Inhibition of Bacillus subtilis and Listeria innocua by nisin in combination with some naturally occurring organic compounds.

The application of combined preservative factors (hurdle technology) is very effective in controlling the growth of food spoilage and foodborne pathogenic bacteria. Antimicrobial activity of nisin alone and in combination with some natural organic compounds (carvacrol, cinnamic acid, eugenol, diacetyl, and thymol) on the growth of gram-positive bacteria Bacillus subtilis and Listeria innocua was-investigated. All the organic compounds tested exhibited antimicrobial activity against the microorganisms used; however, the MICs varied between 0.8 and 15.0 mM depending on the potency of the compound or the sensitivity of the target strain. Investigation of the interaction between the organic compounds and nisin against the test organisms revealed different patterns, varying from synergistic to antagonistic. Combinations of nisin with carvacrol, eugenol, or thymol resulted in synergistic action against both test organisms. Activity of nisin and cinnamic acid together was synergistic against L. innocua, but only additive against B. subtilis. In contrast, the combination of diacetyl and nisin resulted in an antagonistic effect against both test organisms. This study highlights the potential of the combination of these compounds with nisin to inhibit pathogen growth in food.

Identification of yeasts isolated from Nigerian sugar cane peels.

A pilot study was carried out to determine the yeast species associated with sugar cane peels recovered from three separate dumping sites found in Ojo, a typical market town in Nigeria. Species identities were determined by 26S ribosomal DNA (rDNA) sequencing. By using this method, a total of 6 different yeasts were identified, with Candida tropicalis, a recognised human yeast pathogen, being the species most frequently isolated from these dumping sites. The implications of this and other findings of the study, the apparent first of its kind, are discussed in detail.

Activity of natural antimicrobial compounds against Escherichia coli and Salmonella enterica serovar Typhimurium.

AIMS: The objective of this study was to evaluate the inhibitory activity of several natural organic compounds alone or in combination with nisin against Escherichia coli and Salmonella Typhimurium. METHODS AND RESULTS: The minimum inhibitory concentration (MIC) of five natural organic compounds were determined, and the effect of their combinations with nisin was evaluated by the checkerboard assay using the Bioscreen C. As expected, nisin by itself showed no inhibition against either of the Gram-negative bacteria. Thymol was found to be the most effective with the lowest MIC values of 1.0 and 1.2 mmol 1-1 against Salm. Typhimurium and E. coli, respectively. After thymol, the antimicrobial order of the natural organic compounds was carvacrol > eugenol > cinnamic acid > diacetyl. However, the combination of nisin with the natural organic compounds did not result in the enhancement of their antimicrobial activities. On the contrary, combination of nisin with diacetyl against Salm. Typhimurium resulted in an antagonism of diacetyl activity. CONCLUSIONS: While the individual natural organic compounds showed inhibitory activity against the two Gram-negatives, their combinations with nisin showed no improvement of antimicrobial activity. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the potential of the natural organic compounds to control E. coli and Salm. Typhimurium.

The use of a starter culture in the fermentation of cassava for the production of "kivunde", a traditional Tanzanian food product.

Three cassava fermentation methods (spontaneous fermentation, back-slopping and the use of starter culture) for the production of kivunde, executed in three trials at 30 degrees C, were compared in terms of cyanide level reduction, microbiology and product quality improvement. Among the isolates from spontaneously fermented cassava batches, four strains were selected on the basis of their enzymatic activities and acid production. All were identified as Lactobacillus plantarum and were used as starters in this study. Lowest residual cyanide levels were detected after 120 h fermentation time in samples fermented with the starter culture and were below the maximum value of 10 mg/kg recommended by the Codex/FAO for cassava flour. This finding seems to be related to the alpha-glucosidase activity of the inoculated strains of which API-zyme (Bio-Merieux) tests showed activities of between 20 and > or = 40 nmol/4 h. The total residual cyanide levels of the spontaneous and back-slopping fermentations at 96 h were respectively 43.5 and 47.7 mg/kg dry weight of cassava. Extension of the fermentation period to 5 days, lead to further substantial reduction in the residual cyanide level in both these processes, but not below the recommended maximum value as in the case of starter culture fermented products. The spontaneous and back-slopping fermented cassava showed signs of deterioration after 3 days of fermentation. There was a sharp drop of pH and an increase of titratable acidity for all three batches during the first 48 h followed by a slow rise of pH and drop in titratable acidity towards the end of fermentation. The samples fermented with the starter culture had a smooth texture and pleasant fruity aroma, as opposed to the course and dull appearance and more complex flavour of the samples of spontaneous and back-slopping batches. During fermentation with starter culture, Enterobacteriaceae and yeasts and moulds could not be isolated throughout the period of fermentation (detection limit: 10 colony forming units/g). The present findings indicate the suitability of these Lb. plantarum strains as starter cultures for cassava fermentation in the kivunde process. The paper highlights the potential for the improvement of a traditional African fermented food (kivunde) through the use of a starter culture.

