Microbial Dynamics during labneh Ambaris Production in Earthenware Jars.

The responses of various microbial populations to modifications in the physicochemical properties of a food matrix, as well as interactions between these populations already present, are the main factors that shape microbial dynamics in that matrix. This work focused on the study of microbial dynamics during labneh Ambaris production, a traditional Lebanese concentrated fermented goat milk made in jars during 3 months. This was assessed in two earthenware jars at a production facility. DNA metabarcoding of the ITS2 region as well as the V3-V4 region of the 16S rRNA gene was used to characterize the fungal and bacterial communities, respectively. Viable bacterial isolates were also identified by Sanger sequencing of the V1-V4 region of the 16S rRNA gene. Our results showed that the dominant microorganisms identified within labneh Ambaris (Lactobacillus kefiranofaciens, Lentilactobacillus kefiri, Lactococcus lactis, Geotrichum candidum, Pichia kudriavzevii and Starmerella sp.) settle early in the product and remain until the end of maturation with varying abundances throughout fermentation. Microbial counts increased during early fermentation stage, and remained stable during mid-fermentation, then declined during maturation. While microbial compositions were globally comparable between the two jars during mid-fermentation and maturation stages, differences between the two jars were mainly detected during early fermentation stage (D0 until D10). No significant sensorial differences were observed between the final products made in the two jars. Neither coliforms nor Enterobacteriaceae were detected in their viable state, starting D7 in both jars, suggesting the antimicrobial properties of the product.

Microbial communities and main features of labneh Ambaris, a traditional Lebanese fermented goat milk product.

Labneh Ambaris is a traditional Lebanese dairy product typically made using goat milk in special earthenware jars. Its production is characterized by the regular additions of milk and coarse salt, all while draining the whey throughout a process that lasts for a minimum of 2 mo. In this study, 20 samples of labneh Ambaris, all produced by spontaneous fermentation, were studied. They were collected at the end of fermentation from different regions in Lebanon. Physicochemical and sensory properties were studied and microbial diversity was analyzed using culture-dependent and independent techniques. The V3-V4 region of the 16S rRNA gene and the ITS2 region were sequenced by DNA metabarcoding analyses for the identification of bacteria and yeast communities, respectively. Out of 160 bacterial and 36 fungal taxa, 117 different bacterial species and 24 fungal species were identified among all labneh Ambaris samples studied. The remaining ones were multi-affiliated and could not be identified at the species level. Lactobacillus was the dominant bacterial genus, followed by Lentilactobacillus, Lactiplantibacillus, Lacticaseibacillus, and Lactococcus genera, whereas Geotrichum and Pichia were the dominant fungal genera. The 20 samples tested had varying levels of salt, protein, and fat contents, but they were all highly acidic (mostly having a pH < 4). According to the sensory scores generated by classical descriptive analysis, all samples were described as having basic similar characteristics such as goat smell and flavor, but they could be differentiated based on various intensities within the same descriptors like salty and acidic. This work could be considered as a base toward obtaining a quality label for labneh Ambaris.

Artisanal Household Milk Pasteurization Is Not a Determining Factor in Structuring the Microbial Communities of Labneh Ambaris: A Pilot Study.

Labneh Ambaris is a traditional Lebanese dairy product traditionally made using raw goat's milk in earthenware jars, but recently the use of artisanally pasteurized milk was introduced for safety reasons. In this study, 12 samples of labneh Ambaris were studied, six made using raw goat's milk and six others using artisanally pasteurized goat's milk. These samples were collected during fermentation and their microbial compositions were analyzed. The 16S V3-V4 and the ITS2 regions of the rDNA were sequenced by DNA metabarcoding analyses for the identification and comparison of bacterial and fungal communities, respectively. The samples had high microbial diversity but differences in samples microbiota were unrelated to whether or not milk was pasteurized. The samples were consequently clustered on the basis of their dominant bacterial or fungal species, regardless of the milk used. Concerning bacterial communities, samples were clustered into 3 groups, one with a higher abundance of Lactobacillus helveticus, another with Lactobacillus kefiranofaciens as the dominant bacterial species, and the third with Lentilactobacillus sp. as the most abundant species. Species belonging to the Enterobacteriaceae family were detected in higher abundance in all raw milk samples than in artisanally pasteurized milk samples. As for fungal communities, the samples were clustered into two groups, one dominated by Geotrichum candidum and the other by Pichia kudriavzevii.

