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Production of biogenic amines by lactic acid bacteria: screening by PCR, thin-layer chromatography, and high-performance liquid chromatography of strains isolated from wine and must.

Biogenic amines are frequently found in wine and other fermented food. We investigated the ability of 133 strains of lactic acid bacteria isolated from musts and wines of different origins to produce histamine, tyramine, and putrescine. We detected the genes responsible for encoding the corresponding amino acid decarboxylases through PCR assays using two primer sets for every gene: histidine decarboxylase (hdc), tyrosine decarboxylase (tdc), and ornithine decarboxylase (odc); these primers were taken from the literature or designed by us. Only one strain of Lactobacillus hilgardii was shown to possess the hdc gene, whereas four strains of Lactobacillus brevis had the tdc gene. None of the Oenococcus oeni strains, the main agents of malolactic fermentation, was a biogenic amine producer. All PCR amplicon band-positive results were confirmed by thin-layer chromatography and high-performance liquid chromatography analyses.

Base Sequence↗

Screening of lactic acid bacteria for bile salt hydrolase activity.

Bile salt hydrolysis is an important metabolic reaction in the bile salt metabolism of mammals. This reaction has a facilitating effect for bile salt excretion but can also be involved in various illnesses. In recent years interest has increased to use bile salt hydrolysis to influence the cholesterol metabolism of humans and farm animals. To understand the distribution and range of bile salt hydrolase activity in lactic acid bacteria, we screened more than 300 strains of the genera Bifidobacterium and Lactobacillus and the species Lactococcus lactis, Leuconostoc mesenteroides, and Streptococcus thermophilus. Results obtained for 273 strains showed that bile salt hydrolase activity is common in Bifidobacterium and Lactobacillus but absent in L. lactis, Leu. mesenteroides, and S. thermophilus. Nearly all bifidobacteria species and strains have bile salt hydrolase activity, whereas this activity can only be found in selected species of lactobacilli. A strong correlation can be observed between the habitat of a genus or species and the presence of bile salt hydrolase activity. Most often bile salt hydrolase activity is found in strains that have been isolated from the intestines or from feces from mammals--an environment rich in conjugated and unconjugated bile acids. Strains and species from other habitats like milk or vegetables--environments from which bile salts are absent--do normally not have bile salt hydrolase activity. In two independent assays, we established that bile salt hydrolase activity in bifidobacteria is, in general, much higher than in lactobacilli.

Amidohydrolases↗

Screening of lactic acid bacteria from fermented vegetables by carbohydrate profiling and PCR-ELISA.

AIMS: The aim of this study was to identify potential souring agents, isolated from fermented plant material, by API 50 CHL assay and a molecular method based on polymerase chain reaction and colorimetric hybridization (PCR-ELISA). METHODS AND RESULTS: Forty-two strains of lactic acid bacteria derived from plant material were screened by taking advantage of API 50 CHL and PCR-ELISA. Oligonucleotide probes used for hybridization in PCR-ELISA were specific for lactobacilli, the Leuconostoc family, Lactobacillus pentosus/plantarum and Lactobacillus brevis. The hybrides were detected by a colour-developing reaction. Bacteria isolated from fermented cucumbers were identified as Lact. plantarum-related (Lact. plantarum and Lact. pentosus) and Leuconostoc species. Most of the strains isolated from sauerkraut were identified as Lact. pentosus/plantarum. CONCLUSIONS: Complementary results were obtained in the identification of bacterial strains, isolated from fermented cucumbers and sauerkraut, by API 50 CHL and PCR-ELISA. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR-ELISA proved to be suitable for the screening of large numbers of bacterial isolates from fermented vegetables. This will be useful for the identification of strains suitable for the design of starter cultures for the fermentation of plant material.

Carbohydrates↗

Production of antimicrobial substances by lactic acid bacteria II: screening bacteriocin-producing strains with probiotic purposes and characterization of a Lactobacillus bacteriocin.

