Detection Of Lactic Acid Bacteria Research Articles

Detection of Lactic Acid Bacteria in Metasandstone and Limestone Caves of Chapada Diamantina, Brazil

Detection of Lactic Acid Bacteria in Metasandstone and Limestone Caves of Chapada Diamantina, Brazil

Detection of Lactic Acid Bacteria (LAB) In Soaking Water of Arabica Coffee Cherries on Mount Sindoro

Coffee is one of the major commodities in Indonesia's agricultural sector, where Indonesia is the fourth largest coffee producer in the world. Arabica coffee is the only type of coffee produced on Mount Sindoro. The used water is wasted in the sewers when washing and sorting coffee. Even though the skin covering the cherry flesh is likely to contain natural microorganism life since the coffee cherries grow during ripening on the tree. Therefore, this study aims to determine whether there is the presence or absence of Lactic Acid Bacteria (LAB) groups in the water used to soak the Arabica coffee cherries. The research method was carried out with 200 ml of water samples soaked in Arabica coffee cherries for 4 hours, isolating microorganisms from the 10-1 to 10-3 dilution series of soaking water using the spread plate method and reinoculation to obtain pure LAB cultures, then detected macroscopically through observation of colony morphology and microscopically detected, including physiological and biochemical reaction tests. Data analysis was conducted in a fully descriptive-qualitative manner concerning Bergey's Manual of Determinative Bacteriology and other books supporting the detection of the BAL groups obtained. The study results obtained three isolates, but only one isolate was detected as LAB. The isolate belongs to the Streptococcaceae family's LAB group- the Lactococcus genus.

Isolation and some basic characteristics of lactic acid bacteria from beetroot (Beta vulgaris L.) —A preliminary study

The carbohydrate content of fruit and vegetable juices has made them a suitable environment for the growth of lactic acid bacteria. Various studies have shown that beetroot can be a good substrate for the production of probiotic drinks for consumers. Therefore, this study was performed to isolate and identify lactic acid bacteria from beetroot juice. Beetroot juice was prepared from two kilograms of washed beetroot with a cold press. Sterile samples of beetroot juice were first enriched in MRS broth and then cultured on MRS agar. Gram and catalase tests were used for the initial detection of lactic acid bacteria. Then, to identify the species of lactic acid bacteria, biochemical confirmation tests such as the melting of gelatin, and the growth of bacteria has been studied in the ideal condition that is 15-45° C, sugars fermentation, gas production from glucose, and growth in at different concentrations of salt were performed. The final verification of species was done using polymerase chain reaction-amplified 16S rRNA-specific DNA. All isolates formed creamy white round colonies on MRS agar medium, were gram-positive, were able to grow in anaerobic conditions, and were also rod-shaped and catalase-negative. Based on biochemical tests, four types of lactic acid bacteria (Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus salivarius, and Lactobacillus acidophilus) were identified. The findings of this study indicated the presence and diversity of lactic acid bacteria in beet juice, which can be a potential platform for the production of probiotic beetroot juice as a non-dairy product.

Rapid detection of lactic acid bacteria by lateral flow immunochromatographic assay

Lactic acid bacteria (LAB) cause quality deterioration including acidification and production of off-flavor components in food. To rapidly detect LAB, a lateral flow immunochromatographic assay (LFIA) was developed. An antibody (Ab) was obtained using Lactiplantibacillus plantarum as an immunogen, and the LFIA was performed based on this Ab. This showed positive reactions with LAB and a wide range of Gram-positive bacteria. An acid producing ability test using a semiconductor pH sensor was performed in combination with the LFIA. The bacteria testing positive in both the pH test (pH-positive) and the LFIA (LFIA-positive) were LAB. Twenty-three species of LAB in 13 genera could be detected specifically. Fresh foods and their equivalent spoiled foods were cultured for 0, 12, and 24 h, and the time required for the cultures to show LFIA-positive and pH-positive results was investigated. Of the 51 foods, 42 showed shorter culture times for the spoiled food than for the fresh food (spoilage detection rate: 82%). Amplicon analysis detected LAB including Leuconostoc citreum and Leuconostoc gelidum in spoiled foods. As this novel method can detect LAB in approximately 1 h, its use should make it possible to judge the freshness of food before shipping or sale.

A Novel qPCR Method for the Detection of Lactic Acid Bacteria in Fermented Milk.

