Irena Rogelj

Isolation and characterization of two bacteriocins of Lactobacillus acidophilus LF221

Lactobacillus acidophilus LF221 produced bacteriocin-like activity against different bacteria including some pathogenic and food-spoilage species. Besides some lactic acid bacteria, the following species were inhibited: Bacillus cereus, Clostridium sp., Listeria innocua, Staphylococcus aureus, Streptococcus D. L. acidophilus LF221 produced at least two bacteriocins, acidocin LF221 A and acidocin LF221 B, which were purified by ammonium sulphate precipitation, ion-exchange chromatography, hydrophobic interaction and reverse-phase FPLC. The antibacterial substances were heat-stable, sensitive to proteolytic enzymes (trypsin, pepsin, pronase, proteinase K) and migrated as 3500- to 5000-Da proteins on sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The sequences of 46 amino-terminal amino acid residues of peptide A and 35 of peptide B were determined. Among the residues identified, no modified amino acids were found. No significant homology was found between the amino acid sequences of acidocin LF221 A and other bacteriocins of lactic acid bacteria and 26% homology was found between acidocin LF221 B and brevicin 27. L. acidophilus LF221 may be of interest as a probiotic strain because of its human origin and inhibition of pathogenic bacteria, especially Clostridium difficile.

Bacteriocin complex of Lactobacillus acidophilus LF221 — production studies in MRS media at different pH values and effect against Lactobacillus helveticus ATCC 15009

Abstract The production of a bacteriocin complex by Lactobacillus acidophilus LF221 at different pH values in MRS broth (5·5, 6, 6·5 and 7) and the mode of action against Lactobacillus helveticus ATCC 15009 were studied. Adsorption of bacteriocins to the producer cells and induction by its own bacteriocins or by sensitive cells was also examined. Bacteriocins were produced during the logarithmic phase of growth. The highest amount of bacteriocin complex (106 AU/ml) was detected in MRS broth at pH 6·5. The bacteriocin complex and biomass production were not directly related. The production of bacteriocin complex was not reduced by low inoculum (4 × 10 −6 ) and was not affected by the cells of indicator strain or by its own bacteriocins. No bacteriocins adsorbed to the cells of the producer at any pH tested. The bacteriocin complex at a concentration of 100 AU/ml was bactericidal for growing cells of L.helveticus ATCC 15009 but not bacteriolytic.

Changes in the microbial composition of raw milk induced by thermization treatments applied prior to traditional Greek hard cheese processing.

The microbiological quality, safety, and composition of mixtures of ewes and goats milk (90:10) used for cheesemaking were evaluated before and after thermization at 60 and 67 degrees C for 30 s. Such mild thermal treatments are commonly applied to reduce natural contaminants of raw milk before processing for traditional hard Greek cheeses. Raw milk samples had an average total bacterial count of 7.3 log CFU/ml; most of these bacteria were lactic acid bacteria (LAB) and pseudomonads. The LAB flora of raw milk was dominated by enterococci (40.8%), followed by lactococci (20.4%), leuconostocs (18.4%), and mesophilic lactobacilli (10.2%). Enterococcus faecalis (30.1%) and Enterococcus faecium (13.7%) were the most common LAB isolates, followed by Enterococcus durans, Lactococcus lactis subsp. lactis, Lactobacillus plantarum, and Leuconostoc lactis. Thermization at 60 degrees C for 30 s was effective for reducing raw milk contamination by enterobacteria (5.1 log CFU/ml), coagulase-positive staphylococci (3.3 log CFU/ml), and Listeria (present in 25-ml samples) to safe levels, but it also reduced mesophilic lactococci, leuconostocs, lactobacilli, and selected enterococci (72.0%) in thermized milk. Thermization at 67 degrees C for 30 s had a major inactivation effect on all bacterial groups. Two nisin-producing L. lactis subsp. lactis strains (M78 and M104) were isolated from raw milk, but neither nisin-producing nor other bacteriocin-producing LAB strains were isolated from thermized milk. Thus, thermization treatments control harmful bacteria but also may have a negative impact on milk quality by reducing desirable LAB and the biodiversity of raw milk bacteria overall, inactivating potentially protective LAB strains and enhancing the ability of potentially pathogenic enterococci to grow in fresh cheese curds.

