scholarly journals Unprecedented Diversity of Lactococcal Group 936 Bacteriophages Revealed by Amplicon Sequencing of the Portal Protein Gene

Viruses ◽  
2019 ◽  
Vol 11 (5) ◽  
pp. 443 ◽  
Author(s):  
Cyril Alexander Frantzen ◽  
Helge Holo
Keyword(s):  
Protein Gene ◽  
Preventive Measures ◽  
Starter Culture ◽  
Variable Region ◽  
Amplicon Sequencing ◽  
Starter Cultures ◽  
Diversity Analysis ◽  
Portal Protein ◽  
Culture Independent ◽  
Cheese Production

Lactococcus lactis is one of the most important bacteria in dairy fermentations, being used in the production of cheese and buttermilk. The processes are vulnerable to phage attacks, and undefined mixtures of lactococcal strains are often used to reduce the risk of bacteriophage caused fermentation failure. Other preventive measures include culture rotation to prevent phage build-up and phage monitoring. Phage diversity, rather than quantity, is the largest threat to fermentations using undefined mixed starter cultures. We have developed a method for culture independent diversity analysis of lytic bacteriophages of the 936 group, the phages most commonly found in dairies. Using, as a target, a highly variable region of the portal protein gene, we demonstrate an unprecedented diversity and the presence of new 936 phages in samples taken from cheese production. The method should be useful to the dairy industry and starter culture manufacturers in their efforts to reduce phage problems.

scholarly journals Lactococcus lactisDiversity in Undefined Mixed Dairy Starter Cultures as Revealed by Comparative Genome Analyses and Targeted Amplicon Sequencing ofepsD

2017 ◽  
Vol 84 (3) ◽  
Author(s):  
Cyril A. Frantzen ◽  
Hans Petter Kleppen ◽  
Helge Holo
Keyword(s):  
Lactococcus Lactis ◽  
Starter Culture ◽  
Amplicon Sequencing ◽  
Core Gene ◽  
Starter Cultures ◽  
Content Type ◽  
Genome Analyses ◽  
Dairy Starter Cultures ◽  
Rotation Strategy ◽  
Cheese Production

ABSTRACTUndefined mesophilic mixed (DL) starter cultures are used in the production of continental cheeses and contain unknown strain mixtures ofLactococcus lactisand leuconostocs. The choice of starter culture affects the taste, aroma, and quality of the final product. To gain insight into the diversity ofLactococcus lactisstrains in starter cultures, we whole-genome sequenced 95 isolates from three different starter cultures. Pan-genomic analyses, which included 30 publically available complete genomes, grouped the strains into 21L. lactissubsp. lactisand 28L. lactissubsp.cremorislineages. Only one of the 95 isolates grouped with previously sequenced strains, and the three starter cultures showed no overlap in lineage distributions. The culture diversity was assessed by targeted amplicon sequencing usingpurR, a core gene, andepsD, present in 93 of the 95 starter culture isolates but absent in most of the reference strains. This enabled an unprecedented discrimination of starter cultureLactococcus lactisand revealed substantial differences between the three starter cultures and compositional shifts during the cultivation of cultures in milk.IMPORTANCEIn contemporary cheese production, standardized frozen seed stock starter cultures are used to ensure production stability, reproducibility, and quality control of the product. The dairy industry experiences significant disruptions of cheese production due to phage attacks, and one commonly used countermeasure to phage attack is to employ a starter rotation strategy, in which two or more starters with minimal overlap in phage sensitivity are used alternately. A culture-independent analysis of the lactococcal diversity in complex undefined starter cultures revealed large differences between the three starter cultures and temporal shifts in lactococcal composition during the production of bulk starters. A better understanding of the lactococcal diversity in starter cultures will enable the development of more robust starter cultures and assist in maintaining the efficiency and stability of the production process by ensuring the presence of key bacteria that are important to the characteristics of the product.

scholarly journals Lactococcus lactis Diversity Revealed by Targeted Amplicon Sequencing of purR Gene, Metabolic Comparisons and Antimicrobial Properties in an Undefined Mixed Starter Culture Used for Soft-Cheese Manufacture

Foods ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 622
Author(s):  
Sabrina Saltaji ◽  
Olivier Rué ◽  
Valérie Sopena ◽  
Sophie Sablé ◽  
Fatoumata Tambadou ◽  
...  
Keyword(s):  
Microbial Diversity ◽  
Starter Culture ◽  
Antimicrobial Properties ◽  
Amplicon Sequencing ◽  
Antimicrobial Activities ◽  
Operational Taxonomic Units ◽  
Soft Cheese ◽  
Culture Independent ◽  
Identification Of Species ◽  
Species Specific

