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Optimization of Lactic Acid Production by Lactic Acid Bacteria Isolated from Some Traditional Fermented Food in Nigeria Article · January 2009

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Pakistan Journal of Nutrition 8 (5): 611-615, 2009 ISSN 1680-5194 © Asian Network for Scientific Information, 2009

Optimization of Lactic Acid Production by Lactic Acid Bacteria Isolated from Some Traditional Fermented Food in Nigeria I.A. Adesokan1, B.B. Odetoyinbo1 and B.M. Okanlawon2 Department of Biology, The Polytechnic, Ibadan, Oyo State, Nigeria 2 Department of Biomedical Sciences, Ladoke Akintola University of Technology, Ogbomoso, Oyo State, Nigeria 1

Abstract: Seven species of Lactic Acid Bacteria (LAB) namely L. fermentum, L. casei, L. brevis, L delbrueckii, L. acidophilus, L. plantarum and Leuconostoc mesenteroides were isolated from ogi, burukutu and retted cassava (fufu). The isolates were screened for quantitative production of lactic acid using normal MRS broth and modified MRS broth under varying conditions of growth such as temperature and pH and influence of carbon and nitrogen sources. It was observed that all the test isolates best utilized glucose and yeast extract at concentrations of 20 g LG1 and 5 g LG1 respectively for production of lactic acid at optimum temperature of 30°C and pH of 5.5. L. plantarum produced the highest quantity (2.95±0.32 g LG1) of lactic acid while L. delbrueckii produce the lowest (0.89±0.07 g LG1). Lactic acid produced by L. fermentum L. delbruekii and L. plantarum had the highest inhibitory activity against pathogenic microorganisms such as Staphylococcus aureus, E. coli and Pseudomonas species with zone of inhibition ranging between 8 and 20 mm. Key words: Lactic Acid Bacteria, cultural conditions, antagonistic activity and lactic acid acid by fermentation using lactic acid bacteria. This present work is aimed at providing such information.

INTRODUCTION Many chemical substances are constantly involved in the biochemical processes going on in living systems. Among these, lactic acid serves as an important metabolite of an equally important energy yielding process (Prescott et al., 2005). Lactic acid is a carboxylic acid with the chemical formular CH3CHOHCOOH and is a colourless liquid organic acid (Narayanan et al., 2004). It is a widely used chemical that has found application in many industries and various commercial purposes. It is used in leather tanning and textile dyeing and in making inks, solvents and lacquers (Narayanan et al., 2004). Lactic acid is also used as acidulate, flavoring, pH buffering agent or inhibitor of bacterial spoilage in many processed foods. The esters of lactic acid are used in baking foods, as emulsifying agents (Datta, 1995; Narayanan et al., 2004). The production of lactic acid can be done in two ways which are chemical synthesis and carbohydrate fermentation (Narayanan et al., 2004). The method of carbohydrate fermentation is relatively cheaper and so is preferable. Lactic acid is an organic acid that is produced as a result of fermentation metabolism by the lactic acid bacteria (Brook and Madigan, 1991). The fermentative production of lactic acid is advantageous over the synthetic production in that by choosing a stream of lactic acid bacteria that produces only one isomer of lactic acid an optically pure product can be obtained. However, there is still only little or no information on influence of cultural conditions on the production of lactic

MATERIALS AND METHODS Sample collection: Some Nigerian freshly fermented food products namely ogi, burukntu and retted cassava (fufu) were purchased at Iwo road and Army barracks (Eleyele), all located in Ibadan, Oyo state, Nigeria. These samples were brought to the laboratory in sterile containers for immediate analysis Isolation of Lactic Acid Bacteria: Lactic Acid Bacteria were isolated from ogi, burukutu and retted cassava. Sampling and isolation were carried out as described by Halm et al. (1993). Isolates were identified according to Kandler and Weiss (1986). Based on Gram staining, catalase test, growth at 15°C and 45°C, spore staining, motility test and other biochemical tests such as indole production, Voges-Proskauer test, oxidase test, methyl red, production of ammonia from arginine, growth in 4% NaCl and carbohydrate fermentation pattern. Lactic acid production: Lactic acid production by fermentation process was carried out by inoculating the test organisms in MRS broth and incubated at 37°C for 72 hours. Cultures were centrifuged at 3000 rpm for 15 minutes. To the supernatant, 5 mg mLG1 each of catalase and protease was added to neutralise the influence of H202 and bacteriocin respectively. Known volume of the supernatant was used for the titration at 24 hours interval to determine the quantity of lactic acid produced.

