Lactic Acid Bacteria Contamination Research Articles

Development of a quantitative microbiological spoilage risk assessment (QMSRA) model for cooked ham sliced at retail

A quantitative microbiological spoilage risk assessment model (QMSRA) for cooked ham sliced at retail was developed based on a stochastic growth model for lactic acid bacteria (LAB), which are considered as the specific spoilage organisms (SSO), and a “spoilage-response” relationship characterizing the variability in consumer's perception of spoilage. In a simulation involving 10,000 cooked ham purchases, the QMSRA model predicted a median of zero spoilage events for up to 4.5 days of storage. After storage times of 5 and 6 days, the model predicted 1,790 and 8,570 spoilage events, respectively. A sensitivity analysis showed that domestic storage temperature was the most significant factor affecting LAB concentration in cooked ham, followed by the LAB contamination level at slicing. A scenario analysis was performed testing better temperature control of consumer's refrigerators, better hygiene conditions during slicing and a combination of the two strategies. Among the tested scenarios, a 2 log reduction in the LAB contamination at slicing combined with a 2 °C decrease in domestic storage temperature resulted in zero risk of spoilage for up to 12 days of storage. The QMSRA model developed in the present study can be a useful tool for quality management decisions.

Novel endolysin LysMP for control of Limosilactobacillus fermentum contamination in small-scale corn mash fermentation

BackgroundTraditional bioethanol fermentation industries are not operated under strict sterile conditions and are prone to microbial contamination. Lactic acid bacteria (LAB) are often pervasive in fermentation tanks, competing for nutrients and producing inhibitory acids that have a negative impact on ethanol-producing yeast, resulting in decreased yields and stuck fermentations. Antibiotics are frequently used to combat contamination, but antibiotic stewardship has resulted in a shift to alternative antimicrobials.ResultsWe demonstrate that endolysin LysMP, a bacteriophage-encoded peptidoglycan hydrolase, is an effective method for controlling growth of LAB. The LysMP gene was synthesized based on the prophage sequence in the genome of Limosilactobacillus fermentum KGL7. Analysis of the recombinant enzyme expressed in E. coli and purified by immobilized metal chelate affinity chromatography (IMAC) showed an optimal lysis activity against various LAB species at pH 6, with stability from pH 4 to 8 and from 20 to 40 °C up to 48 h. Moreover, it retains more than 80% of its activity at 10% ethanol (v/v) for up to 48 h. When LysMP was added at 250 µg/mL to yeast corn mash fermentations containing L. fermentum, it reduced bacterial load by at least 4-log fold compared to the untreated controls and prevented stuck fermentation. In comparison, untreated controls with contamination increased from an initial bacterial load of 1.50 × 107 CFU/mL to 2.25 × 109 CFU/mL and 1.89 × 109 CFU/mL after 24 h and 48 h, respectively. Glucose in the treated samples was fully utilized, while untreated controls with contamination had more than 4% (w/v) remaining at 48 h. Furthermore, there was at least a fivefold reduction in lactic acid (0.085 M untreated contamination controls compared to 0.016 M treated), and a fourfold reduction in acetic acid (0.027 M untreated contamination controls vs. 0.007 M treated), when LysMP was used to treat contaminated corn mash fermentations. Most importantly, final ethanol yields increased from 6.3% (w/v) in untreated contamination samples to 9.3% (w/v) in treated contamination samples, an approximate 50% increase to levels comparable to uncontaminated controls 9.3% (w/v).ConclusionLysMP could be a good alternative to replace antibiotics for mitigation of LAB contamination in biofuel refineries.

Rapid Single-Cell Detection of Beer-Contaminating Lactic Acid Bacteria Using Bioluminescence/Rapid Microbe Detection.

The rapid microbe detection (RMD) method can detect a trace amount of adenosine triphosphate (ATP) equivalent to that generated by single cell of lactic acid bacteria (LAB). For the improved detection of LAB contamination in beer without cultivation, it is necessary to eliminate the influence of beer-derived ATP and to improve the signal-to-noise ratio. In this protocol, the beer sample is filtered using a membrane filter, thereby avoiding the formation of beer foam to the fullest extent. By washing the beer components remaining on the filter with an ethanol solution and a weakly alkaline solution and then culturing the filter in an agar medium, the beer-derived ATP remaining on the filter can be removed and the ATP of LAB cells is increased. As a result, the signal-to-noise ratio of the RMD method can be dramatically improved.

