To culture or not to culture: careful assessment of metabarcoding data is necessary when evaluating the microbiota of a modified-atmosphere-packaged vegetarian meat alternative throughout its shelf-life period.

BACKGROUND: As the increased consumption of ready-to-eat meat alternatives is a fairly recent trend, little is known about the composition and dynamics of the microbiota present on such products. Such information is nonetheless valuable in view of spoilage and food safety prevention. Even though refrigeration and modified-atmosphere-packaging (MAP) can extend the shelf-life period, microbial spoilage can still occur in these products. In the present study, the microbiota of a vegetarian alternative to poultry-based charcuterie was investigated during storage, contrasting the use of a culture-dependent method to a culture-independent metagenetic method. RESULTS: The former revealed that lactic acid bacteria (LAB) were the most abundant microbial group, specifically at the end of the shelf-life period, whereby Latilactobacillus sakei was the most abundant species. Metabarcoding analysis, in contrast, revealed that DNA of Xanthomonas was most prominently present, which likely was an artifact due to the presence of xanthan gum as an ingredient, followed by Streptococcus and Weissella. CONCLUSIONS: Taken together, these results indicated that Lb. sakei was likely the most prominent specific spoilage organisms (SSO) and, additionally, that the use of metagenetic analysis needs to be interpreted with care in this specific type of product. In order to improve the performance of metagenetics in food samples with a high DNA matrix but a low bacterial DNA load, selective depletion techniques for matrix DNA could be explored.

Metagenomics-based approach for studying and selecting bioprotective strains from the bacterial community of artisanal cheeses.

A metagenome-based approach was used to assess the taxonomic affiliation and functional potential for bacteriocin production of the bacterial community in cow's milk artisanal cheeses from Northwestern Argentina. Three different samples were analyzed by high-throughput sequencing of the V4 region of the 16S rRNA gene and shotgun metagenomics. Taxonomic analysis showed that cheese A and C were quite similar whereas cheese B displayed a rather different bacterial composition. Overall, two families, Streptococceae and Enterococceae, dominated the artisanal cheese microbiota, being the former family prevalent in cheese B and the later family the most important in samples A and C. Besides the usual species associated to cheeses, a number of bacterial taxa that have not been previously found in Argentinean artisanal cheeses were reported in the present work such as Macrococcus caseolyticus and Streptococcus macedonicus Functional metagenomics analysis using the bacteriocin mining software BAGEL3, identified 2 ORFs encoding antimicrobial peptides in cheese B and 42 different peptides in sample C. The bacteriocin genes found showed good correlation with taxonomy. Based on the microbial diversity and functional features found through shotgun metagenomic sequencing, a culture-dependent approach was applied aiming to isolate bacteriocin-producing bacteria able to inhibit the growth of the foodborne pathogen Listeria monocytogenes. From 151 bacterial colonies derived from the cheese samples, 10 were associated to high anti-Listeria activity. Based on partial 16S rRNA gene sequencing and RAPD-PCR analysis, all bacteriocinogenic isolates were identified as Enterococcus faecium. Finally, we carried out a pilot experiment with L. monocytogenes-contaminated cheese using one of the enterococcal isolates as a bioprotective adjunct culture. The use of E. faecium CRL1879 during artisanal cheese manufacturing did not alter the main organoleptic properties of the cheese and ensured an efficient control of the foodborne pathogen up to 30 days. This finding supports the use of E. faecium CRL1879 as an adjunct culture in the cheese-making process with a combination of both safety and minimal processing.

The cocoa bean fermentation process: from ecosystem analysis to starter culture development.

Cocoa bean fermentation is still a spontaneous curing process to facilitate drying of nongerminating cocoa beans by pulp removal as well as to stimulate colour and flavour development of fermented dry cocoa beans. As it is carried out on farm, cocoa bean fermentation is subjected to various agricultural and operational practices and hence fermented dry cocoa beans of variable quality are obtained. Spontaneous cocoa bean fermentations carried out with care for approximate four days are characterized by a succession of particular microbial activities of three groups of micro-organisms, namely yeasts, lactic acid bacteria (LAB) and acetic acid bacteria (AAB), which results in well-fermented fully brown cocoa beans. This has been shown through a plethora of studies, often using a multiphasic experimental approach. Selected strains of several of the prevailing microbial species have been tested in appropriate cocoa pulp simulation media to unravel their functional roles and interactions as well as in small plastic vessels containing fresh cocoa pulp-bean mass to evaluate their capacity to dominate the cocoa bean fermentation process. Various starter cultures have been proposed for successful fermentation, encompassing both cocoa-derived and cocoa nonspecific strains of (hybrid) yeasts, LAB and AAB, some of which have been implemented on farms successfully.

