Charakterisierung einiger Bacteriocine im Hinblick auf mögliche Anwendungen im Lebensmittelbereich. 1. Mitt. Stammauswahl

Characterization of some bacteriocins with regard to possible applications in food industry. Part 1. Selection of strains. The ability to produce bacteriocins is common in all genera of lactic acid bacteria. Thirty-four out of 223 strains of lactic acid bacteria from the culture collection of the University Potsdam produced bacteriocins active against one or more indicator strains. Seventeen Gram-positive and Gram-negative strains, frequent occur as spoilage bacteria in food industry, were chosen as indicator strains, and 11 of them were inhibited by one or several bacteriocin producers. Most of the bacteriocin producers belong to the genera Enterococcus or Lactobacillus. Bacteriocins derived from Enterococcus faecium and Pediococcus acidilactici exhibited the widest inhibitory spectrum. The properties of Enterococcus faecium 10.051 and Enterococcus faecium 10.211 bacteriocins were further investigated to evaluate their potential use as natural food preservatives. The bacteriocin produced by Enterococcus faecium 10.211 exhibited a very wide inhibitory spectrum against food-spoiling strains. It was heat stable (100 °C for 60 min) and stable in a wide range of pH (1.2–10.0). The bacteriocin produced by Enterococcus faecium 10.051 showed a smaller inhibitory spectrum, and it was less stable against temperature and pH as the bacteriocin from strain 10.211. Both bacteriocins were inactivated by proteinase K. pronase E, and α-chymotrypsin. Bacteriocin from strain 10.211 will be the object of further investigations for application of bacteriocins in food industry.     

Incidence and antibiotic susceptibility of bacteriocin‐producing lactic acid bacteria from dairy products

Screening for bacteriocin production by strains of lactic acid bacteria (LAB) from local dairy products in Iran resulted in the detection of 10 bacteriocin‐producing strains. Among 105 isolated, 10 bacteriocin producers were phenotypically and genotypically identified as Enterococcus spp. The antimicrobial compounds produced by these novel strains were inactivated by trypsin, proteinase k. These bacteriocins also were active in a wide range of pH and temperature values, and inhibited not only the closely related LAB, but also Listeria monocytogenes.     

Microbial and Physicochemical Characterization of the Horse Meat in Fermented Sausage

In this study, 150 lactic acid bacterial strains, originating from horse meat, were subjected to a step-by-step screening and characterization to search for potential protective cultures to be used in the meat industry. Strains were first tested for their homofermentation and salt tolerance. Second, the antibacterial capacities towards Listeria monocytogenes were determined in an agar spot test. In total, 50% of the tested strains were inhibitory towards Listeria monocytogenes. However, only 12 strains produced a bacteriocin. Finally, three strains with strong bacteriocin activity were competitively evaluated by comparing their growth rate, acidifying character and lactic acid production at 15°C under anaerobic conditions in a liquid broth. All three strains had a fast growth rate with rapid acidification caused by the production of high levels of lactic acid. Based on this Lactobacillus sakei was used as starter culture for producing sausage horse meat. In this study, fermentations were followed analyzing the microbiological and physicochemical aspects of this product. The sausages were characterized based on microbial activity of lactic acid bacteria that resulted in a product with a final pH of about 4.56. No Listeria monocytogenes, Salmonella spp. nor Sulfite reducing clostridia were ever isolated from the raw materials or the fermented sausages during the maturation, reflecting the safety of this product. The final water activity of the product was 0.85. Lactobacillus sakei was efficient in reducing amine production since this strain caused a quick pH drop during sausage fermentation.     

Effect of Extrinsic Parameters on the Production of a Bacteriocin by Lactobacillus buchneri,Isolated from Mexican Raw Sausages

Bacteriocins are antimicrobial peptides acting on certain pathogens and spoilage microorganisms. They have recently been considered as natural alternative to food preservatives. The objective of this work was to study the inhibition spectrum of a bacteriocin produced by Lactobacillus buchneri, isolated from a Mexican sausage, against Listeria monocytogenes and several lactic acid bacteria, and the influence of several extrinsic parameters on bacteriocin activity. Culture media (MRS or APT) had no significant effect on bacteriocin production (p < 0.060) although higher activity was observed in MRS (220 AU/mL). Conversely, temperature significant affected production (p > 0.001). Bacteriocin activity was also increased by 100% nitrogen atmosphere (180 AU/mL) as compared to N₂/CO₂ (50:50) (150 AU/mL) and 100% CO₂ (60 AU/mL). Maximum activity occurred at the end of the exponential growth phase. This bacteriocin inhibited some Listeria monocytogens strains and several lactic acid bacteria, mainly Lactobacillus sp.     