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Pak. J. Nutr., 8 (5): 611-615, 2009 Table 1: Percentage occurrence of LAB isolated from selected Nigerian fermented foods Isolates Substrate Number of isolates Lactobacillus brevis Sorghum ‘ogi’ 1 Burukutu 1 Lactobacillus fermentum Maize ‘ogi’ 1 Sorghum ogi 1 Retted cassava 1 (fufu) Burukutu 1 Leuconostoc messenteroides Burukutu 1 Lactobacillus plantarum Maize ogi 6 Sorghum ogi 5 Retted cassava 5 Burukutu 4 Lactobacillus casei Maize ogi 2 Retted cassava(fufu) 1 Burukutu 1 Lactobacillus acidophilus Maize ogi 2 Lactobacillus delbruiekii Sorghum ogi 2 Burukutu 2

Quantitative estimation of lactic acid: To 25 mL of the supernatant of the test organisms, three drops of phenolphthalein were added as indicator. From a burette 0.1M NaOH was slowly added to the samples until a pink color appeared. Each mL of 0.1M NaOH is equivalent to 90.08 mg of lactic acid (A.O.A.C., 1990).

Percentage (%) 2.70 2.70 2.70 2.70 2.70 2.70 2.70 16.24 13.53 13.53 10.80 5.40 2.70 2.70 5.40 5.40 5.40

separately into the basal media and sterilized at 121°C for 15 mins. 18 hrs old culture of the isolate were inoculated at 37°C. Samples were collect at 24 hrs interval from these for quantitative determination of lactic acid as stated above. Antagonistic activity of lactic acid produced by the LAB isolates: A well diffusion assay was employed. Overnight broth culture of indicator organism was used to inoculate nutrient agar plate. 6 mm diameter holes were created in the inoculated nutrient agar plate using cork borer. Lactic acid produced by LAB was dispensed into each of the holes. A pre-incubations period of 5hrs was allowed at 28°C to allow the proper diffusion of the Lactic acid dispensed. This was followed by incubation aerobically at 37°C for 24hrs. The zone of inhibition was observed and recorded.

Effect of temperature on lactic acid production: Sterilized MRS broth was inoculated with 18 hours old cultures of the isolates. The tubes were incubated at 4°C, 30°C and 45°C. Samples were collected at 24 hours interval and lactic acid produced quantified as stated above. Effect of pH on lactic acid production: Sterilized MRS broth with pH adjusted to pH 4 and pH 7 were inoculated with 18 hours old cultures of the isolates and incubated at 37°C for 72 hours. Samples were collected at 24 hours interval and lactic acid produced quantified as stated above.

RESULTS Seven different species of LAB were isolated from Ogi, burukutu and retted cassava samples and identified on the basis of Gram staining, catalase test, spore staining, sugar fermentation pattern and other biochemical tests. They were identified as Lactobacillus plantarum, L. fermentum, L. casei, L. acidophilus, L. delbrueckii, L. brevis and Leuconostoc mesenteroides. Table 1 shows the percentage occurrence of each of the isolates in the fermented foods. L. plantarum had the highest percentage occurrence of all the LAB isolated. It was observed that it had 16.22% occurrence in maize ogi while in burukutu it had 10.80%. The isolate with the least occurrence was Leuconostoc mesenteroides, which was only isolated from burukutu with 2.70% occurrence. The LAB species were screened for the quantitative production of lactic acid using normal MRS broth and modified MRS broth. In the normal MRS broth it was observed that L. plantarum produced the highest

Effects of glucose and mannitol as carbon sources on lactic acid production: This was done by preparing 2 batches of MRS broth one containing 2% glucose and the other containing 2% manitol. The media were sterilized by steaming at 110°C for 15 mins. Eighteen hours old culture of the isolates were inoculated into the media and incubated at 37°C. Samples were collected at 24hrs interval for quantitative determinations of lactic acid as described above. Effects of Nitrogen sources on Lactic acid production: A basal medium containing 20 g of dextrose, 1.0 mL tween 80, 2.0 g of dipotassium hydrogen phosphate, 5.0 g of sodium acetate, 0.2 g of magnesium sulphate, 0.05 g of manganese sulphate in 1 liter of distilled water was prepared. 5g of each of nitrogen sources namely casein, yeast extract and potassium nitrate was added

