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.

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.

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.

Variations in Early Response of Grapevine Wood Depending on Wound and Inoculation Combinations with Phaeoacremonium aleophilum and Phaeomoniella chlamydospora.

Defense mechanisms in woody tissue are poorly understood, especially in vine colonized by trunk pathogens. However, several investigations suggest that molecular mechanisms in the central tissue of Vitis vinifera L. may be involved in trunk-defense reactions. In this work, the perception of Phaeoacremonium aleophilum and Phaeomoniella chlamydospora alone or together were investigated in cuttings of Cabernet Sauvignon trunks. Plant responses were analyzed at the tissue level via optical microscopy and at the cellular level via plant-gene expression. The microscopy results revealed that, 6 weeks after pathogen inoculation, newly formed vascular tissue is less developed in plants inoculated with P. chlamydospora than in plants inoculated with P. aleophilum. Co-inoculation with both pathogens resulted in an intermediate phenotype. Further analysis showed the relative expression of the following grapevine genes: PAL, PR10.3, TL, TLb, Vv17.3, STS, STS8, CWinv, PIN, CAM, LOX at 10, 24, 48, and 120 h post-inoculation (hpi). The gene set was induced by wounding before inoculation with the different pathogens, except for the genes CAM and LOX. This response generated significant noise, but the expression of the grapevine genes (PAL, PR10.3, TL, TLb, Vv17.3, STS, STS8, CWinv, and PIN) still differed due to perception of mycelium by the plant. Furthermore, at 48 hpi, the induction of PAL and STS8 differs depending on the pathogen, and a specific pattern emerges from the different inductions associated with the different treatments. Based on these results, we conclude that V. vinifera L. trunk perceives the presence of pathogens differently depending on the inoculated pathogen or even on the combination of co-inoculated pathogens, suggesting a defense orchestration in the perennial organs of woody plants.

A method to detect and quantify Phaeomoniella chlamydospora and Phaeoacremonium aleophilum DNA in grapevine-wood samples.

Grapevines are sensitive to a wide range of fungal pathogens, including agents such as Phaeomoniella chlamydospora and Phaeoacremonium aleophilum that cause tracheomycosis. In the present study, a procedure for DNA extraction from grapevine woody tissue is first evaluated and shown to be suitable for quantitative analysis. Next, a multiplex real-time PCR method targeting the ß-tubulin gene of the pathogens and the actin gene of plant material is developed and its quantitative capability is verified. This protocol was evaluated in inoculated grapevine-wood samples and in young vines from a nursery and was found to be reliable and highly specific. Results obtained from inoculated cuttings show that the fungal colonization process must be considered regardless of the wood phenotype. An analysis of samples of young vines from the nursery shows that a high rate of contamination occurs at the basis of plants and that this contamination is associated with low quantitative values. This finding provides evidence that in vine nurseries, these fungi may be efficient soil-borne pathogens.