Occurrence of nisin Z production in Lactococcus lactis BFE 1500 isolated from wara, a traditional Nigerian cheese product.

Screening for bacteriocin production of 500 strains of lactic acid bacteria (LAB) from various African fermented foods resulted in the detection of a bacteriocin producing Lactococcus lactis (BFE 1500) isolated from a dairy product called wara. The bacteriocin inhibited not only the closely related LAB, but also strains of Listeria monocytogenes, Listeria innocua, Clostridium butyricum, Clostridium perfringens, Bacillis cereus and Staphylococcus aureus. It was heat stable even at autoclaving temperature (121 degrees C for 15 min) and was active over a wide pH range (2-10), but highest activity was observed in the lower pH range. The bacteriocin was inactivated by alpha-chymotrypsin and proteinase K, but not by other proteases. Growth kinetic assay indicated stronger growth inhibition by the bacteriocin produced by Lc. lactis BFE 1500 on L. monocytogenes WS 2250 and B. cereus DSM 2301 than with the nisin A producing strain DSM 20729. Polymerase chain reaction indicated the presence of the nisin operon in strain BFE 1500 and sequencing of its structural gene showed that Lc. lactis BFE 1500 produced the natural nisin variant, nisin Z, as indicated by the substitution of asparagine residue instead of histidine at position 27. The genetic determinants for bacteriocin production in strain BFE 1500 are located on a conjugative transposon. The ability of the bacteriocin produced by Lc. lactis BFE 1500 to inhibit a wide range of food-borne pathogens is of special interest for food safety, especially in the African environment with perennial problems of poor food hygiene.

Inhibition of Listeria monocytogenes by plantaricin NA, an antibacterial substance from Lactobacillus plantarum.

A Lactobacillus plantarum of vegetable origin produced a bacteriocin inhibitor to Listeria monocytogenes. The antimicrobial agent was inactivated by proteolytic enzymes, was resistant to heat (100 degrees C for 30 min) and stable over a wide pH range (pH 2-10), and displayed a bactericidal mode of action. Growth inhibition of L. monocytogenes depend on bacteriocin concentration. The antilisterial efficiency depended on the strain of L. monocytogenes used but was not influenced by the growth phase of this strain. A decrease in absorbance overtime, indicative of cell lysis, was also observed. The significance of the results is discussed in relation to the potential of the bacteriocin in controlling Listeria-associated food-borne hazards in foods.

Plantaricin D, a bacteriocin produced by Lactobacillus plantarum BFE 905 ready-to-eat salad.

Lactobacillus plantarum BFE 905 isolated from 'Waldorf' salad produced a bacteriocin termed plantaricin D which was active against Lact. sake and Listeria monocytogenes strains. Plantaricin D was heat stable, retaining activity after heating at 121 degrees C. The bacteriocin was inactivated by alpha-chymotrypsin, trypsin, pepsin and proteinase K, but not by papain and other non-proteolytic enzymes tested. Plantaricin D was stable at pH values ranging from 2.0 to 10.0. The bacteriocin inhibited growth of L. monocytogenes in automated turbidity assays. Although Lact. Plantarum BFE 905 harboured plasmids ranging in size from 3 to 55 kilobase pairs, loss of bacteriocin production could not be correlated with plasmid loss. A role for bacteriocin-producing Lact. plantarum of vegetable origin in assuring the safety of vegetable foods is suggested.

