New strains are desirable to diversify flavour of fermented dairy products. The objective of this study was to evaluate the potential of Leuconostoc spp. and Lactobacillus spp. in the production of ...aroma compounds by metabolic fingerprints of volatiles. Eighteen strains, including five Lactobacillus species (Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus sakei) and three Leuconostoc species (Leuconostoc citreum, Leuconostoc lactis, and Leuconostoc mesenteroides) were incubated for 5 weeks in a curd-based slurry medium under conditions mimicking cheese ripening. Populations were enumerated and volatile compounds were analysed by headspace trap gas chromatography–mass spectrometry (GC–MS). A metabolomics approach followed by multivariate statistical analysis was applied for data processing and analysis. In total, 12 alcohols, 10 aldehydes, 7 esters, 11 ketones, 5 acids and 2 sulphur compounds were identified. Very large differences in concentration of volatile compounds between the highest producing strains and the control medium were observed in particular for diacetyl, 2-butanol, ethyl acetate, 3-methylbutanol, 3-methylbutanoic acid and 2-methylbutanoic acid. Some of the characterized strains demonstrated an interesting aromatizing potential to be used as adjunct culture.
The aim of this study was to explore diversity and dynamic of indigenous LAB strains associated with a long ripened hard cheese produced from raw milk and undefined natural starter such as PDO Grana ...Padano cheese. Samples of milk, curd, natural whey culture and cheeses (2nd, 6th, 9th and 13th months of ripening) were collected from 6 cheese factories in northern Italy. DNA was extracted from each sample and from 194 LAB isolates. tRNAAla-23S rDNA-RFLP was applied to identify isolates. Strain diversity was assessed by (GTG)5 rep-PCR and RAPD(P1)-PCR. Finally, culture-independent LH-PCR (V1–V2 16S-rDNA), was considered to explore structure and dynamic of the microbiota. Grana Padano LAB were represented mainly by Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus delbrueckii, Lactobacillus helveticus and Pediococcus acidilactici, while the structure and dynamic of microbiota at different localities was specific. The strength of this work is to have focused the study on isolates coming from more than one cheese factories rather than a high number of isolates from one unique production. We provided a valuable insight into inter and intraspecies diversity of typical LAB strains during ripening of traditional PDO Grana Padano, contributing to the understanding of specific microbial ecosystem of this cheese.
•Polyphasic molecular approach was applied to identify LAB associated with GP cheese.•CD approach revealed higher biodiversity.•CI approach provided better insight into population dynamic.•Lactobacillus rhamnosus was found to be the predominant isolate in cheese.•For the first time, Pediococcus acidilactici, was isolated from GP NWC.
The aim of the study was to evaluate intercluster and intracluster phenotypic and genetic diversity of Leuconostoc spp. PFGE and RAPD-PCR were used to evaluate molecular diversity. Carbohydrate ...metabolism of the isolates was evaluated using API 50CH. The study demonstrated that genetically closely related isolates will not necessarily demonstrate the same phenotypic relatedness, indicating that phenotypic traits are isolate specific. Therefore, genetic selection of isolates by clustering for further phenotypic characterisation of representatives, might lead to loss of important phenotypic diversity. Furthermore, PFGE was shown to be an efficient molecular tool for the differentiation of Leuconostoc mesenteroides subsp. cremoris and Leuconostoc pseudomesenteroides, that has not been reported previously. Additionally, amygdaline, arbutine, l-arabinose and mannitol can be important fermentation traits for phenotypic differentiation of Ln. mesenteroides subsp. cremoris and Ln. pseudomesenteroides.
The characteristics of bacterial populations in raw milk at the time of processing has a significant influence on shelf-life, organoleptic quality, spoilage and yields of raw milk, processed milk as ...well as on the other dairy products. Unfortunately, cold and extended storage of raw milk, as a common practice in dairy sector today, favour the growth of psychrotrophic bacteria. Therefore, their count in the refrigerated milk is more than the ideal limit of 10 % of the mesophilic count. Psychrotrophic bacteria are generally able to form extracellular or intracellular thermo-resistant enzymes (proteases, lipases, phospolipases) which can contribute to milk and dairy products spoilage. In addition, besides exhibiting spoilage features, some species belonging to the psychrotrops are considered as emerging pathogens that carry innate resistance to antibiotics or produce toxins. In sense of quality, psychrotrophic bacteria have become major problem for today’s dairy industry as leading cause in spoilage of cold-storage milk and dairy products. This review article focuses on the impact of psychrotrops on quality problems associated with raw milk as well as on th final dairy products. Means of controlling the dominant psychrotrophic species responsible for undesirable activities in milk and dairy products were also discussed.
Microorganisms play an important role in the development of cheese flavor. The aim of this study was to develop an approach to facilitate screening of various cheese-related bacteria for their ...ability to produce aroma compounds. We combined i) curd-based slurry medium incubated under conditions mimicking cheese manufacturing and ripening, ii) powerful method of extraction of volatiles, headspace trap, coupled to gas chromatography-mass spectrometry (HS-trap-GC-MS), and iii) metabolomics-based method of data processing using the XCMS package of R software and multivariate analysis. This approach was applied to eleven species: five lactic acid bacteria (Leuconostoc lactis, Lactobacillus sakei, Lactobacillus paracasei, Lactobacillus fermentum, and Lactobacillus helveticus), four actinobacteria (Brachybacterium articum, Brachybacterium tyrofermentans, Brevibacterium aurantiacum, and Microbacterium gubbeenense), Propionibacterium freudenreichii, and Hafnia alvei. All the strains grew, with maximal populations ranging from 7.4 to 9.2 log (CFU/mL). In total, 52 volatile aroma compounds were identified, of which 49 varied significantly in abundance between bacteria. Principal component analysis of volatile profiles differentiated species by their ability to produce ethyl esters (associated with Brachybacteria), sulfur compounds and branched-chain alcohols (H. alvei), branched-chain acids (H. alvei, P. freudenreichii and L. paracasei), diacetyl and related carbonyl compounds (M. gubbeenense and L. paracasei), among others.