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Pak. J. Nutr., 8 (5): 611-615, 2009 Table 2: Quantity of lactic acid produced (g LG1) by the test isolates Incubation Period (hrs) -----------------------------------------------------------------------------------------------------------------------------Isolates 24 48 72 96 Lactobacillus brevis 1.09±0.01a 1.48±0.09b 1.37±0.02c 0.98±0.09d Lactobacillus fermentum 0.89±0.01ab 1.45±0.04bb 1.14±0.03cb 0.71±0.01db Lactobacillus delbrueckii 0.65±0.04ad 0.89±0.07bc 0.59±0.063cc 0.28±0.06dc Leuconostoc messenteriodes 1.35±0.04ad 1.67±0.09bd 1.57±0.02cd 1.37±0.02dd Lactobacillus plantarum 0.85±0.04ae 2.95±0.32be 2.57±0.09ce 2.27±0.02de Values are means (n = 5) ± standard deviation (SD), Mean values in the same column and row followed by the same letter are not significantly different according to Duncans Multiple Ranges Test (p < 0.05) Table 3:

Effect of varied temperature on the quantity of lactic acid (g LG1) produced by lactic acid bacteria Temperature ----------------------------------------------------------Isolates 4°C 30°C 45°C Lactobacillus brevis 0.95±0.05a 1.57±0.19b 1.35±0.03c Lactobacillus fermentum 0.64±0.13ab 1.32±0.18bb 0.81±0.14cb Lactobacillus delbrueckii 0.77±0.17ad 0.82±0.05bc 0.81±0.063cc Leuconostoc messenteriodes 1.35±0.12ad 1.71±0.04bd 0.99±0.10cd Lactobacillus plantarum 1.74±0.03ae 2.96±0.16be 1.07±0.10ce Values are means (n = 5) ± standard deviation (SD), Mean values in the same column and row followed by the same letter are not significantly different according to Duncans Multiple Ranges Test (p < 0.05)

using yeast extract as nitrogen source. When casein was used as nitrogen source, L. plantarum produced 1.68±0.08 g LG1 of lactic acid in 48 hrs. However, when potassium nitrate was used as nitrogen source 0.85±0.04g LG1 of lactic acid was produced by L. plantarum in 48 hrs (Table 6). Antagonistic activity of lactic acid produced by the test isolates against some pathogenic organisms such as Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, Escherichia coli and Proteus mirabilis was investigated. Lactic acid produced by L. fermentum and L delbrueckii; had the highest inhibitory activity against Staphylococcus aureus with 20 mm zone of inhibition. The lactic acid produced by all the test isolates inhibited S. aureus. However, lactic acid produced by all the test isolates had no inhibitory activity against Candida albicans and Proteus merabilis (Table 7).

quantity of lactic acid (2.95±0.32 g LG1) at 48hrs compared to all other LAB species used in this work with L. delbrueckii having the lowest yield (0.89±0.07 g LG1) of lactic acid at 96 hrs incubation period (Table 2). The influence of temperature on the production of lactic acid by the test organisms was investigated. It was observed that the highest yield (2.96±0.16 g LG1) of lactic acid was produced by L. plantarum at 30°C is 48 hrs while the lowest (0.64±0.13 g LG1) was produced by L. fermentum at 4°C in 24 hrs incubation period (Table 3). The influence of pH on the production of Lactic acid by the test isolates was investigated. It was observed that the highest yield (2.96±0.05 g LG1) of lactic acid was produced by L. plantarum at pH 5.5 in 48 hrs incubation period. At pH 7.0, reduction in the production of lactic acid was significant (P = 0.05) when compared to that produced at pH 5.5 (Table 4). The entire test isolates best utilized glucose for lactic acid production as compared to mannitol when used as a carbon source in this work. L. plantarum produced the highest (2.98±0.03 g LG1) quantity of lactic acid in 48 hrs when glucose was used as carbon source. However, when mannitol was used as carbon source, L. plantarum produced 2.45±0.04 g LG1 of lactic acid in 48hrs. Moreover, Leuconostoc messenteroides produced 1.69±0.08 g LG1 of lactic acid when glucose was used as carbon source while when mannitol was used as carbon source it produced 1.00±0.07 g LG1 of lactic acid in 48 hrs (Table 5). All the test isolates best utilized yeast extract for lactic acid production as compared to other nitrogen sources used in this work. The highest quantity (2.98±0.30 g LG1) of lactic acid was produced by L. plantarum in 48 hrs