Assessment of a bacteriocin-producing Lactobacillus strain in the control of spoilage of a cereal-based African fermented food.

As a part of a program to develop starter cultures aiding in the spoilage control and sanitation of African fermented foods, a cereal-based food ('ogi' and its solid form 'agidi' or 'eko') was prepared using a bacteriocin-producing Lactobacillus strain as the starter culture. The survival of an enterotoxigenic Escherichia coli strain was investigated in the naturally fermented food and in food fermented with the starter bacteriocin-producing Lactobacillus strain. An inhibition of E. coli was observed within 2 h of incubation in 'ogi' fermented with the bacteriocin producing strain. After 6 h, the viable count of E. coli in locally fermented 'ogi' was log 6.41 (2.54 X 10(6) CFU/mL), whereas in 'ogi' fermented with the bacteriocin producer it was reduced to log 1.70 (0.5 x 10(2) CFU/mL). Comparison of the shelf life of 'agidi' prepared from the naturally fermented food with that bacteriocin-producing starter culture showed that the latter had a better shelf life (kept for 11 d before spoilage occurred as compared with 7 d for the natural one). The results are discussed in terms of the potential of bacteriocin-producing cultures in the control and retardation of spoilage and food-forne infections in some African fermented foods.

Bacteriocins of Lactobacillus plantarum strains from fermented foods.

Bacteriocin-producing strains may be used as protective cultures to improve the microbial safety of foods. The crude or purified form of these antimicrobial agents may also be applied directly as food preservative. This review gives survey of the different bacteriocins produced by Lactobacillus plantarum isolated from fermented food products with particular emphasis on their genetic and biochemical properties. A number of bacteriocins are produced by L. plantarum. These include plantaricin B, plantaricin BN, plantaricin A, plantaricin C, plantaricin S and T, plantaricin F, plantaricin C19 and SA6 and other unnamed bacteriocins. However, with the exception of plantaricin A, information on the genetic and biochemical characteristics of L. plantarum bacteriocins is still scant.

Studies on an amylolytic strain of Saccharomyces cerevisiae isolated from yam tuber.

An amylolytic yeast strain identified as Saccharomyces cerevisiae was isolated from yam tuber. Studies on the effect of physical agents such as pH and temperature on the activity of the amylase showed that the optimum pH and temperature for the amylase produced by the S. cerevisiae were 5.0 and 60 degrees C, respectively. Heavy metal resistance study on the amylolytic yeast strain indicated different levels of resistance to copper (6.0 mM), zinc (3.0 mM) and manganese (15.0 mM) ions. The results are discussed in relation to the potential use of an amylolytic yeast in the brewing industry in Nigeria.

Plasmid profiles and antibiotic susceptibility patterns of Staphylococcus aureus isolates from Nigeria.

In an investigation into the problems of infections due to Staphylococcus aureus in Nigeria, 100 strains were isolated from various hospitals in Lagos. The strains were screened for the presence of plasmids and for susceptibility to antimicrobial agents. Plasmids were extracted by modification of the method of Takahashi and Nagono[1]. The plasmids were diverse in nature. The strains were found to be highly resistant to commonly prescribed antibiotics.

Studies on bacteriocinogenic Lactobacillus isolates from selected Nigerian fermented foods.

