scholarly journals Bifidobacterial Distribution Across Italian Cheeses Produced from Raw Milk

2019 ◽  
Vol 7 (12) ◽  
pp. 599 ◽  
Author(s):  
Christian Milani ◽  
Giulia Alessandri ◽  
Leonardo Mancabelli ◽  
Gabriele Andrea Lugli ◽  
Giulia Longhi ◽  
...  
Keyword(s):  
Raw Milk ◽  
Fermented Food ◽  
Species Level ◽  
Rrna Gene ◽  
Bacterial Populations ◽  
Microbial Profiling ◽  
Interactive Network ◽  
Microbe Interactions ◽  
Internally Transcribed Spacer ◽  
Species Specific

Cheese microbiota is of high industrial relevance due to its crucial role in defining the organoleptic features of the final product. Nevertheless, the composition of and possible microbe–microbe interactions between these bacterial populations have never been assessed down to the species-level. For this reason, 16S rRNA gene microbial profiling combined with internally transcribed spacer (ITS)-mediated bifidobacterial profiling analyses of various cheeses produced with raw milk were performed in order to achieve an in-depth view of the bifidobacterial populations present in these microbially fermented food matrices. Moreover, statistical elaboration of the data collected in this study revealed the existence of community state types characterized by the dominance of specific microbial genera that appear to shape the overall cheese microbiota through an interactive network responsible for species-specific modulatory effects on the bifidobacterial population.

scholarly journals Ecology of Lactobacilli Present in Italian Cheeses Produced from Raw Milk

2020 ◽  
Vol 86 (12) ◽  
Author(s):  
Christian Milani ◽  
Federico Fontana ◽  
Giulia Alessandri ◽  
Leonardo Mancabelli ◽  
Gabriele Andrea Lugli ◽  
...  
Keyword(s):  
Raw Milk ◽  
Species Level ◽  
Starter Cultures ◽  
Rrna Gene ◽  
Functional Studies ◽  
Internally Transcribed Spacer ◽  
Main Components ◽  
Species Specific ◽  
Negative Interactions

ABSTRACT Among the bacterial genera that are used for cheese production, Lactobacillus is a key taxon of high industrial relevance that is commonly present in commercial starter cultures for dairy fermentations. Certain lactobacilli play a defining role in the development of the organoleptic features during the ripening stages of particular cheeses. We performed an in-depth 16S rRNA gene-based microbiota analysis coupled with internally transcribed spacer-mediated Lactobacillus compositional profiling of 21 common Italian cheeses produced from raw milk in order to evaluate the ecological distribution of lactobacilli associated with this food matrix. Statistical analysis of the collected data revealed the existence of putative Lactobacillus community state types (LCSTs), which consist of clusters of Lactobacillus (sub)species. Each LCST is dominated by one or two taxa that appear to represent keystone elements of an elaborate network of positive and negative interactions with minor components of the cheese microbiota. The results obtained in this study reveal the existence of peculiar cheese microbiota assemblies that represent intriguing targets for further functional studies aimed at dissecting the species-specific role of bacteria in cheese manufacturing. IMPORTANCE The microbiota is known to play a key role in the development of the organoleptic features of dairy products. Lactobacilli have been reported to represent one of the main components of the nonstarter bacterial population, i.e., bacteria that are not deliberately added to the milk, harbored by cheese, although the species-level composition of this microbial population has never been assessed in detail. In the present study, we applied a recently developed metagenomic approach that employs an internally transcribed spacer to profile the Lactobacillus population harbored by cheese produced from raw milk at the (sub)species level. The obtained data revealed the existence of particular Lactobacillus community state types consisting of clusters of Lactobacillus (sub)species that tend to cooccur in the screened cheeses. Moreover, analysis of covariances between members of this genus indicate that these taxa form an elaborate network of positive and negative interactions that define specific clusters of covariant lactobacilli.

scholarly journals Deciphering the Bifidobacterial Populations within the Canine and Feline Gut Microbiota

2020 ◽  
Vol 86 (7) ◽  
Author(s):  
Giulia Alessandri ◽  
Christian Milani ◽  
Leonardo Mancabelli ◽  
Giulia Longhi ◽  
Rosaria Anzalone ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Companion Animals ◽  
Well Being ◽  
Research Area ◽  
Species Level ◽  
Species Number ◽  
Rrna Gene ◽  
Social Relevance ◽  
Fecal Samples ◽  
Internally Transcribed Spacer