Microbial ecology of sourdough fermentations: diverse or uniform?

Sourdough is a specific and stressful ecosystem inhabited by yeasts and lactic acid bacteria (LAB), mainly heterofermentative lactobacilli. On the basis of their inocula, three types of sourdough fermentation processes can be distinguished, namely backslopped ones, those initiated with starter cultures, and those initiated with a starter culture followed by backslopping. Typical sourdough LAB species are Lactobacillus fermentum, Lactobacillus paralimentarius, Lactobacillus plantarum, and Lactobacillus sanfranciscensis. Typical sourdough yeast species are Candida humilis, Kazachstania exigua, and Saccharomyces cerevisiae. Whereas region specificity is claimed in the case of artisan backslopped sourdoughs, no clear-cut relationship between a typical sourdough and its associated microbiota can be found, as this is dependent on the sampling, isolation, and identification procedures. Both simple and very complex consortia may occur. Moreover, a series of intrinsic and extrinsic factors may influence the composition of the sourdough microbiota. For instance, an influence of the flour (type, quality status, etc.) and the process parameters (temperature, pH, dough yield, backslopping practices, etc.) occurs. In this way, the presence of Lb. sanfranciscensis during sourdough fermentation depends on specific environmental and technological factors. Also, Triticum durum seems to select for obligately heterofermentative LAB species. Finally, there are indications that the sourdough LAB are of intestinal origin.

Expression of the arginine deiminase pathway genes in Lactobacillus sakei is strain dependent and is affected by the environmental pH.

The adaptation of Lactobacillus sakei to a meat environment is reflected in its metabolic potential. For instance, the ability to utilize arginine through the arginine deiminase (ADI) pathway, resulting in additional ATP, represents a competitive benefit. In L. sakei CTC 494, the arc operon (arcABCTDR) shows the same gene order and organization as that in L. sakei 23K, the genome sequence of which is known. However, differences in relative gene expression were found, and these seemed to be optimal in different growth phases, namely, the highest relative gene expression level was in the end exponential growth phase in the case of L. sakei CTC 494 and in the mid-exponential growth phase of L. sakei 23K. Also, the environmental pH influenced the relative expression level of the arc operon, as shown for L. sakei CTC 494, with the highest relative expression level occurring at the optimal pH for growth (pH 6.0). Deviations from this optimal pH (pH 5.0 and pH 7.0) resulted in an overall decline of the relative expression level of all genes of the arc operon. Furthermore, a differential relative expression of the individual genes of the arc operon was found, with the highest relative gene expression occurring for the first two genes of the arc operon (arcA and arcB). Finally, it was shown that some L. sakei strains were able to convert agmatine into putrescine, suggesting an operational agmatine deiminase pathway in these strains, a metabolic trait that is undesirable in meat fermentations. This study shows that this metabolic trait is most probably encoded by a previously erroneously annotated second putative arc operon.

Linoleate isomerase activity occurs in lactic acid bacteria strains and is affected by pH and temperature.

AIMS: To investigate the ability of lactic acid bacteria (LAB) to convert linoleic acid (LA) and α-linolenic acid (α-LNA) to conjugated linoleic acid (CLA) and conjugated linolenic acid (CLNA), respectively. To assess pH and temperature influences on CLA and CLNA production by Lactobacillus sakei LMG 13558. METHODS AND RESULTS: A screening of 48 LAB yielded one Lactobacillus curvatus, five Lactobacillus plantarum and four Lact. sakei strains displaying linoleate isomerase (LAI) activity. CLNA conversion percentages varied largely (1-60%). CLA conversion, occurring in three strains, was lower (2-5%). The LAI gene sequences of the ten LAI-positive strains shared 75-99% identity with the LAI gene sequence of a Lact. plantarum AS1.555. At pH 6.2, CLA and CLNA production by Lact. sakei LMG 13558 was higher at 30°C than at 20 and 25°C. At pH 5.5 (30°C) or 37°C (pH 6.2), LA was not converted and α-LNA only slightly converted. CONCLUSIONS: LAB show strain-dependent LAI activity. Production of CLA and CLNA is affected by pH and temperature, as shown for Lact. sakei LMG 13558. SIGNIFICANCE AND IMPACT OF THE STUDY: Several LAB produce CLA and/or CLNA, as shown for Lact. sakei and Lact. curvatus for the first time. These findings offer potential for the manufacturing of fermented functional foods.

