reported to develop adverse sensitivity reactions to chemical based preservatives. The Lactic acid bacteria (LAB) which are used generally as starter cultures for food fermentation are considered as having the potential to bridge this gap (Budde et al. 2003; Delves-Broughton 2005). They produce antimicrobial compounds that demonstrate antagonism toward the closely related species. The LAB in 'ugba' improve fermentation and also enhance the product storage, quality and safety by restricting the spoilage microflora from spoiling the food condiment and at the same time eliminating the pathogens such as L.monocytogenes, S. aureus and E.coli (Alor and Okonkwo 2021). Lactic acid bacteria (LAB) are heterogenous group of bacteria which playa significant role in a variety of fermentation processes. They ferment food carbohydrates and produce lactic acid as the main product of fermentation. In addition, their degradation of proteins and lipids and production of various alcohols, aldehydes, acids, esters and sulphur compounds contribute to the specific flavour development indifferent fermented food products.(Sindhu and Khetarpaul 2001) While many scientific reports are available on 'ugba', such studies have thus far addressed mainly the microorganisms associated with the fermentation processes of the seed as well as systems for optimization of the fermentation conditions (Odibo et al. 2008). The effect of processing techniques on the nutritive quality of ugba has also been reported by Okonkwo and Alor 2021 while the proximate composition and properties of the seeds have also received modest scientific attention (Odibo et al 2008). There is presently, paucity of scientific information on the ecological contribution of the LAB and bacteriocins for the safety and bio preservation of the food condiments.
Bacteriocins are extracellularly produced primary compounds of bacterial ribosomal synthesis which have a somewhat narrow range of bactericidal activity (Caplice and Fitzgerald 1999). They are active against other bacteria despite varying greatly in the chemical nature and mode of action. Bacteriocins have significant advantage over the established antibiotics inbeing easily degraded by the digestive enzymes without the risk of interference with the normal tract ecology (Caplice and Fitzgerald 1999). Bacteriocin producing LAB have been generally recognized as safe (GRAS) status and have been shown to fortify the barrier function of the gut microflora as well as promote the non-specific improvement of the immune system of man and animals (Tome et al. 2008).
The problem of chemotherapy and its associated side effects together with multiple drug resistance by infectious agents has led to the search for local foods that can serve as functional foods with dual role of protection from pathogenic agents as well as high nutrient based diet. Hence ugba (P macrophylla) which is generally consumed particularly in southeast Nigeria is investigated for its role in the isolation of lactic acid bacteria for bacteriocin production.
Conclusion
This study shows that Lactobacillus plantarum isolated from ugba has great potential for exploitation in food safety and preservation as a result of its bacteriocin content. Thus this locally fermented food plays a dual role of protection from pathogenic agents as well as serving as a functional food. It is therefore recommended that everybody should begin to consume ugba on a regular basis.