ABSTRACT During the course of evolution, dogs and cats have been subjected to extensive domestication, becoming the principal companion animals for humans. For this reason, their health care, including their intestinal microbiota, is considered of considerable importance. However, the canine and feline gut microbiota still represent a largely unexplored research area. In the present work, we profiled the microbiota of 23 feline fecal samples by 16S rRNA gene and bifidobacterial internally transcribed spacer (ITS) approaches and compared this information with previously reported data from 138 canine fecal samples. The obtained data allowed the reconstruction of the core gut microbiota of the above-mentioned samples coupled with their classification into distinct community state types at both genus and species levels, identifying Bacteroides, Fusobacterium, and Prevotella 9 as the main bacterial components of the canine and feline gut microbiota. At the species level, the intestinal bifidobacterial gut communities of dogs and cats differed in terms of both species number and composition, as emphasized by a covariance analysis. Together, our findings show that the intestinal populations of cats and dogs are similar in terms of genus-level taxonomical composition, while at the bifidobacterial species level, clear differences were observed, indicative of host-specific colonization behavior by particular bifidobacterial taxa. IMPORTANCE Currently, domesticated dogs and cats are the most cherished companion animals for humans, and concerns about their health and well-being are therefore important. In this context, the gut microbiota plays a crucial role in maintaining and promoting host health. However, despite the social relevance of domesticated dogs and cats, their intestinal microbial communities are still far from being completely understood. In this study, the taxonomical composition of canine and feline gut microbiota was explored at genus and bifidobacterial species levels, allowing classification of these microbial populations into distinct gut community state types at either of the two investigated taxonomic levels. Furthermore, the reconstruction of core gut microbiota coupled with covariance network analysis based on bifidobacterial internally transcribed spacer (ITS) profiling revealed differences in the bifidobacterial compositions of canine and feline gut microbiota, suggesting that particular bifidobacterial species have developed a selective ability to colonize a specific host.

Detection of Antifungal Properties in Lactobacillus paracasei subsp. paracasei SM20, SM29, and SM63 and Molecular Typing of the Strains

2005 ◽  
Vol 68 (1) ◽  
pp. 111-119 ◽  
Author(s):  
SUSANNE MIESCHER SCHWENNINGER ◽  
UELI VON AH ◽  
BRIGITTE NIEDERER ◽  
MICHAEL TEUBER ◽  
LEO MEILE
Keyword(s):  
Molecular Typing ◽  
Raw Milk ◽  
Rrna Gene ◽  
Sequencing Analysis ◽  
Candida Spp ◽  
Food And Feed ◽  
Carbohydrate Fermentation ◽  
Typing Methods ◽  
Raw Milk Cheese ◽  
Species Specific

Lactobacilli isolated from different food and feed samples such as raw milk, cheese, yoghurt, olives, sour dough, as well as corn and grass silage, were screened for their antifungal activities. Out of 1,424 isolates tested, 82 were shown to be inhibitory to different yeasts (Candida spp. and Zygosaccharomyces bailii) and a Penicillium sp., which were previously isolated from spoiled yoghurt and fruits. Carbohydrate fermentation patterns suggested that a substantial portion, 25%, belonged to the Lactobacillus casei group, including L. casei, L. paracasei, and L. rhamnosus. The isolates SM20 (DSM14514), SM29 (DSM14515), and SM63 (DSM14516) were classified by PCR using species-specific primers to target the corresponding type strains (L. casei, L. paracasei, and L. rhamnosus) as controls. Further molecular typing methods such as randomly amplified polymorphic DNA, pulsed-field gel electrophoresis, and sequencing analysis of the 16S rRNA gene allowed classifying strains SM20, SM29, and SM63 as L. paracasei subsp. paracasei in accordance with the new reclassification of the L. casei group proposed by Collins et al. (Int. J. Syst. Bacteriol. 39:105–108).

scholarly journals Use of DNA Quantification To Measure Growth and Autolysis of Lactococcus and Propionibacterium spp. in Mixed Populations

2006 ◽  
Vol 72 (9) ◽  
pp. 6174-6182 ◽  
Author(s):  
Janneke Treimo ◽  
Gerd Vegarud ◽  
Thor Langsrud ◽  
Knut Rudi
Keyword(s):  
Dna Degradation ◽  
Rrna Gene ◽  
Competitive Pcr ◽  
Single Nucleotide ◽  
Bacterial Populations ◽  
Cheese Ripening ◽  
Mixed Populations ◽  
Species Specific ◽  
Cheese Model ◽  
Cheese Production