The undefined mixed starter culture (UMSC) is used in the manufacture of cheeses. Deciphering UMSC microbial diversity is important to optimize industrial processes. The UMSC was studied using culture-dependent and culture-independent based methods. MALDI-TOF MS enabled identification of species primarily from the Lactococcus genus. Comparisons of carbohydrate metabolism profiles allowed to discriminate five phenotypes of Lactococcus (n = 26/1616). The 16S sequences analysis (V1–V3, V3–V4 regions) clustered the UMSC microbial diversity into two Lactococcus operational taxonomic units (OTUs). These clustering results were improved with the DADA2 algorithm on the housekeeping purR sequences. Five L. lactis variants were detected among the UMSC. The whole-genome sequencing of six isolates allowed for the identification of the lactis subspecies using Illumina® (n = 5) and Pacbio® (n = 1) technologies. Kegg analysis confirmed the L. lactis species-specific niche adaptations and highlighted a progressive gene pseudogenization. Then, agar spot tests and agar well diffusion assays were used to assess UMSC antimicrobial activities. Of note, isolate supernatants (n = 34/1616) were shown to inhibit the growth of Salmonella ser. Typhimurium CIP 104115, Lactobacillus sakei CIP 104494, Staphylococcus aureus DSMZ 13661, Enterococcus faecalis CIP103015 and Listeria innocua CIP 80.11. Collectively, these results provide insightful information about UMSC L. lactis diversity and revealed a potential application as a bio-protective starter culture.

scholarly journals Detection and Characterization of Streptococcus thermophilus Bacteriophages by Use of the Antireceptor Gene Sequence

2005 ◽  
Vol 71 (10) ◽  
pp. 6096-6103 ◽  
Author(s):  
Ana G. Binetti ◽  
Beatriz Del Río ◽  
M. Cruz Martín ◽  
Miguel A. Álvarez
Keyword(s):  
Starter Culture ◽  
Dairy Industry ◽  
Pcr Amplification ◽  
Variable Region ◽  
Streptococcus Thermophilus ◽  
Resistance Mechanisms ◽  
Starter Cultures ◽  
Phage Types ◽  
Specific Pcr

ABSTRACT In the dairy industry, the characterization of Streptococcus thermophilus phage types is very important for the selection and use of efficient starter cultures. The aim of this study was to develop a characterization system useful in phage control programs in dairy plants. A comparative study of phages of different origins was initially performed based on their morphology, DNA restriction profiles, DNA homology, structural proteins, packaging mechanisms, and lifestyles and on the presence of a highly conserved DNA fragment of the replication module. However, these traditional criteria were of limited industrial value, mainly because there appeared to be no correlation between these variables and host ranges. We therefore developed a PCR method to amplify VR2, a variable region of the antireceptor gene, which allowed rapid detection of S. thermophilus phages and classification of these phages. This method has a significant advantage over other grouping criteria since our results suggest that there is a correlation between typing profiles and host ranges. This association could be valuable for the dairy industry by allowing a rational starter rotation system to be established and by helping in the selection of more suitable starter culture resistance mechanisms. The method described here is also a useful tool for phage detection, since specific PCR amplification was possible when phage-contaminated milk was used as a template (detection limit, 105 PFU ml−1).

scholarly journals Lacticaseibacillus rhamnosus Impedes Growth of Listeria spp. in Cottage Cheese through Manganese Limitation

Foods ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1353
Author(s):  
Lieke A. van Gijtenbeek ◽  
Quinn Singer ◽  
Louise E. Steffensen ◽  
Shannon Neuens ◽  
Helle S. Guldager ◽  
...  
Keyword(s):  
Dairy Products ◽  
Competitive Exclusion ◽  
Starter Culture ◽  
Streptococcus Thermophilus ◽  
Starter Cultures ◽  
Growth Potential ◽  
Cottage Cheese ◽  
Dairy Starter Cultures ◽  
Dairy Starter ◽  
Cheese Production

Acidification and nutrient depletion by dairy starter cultures is often sufficient to prevent outgrowth of pathogens during post-processing of cultured dairy products. In the case of cottage cheese, however, the addition of cream dressing to the curd and subsequent cooling procedures can create environments that may be hospitable for the growth of Listeria monocytogenes. We report on a non-bacterio-cinogenic Lacticaseibacillus rhamnosus strain that severely limits the growth potential of L. monocytogenes in creamed cottage cheese. The main mechanism underlying Listeria spp. inhibition was found to be caused by depletion of manganese (Mn), thus through competitive exclusion of a trace element essential for the growth of many microorganisms. Growth of Streptococcus thermophilus and Lactococcus lactis that constitute the starter culture, on the other hand, were not influenced by reduced Mn levels. Addition of L. rhamnosus with Mn-based bioprotective properties during cottage cheese production therefore offers a solution to inhibit undesired bacteria in a bacteriocin-independent fashion.