Pediocin PA-1 and a pediocin producing Lactobacillus plantarum strain do not change the HMA rat microbiota

The bacteriocin pediocin PA-1 has potential use as a food biopreservative, and understanding its effect on the commensal gut microbiota is important for assessment of consumer risks associated with the use of biopreservative cultures. Effects of ingested (i) pediocin PA-1 producing Lactobacillus plantarum DDEN 11007, (ii) the plasmid cured pediocin negative L. plantarum DDEN 12305, or (iii) supernatants of either of these two strains on the composition of the intestinal microbiota of Human Microbiota Associated (HMA) rats were examined by selective cultivation and molecular methods. The culturable microbiota was in all treatments dominated by lactic acid bacteria and coliforms and no changes in the rat commensal microbiota were detected after ingestion of either of the two L. plantarum strains as determined by both culturable methods and molecular methods (DGGE). Both strains were detected in the faeces after ingestion. Pediocin PA-1 did not mediate changes of the rat microbiota, and a biological assay indicated that the bacteriocin was degraded or inactivated during passage through the intestine.     

Lactic acid bacteria as protective cultures against Listeria spp. on cold-smoked salmon

Three bacteriocin producing (Bac⁺) strains of Lactobacillus sakei were used singly and in combination with each other as protective cultures to control the growth of listeria in cold-smoked salmon. Challenge experiments were conducted under practical conditions in a smokehouse. The surface of salmon sides was inoculated with 10⁴ cfu/g of Listeria innocua and 10⁷ cfu/g of Bac⁺ lactic acid bacteria as well as a L. sakei Bac⁻ control. After smoking the counts of listeria and lactic acid bacteria were determined at days 1 and 14. All Bac⁺ L. sakei strains reduced the counts of L. innocua by >2 log units. Strain LTH5754 was an isolate from cold-smoked salmon and achieved even a 5 log reduction of L. innocua within the storage period. In vitro experiments showed, that the Bac⁺ strains were also effective against L. monocytogenes (three strains tested) and L. ivanovii (1 strain). The pH as well the sensorial properties of the smoked salmon were not affected by the L. sakei inocula.     

Optimization and Partial Purification of Bacteriocins from Enterococcus spp. Indigenous to Pakistan

This study was conducted to optimize bacteriocin producing enterococcal strains isolated from indigenous fermented dairy products of Pakistan. Isolates IJ-06, IJ-21, and IJ-31 were identified as Enterococcus faecium and IJ-11 was identified as Enterococcus faecalis. All of these enterococcal isolates were catalase, gelatinase negative, and non-hemolytic on sterile sheep blood. Bacteriocin production trials confirmed E. faecium IJ-06, IJ- 21, and IJ- 31 as the potential producer of bacteriocin showing antimicrobial activity in cell-free supernatant (CFS). E. faecalis IJ-11 displayed activity after partial purification. Optimization of culture conditions for the production of bacteriocin by the selected strains showed that maximum production was achieved at 35–37°C with 1% inoculum after 16 h of incubation and at pH 7–8. The molecular mass of the partially purified enterocins falls in the range of 4.5–4.7 kDa. These enterocins were close to class IIa of bacteriocins and were highly active against Listeria monocytogenes, Bacillus subtilis, Bacillus cereus and other closely related strains.     

Simulation of the effect of sausage ingredients and technology on the functionality of the bacteriocin-producing Lactobacillus sakei CTC 494 strain

Bacteriocin-producing lactic acid bacteria may be applied as novel functional starter cultures for sausage fermentation. In this way, safer and more standardised end products may be obtained. However, it is not clear how such strains behave under sausage fermentation conditions. In this study, the combined effects of typical sausage ingredients and process technology on the functionality of Lactobacillus sakei CTC 494 were simulated by a modelling approach. Under simulated sausage fermentation conditions, the strain was able to produce a considerable amount of bacteriocin. Model simulations indicate that sausage fermentation conditions of temperature and pH favour bacteriocin production, whereas salting and curing with sodium nitrite decrease growth and bacteriocin production. Sodium nitrite inhibits cell growth under its undissociated nitrous acid form, and its inhibitory effect seems to parallel lactic acid production. Whereas oxygen and magnesium levels did not influence bacterial functionality, manganese limitations severely decreased cell growth. Moreover, the presence of large amounts of fat, which is typical for a sausage environment, leads to an apparent bacteriocin inactivation, probably due to adsorption of the bacteriocin molecules from the water phase to the fat particles.     