Optimization of fermentation conditions for the expression of sweet-tasting protein brazzein in Lactococcus lactis.

Aims:  To improve the production of sweet‐tasting protein brazzein in Lactococcus lactis using controlled fermentation conditions.

Expression of nisin genes in cheese—A quantitative real-time polymerase chain reaction approach

The role of bacteriocins in different environments has not been thoroughly explained, mainly because of the difficulties related to the detection of their production. Nisin, an antimicrobial peptide produced by Lactococcus lactis has a long history of safe use in food products and has been studied from many aspects of genetics, biosynthesis, immunity, regulation, and mode of action. Still, some aspects concerning the dynamics of nisin gene expression remain unknown, especially in complex media like cheese. The main objective of the present study was to quantify in a cheese-like medium the expression of nisin genes in L. lactis M78, a well-characterized nisin A producer isolated from raw milk. The expression of all 11 genes involved in nisin biosynthesis was evaluated during cheese production by real-time reverse transcription-PCR. Total RNA was extracted from cheeses using a direct extraction method without prior separation of microbial cells. The M78 strain grew well in experimental cheeses, producing detectable amounts of nisin after 4 h of fermentation. The presence of nisin as an activator modified both the expression of nisin genes and the accumulation of active nisin. Four groups could be distinguished based on gene expression as a function of time: nisA, nisFEG, nisRK and nisBTCIP. Based on nisin-producing strain growth, nisin activity, function of nisin genes, and their location, correlations were established that contribute to the explanation of regulation of nisin biosynthesis and immunity. This study is the first in which the evolution of bacteriocin gene transcripts has been quantified rigorously in a cheese-like medium.

Chromosomal location of the genetic determinants for bacteriocins produced by Lactobacillus gasseri K7

The production of similar or even identical bacteriocins by different lactic acid bacteria is not a rare event. To take advantage of this finding, genetic determinants of the Lactobacillus K7 bacteriocins were tested for putative homologies with previously described bacteriocins of the Lactobacillus acidophilus group through polymerase chain reaction (PCR). Among specific primer pairs of seven known bacteriocins, derived from their respective sequences, only acidocin LF221 A and B primers amplified fragments in chromosomal DNA of K7 strain that revealed strong similarity over small regions of LF221 bacteriocins. Treatment of Lactobacillus K7 with ethidium bromide and mitomycin C was ineffective in generating non-bacteriocinogenic derivatives and had no impact on plasmid loss either. Classification studies elucidated Lactobacillus K7 as a member of the Lactobacillus gasseri species.

Fate of Listeria monocytogenes on Fully Ripened Greek Graviera Cheese Stored at 4, 12, or 25°C in Air or Vacuum Packages : In Situ PCR Detection of a Cocktail of Bacteriocins Potentially Contributing to Pathogen Inhibition

The behavior of Listeria monocytogenes on fully ripened Greek Graviera cheese was evaluated. Three batches (A, B, and C) were tested. Batches A and C were prepared with a commercial starter culture, while in batch B the starter culture was combined with an enterocin-producing Enterococcus faecium Graviera isolate. Cheese pieces were surface inoculated with a five-strain cocktail of L. monocytogenes at ca. 3 log CFU/cm2, packed under air or vacuum conditions, stored at 4, 12, or 25 degrees C, and analyzed after 0, 3, 7, 15, 30, 60, and 90 days. L. monocytogenes did not grow on the cheese surface, regardless of storage conditions. However, long-term survival of the pathogen was noted in all treatments, being the highest (P < 0.05) at 4 degrees C under vacuum conditions. Overall, the lower the storage temperature, the higher and longer the survival of L. monocytogenes was. Although enterocin A-specific PCR products were detected in situ in cheese batch B, inhibition of L. monocytogenes by the enterocin-producing strain was not enhanced compared with batches A and C, which also contained enterocin A, but in lower amounts. Additionally enterocins B, P, L50A, and L50B; lactococcin G; and plantaricin A genes were detected in all batches, suggesting that indigenous bacteriocin-producing lactic acid bacteria might contribute to Listeria inhibition in cheese. In conclusion, Graviera cheeses that may be accidentally contaminated in retail at the European Union maximal allowable level of 100 CFU/cm2 or g are at low risk regarding a potential outgrowth of L. monocytogenes, which, however, may survive for a long period during cheese storage.