Bacteriocins have been defined as proteinaceous, bactericidal substances synthesized by bacteria, which usually have a narrow spectrum of activity, only inhibiting strains of the same or closely related species. The term bacteriocin-like substance is applied to antagonistic substances that are not completely defined or do not fit the typical criteria of bacteriocins. They have been reported to inhibit a wide range of both Gram-positive and Gram-negative bacteria as well as fungi. Lactobacillus species are the dominant microorganisms isolated from the vagina of healthy premenopausal women. In this environment, they exert a protective effect against pathogenic microorganisms by different mechanisms such as production of antimicrobial agents, which include organic acids, hydrogen peroxide, and probably bacteriocins. The production of bacteriocins by vaginal lactobacilli has been demonstrated in vitro; however, it is not yet well established whether they are produced in vivo as another antagonistic mechanism exerted by the normal microflora.Bacteriocin-producing bacteria as well as bacteriocins per se are of growing interest as biological controls in the manufacture of beverages and fermented products, mainly in the area of dairy products. These bacteria have also been proposed as probiotic candidates for human or animal use. The objectives of the present chapter are to describe the methods employed for: 1. Detection of production of bacteriocins among vaginal Lactobacillus strains. 2. Characterization of the bacteriocin or bacteriocin-like substances. 3. Study of the kinetics of production and mode of action of bacteriocins. 4. Determination of the inhibition of pathogenic microorganisms by bacteriocin-producing strains in mixed cultures.

Anti-Infective Agents↗

Simple method for extracting plasmid DNA from lactic acid bacteria.

Rapid screening and large-scale plasmid DNA isolation procedures are described for lactic acid bacteria, using glass beads to break cells. The rapid screening procedure allows one to obtain plasmid DNA pellets in less than 1 h. This method has been successfully tested on various bacteria from the genera Lactococcus, Leuconostoc, Lactobacillus, Pediococcus, Streptococcus, Enterococcus and Propionibacterium. This procedure yields plasmid DNA with minor chromosomal and plasmid DNA-degraded form contaminations.

Bacteriological Techniques↗

Influence of pH, salt and nitrite on the heme-dependent catalase activity of lactic acid bacteria.

A screening of commercial starter cultures used for the production of dry sausage showed a maximum heme-dependent catalase activity in the range of 60 mumol/l hematin for Lactobacillus sake, Lactobacillus plantarum, Lactobacillus pentosus and Pediococcus acidilactici. Pseudocatalase activity was not detected. In standard dry sausage production, 2-3% (w/w) nitrite salt (0.6% sodium nitrite per 100 g NaCl) is normally added, which corresponds to 4-6% salt in the water phase. In vitro experiments with L. sake and L. plantarum have shown that such a high concentration of salt caused a significant reduction of catalase activity and bacterial growth. In the case of P. acidilactici, the catalase activity remained constant at a salt concentration up to 6% (w/w); at 7% (w/w) the activity decreased sharply. The pH also affected the catalase activity, which remained constant up to pH 5.1 and decreased dramatically at lower values. The effect of nitrite has also been investigated. L. pentosus and P. acidilactici were not affected by the addition of 160 ppm nitrite (NO2-); L. plantarum, on the other hand, showed a significantly reduced catalase activity. In practice, optimum fermentation characteristics combined with an optimum catalase activity which are not inhibited by salt concentrations higher than 6% (w/v) and a residual nitrite content of about 160 ppm (w/v), are of the utmost importance in screening and selection of lactic acid bacteria for starter cultures.

Catalase↗

Improved screening procedure for biogenic amine production by lactic acid bacteria.

An improved screening plate method for the detection of amino acid decarboxylase-positive microorganisms (especially lactic acid bacteria) was developed. The suitability and detection level of the designed medium were quantitatively evaluated by confirmation of amine-forming capacity using an HPLC procedure. The potential to produce the biogenic amines (BA) tyramine, histamine, putrescine, and cadaverine, was investigated in a wide number of lactic acid bacteria (LAB) of different origin, including starter cultures, protective cultures, type strains and strains isolated from different food products. Also, several strains of Enterobacteriaceae were examined. Modifications to previously described methods included lowering glucose and sodium chloride concentrations, and increasing the buffer effect with calcium carbonate and potassium phosphate. In addition, pyridoxal-5-phosphate was included as a codecarboxylase factor for its enhancing effect on the amino acid decarboxylase activity. The screening plate method showed a good correlation with the chemical analysis and due to its simplicity it is presented as a suitable and sensitive method to investigate the capacity of biogenic amine production by LAB. Tyramine was the main amine formed by the LAB strains investigated. Enterococci, carnobacteria and some strains of lactobacilli, particularly of Lb. curvatus. Lb. brevis and Lb. buchneri, were the most intensive tyramine formers. Several strains of lactobacilli, Leuconostoc spp., Weissella spp. and pediococci did not show any potential to produce amines. Enterobacteriaceae were associated with cadaverine and putrescine formation. No significant histamine production could be detected for any of the strains tested.

