Temperature and respiration affect the growth and stress resistance of Lactobacillus plantarum C17

Aims: The aim of the study is to gain further insight on the respiratory behaviour of Lactobacillus plantarum and its consequences on stress tolerance. Methods and Results: We investigated the effect of temperature and respiration on the growth and stress (heat, oxidative, freezing, freeze-drying) response of Lact. plantarum C17 during batch cultivations. Temperature as well as respiration clearly affected the physiological state of cells, and generally, cultures grown under respiratory conditions exhibited improved tolerance of some stresses (heat, oxidative, freezing) compared to those obtained in anaerobiosis. Our results revealed that the activities in cell-free extracts of the main enzymes related to aerobic metabolism, POX (pyruvate oxidase) and NPR (NADH peroxidase), were significantly affected by temperature. POX was completely inhibited at 37°C, while the activity of NPR slightly increased at 25°C, indicating that in Lact. plantarum, the temperature of growth may be involved in the activation and modulation of aerobic/respiratory metabolism. Conclusions: We confirmed that respiration confers robustness to Lact. plantarum cells, allowing a greater stress tolerance and advantages in the production of starter and probiotic cultures. Significance and Impact of the Study: This is the first study on respiratory metabolism on a strain other than the model strains WCFS1; novel information on the role of temperature in the modulation of aerobic/ respiratory metabolism in Lact. plantarum is presented

Effect of Respiratory Growth on the Metabolite Production and Stress Robustness of Lactobacillus casei N87 Cultivated in Cheese Whey Permeate Medium

Cheese whey permeate (WP) is a low-cost feedstock used for the production of biomass and metabolites from several lactic acid bacteria (LAB) strains. In this study, Lactobacillus casei N87 was cultivated in an optimized WP medium (WPM) to evaluate the effect of anaerobic and respiratory conditions on the growth performances (kinetics, biomass yield), consumption of sugars (lactose, galactose, glucose) and citrate, metabolite production [organic acids, volatile organic compounds (VOCs)] and stress survival (oxidative, heat, freezing, freeze-drying). The transcription of genes involved in the main pathways for pyruvate conversion was quantified through Real Time-PCR to elucidate the metabolic shifts due to respiratory state. Cultivation in WPM induced a diauxic growth in both anaerobic and respiratory conditions, and L. casei N87 effectively consumed the lactose and galactose present in WPM. Genomic information suggested that membrane PTS system and tagatose-6-P pathway mediated the metabolism of lactose and galactose in L. casei N87. Respiration did not affect specific growth rate and biomass production, but significantly altered the pyruvate conversion pathways, reducing lactate accumulation and promoting the formation of acetate, acetoin and diacetyl to ensure the redox balance. Ethanol was not produced under either cultivation. Pyruvate oxidase (pox), acetate kinase (ack), alpha-acetolactate decarboxylase (ald), acetolactate synthase (als) and oxaloacetate decarboxylase (oad) genes were up-regulated under respiration, while L-lactate dehydrogenase (ldh), pyruvate formate lyase (pfl), pyruvate carboxylase (pyc), and phosphate acetyltransferase (pta) were down regulated by oxygen. Transcription analysis was consistent with metabolite production, confirming that POX-ACK and ALS-ALD were the alternative pathways activated under aerobic cultivation. Respiratory growth affected the production of volatile compounds useful for the development of aroma profile in several fermented foods, and promoted the survival of L. casei N87 to oxidative stresses and long-term storage. This study confirmed that the respiration-based technology coupled with cultivation on low-cost medium may be effectively exploited to produce competitive and functional starter and/or adjunct cultures. Our results, additionally, provided further information on the activation and regulation of metabolic pathways in homofermentative LAB grown under respiratory promoting conditions

Evaluation of a differential medium for the preliminary identification of members of the Lactobacillus plantarum and Lactobacillus casei groups

