1,720,968 research outputs found
Comparison of different fluorescent staining for the assessment of physiological state of lactic acid bacteria
No full textSelection of oxidative tolerant mutants from aerobic culture of Lactobacillus plantarum C17
No full textA comparison of fluorescent stains for the assessment of viability and metabolic activity of lactic acid bacteria.
No full textLactic acid bacteria (LAB) are used as starter or probiotic cultures in the food and pharmaceutical industry and, therefore, rapid and accurate methods for the detection of their viability are of practical relevance.
In this study 10 LAB strains, belonging to the genera Enterococcus, Lactococcus, Leuconostoc,
Lactobacillus, Streptococcus and Weissella, were subjected to heat and oxidative stresses and cell
injury or death was assessed comparing different fluorescent probes (Syto 9; Propidium Iodide, PI; 4,6-diamidino-2-phenylindole, DAPI; 5,(6)-carboxyfluorescein diacetate, cFDA) to identify the stain
combination which most reliably allowed the detection of live/metabolically active and dead cells.
Protocols for specimen preparation and staining were optimized and a simple procedure for automated cell counts was developed using NIH ImageJ macros. Cysteine and semi-solid agar solution were efficiently used as anti-fading agent and mounting medium, respectively. The double staining cFDA-PI apparently offered the best and most versatile indication of both cell metabolic activity and membrane integrity. An excellent correlation between manual and automated cell counts for the majority of strain/stain combinations was found.
This work provided a simple protocol for specimen preparation and staining based on the use of safe, easy to prepare and inexpensive reagents as compared to other methods. Additionally, the automated cell count procedure developed can be applied to several bacterial species and allows to increase the number of experimental trials and the reproducibility and sensitivity of the analysis
Evolution of microbial counts and chemical and physico-chemical parameters in high-moisture Mozzarella cheese during refrigerated storage
No full textThe microbiological quality, pH, colour, proteolysis and head space composition (using an electronic
nose) of several commercial brands of high-moisture Mozzarella cheese produced in Italy were evaluated
at the beginning and at the end (5 days) of refrigerated storage in order to evaluate the effect of the
acidification system (direct acid addition or use of starter cultures) and storage on the quality of the
cheese. A high variability was found for most parameters. At the end of storage all parameters were
affected by the mode of acidification and cheese produced by direct acid addition had a significantly
lower microbiological quality; counts of psychrotrophs exceeded 107 cfu/g for most samples and microbial
counts showed a significant correlation with the residual shelf life. Multivariate analysis
confirmed that samples at the beginning and at the end of storage were clearly separated but no
grouping based on the mode of acidification was found. The electronic nose was only partially successful
(80% correct classification) in classifying the cheeses on the basis of storage time or of microbial counts.
This is likely to be due to the variety of brands used in the analysis and to differences in the starter
systems or acidification mode used
Aerobic metabolism and oxidative stress tolerance in the Lactobacillus plantarum group
Full text linkAerobic metabolism and response to oxidative stress and starvation were studied in 11 Lactobacillus plantarum, L. paraplantarum and L. pentosus strains in order to assess the impact of aerobic metabolism on the growth and on the stress response. The strains were grown in aerobiosis without supplementation (AE), with hemin (AEH) or with hemin and menaquinone (AEHM) supplementation and in anaerobiosis (AN) in a complex buffered substrate. Growth rate, biomass yield, glucose and O2 consumption, production of lactic acid and H2O2, catalase activity, oxidative and starvation stress tolerance were evaluated. Aerobic growth increased biomass yield in late stationary phase. Further increase in yield was obtained with both hemin (H) and menaquinone (M) addition. With few exceptions, the increase in biomass correlated with the decrease of lactic acid which, however, decreased in
anaerobic conditions as well in some strains. Addition of H
or H ? M increased growth rate for some strains but reduced the duration of the lag phase. H2O2 production was found only for aerobic growth with no supplementation due to catalase production when hemin was supplemented. To
our knowledge this is the first study in which the advantages
of aerobic growth with H or H ? M in improving tolerance of oxidative stress and long-term survival is demonstrated on several strains of the L. plantarum group.