Expression of the sweet-tasting plant protein brazzein in Escherichia coli and Lactococcus lactis: a path toward sweet lactic acid bacteria

Brazzein is an intensely sweet-tasting plant protein with good stability, which makes it an attractive alternative to sucrose. A brazzein gene has been designed, synthesized, and expressed in Escherichia coli at 30 °C to yield brazzein in a soluble form and in considerable quantity. Antibodies have been produced using brazzein fused to His-tag. Brazzein without the tag was sweet and resembled closely the taste of its native counterpart. The brazzein gene was also expressed in Lactococcus lactis, using a nisin-controlled expression system, to produce sweet-tasting lactic acid bacteria. The low level of expression was detected with anti-brazzein antibodies. Secretion of brazzein into the medium has not led to significant yield increase. Surprisingly, optimizing the codon usage for Lactococcus lactis led to a decrease in the yield of brazzein.

Microbial stability and safety of traditional Greek Graviera cheese: characterization of the lactic acid bacterial flora and culture-independent detection of bacteriocin genes in the ripened cheeses and their microbial consortia.

The microflora of four batches of traditional Greek Graviera cheese was studied at 5 weeks of ripening, and 200 lactic acid bacteria (LAB) isolates were phenotypically characterized and screened for antilisterial bacteriocins. The cheeses were also analyzed for organic acids by high-performance liquid chromatography and for the potential presence of 25 known LAB bacteriocin genes directly in cheese and their microbial consortia by PCR. All batches were safe according to the European Union regulatory criteria for Listeria monocytogenes, Salmonella, enterobacteria, and coagulase-positive staphylococci. The cheese flora was dominated by nonstarter Lactobacillus casei/paracasei (67.5%) and Lactobacillus plantarum (16.3%) strains, whereas few Streptococcus thermophilus (3.8%), Lactococcus lactis subsp. lactis (0.6%), and Leuconostoc (1.9%) organisms were present. Enterococcus faecium (9.4%) and Enterococcus durans (0.6%) were isolated among the dominant LAB from two batches; however, enterococci were present in all batches at 10- to 100-fold lower populations than mesophilic lactobacilli. Sixteen E. faecium isolates produced antilisterial enterocins. In accordance, enterocin B gene was detectable in all cheeses and enterocin P gene was present in one cheese, whereas the consortia of all cheeses contained at least two of the enterocin A, B, P, 31, L50A, and L50B genes. Plantaricin A gene was also amplified from all cheeses. Mean concentrations of lactic, acetic, citric, and propionic acids in the ripened cheeses exceeded 1.5% in total, of which approximately 0.9% was lactate. Thus, organic acid contents constitute an important hurdle factor for inhibiting growth of pathogens in traditional Graviera cheese products, with LAB bacteriocins, mainly enterocins, potentially contributing to increased cheese safety.

Characterization and stability of lactobacilli and yeast microbiota in kefir grains

Characterization and stability of lactobacilli and yeasts from kefir grains using culture-dependent and culture-independent methods were investigated in this study. Culture-dependent analysis, followed by sequencing of 16S ribosomal DNA for bacteria and 26S rRNA gene for yeasts, revealed 3 different species of lactobacilli and yeasts, respectively. The most frequently isolated bacterial species were Lactobacillus kefiranofaciens ssp. kefirgranum, Lb. parakefiri, and Lb. kefiri, whereas yeasts belonged to Kluyveromyces marxianus, Kazachstania exigua, and Rhodosporidium kratochvilovae. This study is the first to report on the presence of R. kratochvilovae in kefir grains. On the other hand, PCR-denaturing gradient gel electrophoresis in the culture-independent method showed that the dominant microorganisms were Lb. kefiranofaciens ssp. kefirgranum, Kl. marxianus and Ka. exigua, but did not reveal bands corresponding to Lb. parakefiri, Lb. kefiri, or R. kratochvilovae. Our results support the necessity of combining more techniques for detailed and reliable study of microbial communities in kefir grains. Another interesting finding confirmed that the detected dominant microbiota of kefir grains is very stable and did not change over experimental time. This finding is important to ensure consistent product quality.