A medium originally developed for differential enumeration of probiotic species in fermented milk (mMRS-BPB, Lee and Lee 2008) was evaluated for its ability to correctly discriminate members of the Lactobacillus plantarum (L. plantarum) and L. casei groups from other species. The medium was tested on 461 strains of lactic acid bacteria (LAB) belonging to eight genera and thirty-five species. Colony morphology was relatively consistent for L. plantarum, L. paraplantarum, L. pentosus, L. paracasei, L. casei, and L. rhamnosus, but, when used alone, was not always sufficient to discriminate these species from other species potentially present in cheese. A procedure based on tree classification was developed to obtain preliminary identification on the basis of colony morphology, cell morphology, and CO2 production from glucose. By combining results of the tree classification procedure and heuristic rules, correct preliminary identification at the species or group level could be obtained in 74.4 % of cases overall, and the percentage of correct identifications was as high as 88-100 % for members of the L. plantarum and L. casei groups. When species belonging to groups that can be easily discriminated by rapid molecular methods were combined, the decision tree allowed to correct identification at the group level for the 95 % of the strains. Logistic regression was used to evaluate the effect of strain, operator, light source, and incubation temperature. Although all factors significantly affected one or more of the characters used for identification, the classification procedure proved to be quite robust. It may be difficult to use mMRS-BPB in the differential enumeration of LAB in cheese, except when species composition is relatively simple; however, it can be used as a simple tool to guide molecular identification in studies focused on the isolation of new strains from cheese

Selection of mutants tolerant of oxidative stress from respiratory cultures of Lactobacillus plantarum C17

Aims: 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

Dynamics of bacterial communities and interaction networks in thawed fish fillets during chilled storage in air

Thawed hake (Merluccius capensis and M. paradoxus) and plaice (Pleuronectes platessa) fillets were used as a model to evaluate the effect of storage temperature (0 or 10 °C) and biological variability (fish species, lot to lot) on bacterial growth kinetics and microbial successions. Both culture dependent methods (plate counts on non- selective and selective media) and culture independent methods (qPCR and 16S rRNA gene metabarcoding) were used. Bacterial counts exceeded 107 cfu/g within 2–3 days at 10 °C and 7–8 days at 0 °C. Plate counts on three media (Plate Count Agar +0.5% NaCl, Iron Agar Lyngby and Pseudomonas Selective medium) and 16S rRNA gene counts estimated by qPCR were highly correlated. Growth was modelled using the D-model and specific growth rate ranged between 0.97 and 1.24 d−1 and 3.54 and 5.90 d−1 at 0 and 10 °C, respectively. The initial composition of the microbiota showed lot-to-lot variation, but significant differences between the two fish species were detected. Alpha diversity significantly decreased during storage. When bacterial counts exceeded 107 cfu/g, the microbiota was dominated by members of the genera Pseudomonas, Psychrobacter, Acinetobacter, Serratia, Flavobacterium, Acinetobacter, Carnobacterium, Brochothrix and Vagococcus. However, Photobacterium and Shewanella, two genera frequently associated with fish spoilage, were either absent or minor components of the microbiota. As expected, storage temperature significantly affected the abundance of several species. The in- ference of microbial association networks with three different approaches (an ensemble approach using the CoNet app, Sparse Correlations for Compositional data, and SParse InversE Covariance Estimation for Ecological Association Inference) allowed the detection of both a core microbiota, which was present throughout storage, and a number of taxa, which became dominant at the end of spoilage and were characterized by a dispropor- tionate amount of negative interactions

Metabolic profiling and stress response of anaerobic and respiratory cultures of Lactobacillus plantarum C17 grown in a chemically defined medium

We investigated the effect of anaerobic (AN) and respiratory conditions (RS; oxygen, hemin and menaquinone) on the growth, enzymatic activities (POX, NOX, NPR), metabolic profile (1H-NMR spectroscopy), and response to oxidative (catalase, tolerance of H2O2) and heat stresses of Lactobacillus plantarum C17 (wild-type) and its natural oxidative stress-tolerant mutant C17-m58, using chemostat cultivation in a modified chemically defined medium (mCDM). Respiratory growth impaired biomass production and tolerance of heat stress in mCDM compared with cultivation in the complex Weissella medium broth (WMB). POX activity, oxygen consumption, and H2O2 tolerance were higher in respiratory cells and in C17-m58. Significant amounts of formate and acetate were found in AN cultures of wild-type and mutant strains, confirming the activation of an anaerobic pyruvate formate lyase–acetate kinase (PFL-ACK) pathway in L. plantarum. The presence of succinate supports the presence of an incomplete TCA cycle in this species. High concentrations of pyruvate were measured in RS supernatants, indicating reduced functionality of POX in vivo (35 °C) and the need to clarify the regulation and control of oxygen metabolism. Adipate was found in respiratory samples, likely because of lipid peroxidation, and its production by respiring lactic acid bacteria may be investigated and exploited for biotechnological uses