The results may have significant technological consequences
for both starter and probiotic production
Aggregation of yeast cells in Lactobacillus plantarum: correlation with msa gene and description of a novel image analysis method
No full textMicrobial community dynamics in thermophilic undefined milk starter cultures
No full textModel undefined thermophilic starter cultures were produced from raw milk of nine pasta-filata cheesemaking plants using a selective procedure based on pasteurization and incubation at high temperature with the objective of studying the microbial community dynamics and the variability in performances under repeated (7-13) reproduction cycles with backslopping. The traditional culture-dependent approach, based on random isolation and molecular characterization of isolates was coupled to the determination of pH and the evaluation of the ability to produce acid and fermentation metabolites. Moreover, a culture-independent approach based on amplicon-targeted next-generation sequencing was employed. The microbial diversity was evaluated by 16S rRNA gene sequencing (V1-V3 regions), while the microdiversity of Streptococcus thermophilus populations was explored by using novel approach based on sequencing of partial amplicons of the phosphoserine phosphatase gene (serB). In addition, the occurrence of bacteriophages was evaluated by qPCR and by multiplex PCR. Although it was relatively easy to select for a community dominated by thermophilic lactic acid bacteria (LAB) within a single reproduction cycle, final pH, LAB populations and acid production activity fluctuated over reproduction cycles. Both culture-dependent and -independent methods showed that the cultures were dominated by either S. thermophilus or Lactobacillus delbrueckii subsp. lactis or by both species. Nevertheless, subdominant mesophilic species, including lactococci and spoilage organisms, persisted at low levels. A limited number of serB sequence types (ST) were present in S. thermophilus populations. L. delbrueckii and Lactococcus lactis bacteriophages were below the detection limit of the method used and high titres of cos type S. thermophilus bacteriophages were detected in only two cases. In one case a high titre of bacteriophages was concurrent with a S. thermophilus biotype shift in the culture. This study largely confirms previous data on the composition of undefined thermophilic starters used for the production of traditional cheeses in Italy but it is the first one to systematically address the dynamics of the cultures under a repeated reproduction regime with backslopping
Selection of mutants tolerant of oxidative stress from respiratory cultures of Lactobacillus plantarum C17
No full textAims: Lactobacillus plantarum is a lactic acid bacterium involved in the production of many fermented foods. Recently, several studies have demonstrated that aerobic or respiratory metabolism in this species leads to improved technological and stress response properties.
Methods and Results: We investigated respiratory growth, metabolite production and stress resistance of Lact. plantarum C17 during batch, fed- batch and chemostat cultivations under respiratory conditions. Sixty mutants were selected for their ability to tolerate oxidative stress using H2O2 and menadione as selective agents and further screened for their capability to growth under anaerobic, respiratory and oxidative stress conditions.
Dilution rate clearly affected the physiological state of cells and, generally, slow-growing cultures had improved survival to stresses, catalase production and oxygen uptake. Most mutants were more competitive in terms of biomass production and ROS degradation compared with wild-type strain (wt) C17 and two of these (C17-m19 and C17-m58) were selected for further experiments.
Conclusions: This work confirms that, in Lact. plantarum, respiration and low growth rates confer physiological and metabolic advantages compared with anaerobic cultivation.
Significance and Impact of the Study: Our strategy of natural selection successfully provides a rapid and inexpensive screening for a large number of strains and represents a food-grade approach of practical relevance in the production of starter and probiotic culture
The microbiota of high-moisture mozzarella cheese produced with different acidification methods
No full textThe microbiota of high-moisture Mozzarella cheese made from cow's milk and produced with different acidification methods was evaluated at the end of refrigerated storage by pyrosequencing of the 16S rRNA gene. The cheeses were clearly separated on the basis of the acidification methods. Cheeses produced with the addition of starters were dominated by Streptococcus thermophilus, but a variety of lactic acid bacteria and spoilage microorganisms appeared at low levels (0.01-1%). Cheeses produced by direct addition of citric acid were dominated by a diverse microbiota, including both lactic acid bacteria and psychrotrophic γ-proteobacteria. For five brands the acidification system was not declared on the label: the microbiota was dominated by thermophilic lactic acid bacteria (S. thermophilus, Lactobacillus delbrueckii, Lactobacillus helveticus) but a variety of other subdominant lactic acid bacteria, psychrotrophs and Enterobacteriaceae were present, with a diversity comparable or higher to cheeses produced by direct acid addition. This led to the conclusion that undefined starters were used for acidification. Both ordination methods and network analysis were used for the representation of beta-diversity: matrix cluster analysis, principal coordinate analysis and OTU networks uncovered different aspects of the microbial community structure. For three cheese brands both biological replicates (cheeses from different lots) and technical replicates (replicate cheeses from the same lot) were analyzed. Repeatability was acceptable for OTUs appearing at frequencies >1%, but was low otherwise. A linear mixed model showed that the starter system was responsible for most differences related to dairies, while difference due to psychrotrophic contaminants was more related to lot-to-lot variability
- …