DISCUSSION In this study a total of 7 species of lactic acid bacteria were isolated from some traditionally fermented food in Nigeria which include burukutu, retted cassava (fufu) and ogi. According to Odunfa and Adeyele (1985), members of lactic acid bacteria can be detected in a variety of habitats including fermented foods. The isolates were identified with reference to Sneath (1986) on the basis of morphological and biochemical characteristics. The lactic acid bacteria were identified as L. fermentum, L. casei, L. brevis, L delbruckii, L. acidophilus, L. plantarum and Leuconostoc mesenteroides. The predominant LAB species isolated was L. plantarum. This agrees with the findings of Akinrele (1970) Odunfa and Adeyele (1985) who reported predominance of this organism in spontaneous cereal fermentation. This species of LAB was found to be superior to most other LAB in lowering pH and producing growth inhibitory concentration of lactic acid. All the test isolates produced lactic acid with peak production at 48 h of incubation period. L. plantarum produced the largest quantity of lactic acid while L. delbrueckii produced the lowest. Lactic acid produced by the LAB isolates has inhibitory activity against some pathogenic microorganisms used as indicator organisms in this work with Lactobacillus

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Pak. J. Nutr., 8 (5): 611-615, 2009 Table 4: Effect of pH on lactic acid production (g LG1) by the test isolates pH -----------------------------------------------------------------------------------------------------------------------------------------------------------------4.0 5.5 7.0 ------------------------------------------------------------------------------------------------------------------------------------------------------Incubation Period (hr) 24 48 72 24 48 72 24 48 72 Isolates Lactobacillus brevis 0.73±0.20a 1.35±0.16b 0.90±0.03c 1.05±0.10d 1.63±0.14e 1..50±031f 0.67±0.69g 1.35±0.04h 1.32±0.11I Lactobacillus fermentum 0.40±0.20ab 1.07±0.12b 1.00±0.07cb 0.86±0.16bd 1..34±0.21eb 1..20±0.31fb 0.45±0.14gb 0.74±0.10hb 0.70±0.19ib Lactobacillus delbrueckii 1.83±0.22ac 0.87±0.03bc 1.11±0.11cc 0.63±0.14dc 0..98±0.10ec 0.90±0.16fc 0..23±0.18gc 0.83±0.14hc 0.70±0.10ic Leuconostoc messenteriodes 1.13±0.13ad 1.73±0.12db 1.60±0.04cd 1.45±0.17dd 1.87±0.11ed 0..90±0.05fd 1.66±0.02ld 1.53±0.02ld 1..33±0.10id Lactobacillus plantarum 2.26±0.16ae 2.78±0.12be 0.60±0.01cc 0.84±0.14de 2..96±0.05ee 2.60±0.12fe 1.69±0.18he 2.36±0.18he 2.00±0.20ie Values are means (n = 5) ± standard deviation (SD). Mean values in the same column and row followed by the same letter are not significantly different according to Duncans Multiple Ranges Test (p < 0.05) Table 5: Effect of carbon sources on lactic acid production (g LG1) by the test isolates Glucose Mannitol ---------------------------------------------------------------------------------------------------------------------------------------------Isolates 24 48 72 24 48 72 Lactobacillus brevis 1.05±0.07a 1.52±0.06b 1.32±0.06c 0.65±0.04d 0.86±0.03e 0.94±0.03f Lactobacillus fermentum 0.83±0.05ab 1.32±0.06bb 0.96±0.09cb 0.86±0.03db 0.88±0.01eb 0.66±0.03fd Lactobacillus delbrueckii 0.65±0.4ac 0.92±0.01bc 0.52±0.01cc 0.65±0.04dc 0.65±0.04ec 0.53±0.02fc Leuconostoc messenteriodes 1.30±0.07ad 1.69±0.08bd 1.55±0.03cd 1.00±0.07dd 1.00±0.07ed 0.99±0.07fd Lactobacillus plantarum 0.88±0.01ac 2.98±0.03be 2.65±0.04ce 2.65±0.04de 2.45±0.04ed 2.25±0.04 fe Values are means (n = 5) ± standard deviation (SD), Mean values in the same column and row followed by the same letter are not significantly different according to Duncans Multiple Ranges Test (p < 0.05) Table 6: Effect of nitrogen sources on lactic acid production (g LG1) by the test isolates Casein Yeast extract KNO3 ------------------------------------------------------ -------------------------------------------------------------------------------------------------Incubation period (hr) Incubation period (hr) Incubation period (hr) ------------------------------------------------------ ----------------------------------------------------------------------------------------------------Isolates 24 48 72 24 48 72 24 48 72 Lactobacillus brevis 0..52±0.014a 0.89±0.01b 0.92±0.063c 1.01±0.06d 1.52±0.05e 1.36±0.03f 0.19±0.07g 0.66±0.03h 0.45±i0.4I Lactobacillus fermentum 0.16±0.03ab 0.74±0.03bb 0.54±0.03cb 0.89±0.01bd 1.35±0.04ed 1.05±0.01fb 0.21±0.01gb 0.54±0.03hb 0.19±0.01ib Lactobacillus delbrueckii 0.46±0.03ac 0.65±0.03ac 0.73±0.02cc 0.72±0.02dc 0.85±0.04ec 0.45±0.04c 0.22±0.01gc 0.53±0.02hc 0.47±0.02ic Leuconostoc messenteroides 0.66±0.03ad 0.46±0.04bd 0.53±0.02cd 1.37±0.02dd 1.58±0.16cd 1.45f±0.11d 0.47±0.02gd 0.54±0.03hd 0.54±0.03id Lactobacillus plantarum 0.85±0.04ae 1.68±0.08bd 1.45±0.08de 0.78±0.08de 2.98±0.30ee 2.32±0.27fe 0.66±o.03ge 0.85±0.04he 0.88±0.01ie Values are means (n = 5) ± standard deviation (SD), Mean values in the same column and row followed by the same letter are not significantly different according to Duncans Multiple Ranges Test (p < 0.05)