The number of live lactic acid bacteria (LAB) is an important quality indicator for yogurt, the quantitative testing of LAB has become an important task in the evaluation of product quality and function. By analyzing and comparing the performance of 16S rRNA gene and tuf gene used in absolute quantification, the tuf gene with copy number 1 was selected as the target gene of six LAB. By drawing a standard curve to achieve qualitative and quantitative detection of six strains of LAB, the detection range was found to be 1 × 103–1 × 108 copies/µL. The traditional plate colony count and Flow Cytometry (FCM) were compared with the method of qPCR, which was used in this experiment. Meanwhile, the confocal laser microscope combined with STYO 9 and propidium iodide dyes was used to determine that the content of viable bacteria in the yogurt was more than 90%, which proved that the detection result using qPCR method was closer to the true level of LAB in yogurt. Compared with the existing methods, the method in this study allowed the qualitative and quantitative detection of the six kinds of LAB in yogurt, and the distribution of live and dead bacteria in yogurt could be calculated.

Detection of Lactic Acid Bacteria (LAB) from Local Breed Chicken Gut as Probiotic Agent in Livestock

Detection of Lactic Acid Bacteria (LAB) from Local Breed Chicken Gut as Probiotic Agent in Livestock

Effect of plating and incubation conditions on the detection of lactic acid bacteria after high hydrostatic pressure treatment

ABSTRACTLactic acid bacteria (Leuconostoc mesenteroides, Enterococcus faecalis, and Lactobacillus fermentum) were subjected to high hydrostatic pressures (HHPs) of 400 and 600 MPa at 25°C for 10 min in phosphate-buffered saline. Differential plating methods were applied to evaluate HHP-treated cell populations, assuming that healthy and injured cells during plate incubation survived maximum and minimal stress, respectively. The stress was altered by using several selective media in combination with aerobic or anaerobic incubation at 25°C or 30°C. E. faecalis was detectable after 600 MPa treatment while L. mesenteroides and L. fermentum were nondetectable. Specific combinations of incubation conditions were suggested to determine maximum and minimum viable counts of L. mesenteroides and E. faecalis. The difference between the maximum and minimum counts can be used to evaluate HHP-injured population with reduced risks to overestimate healthy and/or underestimate HHP-injured cells.

English

Camel milk is a suitable substrate for the growth of protective bacterial flora. Detection of lactic acid bacteria producing antimicrobial substances from camel (Camelus dromedarius) milk in south Algeria against some food-borne pathogens is the subject of this work. Morphological, physiological and biochemical tests have identified four Lactobacillus isolates belonging to the following species: Lactobacillus fermentum, Lactobacillus helviticus, Lactobacillus plantarum and Lactobacillus acidophilus. In order to demonstrate the inhibitory effect of these bacteria in vitro, their antagonistic property was tested against six pathogenic strains often involved in food-borne illness: Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Salmonella enteritidis and Shigella flexneri using the disc diffusion method. The antagonistic effect was manifested by the appearance of inhibition zones around the discs. The potential inhibitor was estimated by calculating diameter of inhibition zones which extend from 02 to 16 mm. All Lactobacillus isolates secreted into the culture medium inhibitory substances were able to inactivate the growth of pathogenic strains tested. L. plantarum has shown the largest inhibition zone against S. aureus (16 mm). These two strains were chosen to determine the nature of L. plantarum secreted substances responsible for the antagonistic effect. The obtained results have shown that L. plantarum inhibitory property against S. aureus resulted from the combined effect of several biological agents originating from their metabolic activities, especially organic acids and bacteriocins. Key words: Antagonism, camel milk, Lactobacilli, food borne pathogens, mixed culture, pure culture.

Rapid quantification of live/dead lactic acid bacteria in probiotic products using high-sensitivity flow cytometry

A laboratory-built high-sensitivity flow cytometer (HSFCM) was employed for the rapid and accurate detection of lactic acid bacteria (LAB) and their viability in probiotic products. LAB were stained with both the cell membrane-permeable SYTO 9 green-fluorescent nucleic acid stain and the red-fluorescent nucleic acid stain, propidium iodide, which penetrates only bacteria with compromised membranes. The side scatter and dual-color fluorescence signals of single bacteria were detected simultaneously by the HSFCM. Ultra-high temperature processing milk and skim milk spiked with Lactobacillus casei were used as the model systems for the optimization of sample pretreatment and staining. The viable LAB counts measured by the HSFCM were in good agreement with those of the plate count method, and the measured ratios between the live and dead LAB matched well with the theoretical ratios. The established method was successfully applied to the rapid quantification of live/dead LAB in yogurts and fermented milk beverages of different brands. Moreover, the concentration and viability status of LAB in ambient yogurt, a relatively new yet popular milk product in China, are also reported.