Harnessing diversity of Lactococcus lactis from raw goat milk: Design of an indigenous starter for the production of Rocamadour, a French PDO cheese.

Twenty-four strains of Lactococcus lactis isolated from raw goat milk collected in the Rocamadour PDO area were analysed by MLST typing and phenotypic characterisation. The strains were combined to design an indigenous starter for the production of Rocamadour PDO cheese. The strains were divided into three classes based on their technological properties: acidifying and proteolytic strains in class I (12/24 strains), slightly acidifying and non-proteolytic strains in class II (2/24 strains), and non-acidifying and non-proteolytic strains in class III (10/24 strains). Interestingly, all but three strains (21/24) produced diacetyl/acetoin despite not having citrate metabolism genes, as would classically be expected for the production of these aroma compounds. Three strains (EIP07A, EIP13D, and EIP20B) were selected for the indigenous starter based on the following inclusion/exclusion criteria: (i) no negative interactions between included strains, (ii) ability to metabolize lactose and at least one strain with the prtP gene and/or capable of producing diacetyl/acetoin, and (iii) selected strains derived from different farms to maximise genetic and phenotypic diversity. Despite consisting exclusively of L. lactis strains, the designed indigenous starter allowed reproducible cheese production with performances similar to those obtained with an industrial starter and with the sensory qualities expected of Rocamadour PDO cheese.

Quality assessment of artisanal cheeses in the northwest region of Paraná.

Artisanal unripened cheeses produced in northwestern Paraná, Brazil, were studied for microbiological quality and sensory quality. The cheeses analyzed showed high counts of aerobic mesophilic microorganisms and S. aureus. However, even with the results showing poor microbiological quality, from a sensory point of view, consumers considered cheeses acceptable (high acceptance index). The results may indicate that there is still a lack of training and knowledge of production procedures to reduce microbiological contamination of artisanal cheese produced in northwestern Paraná.

Precise Populations' Description in Dairy Ecosystems Using Digital Droplet PCR: The Case of L. lactis Group in Starters.

Lactococcus lactis group (composed of the lactis and cremoris subspecies, recently reassigned as two distinct species) plays a major role in dairy fermentations. Usually present in starter cultures, the two species enable efficient acidification and improve the organoleptic qualities of the final product. Biovar diacetylactis strains produce diacetyl and acetoin, aromas from the citrate metabolization. As these populations have distinct genomic and phenotypic characteristics, the proportions of each other will affect the final product. Today, there is no quantitative test able to distinguish between the two species and the biovar in dairy ecosystems. In this study, we developed a specific, reliable, and accurate strategy to quantify these populations using, species-, and diacetylactis-specific fluorescent probes in digital droplet PCR assays (ddPCR). Species were distinguished based on three single nucleotide polymorphisms in the glutamate decarboxylase gadB gene, and the citD gene involved in citrate metabolism was used to target the biovar. Used in duplex or singleplex, these probes made it possible to measure the proportion of each population. At 59°C, the probes showed target specificity and responded negatively to the non-target species usually found in dairy environments. Depending on the probe, limit of detection values in milk matrix ranged from 3.6 × 103 to 1.8 × 104 copies/ml. The test was applied to quantify sub-populations in the L. lactis group during milk fermentation with a commercial starter. The effect of temperature and pH on the balance of the different populations was pointed out. At the initial state, lactis and cremoris species represent, respectively, 75% and 28% of the total L. lactis group and biovar diacetylactis strains represent 21% of the lactis species strains. These ratios varied as a function of temperature (22°C or 35°C) and acidity (pH 4.5 or 4.3) with cremoris species promoted at 22°C and pH4.5 compared to at 35°C. The biovar diacetylactis strains were less sensitive to acid stress at 35°C. This methodology proved to be useful for quantifying lactis and cremoris species and biovar diacetylactis, and could complete 16S metagenomics studies for the deeply description of L. lactis group in complex ecosystems.

