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Two-component signal transduction systems as key players in stress responses of lactic acid bacteria.

Lactic acid bacteria (LAB) continue as an important group of gram-positive bacteria that have been extensively exploited in food industries and various biotechnological applications. Some LAB species are, however, opportunistic pathogens and were reported to be associated with overwhelming number of human infections. During the use of LAB in industry or over the course of human infection, these bacteria are exposed to environmental stress. While LAB display adaptive mechanisms to cope with adverse conditions, the regulation of these mechanisms remains to be elucidated. Recent completion of genome sequencing of various LAB strains combined with the development of advanced molecular techniques have enabled the identification of a number of putative two-component signal transduction systems, also known as two-component regulatory systems (2CRS), in LAB. Examining the effect of deleting genes specifying putative 2CRS proteins in these organisms has revealed the involvement of 2CRS in the responses of LAB to different stresses. There are lines of evidence indicating that certain 2CRS may mediate a general stress response in Enterococcus faecalis and Streptococcus pyogenes. This review highlights the influence of 2CRS on the physiology of LAB during optimal growth and survival/growth on exposure to environmental stress.

Lactobacillaceae↗

Aspects of microbiological and chemical quality of turmus, lupin seeds debittered by soaking in water.

Eleven species of spherical lactic acid bacteria (LAB) belonging to the genera Leuconostoc, Lactococcus, Enterococcus and Pediococcus were the predominant microorganisms in 40 samples of turmus, ready-to-eat lupin seeds debittered by boiling and soaking in water. The average counts of the LAB in the 20 winter samples and the 20 summer samples were 7.4 and 8.7 log CFU/g, respectively. The averages of the Enterobacteriaceae counts were 5.1 and 6.6 log CFU/g, respectively, and the 11 species isolated belonged to the genera Enterobacter, Citrobacter, Escherichia and Klebsiella. The average yeast counts in winter and summer samples were 3 and 3.2 log CFU/g, respectively, and the 5 species isolated were in the genera Saccharomyces, Cryptococcus, Rhodotorula and Candida. Although Salmonella was not isolated from any sample and the Staphylococcus aureus count in all samples was < 1 log CFU/g, microbial hazards could be associated with the high Enterobacteriaceae counts and the presence of Escherichia coli. Total alkaloid concentration in 30% of the samples examined was higher than 0.02%, thus making the seeds a potential chemical hazard. Boiling the turmus directly before consumption and discarding the seeds with a bitter taste may help in avoiding some of the microbial and chemical hazards which could be associated with turmus consumption.

Alkaloids↗

Inhibition of Listeria monocytogenes by in situ produced and semipurified bacteriocins of Carnobacterium spp. on vacuum-packed, refrigerated cold-smoked salmon.

Listeria monocytogenes inhibition by Carnobacterium strains and crude bacteriocins on sterile and commercial vacuum-packed cold-smoked salmon stored at 4 degrees C and 8 degrees C was investigated. Carnobacterium piscicola V1 was bactericidal against L. monocytogenes at the two temperatures, whereas Carnobacterium divergens V41 presented a bacteriostatic effect. C. piscicola SF668 delayed L. monocytogenes growth at 8 degrees C and had a bacteriostatic effect at 4 degrees C. Listeria growth was not affected by a non-bacteriocin-producing C. piscicola. Crude extracts of piscicocins were bactericidal at 4 degrees C and 8 degrees C. Listeria growth was delayed by divercin V41 at 8 degrees C and was inhibited at 4 degrees C. Nisin delayed Listeria growth at 8 degrees C and was bacteriostatic at 4 degrees C. The present study demonstrates that L. monocytogenes growth could be prevented on vacuum-packed cold-smoked salmon by Carnobacterium and associated bacteriocins at chilled temperatures. Moreover, no product spoilage could be observed with the use of such bacteriocin-producing strains as demonstrated by good sensorial analyses and low biogenic amine production.

Animals↗

Effects of antioxidants and gamma irradiation on the shelf life of beef patties.