ABSTRACT Autolysis is self-degradation of the bacterial cell wall that results in the release of enzymes and DNA. Autolysis of starter bacteria, such as lactococci and propionibacteria, is essential for cheese ripening, but our understanding of this important process is limited. This is mainly because the current tools for measuring autolysis cannot readily be used for analysis of bacteria in mixed populations. We have now addressed this problem by species-specific detection and quantification of free DNA released during autolysis. This was done by use of 16S rRNA gene single-nucleotide extension probes in combination with competitive PCR. We analyzed pure and mixed populations of Lactococcus lactis subsp. lactis and three different species of Propionibacterium. Results showed that L. lactis subsp. lactis INF L2 autolyzed first, followed by Propionibacterium acidipropionici ATCC 4965, Propionibacterium freudenreichii ISU P59, and then Propionibacterium jensenii INF P303. We also investigated the autolytic effect of rennet (commonly used in cheese production). We found that the effect was highly strain specific, with all the strains responding differently. Finally, autolysis of L. lactis subsp. lactis INF L2 and P. freudenreichii ISU P59 was analyzed in a liquid cheese model. Autolysis was detected later in this cheese model system than in broth media. A challenge with DNA, however, is DNA degradation. We addressed this challenge by using a DNA degradation marker. We obtained a good correlation between the degradation of the marker and the target in a model experiment. We conclude that our DNA approach will be a valuable tool for use in future analyses and for understanding autolysis in mixed bacterial populations.

scholarly journals Bacterial Succession through the Artisanal Process and Seasonal Effects Defining Bacterial Communities of Raw-Milk Adobera Cheese Revealed by High Throughput DNA Sequencing

2020 ◽  
Vol 9 (1) ◽  
pp. 24
Author(s):  
José M. Ruvalcaba-Gómez ◽  
Raúl J. Delgado-Macuil ◽  
Lily X. Zelaya-Molina ◽  
Otoniel Maya-Lucas ◽  
Edmundo Ruesga-Gutiérrez ◽  
...  
Keyword(s):  
High Throughput ◽  
Manufacturing Process ◽  
High Throughput Sequencing ◽  
Raw Milk ◽  
Seasonal Effects ◽  
Rrna Gene ◽  
Process Standardization ◽  
Bacterial Populations ◽  
Different Seasons ◽  
Key Steps

The bacterial community of the artisanal Adobera cheese from Los Altos de Jalisco was described through high-throughput sequencing of 16S rRNA gene libraries. Samples were collected in two different seasons (dry and rainy) during four key steps of the manufacturing process (raw milk, fresh curd, matured curd, and cheese). Bacterial diversity was higher in early steps in comparison with the final elaboration stages. Firmicutes and Proteobacteria were the most abundant phyla, strongly represented by the Streptococcaceae, Enterobacteriaceae and Lactobacillaceae families, and core bacteria genera such as Streptococcus spp., Lactococcus spp., and Lactobacillus spp. Undesirable bacteria, including Pseudomonas spp. and Acinetobacter spp., were also detected in raw milk but almost undetectable at the end of the cheese manufacturing process, and seemed to be displaced by lactic-acid bacteria-related genera. Seasonal effects were observed on the community structure but did not define the core microbiota composition. Predictive metabolism was related to membrane transport, and amino-acid, lipid, and carbohydrate metabolism pathways. Our results contribute to deduce the role of bacteria involved in Adobera cheese manufacturing in terms of the metabolism involved, cheese microbial safety, and how undesirable bacterial populations could be regulated by process standardization as a potential tool to improve safety.

scholarly journals Species-resolved sequencing of low-biomass microbiomes by 2bRAD-M

2020 ◽  
Author(s):  
Zheng Sun ◽  
Shi Huang ◽  
Pengfei Zhu ◽  
Lam Tzehau ◽  
Helen Zhao ◽  
...  
Keyword(s):  
16S Rrna ◽  
Species Level ◽  
Invasive Cancer ◽  
Rrna Gene ◽  
16S Rrna Gene Analysis ◽  
Tissue Samples ◽  
Microbial Profiling ◽  
Formalin Fixed Paraffin Embedded ◽  
Sequencing Strategy ◽  
Metagenome Sequencing