Characterization of a Bacteriocin from Lactobacillus plantarum Strain LR/14

A rhizospheric isolate of a lactic acid bacterium, identified as Lactobacillus plantarum strain LR/14, was characterized to produce a bacteriocin. A supernatant from 20h culture growth was used as the source of bacteriocin. The antimicrobial compound showed remarkable stability at high temperatures (100°C for 30 min and 121°C for 15 min under 15 psi pressure) and to the presence of organic solvents, detergents and surfactants. It was also active in the pH regime of pH 2.0–6.0. Moreover, the compound was stable under different storage temperatures as tested up to 24 months. While antimicrobial function was not lost by catalase or β-glycerophosphate treatment, the same was sensitive to a number of proteolytic enzymes. The crude preparation inhibited not only related strains but also other gram-positive and gram-negative bacteria, such as Staphylococcus aureus, Listeria monocytogenes and urogenic E. coli. Bacteriocinogenic activity co-migrated as a single protein band on tricine-SDS-PAGE with molecular mass of ～3.6 kDa.     

Short Communication: Culture-Independent Detection of Lactic Acid Bacteria Bacteriocin Genes in Two Traditional Slovenian Raw Milk Cheeses and Their Microbial Consortia

Two Slovenian traditional raw milk cheeses, Tolminc (from cows’ milk) and Kraški (from ewes’ milk), were examined for the presence of 19 lactic acid bacteria bacteriocin genes by PCR analysis of total DNA extracts from 9 cheeses and from consortia of strains isolated from these cheeses. Eleven bacteriocin genes were detected in at least one cheese or consortium, or from both. Different cheeses or consortia contained 3 to 9 bacteriocin determinants. Plantaricin A gene determinants were found in all cheese and consortia DNA extracts. Genes for enterocins A, B, P, L50A, and L50B, and the bacteriocin cytolysin were commonly detected, as were genes for nisin. These results indicate that bacteriocinogenic strains of Lactobacillus, Enterococcus, and Lactococcus genera with protective potential are common members of indigenous microbiota of raw milk cheeses, which can be a good source of new protective strains.     

Isolation and characterisation of the bacteriocin-producing Leuconostoc citreum HW02 from malts

Herein, a lactic acid bacterium which produces bacteriocin was isolated from malted barley. The isolated strain was identified by 16 S rRNA analysis and designated Leuconostoc citreum HW02. Bacteriocin HW02 was shown to have growth inhibition against health-threatening Gram-positive microorganisms such as Staphylococcus aureus, Listeria monocytogenes, and Latilactobacillus curvatus. Bacteriocin HW02 was reported to be incapable of inhibiting the growth of health-threatening microorganisms after treatment with proteolytic enzymes; thus, it was indicated that bacteriocin is proteinaceous in nature. Bacteriocin HW02 showed stability against a broad range of temperatures (60–121 °C), pH (2–10), various detergents, or organic solvents. Its antimicrobial activity against the indicator strain, Lactiplantibacillus plantarum NCDO 955, was maximised (320 AU mL⁻¹) midway through the stationary phase at 12 h. The crude bacteriocin HW02 molecular weight was estimated to be about 7.7 kDa. After the treatment of the indicator strain with bacteriocin, the maintenance of the optical density of the culture, the morphological analysis of cell-structure abnormalities, and cell shrinkage implied that bacteriocin showed bactericidal activity.     

Diversity of Streptococcus thermophilus in bacteriocin production; inhibitory spectrum and occurrence of thermophilin genes

The bacteriocin-producing Streptococcus thermophilus strains that can dominate in natural dairy ecosystems, may also enhance safety in products obtained from natural cultures. In this study, we sought to identify bacteriocin production and bacteriocin genes in 75 strains of dairy and plant origin. The strains were tested for antimicrobial activity against pathogens or pathogen models, spoiling bacteria, and lactic acid bacteria associated with dairy products. All strains moderately inhibited Staphylococcus aureus P310, none inhibited Listeria innocua LMG 11387^T or Clostridium tyrobutyricum LMG 1285^T. In addition, 14 were active against one or more indicators in addition to S. aureus P310. Inhibition of other starter bacteria was more common than the inhibition of unwanted microorganisms. The involvement of a proteinaceous compound was ascertained in all cases. Results suggested that the selection of bacteriocinogenic S. thermophilus strains for use in biopreservation must take into account the effects exerted on other lactic acid bacteria.     