To improve the storage safety of two types of ground beef patty popular in Korea (general beef patties and bulgogi patties), we added various antioxidants (200 ppm; including butylated hydroxyanisole, ascorbyl palmitate, alpha-tocopherol, and beta-carotene) to typical formulations of patties, cooked the patties to 70 degrees C, and irradiated them at a dose of 1.5 or 3 kGy. During 30 d of storage at 5 degrees C, the number of aerobic bacteria and lactic acid bacteria were determined using total aerobic plate count and phenyl ethyl alcohol-sucrose agar, respectively. The concentration of thiobarbituric acid was also determined in each type of patty. No colonies were observed in patties irradiated at 3 kGy regardless of which antioxidant was added. In control patties and patties with butylated hydroxyanisole that were irradiated at a dose of 1.5 kGy, growth of microorganisms appeared to be more rapid than in patties with natural antioxidants. The microbiological safety of nonirradiated patties could not be ensured for a period of 20 d. Lipid oxidation was retarded in both types of patty when an antioxidant was added. Ascorbyl palmitate had the strongest antioxidant effect among the natural antioxidants. However, butylated hydroxyanisole was more effective than ascorbyl palmitate when used in an equal amount.

Animals↗

Description of a simple detection assay for in situ production of bacteriocin on meat.

Using a modification of the agar diffusion assay, in situ bacteriocin production on meat was analyzed using cooked meat medium (CMM) and sterile pork tissue (lean and fat) with Carnobacterium piscicola LV17 as the producer and Carnobacterium divergens LV13 as the indicator strains. Contrary to what is observed in APT broth, bacteriocin production by C. piscicola LV17 occurred with growth at low inoculum levels (< or =10(4) CFU/cm2 or g of meat) on disks (10 cm2) of pork fat tissue (pH 6.58) and on CMM particles (pH 7.0) but not on disks of lean tissue (pH 5.61). The assays described in this study do not required sophisticated equipment and would be useful to study bacteriocin production on meat products stored under various conditions.

Bacterial Typing Techniques↗

Characterization of the lactic acid bacteria in ewe's milk and cheese from northwest Argentina.

Indigenous lactic acid bacteria in ewe's milk and artisanal cheese were studied in four samples of fresh raw milk and four 1-month-old cheeses from the provinces of northwest Argentina. Mean growth counts on M17, MRS, and MSE agar media did not show significant differences (P < 0.05) in raw milk and cheeses. Isolates of lactic acid bacteria from milk were identified as Enterococcus (48%), lactococci (14%), leuconostocs (8%), and lactobacilli (30%). All lactococci were identified as Lactococcus lactis (subsp. lactis and subsp. cremoris). Lactobacilli were identified as Lactobacillus plantarum (92%) and Lactobacillus acidophilus (8%). Enterococci (59%) and lactobacilli (41%) were isolated from cheeses. L. plantarum (93%), L. acidophilus (5%), and Lactobacillus casei (2%) were most frequently isolated. L. lactis subsp. lactis biovar diacetylactis strains were considered as fast acid producers. L. lactis subsp. cremoris strains were slow acid producers. L. plantarum and L. casei strains identified from the cheeses showed slow acid production. The majority of the lactobacilli and Lactococcus lactis strains utilized citrate and produced diacetyl and acetoin in milk. Enzyme activities (API-ZYM tests) of lactococci were low, but activities of L. plantarum strains were considerably higher. The predominance of L. plantarum in artisanal cheese is probably important in the ripening of these cheeses due to their physiological and biochemical characteristics.

Animals↗

Growth of Carnobacterium divergens V41 and production of biogenic amines and divercin V41 in sterile cold-smoked salmon extract at varying temperatures, NaCl levels, and glucose concentrations.

A complete factorial design 2(3) was used to study some aspects of Carnobacterium divergens V41 metabolism (growth, biogenic amine production, and divercin V41 production) in sterile cold-smoked salmon extract (SSE) at varying temperatures (3 to 9 degrees C), NaCl levels (2.5 to 6.5%), and glucose concentrations (2 to 6 g liter(-1)). The results showed that temperature and NaCl content were the most influential factors on growth parameters in SSE. Predictive models are suggested for the assessment of C. divergens lag time (t(lag)) and maximum specific growth rate (micro(max)) Among the biogenic amines studied, only tyramine was found to be produced by C. divergens in SSE. Furthermore, we showed that temperature, NaCl, and glucose variations did not greatly affect tyramine and divercin V41 production by the bacteria under the experimental conditions used. Indeed, divercin V41, a bacteriocin from C. divergens V41 that is highly active against some Listeria strains, was produced in SSE even under harsh culture conditions. Similarly, tyramine production in SSE was delayed at 3 degrees C but reached 35 microg ml(-1) in all experiments after 27 days of storage. However, this final tyramine concentration in SSE is low compared with the threshold values of 100 to 800 microg g(-1) reported as the potentially toxic dose in foods. Thus, we have found that C. divergens V41 is a promising strain for the biopreservation of refrigerated cold-smoked salmon.