Prevalence and abundance of lactic acid bacteria in raw milk associated with forage types in dairy cow feeding

Lactic acid bacteria (LAB) found in milk can be responsible for organoleptic defects in cheese. To identify sources of LAB that could potentially develop during cheese making, we evaluated their prevalence and abundance in milk according to the type of forage used in dairy cow feeding. Forages and bulk tank milk were sampled 3 times on 24 farms using either hay alone (control), or grass or legume silage supplemented with corn silage or not. Both types of silage were either non-inoculated or inoculated with commercial preparations containing at least a Lactobacillus buchneri strain along with Lactobacillus casei, Lactobacillus plantarum, Enterococcus faecium, or Pediococcus pentosaceus. Our results indicate that LAB viable counts in milk samples (2.56 log cfu/mL) did not differ according to the type of forage used. A total of 1,239 LAB were isolated and identified by partial 16S rRNA gene sequencing. Although inoculation increased lactobacilli abundance in grass silage by 35%, we did not observe an effect on the LAB profile of milk. Indeed, we found no significant difference in milk LAB prevalence and abundance according to the type of forage (P > 0.05). Moreover, isolates belonging to the L. buchneri group were rarely found in bulk tank milk (3 out of 481 isolates). Random amplified polymorphic DNA typing of 406 LAB isolates revealed the plausible transfer of some strains from silage to milk (~6%). Thus, forage is only a minor contributor to LAB contamination of milk.

ATR-MIR spectroscopy and multivariate analysis in alcoholic fermentation monitoring and lactic acid bacteria spoilage detection

Wine production processes still rely on post-production evaluation and off-site laboratory analyses to ensure the quality of the final product. Here we propose an at-line methodology that combines a portable ATR-MIR spectrometer and multivariate analysis to control the alcoholic fermentation process and to detect wine fermentation problems. In total, 36 microvinifications were conducted, 14 in normal fermentation conditions (NFC) and 22 intentionally contaminated fermentations (ICF) with different lactic acid bacteria (LAB) concentrations. ATR-MIR measurements were collected during alcoholic and malolactic fermentations and relative density, pH, and l-malic acid were analyzed by traditional methods. Partial Least Squares Regression could suitably predict density and pH in fermenting samples (root mean squared errors of prediction of 0.0014 g mL−1 and 0.06 respectively). With regard to ICF, LAB contamination was detected by multivariate discriminant analysis when the difference in l-malic acid concentration between NFC and ICF was in the order of 0.7–0.8 g L−1, before the end of malolactic fermentation. This methodology shows great potential as a fast and simple at-line analysis tool for detecting fermentation problems at an early stage.

Yeast Derived LysA2 Can Control Bacterial Contamination in Ethanol Fermentation.

Contamination of fuel-ethanol fermentations continues to be a significant problem for the corn and sugarcane-based ethanol industries. In particular, members of the Lactobacillaceae family are the primary bacteria of concern. Currently, antibiotics and acid washing are two major means of controlling contaminants. However, antibiotic use could lead to increased antibiotic resistance, and the acid wash step stresses the fermenting yeast and has limited effectiveness. Bacteriophage endolysins such as LysA2 are lytic enzymes with the potential to contribute as antimicrobials to the fuel ethanol industries. Our goal was to evaluate the potential of yeast-derived LysA2 as a means of controlling Lactobacillaceae contamination. LysA2 intracellularly produced by Pichia pastoris showed activity comparable to Escherichia coli produced LysA2. Lactic Acid Bacteria (LAB) with the A4α peptidoglycan chemotype (L-Lys-D-Asp crosslinkage) were the most sensitive to LysA2, though a few from that chemotype were insensitive. Pichia-expressed LysA2, both secreted and intracellularly produced, successfully improved ethanol productivity and yields in glucose (YPD60) and sucrose-based (sugarcane juice) ethanol fermentations in the presence of a LysA2 susceptible LAB contaminant. LysA2 secreting Sacharomyces cerevisiae did not notably improve production in sugarcane juice, but it did control bacterial contamination during fermentation in YPD60. Secretion of LysA2 by the fermenting yeast, or adding it in purified form, are promising alternative tools to control LAB contamination during ethanol fermentation. Endolysins with much broader lytic spectrums than LysA2 could supplement or replace the currently used antibiotics or the acidic wash.

Lactic acid bacteria in dried vegetables and spices

Spices and dried vegetable seasonings are potential sources of bacterial contamination for foods. However, little is known about lactic acid bacteria (LAB) in spices and dried vegetables, even though certain LAB may cause food spoilage. In this study, we enumerated LAB in 104 spices and dried vegetables products aimed for the food manufacturing industry. The products were obtained from a spice wholesaler operating in Finland, and were sampled during a one-year period. We picked isolates (n = 343) for species identification based on numerical analysis of their ribotyping patterns and comparing them with the corresponding patterns of LAB type strains. We found LAB at levels >2 log CFU/g in 68 (65%) of the samples, with the highest counts detected from dried onion products and garlic powder with counts ranging from 4.24 to 6.64 log CFU/g. The LAB identified were predominantly Weissella spp. (61%) and Pediococcus spp. (15%) with Weissella confusa, Weissella cibaria, Weissella paramesenteroides, Pediococcus acidilactici and Pediococcus pentosaceus being the species identified. Other species identified belonged to the genera of Enterococcus spp. (8%), Leuconostoc spp. (6%) and Lactobacillus spp. (2%). Among the LAB identified, Leuconostoc citreum, Leuconostoc mesenteroides and W. confusa have been associated with food spoilage. Our findings suggest that spices and dried vegetables are potential sources of LAB contamination in the food industry.