Preliminary characterization of bacteriocin-like substances from lactic acid bacteria isolated from organic leafy vegetables

This study presents the characterization of new strains of lactic acid bacteria (LAB) from organic vegetables. Forty-five strains of LAB isolated from vegetables were investigated by its antimicrobial activity against taxonomically related microorganisms. Genetic identification of selected LAB was performed by means of PCR method. These strains were Enterococcus faecium, Lactococcus lactis, Enterococcus hirae and Enterococcus canis. Bacteriocin-like substances were active against Gram-positive bacteria and Gram-negative foodborne pathogens (Listeria monocytogenes and Escherichia coli, respectively). The antimicrobial activity of LAB strains was inactivated by the addition of proteases, thus confirming the proteinaceous nature of the inhibition. In all four strains the bacteriocin activity was stable after extended refrigerated storage and freezing-thawing cycles. This fact suggests that bacteriocin produced by the four LAB strains may find application as biopreservatives in minimally processed vegetables.     

Optimisation of bacteriocin production of Lactococcus lactis subsp. lactis MA23, a strain isolated from Boza

Lactococcus lactis subsp. lactis MA23 produces a bacteriocin (6400 AU/mL) that inhibits the growth of many Gram‐positive bacteria but is not active against Gram‐negative bacteria. This bacteriocin inhibits growth of lactococcal strains that are producing nisin, lacticin or lactococcin suggesting it to be different from these bacteriocins. The nutritional requirements and optimal growth conditions for MA23 bacteriocin production were studied with fed‐batch fermentations. The optimal pH, carbon source and nitrogen source for bacteriocin production were pH 6.5, sucrose (0.5%) and yeast extract (1%), respectively.     

Antibacterial and antifungal effect of moringa (Moringa oleifera) seedmeal on marinated smoked African mud catfish (Clarias gariepinus)

The study assessed the antibacterial and antifungal effect of Moringa oleifera seed marinade on smoked Clarias gariepinus stored at ambient temperature for 15 days. Sixty C. gariepinus (15 kg) were gutted, washed, and randomly assigned in triplicate to five treatments containing 0, 5, 10, 15, and 20 g/kg M. oleifera seedmeal in 3.67 g/kg brine solution for 2 hr and later hot smoked for 24 hr. After smoking, the fishes were allowed to cool and packed separately in a transparent Ziploc polythene bag arranged in a box and kept at room temperature. Microbial load and diversity were conducted in the laboratory at 5 days intervals. The fungi isolated were Aspergillus flavus, Aspergillus niger, Saccharomyces cerevisiae, Alternaria alternata, Penicillium citrinium, Neurospora crassa, and Rhizopus stolonifer while Enterobacter spp., Staphylococcus spp., Proteus spp., Streptococcus spp., and Bacillus spp. were the identified bacteria. The optimum effective concentration of moringa seed marinade that acts as anti-bacteria was found best to be 11.2 g/kg of fish moringa marinade. While 12.08 g/kg exhibited the highest anti-yeast effect in C. gariepinus and 10.6 g/kg serves as antifungi. Thus, moringa seed marinade is safe to use as a natural edible food preservative that can protect smoked dried catfish from microbial spoilage.     

Characterisation of anti-Listeria monocytogenes bacteriocins from Enterococcus faecium strains isolated from dairy products

Bacteriocin-producing lactic acid bacteria were isolated from 34 samples of dairy products. Nine bacteriocin producers were phenotypically and genotypically identified as Enterococcus faecium . By means of PCR-techniques, enterocin A was characterised in all of the nine bacteriocin-producing Enterococcus isolates. Enterocin-producing lactic acid bacteria were the most abundant in dairy products collected from different areas in Iran. Maximum bacteriocin production by Enterococcus faecium strains was detected in the stationary phase of growth. Bacteriocins produced by all isolates were found to have anti-listerial activity in sterile milk. The purified bacteriocins were identified as <6.5 kDa peptide by sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE). The molecular weights of bacteriocins were found to be the same in all strains. This bacteriocin might be useful as a natural preservative.     