Environmental pH determines citrulline and ornithine release through the arginine deiminase pathway in Lactobacillus fermentum IMDO 130101.

Sourdough lactic acid bacteria (LAB) need to be adapted to a highly acidic and, therefore, challenging environment. Different mechanisms are employed to enhance competitiveness, among which conversion of arginine into ornithine through the arginine deiminase (ADI) pathway is an important one. A combined molecular and kinetic approach of the ADI pathway in Lactobacillus fermentum IMDO 130101, a highly competitive sourdough LAB strain, identified mechanisms with advantageous technological effects and quantified the impact of these effects. First, molecular analysis of the arcBCAD operon of 4.8 kb revealed the genes encoding the enzymes ornithine transcarbamoylase, carbamate kinase, arginine deiminase, and an arginine/ornithine (A/O) antiporter, respectively, with an additional A/O antiporter 702.5 kb downstream of the ADI operon. The latter could play a role in citrulline transport. Second, pH-controlled batch fermentations were carried out, generating data for the development of a mathematical model to describe the temporal evolution of the three amino acids involved in the ADI pathway (arginine, citrulline, and ornithine) as a result of the activity of these enzymes and transporter(s). Free arginine in the medium was converted completely into a mixture of citrulline and ornithine under all conditions tested. However, the ratio between these end-products and the pattern of their formation showed variation as a function of environmental pH. Under optimal pH conditions for growth, citrulline release and some further conversion into ornithine was observed. When growing under sub-optimal pH conditions, ornithine was the main product of the ADI pathway. These kinetic data suggest a role in adaptation of L. fermentum IMDO 130101 to growth under sub-optimal conditions.

Biodiversity, ecological determinants, and metabolic exploitation of sourdough microbiota.

Sourdough is a microbial ecosystem of lactic acid bacteria (LAB) and yeasts in a matrix of mainly cereal flour and water. Culture-dependent and culture-independent microbiological analysis together with metabolite target analyses of different sourdoughs enabled to understand this complex fermentation process. It is difficult to link the species diversity of the sourdough microbiota with the (geographical) type of sourdough and the flour used, although the type and quality of the latter is the main source of autochthonous LAB in spontaneous sourdough fermentations and plays a key role in establishing stable microbial consortia within a short time. Carbohydrate fermentation targeted towards maltose catabolism, the use of external alternative electron acceptors, amino acid transamination reactions, and/or the arginine deiminase pathway are metabolic activities that favour energy production, cofactor (re)cycling, and/or tolerance towards acid stress, and hence contribute to the competitiveness and dominance of certain species of LAB found in sourdoughs. Also, microbial interactions play an important role. The availability of genome sequences for several LAB species that are of importance in sourdough as well as technological advances in the fields of functional genomics, transcriptomics, and proteomics enable new approaches to study sourdough fermentations beyond the single species level and will allow an integral analysis of the metabolic activities and interactions taking place in sourdough. Finally, the implementation of selected starter cultures in sourdough technology is of pivotal importance for the industrial production of sourdoughs to be used as flavour carrier, texture-improving, or health-promoting dough ingredient.

De novo KCNQ2 mutations in patients with benign neonatal seizures.

Benign familial neonatal convulsions (BFNC) are characterized by unprovoked seizures during the first weeks of life with spontaneous remission after a few months. Mutations have been identified in the voltage-gated potassium ion channels KCNQ2 and KCNQ3. The authors performed a mutation analysis of KCNQ2 and KCNQ3 in six patients of whom four had no family history of neonatal seizures. The authors identified three KCNQ2 mutations in four patients that all arose de novo.