Identification of lactic acid bacteria Enterococcus and Lactococcus by near-infrared spectroscopy and multivariate classification.

Lactic acid bacteria are important in numerous biological processes. The fabrication of cheese, for example, uses the lactic acid bacteria found in raw milk such as Lactococcus lactis as starters to improve the organoleptic properties of milk. Conventional methods to determine the genus and species of lactic acid bacteria isolated from raw milk involve genotyping and phenotyping, which require specific preparation and sample destruction. To improve on this situation, we present herein a simple and non-destructive screening method to discriminate between the Lactococcus and Enterococcus species most commonly found in raw milk (L. lactis, E. durans, E. faecalis, and E. faecium). The bacteria are grown on agar plates and assessed by using near-infrared spectroscopy in a spectral range from 800 to 2777 nm. Principle component analysis loading line plots highlight the inter-genus and inter-species differences at various wavelengths, which are mostly assigned to cell-wall compounds such as polysaccharides. The best artificial neural network identification models give 98.8% and 86.3% classification rates at the genus and species level, respectively, for an external validation set made of 80 samples. These results suggest that near-infrared spectroscopy may be used to identify lactic acid bacteria on agar medium.

Effects of thermized donkey milk with lysozyme activity on altered gut barrier in mice exposed to water-avoidance stress.

Nutrition plays a crucial role in human gut health through the improvement of gut barrier functionality. Donkey milk represents an interesting source of natural antimicrobial factors such as lysozyme. Recently, anti-inflammatory properties of donkey milk lysozyme activity were described in a mouse model of ileitis. The current increase of donkey milk consumption highlights the necessity to propose a healthy milk compliant with microbiological standards. This study aims to define a heat treatment of donkey milk, retaining its high lysozyme activity, and to evaluate its beneficial effects on a gut barrier impairment model due to chronic stress in mice. To perform this experiment, samples of raw donkey milk were collected in 15 distinct French farms. Microbiological analysis and lysozyme content and activity were evaluated for each sample. Then, several heat treatments were carried out to define a time and temperature combination that allowed for both a reduction in the number of total micro-organisms, increasing the shelf-life of the product, and preservation of lysozyme activity. The beneficial effect of heated donkey milk on the gut barrier of mice was evaluated and compared with raw donkey milk. We found that samples of raw donkey milk showed low total mesophilic microbial counts, and no pathogens were detected. Among the different heat-treatment procedures tested, a 2-min, 72°C combination was determined to be the most optimal time and temperature combination to preserve lysozyme activity and increase the shelf-life of donkey milk. Oral administration of this heat-treated donkey milk in mice counteracted chronic stress-induced intestinal damage, illustrated by gut hyper-permeability and low-grade inflammation, similar to raw donkey milk. We have demonstrated for the first time that oral intervention with donkey milk, optimally heat-treated to retain enzymatic lysozyme activity, improves intestinal barrier damage linked to psychological stress in mice.

Donkey milk consumption exerts anti-inflammatory properties by normalizing antimicrobial peptides levels in Paneth's cells in a model of ileitis in mice.