scholarly journals Influence of Environmental Microbiota on the Activity and Metabolism of Starter Cultures Used in Coffee Beans Fermentation

Fermentation ◽  
2021 ◽  
Vol 7 (4) ◽  
pp. 278
Author(s):  
Vanessa Bassi Pregolini ◽  
Gilberto Vinícius de Melo Pereira ◽  
Alexander da Silva Vale ◽  
Dão Pedro de Carvalho Neto ◽  
Carlos Ricardo Soccol
Keyword(s):  
Fermentation Process ◽  
Starter Culture ◽  
Amplicon Sequencing ◽  
Isoamyl Acetate ◽  
Starter Cultures ◽  
Positive Interaction ◽  
Coffee Beans ◽  
Fermentation Tank ◽  
Coffee Farm ◽  
Food Commodities

Microbial activity is an integral part of agricultural ecosystems and can influence the quality of food commodities. During on-farm processing, coffee growers use a traditional method of fermentation to remove the cherry pulp surrounding the beans. Here, we investigated the influence of the coffee farm microbiome and the resulting fermentation process conducted with selected starter cultures (Pichia fermentans YC5.2 and Pediococcus acidilactici LPBC161). The microbiota of the coffee farm (coffee fruits and leaves, over-ripe fruits, cherries before de-pulping, depulped beans, and water used for de-pulping beans) was dominated by Enterobacteriaceae and Saccharomycetales, as determined by llumina-based amplicon sequencing. In addition, 299 prokaryotes and 189 eukaryotes were identified. Following the fermentation process, Pichia and the family Lactobacillaceae (which includes P. acidilactici) represented more than 70% of the total microbial community. The positive interaction between the starters resulted in the formation of primary metabolites (such as ethanol and lactic acid) and important aroma-impacting compounds (ethyl acetate, isoamyl acetate, and ethyl isobutyrate). The success competitiveness of the starters towards the wild microbiota indicated that coffee farm microbiota has little influence on starter culture-added coffee fermentation. However, hygiene requirements in the fermentation process should be indicated to prevent the high microbial loads present in coffee farm soil, leaves, fruits collected on the ground, and over-ripe fruits from having access to the fermentation tank and transferring undesirable aromas to coffee beans.

Potential of lactic acid bacteria from Pico cheese for starter culture development

2019 ◽  
Vol 25 (4) ◽  
pp. 303-317 ◽  
Author(s):  
SP Câmara ◽  
A Dapkevicius ◽  
C Riquelme ◽  
RB Elias ◽  
CCG Silva ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Starter Culture ◽  
Fermented Food ◽  
Starter Cultures ◽  
Rrna Gene ◽  
Culture Development ◽  
Positive Traits ◽  
Leuconostoc Pseudomesenteroides ◽  
Cheese Production

Autochthonous lactic acid bacteria may provide a means of promoting the quality and safety of traditional fermented food products, in particular, artisanal cheeses. Pico cheese is an artisanal, dairy specialty of the Azores in risk of disappearing. Efforts to maintain its quality to the requirements of the modern markets are, thus, necessary. Lactic acid bacteria were isolated from artisanal Pico cheese, identified by sequencing of the 16S rRNA gene, and their potential as starter cultures was evaluated by studying their acidification ability, enzymatic activities (caseinolysis, lipolysis and API-ZYM profile), diacetyl and expolysaccharide production, autolysis, antimicrobial activity against Listeria monocytogenes ATCC 7466, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29523, Pseudomonas aeruginosa ATCC 27853 and Clostridium perfringens ATCC 8357, sensory evaluation of odour formation in milk, syneresis and firmness of the curd. Several of the studied lactic acid bacteria isolates showed interesting properties for practical application as starters in artisanal cheese production. The isolates with the highest number of positive traits and, therefore, the most promising for starter development were Lactococcus lactis ssp. lactis L1C21M1, Lactobacillus paracasei L1B1E3, Leuconostoc pseudomesenteroides L1C1E6, Lactobacillus casei L1A1E5 and L1C1E8.