Animals↗

Control of Brochothrix thermosphacta spoilage of pork adipose tissue using bacteriophages.

Adipose tissue discs were coinoculated with Brochothrix thermosphacta and homologous bacteriophages (phages) to determine the effects these had on phage multiplication, bacterial growth, and off-odor development during storage at 2 degrees C or under simulated retail display at 6 degrees C. In the presence of about 10(5) bacteria/cm2 and an equivalent number of phages, there was a 3-log increase in phage numbers and a 2-log decrease in bacterial numbers, and objectionable off-odors were suppressed during refrigerated storage. Up to 68% of the surviving bacterial population were resistant to phages. The storage life of adipose tissue could be increased from 4 days in controls to 8 days in phage-treated samples by preventing the development of off-odors associated with the growth of B. thermosphacta. Phages may provide a novel approach to extending the storage quality of chilled meats.

Adipose Tissue↗

Inhibition of Listeria monocytogenes in cold-smoked salmon by Carnobacterium piscicola CS526 isolated from frozen surimi.

Strain CS526 was isolated from frozen surimi and identified as a bacteriocin producer that had strong inhibitory activity against Listeria monocytogenes. Strain CS526 was identified as Carnobacterium piscicola by partial 16S rDNA sequence similarity. The ability of this bacteriocinogenic strain and nonbacteriocinogenic C. piscicola JCM5348 to inhibit the growth of L. monocytogenes was examined in culture broth incubated at 12 degrees C and cold-smoked salmon stored at 4, 12, and 20 degrees C. L. monocytogenes viable counts in the culture broth rapidly declined from 10(6) colony-forming units per ml to less than 10 colony-forming units per ml within 1 day at 12 degrees C in the presence of C. piscicola CS526. At 4 and 12 degrees C, inhibition of L. monocytogenes on salmon depended on the initial inoculum level of C. piscicola CS526. However, C. piscicola CS526 was bactericidal to L. monocytogenes within 21 and 12 days at 4 and 12 degrees C in cold-smoked salmon, respectively, even when the initial inoculum levels were low. C. piscicola CS526 suppressed the maximum cell number of L. monocytogenes by two and three log cycles, even at 20 degrees C. However, C. piscicola JCM5348 did not prevent the growth of the pathogen, except at 4 degrees C. Bacteriocin was detected in the samples coinoculated with C. piscicola CS526. The study shows that C. piscicola CS526 might have potential for biopreservation of refrigerated foods against L. monocytogenes.

Animals↗

Antilisterial activity of a Carnobacterium piscicola isolated from Brazilian smoked fish (surubim [Pseudoplatystoma sp.]) and its activity against a persistent strain of Listeria monocytogenes isolated from surubim.

Data on the prevalence and growth of Listeria monocytogenes in lightly preserved fish products from subtropical and tropical regions are very scarce. Our research describes L. monocytogenes that was detected in 5% of the packages of cold-smoked surubim, a native Brazilian freshwater fish that we analyzed, and shows that the strains isolated were of the same random amplified polymorphic DNA subtype as the strains that were isolated from the same factory 4 years earlier. A bacteriocinogenic strain of Carnobacterium piscicola (strain C2), isolated from vacuum-packed cold-smoked surubim, and two C. piscicola strains, isolated from vacuum-packed, cold-smoked salmon, were capable of limiting or completely inhibiting the growth of an L. monocytogenes (strain V2) isolated from surubim in fish peptone model systems incubated at 10 degrees C. Monocultures of L. monocytogenes reached 108 CFU/ml (g), whereas the growth of L. monocytogenes was completely inhibited by C. piscicola C2. The bacteriocinogenic C. piscicola A9b+ and its nonbacteriocinogenic mutant A9b- reduced maximum Listeria levels by 2 to 3 log units. Both bacteriocinogenic C. piscicola strains prevented listerial growth in cold-smoked fish juices (surubim and salmon). Although the carnobacteria grew poorly on cold-smoked surubim at 10 degrees C, the strains were able to reduce maximum Listeria counts by 1 to 3 log units in an artificially inoculated product (surubim). We conclude that Brazilian smoked fish products harbor L. monocytogenes and should be stabilized against the growth of the organism. C. piscicola C2 has the potential for use as a bioprotective culture in surubim and other lightly preserved fish, but further studies are required to optimize its effect.