PURPOSE: In this study, we showed the beneficial effects of donkey milk (DM) on inflammatory damages, endogenous antimicrobial peptides levels and fecal microbiota profile in a mice model of Crohn's disease. Nowadays, new strategies of microbiome manipulations are on the light involving specific diets to induce and/or to maintain clinical remission. Interest of DM is explained by its high levels of antimicrobial peptides which confer it anti-inflammatory properties. METHODS: C57BL/6 mice were orally administered with or without indomethacin for 5 days and co-treated with vehicle, DM or heated DM during 7 days. Intestinal length and macroscopic damage scores (MDSs) were determined; ileal samples were taken off for microscopic damage (MD), lysozyme immunostaining and mRNA α-defensin assessments. Ileal luminal content and fecal pellets were collected for lysozyme enzymatic activity and lipocalin-2 (LCN-2) evaluations. Fecal microbiota profiles were compared using a real-time quantitative PCR-based analysis. RESULTS: Administration of indomethacin caused an ileitis in mice characterized by (1) a decrease in body weight and intestinal length, (2) a significant increase in MDS, MD and LCN-2, (3) a reduction in both α-defensin mRNA expression and lysozyme levels in Paneth's cells reflected by a decrease in lysozyme activity in feces, and (4) a global change in relative abundance of targeted microbial communities. DM treatment significantly reduced almost of all these ileitis damages, whereas heated DM has no impact on ileitis. CONCLUSIONS: DM consumption exerts anti-inflammatory properties in mice by restoring the endogenous levels of antimicrobial peptides which contribute in turn to reduce microbiota imbalance.

From Genome to Phenotype: An Integrative Approach to Evaluate the Biodiversity of Lactococcus lactis.

Lactococcus lactis is one of the most extensively used lactic acid bacteria for the manufacture of dairy products. Exploring the biodiversity of L. lactis is extremely promising both to acquire new knowledge and for food and health-driven applications. L. lactis is divided into four subspecies: lactis, cremoris, hordniae and tructae, but only subsp. lactis and subsp. cremoris are of industrial interest. Due to its various biotopes, Lactococcus subsp. lactis is considered the most diverse. The diversity of L. lactis subsp. lactis has been assessed at genetic, genomic and phenotypic levels. Multi-Locus Sequence Type (MLST) analysis of strains from different origins revealed that the subsp. lactis can be classified in two groups: "domesticated" strains with low genetic diversity, and "environmental" strains that are the main contributors of the genetic diversity of the subsp. lactis. As expected, the phenotype investigation of L. lactis strains reported here revealed highly diverse carbohydrate metabolism, especially in plant- and gut-derived carbohydrates, diacetyl production and stress survival. The integration of genotypic and phenotypic studies could improve the relevance of screening culture collections for the selection of strains dedicated to specific functions and applications.

Phenotypic and genotypic characterization of lactic acid bacteria isolated from raw goat milk and effect of farming practices on the dominant species of lactic acid bacteria.

Lactic acid bacteria, in particular Lactococcus lactis, play a decisive role in the cheese making process and more particularly in lactic cheeses which are primarily produced on goat dairy farms. The objective of this study was therefore to identify the main lactic acid bacteria found in raw goats' milk from three different regions in France and evaluate if certain farming practices have an effect on the distribution of species of lactic acid bacteria in the various milk samples. Identification at genus or species level was carried out using phenotypic tests and genotypic methods including repetitive element REP-PCR, species-specific PCR and 16S rRNA gene sequencing. The distribution of the main bacterial species in the milk samples varied depending on farms and their characteristics. Out of the 146 strains identified, L. lactis was the dominant species (60% of strains), followed by Enterococcus (38%) of which Enterococcus faecalis and Enterococcus faecium. Within the species L. lactis, L. lactis subsp lactis was detected more frequently than L. lactis subsp cremoris (74% vs. 26%). The predominance of L. lactis subsp cremoris was linked to geographical area studied. It appears that the animals' environment plays a role in the balance between the dominance of L. lactis and enterococci in raw goats' milk. The separation between the milking parlor and the goat shed (vs no separation) and only straw in the bedding (vs straw and hay) seems to promote L. lactis in the milk (vs enterococci).