Distiller yeasts producing antibacterial peptides

A new method of controlling lactic acid bacteria contamination was developed with the use of recombinant Saccharomyces cerevisiae strains producing antibacterial peptides. Genes encoding the antibacterial peptides pediocin and plantaricin with codons preferable for S. cerevisiae were synthesized, and a system was constructed for their secretory expression. Recombinant S. cerevisiae strains producing antibacterial peptides effectively inhibit the growth of Lactobacillus sakei, Pediacoccus pentasaceus, Pediacoccus acidilactici, etc. The application of distiller yeasts producing antibacterial peptides enhances the ethanol yield in cases of bacterial contamination. Recombinant yeasts producing the antibacterial peptides pediocin and plantaricin can successfully substitute the available industrial yeast strains upon ethanol production.

Regulation of Lactobacillus plantarum contamination on the carbohydrate and energy related metabolisms of Saccharomyces cerevisiae during bioethanol fermentation

During the industrial bioethanol fermentation, Saccharomyces cerevisiae cells are often stressed by bacterial contaminants, especially lactic acid bacteria. Generally, lactic acid bacteria contamination can inhibit S. cerevisiae cell growth through secreting lactic acid and competing with yeast cells for micronutrients and living space. However, whether are there still any other influences of lactic acid bacteria on yeast or not? In this study, Lactobacillus plantarum ATCC 8014 was co-cultivated with S. cerevisiae S288c to mimic the L. plantarum contamination in industrial bioethanol fermentation. The contaminative L. plantarum-associated expression changes of genes involved in carbohydrate and energy related metabolisms in S. cerevisiae cells were determined by quantitative real-time polymerase chain reaction to evaluate the influence of L. plantarum on carbon source utilization and energy related metabolism in yeast cells during bioethanol fermentation. Contaminative L. plantarum influenced the expression of most of genes which are responsible for encoding key enzymes involved in glucose related metabolisms in S. cerevisiae. Specific for, contaminated L. plantarum inhibited EMP pathway but promoted TCA cycle, glyoxylate cycle, HMP, glycerol synthesis pathway, and redox pathway in S. cerevisiae cells. In the presence of L. plantarum, the carbon flux in S. cerevisiae cells was redistributed from fermentation to respiratory and more reducing power was produced to deal with the excess NADH. Moreover, L. plantarum contamination might confer higher ethanol tolerance to yeast cells through promoting accumulation of glycerol. These results also highlighted our knowledge about relationship between contaminative lactic acid bacteria and S. cerevisiae during bioethanol fermentation.

Monitoring psychrotrophic lactic acid bacteria contamination in a ready-to-eat vegetable salad production environment

A study monitoring lactic acid bacteria contamination was conducted in a company producing fresh, minimally processed, packaged and ready-to-eat (RTE) vegetable salads (stored at 4°C) in order to investigate the reason for high psychrotrophic LAB levels in the products at the end of shelf-life. Initially, high microbial counts exceeding the established psychrotrophic thresholds (>107–108CFU/g) and spoilage manifestations before the end of the shelf-life (7days) occurred in products containing an assortment of sliced and diced vegetables, but within a one year period these spoilage defects became prevalent in the entire processing plant. Environmental sampling and microbiological analyses of the raw materials and final products throughout the manufacturing process highlighted the presence of high numbers of Leuconostoc spp. in halved and unseeded, fresh sweet bell peppers provided by the supplier. A combination of two DNA fingerprinting techniques facilitated the assessment of the species diversity of LAB present in the processing environment along with the critical point of their introduction in the production facility. Probably through air mediation and surface adhesion, mainly members of the strictly psychrotrophic species Leuconostoc gelidum subsp. gasicomitatum and L. gelidum subsp. gelidum were responsible for the cross-contamination of every vegetable handled within the plant.