INHIBITION OF BEER-SPOILAGE LACTIC ACID BACTERIA BY NISIN

149 strains of bacteria, mostly brewery contaminants able to spoil wort or beer, and 12 brewing strains of yeast (8 ale and 4 lager strains) have been screened using a well-test assay for sensitivity to the food preservative, Nisin (E234), Nisin inhibited growth of 92% of the gram-positive strains, predominantly lactic acid bacteria of the genera Lactobacillus and Pediococcus. In contrast, all 32 gram-negative strains tested, except 3 Flavobacter strains, were Nisin-resistant; in addition none of the brewing yeasts showed Nisin-sensitivity. Therefore. Nisin has potential applications in preventing spoilage of worts or beers by lactic acid bacteria.     

CHARACTERIZATION OF A LACTIC ACID BACTERIUM,CARNOBACTERIUM PISCICOLA LK5, WITH ACTIVITY AGAINST LISTERIA MONOCYTOGENES AT REFRIGERATION TEMPERATURES1

A lactic acid bacterium (LK5) originally isolated from raw ground beef was characterized in relation to its ability to inhibit the growth of Listeria monocytogenes. The isolate, which was identified as Carnobacterium piscicola, inhibited the growth of 17 of 21 strains of Listeria (L. monocytogenes, L. innocua, L. ivanovii, L. welshimeri, and L. grayii). Its activity was not due to either acid or hydrogen peroxide production, but was related to the production of a heat stable bacteriocin. The isolate was most active against L. monocytogenes at refrigeration temperatures due to the combined effect of the pathogen's increased susceptibility, LK5's rapid growth rate, and enhanced bacteriocin production at low temperatures. Examination of the effect of inoculum ratios in co-cultures of C. piscicola LK5 and L. monocytogenes Scott A indicated that the lactic acid bacterium was active against L. monocytogenes even when the initial level of the pathogen was 100-fold greater. Evaluation of the impact of oxygen availability, initial pH, and sodium chloride content on the effectiveness of LK5 suggested that the isolate could be used to suppress the growth of Listeria in a variety of refrigerated foods.     

ISOLATION AND PARTIAL CHARACTERIZATION OF A NOVEL BACTERIOCIN PRODUCED BY LACTOCOCCUS LACTIS SSP. LACTIS MC38

This work presents the isolation and partial characterization of a new lactococcal bacteriocin produced by Lactococcus lactis ssp. lactis MC38. The bacteriocin demonstrated broad spectrum of inhibition activity against both pathogenic and food spoilage organisms, and various lactic acid bacteria. This antimicrobial substance appeared to be proteinaceous because its activity was completely inactivated by proteinase K and α‐chymotrypsin. It was heat and pH stable. The apparent molecular mass of the purified bacteriocin, determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, was 8.0 kDa. The amino acid composition of the studied bacteriocin was found to be quite different from known lactococcal bacteriocins. The calculation of the number of amino acid residues in the bacteriocin molecule revealed that it contained 62 amino acids.     

Potential of bacteriocinogenic Lactococcus lactis subsp. lactis inhabiting low pH vegetables to produce nisin variants

Antimicrobial behavior of lactic acid bacteria (LAB) has been explored since many years to assess their ability to produce bacteriocin, a natural preservative, to increase the shelf life of food. This study aims to characterize bacteriocin producing strains of lactic acid bacteria isolated from acidic to slightly acidic raw vegetables including tomato, bell pepper and green chili and to investigate their potential to inhibit food related bacteria. Among twenty nine LAB screened for antimicrobial activity, three exhibited antagonism against closely related bacterial isolates which was influenced by varying temperature and pH. They were identified up to strain level as Lactococcus lactis subsp. lactis TI-4, L. lactis subsp. lactis CE-2 and L. lactis subsp. lactis PI-2 based on 16S rRNA gene sequence. Their spectrum of inhibition was observed against food associated strains of Bacillus subtilis and Staphylococcus aureus. Moreover, L. lactis subsp. lactis PI-2 selected on the basis of higher antimicrobial activity was further evaluated for bacteriocin production which was detected as nisin A and nisin Z. These findings suggest the possible use of L. lactis strains of vegetable origin as protective cultures in slightly acidic as well as slightly alkaline food by the bio-preservative action of bacteriocins.