Aromatic-L-Amino-Acid Decarboxylases↗

[A simple method for screening of highly inhibitory Lactic acid bacteria on Aspergillus flavus growth].

A simple and rapid method for screening of Lactic acid bacteria with high inhibitory power is developed. The methodology employed was the agar-diffusion assay, which was standardized for medium composition, and incubation conditions. The assay was composed of two layers of medium, MRS agar at the bottom of plate, PDA at the top of MRS agar. Lactic acid bacteria culture was inoculated to the media between MRS and PDA, then a known number of mold spores was surface plated on PDA plates, and incubated at 25 degrees C for 72 h.

Aspergillus flavus↗

Toxicity evaluation for an Enterococcus faecium strain TM39 in vitro and in vivo.

Previously, we have screened lactic acid bacteria (LAB) strains from infant feces and evaluated their functional properties. We found a strain of Enterococcus faecium termed as TM39 which is acid and bile tolerant, able to adhere to the intestinal epithelium and with antagonistic activity against Helicobacter pylori. In this study, we demonstrate that strain TM39 is not vancomycin resistant, not invasive to human gastric carcinoma cell line TSGH 9201 and human intestinal epithelial cell line Int-407 in vitro. In addition, we have conducted the in vivo study to evaluate the toxicity of this E. faecium strain TM39 in Wistar rats. For such study, cells of strain TM39 were daily oral administrated with dose of 1 x 10(12), 5 x 10(11) and 2 x 10(10) CFU/kg of body weight, respectively, to the rats for 28 consecutive days. There were no adverse effects on the general condition, behavior, growth, feed and water consumption, hematology, clinical chemistry values, organ weights and histopathologic analysis of the rats. Results of this study demonstrate that consumption of strain E. faecium TM39, even in large quantities, is not associated with any obvious signs of toxicity in Wistar rats.

Animals↗

Characterization of new bacteriocinogenic lactic acid bacteria isolated using a medium designed to simulate inhibition of Listeria by Lactobacillus sakei 2512 on meat.

Bacteriocinogenic bacteria have been proposed to protect food products from Listeria contamination as bioprotective cultures. Lactobacillus sakei 2512 was demonstrated to inhibit the growth of Listeria on sliced cooked ham by challenge test. A liquid medium simulating ham, BHI5L200, was designed in order to select bioprotective strains for meat protection. Two strains were selected, from the 201 lactic acid bacteria screened, that produced bacteriocins at pH 5.8 in BHI5L200. The first one, Leuconostoc pseudomesenteroides 2733, produced a new bacteriocin which was purified and partially characterized. The second, Lactobacillus curvatus 2711, produced sakacin X and was shown to contain sakacin T and sakacin P structural genes. Co-culture experiments in BHI5L200 demonstrated that growth of Listeria was inhibited by L. sakei 2512 as well as by L. curvatus 2711.

Animals↗

Antilisterial activity of lactic acid bacteria isolated from rigouta, a traditional Tunisian cheese.