Animals↗

Formation of biogenic amines throughout the industrial manufacture of red wine.

Changes in biogenic amines (histamine, methylamine, ethylamine, tyramine, phenylethylamine, putrescine, and cadaverine) were monitored during the industrial manufacture of 55 batches of red wine. The origin of these amines in relation to must, alcoholic fermentation, malolactic fermentation, sulfur dioxide addition, and wine aging and the interactions between amines and their corresponding amino acids and pH were statistically evaluated in samples from the same batches throughout the elaboration process. Some amines can be produced in the grape or the musts (e.g., putrescine, cadaverine, and phenylethylamine) or can be formed by yeast during alcoholic fermentation (e.g., ethylamine and phenylethylamine), although quantitatively only very low concentrations are reached in these stages (less than 3 mg/liter). Malolactic fermentation was the main mechanism of biogenic amine formation, especially of histamine, tyramine, and putrescine. During this stage, the increase in these amines was accompanied by a significant decline in their amino acid precursors. Significant correlations between biogenic amine formation and the disappearance of their corresponding amino acids were observed, which clearly supports the hypothesis that malolactic bacteria are responsible for accumulation of these amines in wines. No increase in the concentration of biogenic amines was observed after SO2 addition and during wine aging, indicating that sulfur dioxide prevents amine formation in subsequent stages.

Biogenic Amines↗

[Search of optimum survival conditions for lactic acid bacteria in powders obtained by spray drying of yoghurt].

The paper reports the results of two spray-drying tests carried out on yogurt as such or previously neutralized. The operating conditions of the drying operation were such that the temperature of the product didn't exceed 80 degrees C, and the residual moisture of the powder was about 10%, slightly higher than the B.E.T. monolayer value. The decrease of total counts was three and two decimal reductions respectively for the natural and neutralized product. This latter gave also much higher viability index as determined in a "fermenting activity" test. Also, the analyses, related to the quality of the protein fraction, strongly support the opportunity of neutralization before drying. In storage tests carried out during 8 months the survival data were higher at 5% relative humidity (corresponding to 7-8% humidity) and at 5 degrees C temperature. In all cases the better results were obtained from the powders of neutralized yogurt, even in comparison with a freeze-dried sample obtained from the natural product. The typical shape of the sorption isotherms of these products suggests that the optimum process as regard to microorganisms' survival consists in drying the neutralized product up to residual humidities of 8-10%, followed by storage, in humidity-proof containers, at temperatures of about 5 degrees C.

Dairy Products↗

From DNA sequence to application: possibilities and complications.

The development of sophisticated genetic tools during the past 15 years have facilitated a tremendous increase of fundamental and application-oriented knowledge of lactic acid bacteria (LAB) and their bacteriophages. This knowledge relates both to the assignments of open reading frames (ORF's) and the function of non-coding DNA sequences. Comparison of the complete nucleotide sequences of several LAB bacteriophages has revealed that their chromosomes have a fixed, modular structure, each module having a set of genes involved in a specific phase of the bacteriophage life cycle. LAB bacteriophage genes and DNA sequences have been used for the construction of temperature-inducible gene expression systems, gene-integration systems, and bacteriophage defence systems. The function of several LAB open reading frames and transcriptional units have been identified and characterized in detail. Many of these could find practical applications, such as induced lysis of LAB to enhance cheese ripening and re-routing of carbon fluxes for the production of a specific amino acid enantiomer. More knowledge has also become available concerning the function and structure of non-coding DNA positioned at or in the vicinity of promoters. In several cases the mRNA produced from this DNA contains a transcriptional terminator-antiterminator pair, in which the antiterminator can be stabilized either by uncharged tRNA or by interaction with a regulatory protein, thus preventing formation of the terminator so that mRNA elongation can proceed. Evidence has accumulated showing that also in LAB carbon catabolite repression in LAB is mediated by specific DNA elements in the vicinity of promoters governing the transcription of catabolic operons. Although some biological barriers have yet to be solved, the vast body of scientific information presently available allows the construction of tailor-made genetically modified LAB. Today, it appears that societal constraints rather than biological hurdles impede the use of genetically modified LAB.

Bacteriophages↗

Lactic acid bacteria in the quality improvement and depreciation of wine.