AbstractMicrobiome samples with low microbial biomass or severe DNA degradation remain challenging for amplicon-based (e.g., 16S/18S-rRNA) or whole-metagenome sequencing (WMS) approaches. Here, we introduce 2bRAD-M, a “reduction” metagenome-sequencing strategy that simultaneously produces species-level bacterial, archaeal, and fungal profiles for low-biomass samples, yet is cost-competitive with 16S rRNA gene analysis. For mock communities, 2bRAD-M can accurately generate species-level taxonomic profiles for otherwise hard-to-sequence samples with (i) low biomass of merely 1 pg of total DNA, (ii) high host DNA contamination (99%), and (iii) severely fragmented DNA (50-bp) from degraded samples. Tests of 2bRAD-M on stool, skin and environment-surface samples deliver successful reconstruction of comprehensive, high-resolution microbial profiles with agreement across 16S-rRNA, WMS and existing literature. In addition, it enables microbial profiling in formalin-fixed paraffin-embedded (FFPE) cervical tissue samples which were recalcitrant to conventional approaches due to the low amount and heavy degradation of microbial DNA, and discriminated healthy tissue, pre-invasive cancer and invasive cancer via species-level microbial profiles with 91.1% accuracy. Therefore, 2bRAD-M greatly expands the reach of microbiome sequencing.

Microbial Profiling of Malera and Phab: Starters Used for Preparing Traditional Fermented Foods and Beverages in Himachal Pradesh, India

2019 ◽  
Vol 15 (7) ◽  
pp. 707-711
Author(s):  
Vipasha Sharma ◽  
Sampan Attri ◽  
Rishi Mahajan ◽  
Gunjan Goel
Keyword(s):  
16S Rrna ◽  
Himachal Pradesh ◽  
Fermented Food ◽  
Alcoholic Beverages ◽  
Careful Examination ◽  
Rrna Gene ◽  
Fermented Foods ◽  
Dgge Profile ◽  
Shannon Diversity ◽  
Microbial Profiling

Background: Traditional fermented food preparation uses customary processing methods passed on from generation to generation under natural conditions. These fermented foods use native flora without being aware of the significant role of microbes involved in the fermentation process. Therefore, the present study aimed to determine the bacterial composition of traditional starters used in different fermented food preparations in Himachal Pradesh region India. Methods: The study investigated the bacterial DGGE (Denaturating Gradient Gel Electrophoresis) profile targeting V3 region of 16S rRNA of two traditional starters known as Malera and Phab. The starters are used in the preparation of fermented cereals product known as bhaturoo and alcoholic beverages. The Shannon diversity and richness were calculated from DGGE profile. The 16S rRNA gene sequences of identified bacterial species were deposited in NCBI database. Results: The DGGE profile identified eleven and seven different bacterial strains in Malera and Phab, respectively. The Shannon diversity index of 1.07 and 0.94 was obtained for Malera and Phab, respectively. The bacterial population was dominated by different strains of Bifidobacterium sp. in both the starters along with the presence of non lactic enterobacteriacae members such as Klebsiella sp. and a pathogenic strain of Dickeya chrysanthemi. Conclusion: The study is the first report on microbial profiling of microflora of starters. A careful examination of individual components and method of preparation of the starters should be taken to avoid contamination by pathogens.

scholarly journals Full-Length SSU rRNA Gene Sequencing Allows Species-Level Detection of Bacteria, Archaea, and Yeasts Present in Milk

2021 ◽  
Vol 9 (6) ◽  
pp. 1251
Author(s):  
Isabel Abellan-Schneyder ◽  
Annemarie Siebert ◽  
Katharina Hofmann ◽  
Mareike Wenning ◽  
Klaus Neuhaus
Keyword(s):  
Gene Sequencing ◽  
Raw Milk ◽  
Amplicon Sequencing ◽  
Species Level ◽  
Full Length ◽  
Rrna Gene ◽  
Ssu Rrna ◽  
Ssu Rrna Gene ◽  
Milk Samples ◽  
Rrna Gene Sequencing

Full-length SSU rRNA gene sequencing allows species-level identification of the microorganisms present in milk samples. Here, we used bulk-tank raw milk samples of two German dairies and detected, using this method, a great diversity of bacteria, archaea, and yeasts within the samples. Moreover, the species-level classification was improved in comparison to short amplicon sequencing. Therefore, we anticipate that this approach might be useful for the detection of possible mastitis-causing species, as well as for the control of spoilage-associated microorganisms. In a proof of concept, we showed that we were able to identify several putative mastitis-causing or mastitis-associated species such as Streptococcusuberis, Streptococcusagalactiae, Streptococcusdysgalactiae, Escherichiacoli and Staphylococcusaureus, as well as several Candida species. Overall, the presented full-length approach for the sequencing of SSU rRNA is easy to conduct, able to be standardized, and allows the screening of microorganisms in labs with Illumina sequencing machines.