Identification and Antimicrobial Activity Detection of Lactic Acid Bacteria Isolated from Corn Stover Silage

A total of 59 lactic acid bacteria (LAB) strains were isolated from corn stover silage. According to phenotypic and chemotaxonomic characteristics, 16S ribosomal DNA (rDNA) sequences and recA gene polymerase chain reaction amplification, these LAB isolates were identified as five species: Lactobacillus (L.) plantarum subsp. plantarum, Pediococcus pentosaceus, Enterococcus mundtii, Weissella cibaria and Leuconostoc pseudomesenteroides, respectively. Those strains were also screened for antimicrobial activity using a dual-culture agar plate assay. Based on excluding the effects of organic acids and hydrogen peroxide, two L. plantarum subsp. plantarum strains ZZU 203 and 204, which strongly inhibited Salmonella enterica ATCC 43971T, Micrococcus luteus ATCC 4698T and Escherichia coli ATCC 11775T were selected for further research on sensitivity of the antimicrobial substance to heat, pH and protease. Cell-free culture supernatants of the two strains exhibited strong heat stability (60 min at 100°C), but the antimicrobial activity was eliminated after treatment at 121°C for 15 min. The antimicrobial substance remained active under acidic condition (pH 2.0 to 6.0), but became inactive under neutral and alkaline condition (pH 7.0 to 9.0). In addition, the antimicrobial activities of these two strains decreased remarkably after digestion by protease K. These results preliminarily suggest that the desirable antimicrobial activity of strains ZZU 203 and 204 is the result of the production of a bacteriocin-like substance, and these two strains with antimicrobial activity could be used as silage additives to inhibit proliferation of unwanted microorganism during ensiling and preserve nutrients of silage. The nature of the antimicrobial substances is being investigated in our laboratory.

Detection of lactic acid bacteria in the bovine uterus and vagina by real-time PCR approaches

Detection of lactic acid bacteria in the bovine uterus and vagina by real-time PCR approaches

Heterofermentative process in dry fermented sausages - a case report

In certain circumstances the fermentation process in dry fermented sausages converts to heterofermentation pathway leading to acetic acid and carbon dioxide beside lactic acid. The study describes two cases of undesirable heterofermentation in dry sausages from two different producers. In the sausage samples (n = 7) the pH value and the content of lactic and acetic acids were measured. Microbial analysis focused on quantitative and qualitative detection of lactic acid bacteria. The acetic acid content varied from 24.28 to 67.41 µmol·g-1 dry matter, in the case of samples from the second producer the content of acetic acid (48.45 to 67.41 µmol·g-1 dry matter) was higher than the lactic acid content (20.98 to 29.02 µmol·g-1 dry matter). The lactobacilli strains from the sausages were assigned to the corresponding species by Matrix-Assisted Laser Desorption-Ionization – Time of Flight Mass Spectrometry (MALDI-TOF MS) and classified to three groups according to the sugar fermentation pattern (obligately homofermentative, facultatively heterofermentative and obligately heterofermentative) and they caused the heterofermentation process in the samples of dry fermented sausages. The description of the case of heterofermentation process in dry sausages is unique and there is little information about this topic.

Quantitative detection of lactic acid bacteria in dried sourdoughs using real-time PCR

A real-time PCR system with 16S rRNA gene-targeted group-specific primers was developed to quantitatively detect lactic acid bacteria (LAB) of the genera Lactobacillus, Leuconostoc, Pediococcus, and Weissella in different types of commercially available dried sourdoughs. Despite a high degree of degradation in the DNA isolated from the doughs, the 341-bp 16S rRNA gene fragment of the sourdough LAB biota could specifically be amplified. For dried sourdoughs, the resulting calculated LAB cell counts were determined to be up to 3.7 × 107 cells/g fresh dough, whereas in non-fermented dough acidifiers, used as a control, the calculated LAB cell counts did not exceed 3.6 × 104 cells/g fresh dough. Moreover, the effect of low pH and/or high lactic acid concentrations prevailing in the doughs on the detectability of LAB cells in spray- and roller-dried sourdoughs by PCR was investigated. Drying of non-acidified sourdoughs still permitted to detect the LAB cells by PCR, whereas drying of acidified doughs reduced the detectable cell counts by approximately 5 (spray dried) and 2 (roller dried) orders of magnitudes. Incubation of acidified doughs for 24 h did not affect the detectability of LAB cells in spray-dried doughs but further reduced the detectable cell counts in roller-dried doughs by additional 2 orders of magnitude.