The winemaking process includes two main steps: lactic acid bacteria are responsible for the malolactic fermentation which follows the alcoholic fermentation by yeasts. Both types of microorganisms are present on grapes and on cellar equipment. Yeasts are better adapted to growth in grape must than lactic acid bacteria, so the alcoholic fermentation starts quickly. In must, up to ten lactic acid bacteria species can be identified. They belong to the Lactobacillus, Pediococcus, Leuconostoc and Oenococcus genera. Throughout alcoholic fermentation, a natural selection occurs and finally the dominant species is O. oeni, due to interactions between yeasts and bacteria and between bacteria themselves. After bacterial growth, when the population is over 10(6) CFU/ml, malolactic transformation is the obvious change in wine composition. However, many other substrates can be metabolized. Some like remaining sugars and citric acid are always assimilated by lactic acid bacteria, thus providing them with energy and carbon. Other substrates such as some amino acids may be used following pathways restricted to strains carrying the adequate enzymes. Some strains can also produce exopolysaccharides. All these transformations greatly influence the sensory and hygienic quality of wine. Malic acid transformation is encouraged because it induces deacidification. Diacetyl produced from citric acid is also helpful to some extent. Sensory analyses show that many other reactions change the aromas and make malolactic fermentation beneficial, but they are as yet unknown. On the contrary, an excess of acetic acid, the synthesis of glucane, biogenic amines and precursors of ethylcarbamate are undesirable. Fortunately, lactic acid bacteria normally multiply in dry wines; moreover some of these activities are not widespread. Moreover, the most striking trait of wine lactic acid bacteria is their capacity to adapt to a hostile environment. The mechanisms for this are not yet completely elucidated. Molecular biology has provided some explanations for the behaviour and the metabolism of bacteria in wine. New tools are now available to detect the presence of desirable and undesirable strains. Even if much remains unknown, winemakers and oenologists can nowadays better control the process. By acting upon the diverse microflora and grape musts, they are more able to produce healthy and pleasant wines.

Carbohydrate Sequence↗

[The bacteriocinogenic and lysozyme-synthesizing activity of lactobacilli].

It was shown that lactic acid bacteria isolated from different sources produced along with lactic acid the bacteriocine substances. Strains producing bacteriocines were mainly isolated from digestive tract of people and calves. Bacteriogenic properties have been shown more frequently in the case of lactobacilli (7.8%) more rarely--in coccoid forms of lactic acid bacteria (4.5%). At the same time bacteriocine activity was higher in the case of Enterococcus and depended both on the strain type and cultivation conditions. Synthesis of bacteriocines started from the hours of phase of the culture growth and reached maximum at the beginning of stationary phase. The bacteriocine accumulation rate depended on composition of the growth medium.

Animals↗

The relationship between nitro group reduction and the intestinal microflora.

The capacity of rats to reduce a 25-mg dose of p-nitrobenzoic acid (PNBA) was measured by quantifying the amount of this compound recovered in the urine as p-aminobenzoic acid (PABA) and its conjugates. It was found that germfree rats converted approximately 1% of PNBA to PABA; in conventional rats the conversion was approximately 25%. Various bacteria isolated from the rat cecum were selectively associated with germfree rats and it was demonstrated that these bacteria colonized their gastrointestinal tracts. In assoication with Lactobacillus plantarum, the conversion of PNBA to PABA increased to 3.9%. When these rats were further associated with Clostridium sp. and Streptoccocus faecalis, the conversion increased to approximately 12%. A general correlation was found between the capacity of constituents of the microflora to reduce PNBA in vitro and when associated with the germfree rat. Cecectomy, which removes a substantial portion of the microflora of the rat, decreases the capacity of the conventional rat to reduce PNBA. Similar experiments with p-nitrobenzenesulfonamide indicate that this compound is also largely reduced by the flora. Evidence that the reduction of the nitro group in these compounds is carried out by the intestinal microflora explains previous observations in which the reduction of these compounds in rats did not correlate with the activity of liver enzymes putative for these reactions.

Aminobenzoates↗

[Study of the intestinal microflora in residents of the city of Vladikavkaz].

Microbiological study revealed high occurrence of dysbacteriosis with the decreased amount of Escherichia coli (80.9%) and the presence of E. coli hemolytic forms (76.6%). Rather often the elevated content of E. coli with decreased fermentative properties was noted (51.1%). In all cases E. coli with decreased biochemical activity had hemolytic activity, which was accompanied by the deficiency of bifidobacteria (79.2% of cases) and the combined deficiency of lactic acid bacteria and bifidobacteria (75% of cases).

Bifidobacterium↗