AIMS: Screening for lactic acid bacteria (LAB) producing bacteriocins and other antimicrobial compounds is of a great significance for the dairy industry to improve food safety. METHODS AND RESULTS: Six-hundred strains of LAB isolated from 'rigouta', a Tunisian fermented cheese, were tested for antilisterial activity. Eight bacteriocinogenic strains were selected and analysed. Seven of these strains were identified as Lactococcus lactis and produced nisin Z as demonstrated by mass spectrometry analysis of the purified antibacterial compound. Polymerase chain reaction experiments using nisin gene-specific primers confirmed the presence of nisin operon. Plasmid profiles analysis suggests the presence of, at least, three different strains in this group. MMT05, the eighth strain of this antilisterial collection was identified, at molecular level, as Enterococcus faecalis. The purified bacteriocin produced by this strain showed a molecular mass of 10 201.33 +/- 0.85 Da. This new member of class III bacteriocins was termed enterocin MMT05. CONCLUSIONS: Seven lactococcal strains producing nisin Z were selected and could be useful as bio-preservative starter cultures. Additional experiments are needed to evaluate the promising strain MMT05 as bio-preservative as Enterococci could exert detrimental or beneficial role in foods. SIGNIFICANCE AND IMPACT OF THE STUDY: Only a few antibacterial strains isolated from traditional African dairy products were described. The new eight strains described herein contribute to the knowledge of this poorly studied environment and constitute promising strains for fermented food safety.

Bacteriocin Plasmids↗

Microbial ecology of fresh pork stored under modified atmosphere at -1, 4.4 and 10 degrees C.

The prevalent bacteria on fresh pork packaged in modified atmosphere with elevated CO2 were determined by selection of representative colonies from the greatest dilution of meat samples. The pork samples were stored in two packaging films of different oxygen permeability at three storage temperatures. Strains were classified and those identified as lactic acid bacteria were screened for production of inhibitory substances. The types of bacteria isolated from samples stored in the two packaging films were similar. Storage temperature influenced the type of bacteria that dominated the microbial population. At 10 degrees C the prevalent microflora consisted of aeromonads, Enterobacteriaceae and lactic acid bacteria but at 4.4 and -1 degrees C, aeromonads, Brochothrix thermosphacta and lactic acid bacteria dominated. Listeriae were detected as part of the prevalent microflora on samples stored at -1 degree C, but not on samples stored at 4.4 or 10 degrees C. Species of lactic acid bacteria dominating the microflora were influenced by growth medium. The majority of isolates taken from Plate Count agar were carnobacteria whereas those from Lactobacilli MRS agar were homofermentative lactic acid bacteria. Of the 538 lactic acid bacteria isolates screened for production of inhibitory substances, 162 strains showed deferred inhibition toward a range of lactic acid bacteria and nonlactic acid bacteria indicator strains.

Animals↗

Screening of lactic-acid bacteria from South African barley beer for the production of bacteriocin-like compounds.

Strains of Lactobacillus paracasei subsp. paracasei (strain ST11BR), L. pentosus (strain ST151BR), L. plantarum (strain ST13BR), and Lactococcus lactis subsp. lactis (strain ST34BR) producing bacteriocin-like peptides were isolated from barley beer produced in the Western, Northern and Eastern provinces of South Africa. The peptides (bacST11BR, bacST151BR, bacST13BR and bacST34BR) lost their activity after treatment with proteinase K, a proteinase, papain, chymotrypsin, trypsin, pepsin and pronase, but not when they were treated with alpha-amylase, suggesting that the peptides are not glycosylated. The peptides inhibited the growth of Lactobacillus casei, L. sakei, Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecalis, but not Enterobacter cloacae, Lactobacillus bulgaricus subsp. delbrueckii, L. plantarum, L. salivarius, Listeria innocua, Staphylococcus aureus, Streptococcus uberis, S. agalactiae, S. caprinus and S. pneumoniae. Peptides bacST11BR and bacST13BR differed from the other 2 peptides by failing to kill Klebsiella pneumoniae and one of the E. coli strains. Peptides were stable after 2 h of incubation at pH 2.0-12.0, and after 90 min at 100 degrees C. When autoclaved (121 degrees C, 20 min), only bacST13BR lost its activity. The bacteriocin-like peptides were produced at a growth temperature of 30 degrees C, but not at 37 degrees C.

Bacteriocins↗

Acid and bile tolerance of spore-forming lactic acid bacteria.