Phylogenetic group- and species-specific oligonucleotide probes for single-cell detection of lactic acid bacteria in oral biofilms.

BackgroundThe purpose of this study was to design and evaluate fluorescent in situ hybridization (FISH) probes for the single-cell detection and enumeration of lactic acid bacteria, in particular organisms belonging to the major phylogenetic groups and species of oral lactobacilli and to Abiotrophia/Granulicatella.ResultsAs lactobacilli are known for notorious resistance to probe penetration, probe-specific assay protocols were experimentally developed to provide maximum cell wall permeability, probe accessibility, hybridization stringency, and fluorescence intensity. The new assays were then applied in a pilot study to three biofilm samples harvested from variably demineralized bovine enamel discs that had been carried in situ for 10 days by different volunteers. Best probe penetration and fluorescent labeling of reference strains were obtained after combined lysozyme and achromopeptidase treatment followed by exposure to lipase. Hybridization stringency had to be established strictly for each probe. Thereafter all probes showed the expected specificity with reference strains and labeled the anticipated morphotypes in dental plaques. Applied to in situ grown biofilms the set of probes detected only Lactobacillus fermentum and bacteria of the Lactobacillus casei group. The most cariogenic biofilm contained two orders of magnitude higher L. fermentum cell numbers than the other biofilms. Abiotrophia/Granulicatella and streptococci from the mitis group were found in all samples at high levels, whereas Streptococcus mutans was detected in only one sample in very low numbers.ConclusionsApplication of these new group- and species-specific FISH probes to oral biofilm-forming lactic acid bacteria will allow a clearer understanding of the supragingival biome, its spatial architecture and of structure-function relationships implicated during plaque homeostasis and caries development. The probes should prove of value far beyond the field of oral microbiology, as many of them detect non-oral species and phylogenetic groups of importance in a variety of medical conditions and the food industry.

Protocols for the isolation and detection of lactic acid bacteria with bacteriocinogenic potential

The objective of this study was to evaluate culture media and methodologies for isolation and detection of lactic acid bacteria (LAB) capable to produce bacteriocin-like substances. Samples of milk and cheese were pour plated on de Mann-Rogosa-Sharpe agar (MRS) and Kang-Fung-Sol agar (KFS) (both at 35 °C/48 h, under anaerobiosis), from which 389 and 256 LAB cultures were selected. The antagonistic activity of them was evaluated using the spot-on-the-lawn and two culture media: brain-heart infusion agar with catalase (BHI + C) and M17 (both at 35 °C/24 h). The proteinaceous nature of the antagonistic cultures was verified using: spot-on-the-lawn (MRS, 25 °C/24 h, under anaerobiosis) and well-diffusion (cultures amplified on modified MRS broth at 25 °C/24 h, and then neutralized using NaOH). Listeria monocytogenes ATCC 7644 was used as indicator. A larger number of antagonist cultures were isolated from MRS (83 by M17 and 65 by BHI + C) in comparison to KFS (24 by M17 and 15 by BHI + C). The spot-on-the-lawn identified a higher frequency of LAB capable of producing bacteriocin-like substances. MRS was considered to be the best culture media for the isolation of LAB capable to produce bacteriocin-like substances, activity that was better identified using the spot-on-the-lawn methodology.

Identification of the lactic acid bacteria in Kimchi according to initial and over-ripened fermentation using PCR and 16S rRNA gene sequence analysis

This paper aimed to identify the lactic acid bacteria species involved in kimchi fermentation at different fermentation periods by using culture-independent 16S rRNA gene clone libraries, and develop polymerase chain reaction for the detection of lactic acid bacteria (LAB). We investigated 6 commercially produced kimchi samples, including kimchi at an initial stage of fermentation and kimchi that was fermented to an over-ripened stage. The results of our study show that the analysis with cultureindependent 16S rRNA gene clone libraries could successfully identify 134 clones, 11 species, including Weissella, Lactobacillus, Pediococcus, and Leuconostoc from the 6 commercial kimchi samples. Weisella koreensis and Lactobacillus brevis were the predominant LAB in the initial stage of kimchi fermentation at 4°C (pH 4.96–5.27 and acidity 0.81–0.88), and Leuconostoc gelidum and Lactobacillus sakei subsp. sakei may play an important role in kimchi fermentation at the over-ripened stage (pH 3.61–3.91 and acidity 1.70–1.79).