Evaluation of a differential medium for the preliminary identification of members of the Lactobacillus plantarum and Lactobacillus casei groups

A medium originally developed for differential enumeration of probiotic species in fermented milk (mMRS- BPB, Lee and Lee 2008) was evaluated for its ability to correctly discriminate members of the Lactobacillus plantarum (L. plantarum) and L. casei groups from other species. The medium was tested on 461 strains of lactic acid bacteria (LAB) belonging to eight genera and thirty-five spe- cies. Colony morphology was relatively consistent for L. plantarum, L. paraplantarum, L. pentosus, L. paracasei, L. casei, and L. rhamnosus, but, when used alone, was not always sufficient to discriminate these species from other species potentially present in cheese. A procedure based on tree classification was developed to obtain preliminary iden- tification on the basis of colony morphology, cell morphology, and CO2 production from glucose. By combining results of the tree classification procedure and heuristic rules, correct preliminary identification at the species or group level could be obtained in 74.4 % of cases overall, and the percentage of correct identifications was as high as 88-100 % for members of the L. plantarum and L. casei groups. When species belonging to groups that can be easily discriminated by rapid molecular methods were combined, the decision tree allowed to correct identification at the group level for the 95 % of the strains. Logistic regression was used to evaluate the effect of strain, operator, light source, and incubation temperature. Although all factors significantly affected one or more of the characters used for identification, the classification procedure proved to be quite robust. It may be difficult to use mMRS- BPB in the differential enumeration of LAB in cheese, except when species composition is relatively simple; however, it can be used as a simple tool to guide molecular identification in studies focused on the isolation of new strains from cheese

A survey of non-starter lactic acid bacteria in traditional cheeses: Culture dependent identification and survival to simulated gastrointestinal transit

Cultivable NSLAB in traditional Pasta filata and ewes' milk cheeses were studied by both PCR-DGGE of cells from Rogosa agar and by isolation and molecular identification after a simulated gastric juice (SGJ) treatment of the cheese. Two to six species were retrieved from each sample. The majority of isolates were identified as Lactobacillus paracasei, Lactobacillus fermentum, Lactobacillus rhamnosus, Enterococcus or Pediococcus. Bile tolerance and bile salt hydrolase (BSH) activity were tested on 88 strains: 64% were able to grow with ≥0.15% bile and 40% were BSH positive. The effect of simulated digestion was tested on 15 strains. Inactivation ranged from 0.15 to 2.93log cycles; most of the lethality was associated with pancreatic juice treatment. Although SGJ treatment alone may not provide a correct estimate of tolerance to gastrointestinal transit, it allowed selection of strains with a high tolerance to gastric juice, which may be tested as probiotic candidates

Effect of respirative and catalase-positive Lactobacillus casei adjuncts on the production and quality of Cheddar-type cheese

Lactobacillus casei N87 and L. casei N2014, cultivated in anaerobic and respiratory conditions, were used as adjuncts for the production of Cheddar-type cheeses. Cheese inoculated only with a defined lactococcal starter was used as control. After 14, 30, 60, 120 and 180 days of ripening, microbial counts, gross composition, proteolysis, production of volatile organic compounds, radical scavenging activity and lipid and protein oxidation were evaluated. The addition of anaerobic or respirative cultures of L. casei N87 and N2014 did not affect cheese composition and primary proteolysis. Respirative cells increased the production of free amino acids, peptides, diacetyl and acetoin and reduced the content of free radicals as well as lipid and protein oxidation. The lower redox potential in Cheddar-type cheeses produced with respirative cells also contributed to the reduction of oxidation processes. Respirative strains of L. casei could be successfully used to improve the organoleptic and nutritional properties of dairy product