scholarly journals Sarocladium and Lecanicillium Associated with Maize Seeds and Their Potential to Form Selected Secondary Metabolites

Biomolecules ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 98
Author(s):  
Lidia Błaszczyk ◽  
Agnieszka Waśkiewicz ◽  
Karolina Gromadzka ◽  
Katarzyna Mikołajczak ◽  
Jerzy Chełkowski
Keyword(s):  
Large Subunit ◽  
Solid Substrate ◽  
Rrna Gene ◽  
Decenoic Acid ◽  
Bioactive Substances ◽  
Lecanicillium Lecanii ◽  
Maize Seeds ◽  
Internally Transcribed Spacer ◽  
First Time

The occurrence and diversity of Lecanicillium and Sarocladium in maize seeds and their role in this cereal are poorly understood. Therefore, the present study aimed to investigate Sarocladium and Lecanicillium communities found in endosphere of maize seeds collected from fields in Poland and their potential to form selected bioactive substances. The sequencing of the internally transcribed spacer regions 1 (ITS 1) and 2 (ITS2) and the large-subunit (LSU, 28S) of the rRNA gene cluster resulted in the identification of 17 Sarocladium zeae strains, three Sarocladium strictum and five Lecanicillium lecanii isolates. The assay on solid substrate showed that S. zeae and S. strictum can synthesize bassianolide, vertilecanin A, vertilecanin A methyl ester, 2-decenedioic acid and 10-hydroxy-8-decenoic acid. This is also the first study revealing the ability of these two species to produce beauvericin and enniatin B1, respectively. Moreover, for the first time in the present investigation, pyrrocidine A and/or B have been annotated as metabolites of S. strictum and L. lecanii. The production of toxic, insecticidal and antibacterial compounds in cultures of S. strictum, S. zeae and L. lecanii suggests the requirement to revise the approach to study the biological role of fungi inhabiting maize seeds.

scholarly journals Amplicon-sequencing of raw milk microbiota: impact of DNA extraction and library-PCR

2021 ◽  
Author(s):  
Annemarie Siebert ◽  
Katharina Hofmann ◽  
Lena Staib ◽  
Etienne V. Doll ◽  
Siegfried Scherer ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Dna Extraction ◽  
Raw Milk ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Bacterial Dna ◽  
Microbiome Analysis ◽  
Eukaryotic Dna ◽  
Selective Lysis ◽  
The Impact

Abstract The highly complex raw milk matrix challenges the sample preparation for amplicon-sequencing due to low bacterial counts and high amounts of eukaryotic DNA originating from the cow. In this study, we optimized the extraction of bacterial DNA from raw milk for microbiome analysis and evaluated the impact of cycle numbers in the library-PCR. The selective lysis of eukaryotic cells by proteinase K and digestion of released DNA before bacterial lysis resulted in a high reduction of mostly eukaryotic DNA and increased the proportion of bacterial DNA. Comparative microbiome analysis showed that a combined enzymatic and mechanical lysis procedure using the DNeasy® PowerFood® Microbial Kit with a modified protocol was best suitable to achieve high DNA quantities after library-PCR and broad coverage of detected bacterial biodiversity. Increasing cycle numbers during library-PCR systematically altered results for species and beta-diversity with a tendency to overrepresentation or underrepresentation of particular taxa. To limit PCR bias, high cycle numbers should thus be avoided. An optimized DNA extraction yielding sufficient bacterial DNA and enabling higher PCR efficiency is fundamental for successful library preparation. We suggest that a protocol using ethylenediaminetetraacetic acid (EDTA) to resolve casein micelles, selective lysis of somatic cells, extraction of bacterial DNA with a combination of mechanical and enzymatic lysis, and restriction of PCR cycles for analysis of raw milk microbiomes is optimal even for samples with low bacterial numbers. Key points • Sample preparation for high-throughput 16S rRNA gene sequencing of raw milk microbiota. • Reduction of eukaryotic DNA by enzymatic digestion. • Shift of detected microbiome caused by high cycle numbers in library-PCR.

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