•An approach suitable to evaluate the aroma potential of very diverse bacteria.•HS-trap/GC-MS method demonstrated to be efficient tool for volatile extraction.•Metabolomics-based workflow of data processing using XCMS facilitates data analysis.
Flavor is an important sensory property of fermented food products, including cheese, and largely results from the production of aroma compounds by microorganisms. Propionibacterium freudenreichii is ...the most widely used species of dairy propionibacteria; it has been implicated in the production of a wide variety of aroma compounds through multiple metabolic pathways and is associated with the flavor of Swiss cheese. However, the ability of other dairy propionibacteria to produce aroma compounds has not been characterized. This study sought to elucidate the effect of interspecies and intraspecies diversity of dairy propionibacteria on the production of aroma compounds in a cheese context. A total of 76 strains of Propionibacterium freudenreichii, Propionibacterium jensenii, Propionibacterium thoenii, and Propionibacterium acidipropionici were grown for 15days in pure culture in a rich medium derived from cheese curd. In addition, one strain each of two phylogenetically related non-dairy propionibacteria, Propionibacterium cyclohexanicum and Propionibacterium microaerophilum were included. Aroma compounds were analyzed using headspace trap-gas chromatography-mass spectrometry (GC–MS). An analysis of variance performed on GC–MS data showed that the abundance of 36 out of the 45 aroma compounds detected showed significant differences between the cultures. A principal component analysis (PCA) was performed for these 36 compounds. The first two axes of the PCA, accounting for 60% of the variability between cultures, separated P. freudenreichii strains from P. acidipropionici strains and also differentiated P. freudenreichii strains from each other. P. freudenreichii strains were associated with greater concentrations of a variety of compounds, including free fatty acids from lipolysis, ethyl esters derived from these acids, and branched-chain acids and alcohols from amino acid catabolism. P. acidipropionici strains produced less of these compounds but more sulfur-containing compounds from methionine catabolism. Meanwhile, branched-chain aldehydes and benzaldehyde were positively associated with certain strains of P. jensenii and P. thoenii. Moreover, the production of compounds with a common origin was correlated. Compound abundance varied significantly by strain, with fold changes between strains of the same species as high as in the order of 500 for a single compound. This suggests that the diversity of dairy propionibacteria can be exploited to modulate the flavor of mild cheeses.
•78 propionibacteria strains screened for their production of cheese aroma compounds•Aroma compounds produced from fermentation, amino acid catabolism and lipolysis•P. freudenreichii strains associated with greater diversity of aroma compounds•Great variation between strains, with specific features for each species•Dairy propionibacteria have the potential to modulate the flavor of mild cheeses.
Kefir grains represent the unique microbial community consisting of bacteria, yeasts, and sometimes filamentous moulds creating complex symbiotic community. The complexity of their physical and ...microbial structures is the reason that the kefir grains are still not unequivocally elucidated. Microbiota of kefir grains has been studied by many microbiological and molecular approaches. The development of metagenomics, based on the identification without cultivation, is opening new possibilities for identification of previously nonisolated and non-identified microbial species from the kefir grains. Considering recent studies, there are over 50 microbial species associated with kefir grains. The aim of this review is to summarise the microbiota composition of kefir grains. Moreover, because of technological and microbiological significance of the kefir grains, the paper provides an insight into the microbiological and molecular methods applied to study microbial biodiversity of kefir grains.
Culture-independent molecular tools have been introduced into food microbiology during the last ten years. Most of them are based on the amplification of a bulk bacterial DNA extracted directly from ...a sample, the targeting of a selected gene, or a variable region of the selected gene. Many studies have explored indigenous lactic acid bacteria in dairy products by culture-independent molecular approaches. It is well known that indigenous microbiota significantly contribute to the uniqueness of artisanal cheeses. However, there is no molecular method that can provide complete qualitative and quantitative insight into the microbiota associated with a certain ecosystem. Therefore, a combination of molecular approaches should be applied to get a more objective picture of the microbiota. This paper aims to present the most widely used culture-independent molecular tools for identifying lactic acid bacteria in dairy products. Key words: culture-independent identification, molecular methods, lactic acid bacteria, dairy products
Karakačanski skakutanac is an artisanal fresh sheep cheese produced on a small scale in a limited area of eastern Croatia. It is manufactured from unrefrigerated raw sheep milk immediately after ...milking, without the addition of starter culture, and coagulated with industrial rennet. To date, no microbiological or molecular characterization of the biodiversity of the microbiota has been performed. The objective of this study was to obtain an initial insight into the biodiversity of the microbial community associated with this cheese during the production season and shelf life period. Eleven cheeses were obtained from a dairy farm at 14-day intervals during the lactation period of east Friesian sheep in 2007. Bacterial DNA was isolated directly from cheese on the first, second and third day of the cheese shelf life, resulting in a total of 33 DNA samples. Extracted DNA was used as a template for PCR-ARDRA and PCR-DGGE analysis. The use of dual culture-independent approaches revealed similar results and indicated predominance of Lactococcus lactis.
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