Uracil evolution in tomato pulp inoculated with different microbial strains during a long incubation time

Uracil has been previously proposed as a successful index of lactic acid bacteria (LAB) contamination in tomato products. The uracil produced in tomato pulp, as a consequence of uridine hydrolysis by different LAB strains ( Lactobacillus brevis, Lactobacillus fermentum, Lactobacillus plantarum, Leuconostoc mesenteroides, and Pediococcus spp.), remained constant over the entire incubation time (180 days), confirming uracil as a good index of LAB contamination. An anaerobic spore-forming microorganism ( Clostridium pasteurianum) was also able to produce uracil. In order to deepen the understanding of uracil formation mechanisms, uracil and uridine were determined in De Man Rogosa Sharpe (MRS) uridine-enriched broth inoculated with some LAB strains: the maximum level of uracil formation was followed by a total or partial uracil decrease. Only under stressful conditions, such as reduced glucose concentrations (0.2–0.02%) in culture media, did LAB strains not degrade or use the uracil produced, as observed in tomato pulp.

Role of broiler carcasses and processing plant air in contamination of modified-atmosphere-packaged broiler products with psychrotrophic lactic acid bacteria.

Some psychrotrophic lactic acid bacteria (LAB) are specific meat spoilage organisms in modified-atmosphere-packaged (MAP), cold-stored meat products. To determine if incoming broilers or the production plant environment is a source of spoilage LAB, a total of 86, 122, and 447 LAB isolates from broiler carcasses, production plant air, and MAP broiler products, respectively, were characterized using a library of HindIII restriction fragment length polymorphism (RFLP) patterns of the 16 and 23S rRNA genes as operational taxonomic units in numerical analyses. Six hundred thirteen LAB isolates from the total of 655 clustered in 29 groups considered to be species specific. Sixty-four percent of product isolates clustered either with Carnobacterium divergens or with Carnobacterium maltaromaticum type strains. The third major product-associated cluster (17% of isolates) was formed by unknown LAB. Representative strains from these three clusters were analyzed for the phylogeny of their 16S rRNA genes. This analysis verified that the two largest RFLP clusters consisted of carnobacteria and showed that the unknown LAB group consisted of Lactococcus spp. No product-associated LAB were detected in broiler carcasses sampled at the beginning of slaughter, whereas carnobacteria and lactococci, along with some other specific meat spoilage LAB, were recovered from processing plant air at many sites. This study reveals that incoming broiler chickens are not major sources of psychrotrophic spoilage LAB, whereas the detection of these organisms from the air of the processing environment highlights the role of processing facilities as sources of LAB contamination.

PCR-TTGE and RAPD-PCR Techniques to AnalyzeSaccharomyces cerevisiaeandSaccharomyces carlsbergensisIsolated from Craft Beers

In the Friuli Venezia Giulia region (North East of Italy) the production of craft beers has been increasing constantly. Usually microbreweries use yeasts supplied by Italian or foreign industrial breweries for beer production. Yeast species are often not known, moreover the vitality, the viability, the physiological state and the number of generation are not known. To improve the quality of the final product it is important to evaluate the quality of the yeast strain used and the lactic acid bacteria contamination. Various molecular methods have been developed to compare genetic characteristics of yeast strains used in beer and wine production. The methods proposed in this work, PCR-TTGE and RAPD-PCR techniques, allow the comparison of specific DNA sequences to identify and/or characterize yeast strains. The molecular methods are faster than traditional methods and they allowed the identification of the strains analysed as S. cerevisiae and the intraspecies differentiation among yeast strains tested within 8 h after cell growth.

Uracil as an Index of Lactic Acid Bacteria Contamination of Tomato Products

The aim of this research was to evaluate the suitability of uracil as an hygienic quality index of tomato products. Whereas uridine was naturally present throughout tomato fruits' ripening, uracil appeared only after microbial contamination. In tomato pulp inoculated with nine different microbial strains, all five lactic acid bacteria (LAB) studied released relevant quantities of uracil (150-1040 mg/kg of dm), with a correlated partial or total decrease of uridine. Uracil production by yeasts and molds was very low or nonexistent; the starting uridine concentration (approximately 960 mg/kg of dm) remained constant or increased. Uracil thermostability was also verified. Twenty-six samples of tomato paste (30 degrees Brix) were collected from bag-in-drums produced in an industrial processing plant, some with evident swelling symptoms. All of the samples with high microbial count presented uracil. Uracil was also present in samples with microbial contamination under the detection limit and Howard mold count below legislation limits, implying the reprocessing, at least partial, of altered tomato product. The results indicate that uracil presence in tomato products is an index of LAB contamination that has occurred before heat treatment.

Ropy slime-producing lactic acid bacteria contamination at meat processing plants

At three Finnish meat processing plants the processing rooms, meat trimmings and carcasses were examined for the presence of ropy slime-producing lactic acid bacteria. Bacterial isolates similar to ropy slime-producing lactobacilli strains able to produce ropy slime on meat products, were recovered from the processing rooms and meat trimmings, indicating that these rooms and raw materials are a source of contamination for ropy bacteria. The ability to produce ropy slime would appear to be a common characteristic of lactobacilli, since altogether 10 different ropy lactobacilli groups were isolated in this study.