Table 7: Antagonistic activity of LAB isolates against some pathogenic organisms Isolates Pseudomonas aeruginosa Staphylococcus aureus Candida albican L. plantarum + (12mm.) + (8mm) _ L. fermentum + (16mm) + (20mm) _ L. casei + (12mm) + (8mm) _ L. acidophilus _ + (7mm) _ Leuconostoc + (8mm) + (17mm) _ mesnteroides L. delbruecki _ + (20mm) _ L. brevis _ + (10mm) _ Keys: + Inhibition, _ No inhibition

delbrueckii and L. fermentum having the highest inhibitory activity against Staphylococcus aureus. According to Ogunbanwo et al. (2004), LAB have potentials to inhibit the growth of pathogenic and spoilage bacteria and the possibility exists for using them to improve the shelf life of different foods. Their antagonistic property is attributed to the low pH, the undissociated acid and production of other primary and secondary antimicrobial metabolites (Ten Brink et al., 1994). As the incubation period increased, the quantity of lactic

Escherichia coli _ _ _ + (7mm) _ + (17mm) _

Proteus mirabilis _ _ _ _ _ _ _

acid produced also increased until there was a gradual decline after 48 h of incubation. The influence of temperature, pH, carbon and nitrogen sources on the production of lactic acid by the test isolates was investigated. The optimum temperature and pH for the production of lactic acid by the test isolates was at 30°C and pH 5.5. All the test isolates best utilized glucose as carbon source for production of lactic acid when compared to mannitol. LAB convert glucose to lactic acid with 100% yield. However, glucose is the only one of many sugars