Ten bacteriocin-producing (bacteriocinogenic) Lactobacillus isolates obtained from three Nigerian fermented foods namely: kenkey, ogi and wara were tested against the following indicator organisms: Lactobacillus plantarum and food borne pathogens comprising enterotoxigenic Escherichia coli, enterohaemorrhagic Escherichia coli, Serratia, Pseudomonas, Vibrio cholerae, Aeromonas sobria, Aeromonas cavice, Salmonella typhimurium, Plesiomonas shigelloides and Staphylococcus aureus. All the bacteriocinogenic Lactobacillus were found to inhibit L. plantarum while some inhibited some of the food borne pathogens listed above. The antimicrobial activities of bacteriocins from L. plantarum KKY12 and L. casei OGM12 were caused by proteins detectable in the culture liquids. They are designated Plantacin N and Caseicin A and they have narrow antimicrobial spectra. Plantacin N from L. plantarum KKY12 was active against L. plantarum, Pseudomonas, Aeromonas sobria and Aeromonas cavice whereas Caseicin A from L. casei OGM12 inhibited L. plantarum, enterotoxigenic Escherichia coli and Vibrio cholerae. Caseicin A is stable at 121 degrees C/15 mins, and both are inactivated by proteolytic enzymes. The bacteriocinogenic properties of the local isolates of Lactobacillus can help to reduce hygienic risk and the spoilage of fermented foods.

Bacteriocin production by Enterococcus faecium NA01 from 'wara'--a fermented skimmed cow milk product from west Africa.

An Enterococcus faecium strain from Nigerian fermented skimmed cow milk ('wara') produced bacteriocin inhibitory towards Lactobacillus, Enterococcus and Listeria strains. The bacteriocin (designated enterocin 01) was inactivated by proteases, heat-stable at 100 degrees c and active at pH 2.0-6.0. The Ent. faecium isolate harboured plasmids of ca 36.3 and 23.1 kb. Curing experiments with ethidium bromide resulted in a bacteriocin-negative mutant which had not lost immunity to the bacteriocin. Slight differences in plasmid profiles between wild-type and mutant indicated a possible plasmid-coded bacteriocin production.

Production of DogiK: an improved Ogi (Nigerian fermented weaning food) with potentials for use in diarrhoea control.

As part of a programme to formulate foods to aid the control of diarrhoeal diseases, an improved ogi (the commonest weaning food in Nigeria) named DogiK has been developed. DogiK was produced by using Lactobacillus starter cultures with antimicrobial activity against diarrhoeagenic bacteria and also possessing amylolytic activity. The survival of diarrhoeagenic bacteria was investigated in locally-fermented ogi and in DogiK. The foods were inoculated with cell suspensions of Salmonella, Shigella, Campylobacter, Aeromonas, Pleisiomonas, Enteropathogenic and Enterotoxigenic Escherichia coli, Yersinia enterocolitica, and Vibrio cholerae. None of the diarrhoeagenic bacteria were detected in DogiK after 6 h whereas in the local ogi Salmonella, E. coli, and Shigella survived for 24 h or more, but showed a sharp decrease in numbers, while V. cholerae survived for 12 h. DogiK is active whether cooked or uncooked and exhibited inhibition of pathogens at neutral pH. It gives consistent quality. Preliminary investigation indicates possession of a better shelf life. Thus, DogiK may have a potential use in the prevention and treatment of diarrhoea.

Plasmid profiles of bacteriocin-producing Lactobacillus isolates from African fermented foods.

Fourteen of 200 Lactobacillus isolates from African fermented foods, viz. 'wara', 'kenkey', 'ugba', 'ogi', 'kunuzarki', 'fufu' and 'iru' were found to produce bacteriocins against L. plantarum and only three bacteriocinogenic isolates inhibited some of the food pathogens. Plasmid analysis of the 14 bacteriocin-producing lactobacilli showed that only 5 isolates harbored plasmids ranging in size from 3.1 to 55.5 kb.

Resistance to heavy metals by some Nigerian yeast strains.

The heavy metal resistance of yeasts isolated from sugary substrates such as orange, palm wine and pineapple and identified as Saccharomyces carlsbergensis and S. cerevisiae was studied. The yeast isolates were tested against different concentrations of cadmium, copper, manganese, silver and zinc salts ranging from 1 to 20 mmol/L. Local yeasts showed resistance to 3-15 mmol/L cadmium, 18-20 copper, 16-20 manganese, 1-9 silver and 16-19 for zinc. The significance of the results is discussed in relation to the effects of heavy metals on growth of microorganisms and selection of yeasts for the brewing industry in Nigeria.