Criteria for screening probiotics such as bile tolerance and resistance to acids were studied with 13 spore-forming lactic acid producing bacteria. Different strains of Sporolactobacillus, Bacillus laevolacticus, Bacillus racemilacticus and Bacillus coagulans grown in MRS broth were subjected to low pH conditions (2, 2.5 and 3) and increasing bile concentrations. Among these microorganisms, Bacillus laevolacticus DSM 6475 and all Sporolactobacillus strains tested except Sporolactobacillus racemicus IAM 12395, were resistant to pH 3. Only Bacillus racemilacticus and Bacillus coagulans strains were tolerant to bile concentrations over 0.3% (w/v).

Bacillus↗

Screening of bile resistance and bile precipitation in lactic acid bacteria and bifidobacteria.

A modification of the ecometric method was developed for a rapid screening of bile resistance in lactic acid bacteria and bifidobacteria. Validation of the MEM bile assay (modified ecometric method) was performed comparing the bile resistance index (RIbile) and the bile resistance ratio (R%). Most Bifidobacterium strains assayed were bile sensitive (83.3%), while only 62.1% of lactobacilli showed that behavior. Some bifidobacterial strains (55.6%) showed a crystalline precipitate when grown on solid medium supplemented with 0.5% ox bile. The crystalline structures produced by B. pseudolongum CIDCA 531 were isolated and analyzed by optical and scanning electron microscopy, thin-layer chromatography, melting point, and specific cholesterol reactions. Those studies confirmed the presence of cholesterol in these crystalline structures. On the other hand, none of the lactobacilli and streptococci studied had the ability to produce crystalline precipitates.

Bifidobacterium↗

NAD-specific 6-phosphogluconate dehydrogenase in lactic acid bacteria.

6-Phosphogluconate dehydrogenase was screened in for cell-free extracts from seventeen strains of lactic acid bacteria. Three types of the enzyme could be classified according to the coenzyme specificity: NAD-specific, NADP-specific, and non-specific types. The two strains that had a heterofermentative pathways had an NAD-specific 6-phosphogluconate dehydrogenase. This type had higher enzyme activity per proteins in the cell-free extracts, and more specificity to NAD compared to the other types of the enzyme in the lactic acid bacteria studied.

Cell-Free System↗

Resistance screening essay of wine lactic acid bacteria on lysozyme: efficacy of lysozyme in unclarified grape musts.

In wine making, the bacteriolytic activity of lysozyme has primarily been used to control the malolactic fermentation in wines. The use of lysozyme in musts before settling and the beginning of the alcoholic fermentation to inhibit the growth of lactic acid bacteria could be very beneficial. In a resistance test carried out in MT/b broth, lysozyme had greater antimicrobial activity toward Oenococcus oeni than Lactobacillus species. Several strains of wine bacteria belonging to Oenococcus proved sensitive to the bacteriolytic activity of lysozyme at low concentrations in both synthetic medium (MT/b) (50 mg/L), white must, or red must made with or without the skins (100 mg/L). Lactobacillus and Pediococcus strains survived at lysozyme concentrations of 200-500 and 500 mg/L, respectively, in MT/b and musts. Suspended solids in unclarified musts may strongly bind to lysozyme thereby causing its removal by filtration or centrifugation. One hour after lysozyme was added to musts, it was quantified by HPLC and found after centrifugation to be 40-50% and only 10% in musts made with or without the skins, respectively. Although appreciable amounts of lysozyme were bound to wine components, this did not appear to be a serious hindrance to lysozyme activity.

Enzyme Stability↗

Selection of Escherichia coli-inhibiting strains of Lactobacillus paracasei subsp. paracasei.

The aim of this study was to select Escherichia coli-inhibiting strains among lactic acid bacteria. On the basis of phenotypical and technological characteristics, 20 strains of lactic acid bacteria were screened from a total of 225 isolates, obtained from nine samples of artisanal Caprino d'Aspromonte cheese, made from raw goats' milk. The antagonistic activity of these 20 strains was detected in plates against three different strains of E. coli. Two strains of Lactobacillus paracasei subsp. paracasei showed a marked anti-E. coli activity against all three strains tested; the other lactic acid bacteria did not exhibit inhibiting activity. The E. coli inhibition can be ascribed to production of bacteriocin-like compounds. The use of L. paracasei subsp. paracasei strains to increase the safety of the cheeses made from raw milk is recommended because these cultures strongly inhibit E. coli, without foreseeable adverse sensory changes.

Animals↗