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Pak. J. Nutr., 8 (5): 611-615, 2009 found in nature; more complex pentose sugars such as L-arabinose (arabinose), D-ribose and D-xylose (xylose) under normal conditions are not degraded by lactic acid bacteria. All the test isolates preferred yeast extract for lactic acid production compared to all other nitrogen sources used in this work. Lactic acid bacteria require complex nitrogen sources which is very important in lactic acid production and as growth factors (Suma et al., 1999). However proteolytic activity is first required before nitrogen can be consumed. Because of this slow catabolism, a state of nitrogen limitations is created when using urea, resulting in the suppression of possible metabolic regulatory mechanisms such as the repression of catabolic enzymes and amino acid transport (Aharonowitz, 1980). A slow metabolism rate also related to a low specific growth rate, which could lead to reduction in lactic acid production. Antagonistic activity of lactic acid produced by the Lactic acid bacteria was investigated in this work. All the test isolates showed antagonistic activities against one or more pathogenic organisms used in this work. This agrees with the work of Ten Brink et al. (1994), who reported that lactic acid bacteria inhibit microbes due to lactic acid and bacteriocin production. Adams and Hall (1988), also reported that inhibitory action of lactic acid bacteria is due to the production of lactic acid in addition to other factors. In conclusion, Lactic acid bacteria, generally regarded as safe, will produce lactic acid optimally at 30°C and pH 5 in the modified MRS broth containing 2% glucose and 2% yeast extract. The lactic acid produced by these organisms has antagonistic activity against pathogenic microorganisms associated with food, thereby serving as a means of preservation of food fermented with these organisms and invariably serving as a prophylactic means of checking proliferation of intestinal pathogens.

Akinrele, I.A., 1970. Fermentation of maize during the preparation of a traditional African starch cake food. J. Sci. Food Agri., 21: 269. Brook, T.M.T. and Madigan, 1991. Host-microbe relationships and disease processes In Biology of Microorganisms; 6th Edition Prentice hall publicity Inc., pp: 379-380. Datta R., 1995. Technological and economical potential of polylactic acid and lactic acid derivatives. FEMS Microbiol. Rev., 16: 221-231. Halm, M., A. Lillie, A.K. Sorensen and M. Jakobsen, 1993. Microbiological and aromatic characteristics of fermented maize doughs for kenkey production in Ghana. Inter. J. Food Microbiol. Biotechnol., 12: 531536. Kandler, O. and N. Weiss, 1986. Regular Nonsporing Grampositive rods. In Sneath, P, Mar, N.S. Sharpe M.E and Heltt, J (edds.) Bergey’s Manual of Systematic Bacteriol. Baltimore: Williams and Wilkins, pp: 1209-1234. Narayanan, N., Roychoudhury and A. Srivastava, 2004. Isolation of adh mutant of lactobacillus rhamnosus for production of L (+) lactic acid. Electronic journal of biotechnology [online]. 15 April 2004. vol. 7no. 1] available from internet: http//www. ejbiotecnology. info/vol7/issue1/full/7/index.html.issn07173458. Odunfa, S.A. and S. Adeyele, 1985. Microbiological changes during the traditional production of ogi baba, a West African fermented sorghum curl. J. Cereal Sci., 3: 173-180. Ogunbanwo, S.T., A.I. Sanni and A.A. Onilude, 2004. Effect of bacteriocinogenic Lactobacillus spp. On the shelf life of fufu, atraditional fermented cassava product. World J. Microbiol. Biotechnol., 20: 57-63. Prescott, M. Harley and O. Kelvin, 2005. Microbiology. McGraw Hill Inc. International edition, 511-517. Sneath, R.H.A., 1986. Classification of Microorganisms in Essays in Microbiology (Eds Norris, J.R. and M.H. Richman), Wiley Lenden. Suma, M.V., M.S. Uma Maheshwar and M.C. Mistra, 1999. Effect of nitrogen source on lactic acid production in Abstracts of papers International Workshop on Lactic acid bacteria. Central Food Technol. Reseach Institute, Mysore, India, pp: 18. Ten Brink, B., M. Minekus, J.M.B.M. Vander Vossen, R.J. Leer and J.H.J. Huis, 1994. Antimicrobial activity of Lactobacilli: preliminary characterization and optimization of production of acidecin B., a novel bacteriocin produced by L. acidophilus M46. J. Applied Bacteriol., 77: 140-148.

ACKNOWLEDGEMENT The authors are grateful for the technical assistance of Dr. S.T. Ogunbanwo, Department of Botany and Microbiology, University of Ibadan, Nigeria.

REFERENCES A.O.A.C., 1990. Official methods of the analysis of the A.O.A.C, 5th Edition. A.O.A.C. Alington, Virginia, U.S.A. Adams, M.R., Hall, 1988. Growth inhibition of food borne pathogens by lactic acid and acetic acid and their mixtures. Int. J. Food Sci. Tehnol., 23: 287-292. Aharonowitz, Y., 1980. Nitrogen metabolite regulation of antibiotic biosynthesis. Annu. Rev. Microbiol., 34: 209-233.

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