Microbiological Quality and Safety of Raw Milk and Soft Cheese and Detection of Autochthonous Lactic Acid Bacteria with Antagonistic Activity AgainstListeria monocytogenes,SalmonellaSpp., andStaphylococcus aureus

This study aimed to characterize the microbiological quality and safety of raw milk and soft cheese, verifying possible associations between microbial populations, and the detection of lactic acid bacteria (LAB) with antagonistic activity against foodborne pathogens. Raw milk (n = 36) and soft cheese (n = 18) samples were collected and submitted for the analysis of mesophilic aerobes, total coliforms, Escherichia coli, LAB, coagulase-positive Staphylococcus (CPS), Listeria monocytogenes, and Salmonella spp. In all, 389 LAB isolates were randomly selected and submitted for antagonistic tests against L. monocytogenes, St. aureus, Salmonella Typhimurium, and Lactobacillus sakei. The samples presented high counts of mesophilic aerobes, total coliforms, and LAB, and also high and significant correlation indices between these populations. Low levels of CPS and E. coli were observed, as well as an absence of Salmonella spp. and L. monocytogenes. A substantial portion of the analyzed samples presented LAB cultures with antagonistic activity, but not against Salmonella Typhimurium. The obtained results indicate the antimicrobial potential of the autochthonous microbiota of raw milk and soft cheese. Despite the spoilage potential, the LAB present in the studied food products can be isolated and properly characterized as antagonistic cultures, to be used in bioconservation studies for pathogen control in foods.

HLP Medium for the Detection of Lactic Acid Bacteria (International Method)

HLP Medium for the Detection of Lactic Acid Bacteria (International Method)

Novel multiplex PCR for the detection of lactic acid bacteria during kimchi fermentation

We developed a multiplex PCR assay for the detection of lactic acid bacteria (LAB) species, and used it to examine the LAB species involved in kimchi fermentation. The LAB profile during kimchi fermentation varied with pH and acidity. Leuconostoc mesenteroides was observed during early fermentation (pH 5.64–4.27 and acidity 0.48–0.89%), and Lactobacillus sakei become dominant later in fermentation (pH ≤4.15 and acidity ≥0.98%). The efficiency of the multiplex PCR ranged from 86.5% at day 0 (pH 6.17 and acidity 0.24%) to 100% at day 96 (pH 4.16 and acidity 1.14). This multiplex PCR assay will facilitate study of the microbial ecosystem of kimchi and its impact on kimchi fermentation.

Molecular characterisation of the intestinal microbiota of farmed Atlantic salmon (Salmo salar L.)

In this study, the intestinal microbiota of farmed Atlantic salmon (Salmo salar L.) was examined with traditional culture-based techniques, and by molecular analysis of the 16S rDNA. The aim of the study was to evaluate the use of cultivation-based and molecular-based techniques, and compare the results obtained by the two methods. Samples were collected from the fore-, mid- and hind-gut, respectively. PCR and denaturing gradient gel electrophoresis (DGGE) analyses were performed on DNA extracted directly from the gut content and from bacteria cultivated on Tryptic Soy Agar (TSA). Population fingerprints of the predominant microbiota were generated by DGGE analysis of universal V3 16S rDNA PCR amplicons, and distinct bands from DGGE were sequenced. Results show that the salmon intestine was dominated by Lactobacillus spp., Lactococcus sp., Bacillus sp., Photobacterium phosphoreum, Acinetobacter sp., Pseudomonas sp. and Vibrio sp. Molecular analyses of samples from direct DNA extraction enabled detection of lactic acid bacteria. Prior cultivation detected Vibrio sp., Acinetobacter sp. and Pseudomonas sp., which were not detected by the direct analyses. In addition to the PCR–DGGE approach, 50 TSA isolates from the hind-gut were pure cultured and identified by partial sequencing of the 16S rDNA. Vibrio spp. and P. phosphoreum dominated among these isolates. The data provided demonstrate the use of PCR–DGGE as an alternative method for studying the intestinal microbiota of fish.