Development of biotechnology for gluten-free sourdough bakery products with a new microbial composition

2020 ◽  
Vol 29 (12) ◽  
pp. 59-63
Author(s):  
O.I. Parakhina ◽  
◽  
M.N. Lokachuk ◽  
L.I. Kuznetsova ◽  
E.N. Pavlovskaya ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Starter Culture ◽  
Comparative Assessment ◽  
Starter Cultures ◽  
Bakery Products ◽  
Gluten Free ◽  
Microbial Composition ◽  
Fermentative Activity ◽  
Theoretical Foundations

The research was carried out within the framework of the theme of state assignment № 0593–2019–0008 «To develop theoretical foundations for creating composite mixtures for bakery products using physical methods of exposure that ensure homogeneity, stability of mixtures and bioavailability of nutrients, to optimize diets population of Russia». The data on the species belonging of new strains of lactic acid bacteria and yeast isolated from samples of good quality gluten-free starter cultures are presented. A comparative assessment of the antagonistic and acid-forming activity of strains of lactic acid bacteria and the fermentative activity of yeast was carried out. The composition of microbial compositions from selected strains of LAB and yeast was developed. The influence of the starter culture on the new microbial composition on the physicochemical, organoleptic indicators of the bread quality and resistance to mold and ropy-disease was investigated.

scholarly journals The Diversity of Root-Associated Endophytic Fungi from Four Epiphytic Orchids in China

Diversity ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 197
Author(s):  
Tao Wang ◽  
Miao Chi ◽  
Ling Guo ◽  
Donghuan Liu ◽  
Yu Yang ◽  
...  
Keyword(s):  
Endophytic Fungi ◽  
Southern China ◽  
Culture Method ◽  
Amplicon Sequencing ◽  
Community Diversity ◽  
Mycorrhizal Symbiosis ◽  
Epiphytic Orchids ◽  
Culture Independent ◽  
Almost All

Root-associated endophytic fungi (RAF) are found asymptomatically in almost all plant groups. However, little is known about the compositions and potential functions of RAF communities associated with most Orchidaceae species. In this study, the diversity of RAF was examined in four wild epiphytic orchids, Acampe rigida, Doritis pulcherrima, Renanthera coccinea, and Robiquetia succisa, that occur in southern China. A culture-independent method involving Illumina amplicon sequencing, and an in vitro culture method, were used to identify culturable fungi. The RAF community diversity differed among the orchid roots, and some fungal taxa were clearly concentrated in a certain orchid species, with more OTUs being detected. By investigating mycorrhizal associations, the results showed that 28 (about 0.8%) of the 3527 operational taxonomic units (OTUs) could be assigned as OMF, while the OTUs of non-mycorrhizal fungal were about 99.2%. Among the OMFs, Ceratobasidiaceae OTUs were the most abundant with different richness, followed by Thelephoraceae. In addition, five Ceratobasidium sp. strains were isolated from D. pulcherrima, R. succisa, and R. coccinea roots with high separation rates. These culturable Ceratobasidium strains will provide materials for host orchid conservation and for studying the mechanisms underlying mycorrhizal symbiosis.

scholarly journals Culture-Independent and Culture-Dependent Characterization of the Black Soldier Fly Gut Microbiome Reveals a Large Proportion of Culturable Bacteria with Potential for Industrial Applications

2021 ◽  
Vol 9 (8) ◽  
pp. 1642
Author(s):  
Dorothee Tegtmeier ◽  
Sabine Hurka ◽  
Sanja Mihajlovic ◽  
Maren Bodenschatz ◽  
Stephanie Schlimbach ◽  
...  
Keyword(s):  
Gut Microbiome ◽  
Amplicon Sequencing ◽  
Industrial Applications ◽  
Culture Collection ◽  
Rrna Gene ◽  
Organic Substrates ◽  
Culturable Bacteria ◽  
Black Soldier Fly ◽  
Culture Independent ◽  
Culture Dependent

Black soldier fly larvae (BSFL) are fast-growing, resilient insects that can break down a variety of organic substrates and convert them into valuable proteins and lipids for applications in the feed industry. Decomposition is mediated by an abundant and versatile gut microbiome, which has been studied for more than a decade. However, little is known about the phylogeny, properties and functions of bacterial isolates from the BSFL gut. We therefore characterized the BSFL gut microbiome in detail, evaluating bacterial diversity by culture-dependent methods and amplicon sequencing of the 16S rRNA gene. Redundant strains were identified by genomic fingerprinting and 105 non-redundant isolates were then tested for their ability to inhibit pathogens. We cultivated representatives of 26 genera, covering 47% of the families and 33% of the genera detected by amplicon sequencing. Among these isolates, we found several representatives of the most abundant genera: Morganella, Enterococcus, Proteus and Providencia. We also isolated diverse members of the less-abundant phylum Actinobacteria, and a novel genus of the order Clostridiales. We found that 15 of the isolates inhibited at least one of the tested pathogens, suggesting a role in helping to prevent colonization by pathogens in the gut. The resulting culture collection of unique BSFL gut bacteria provides a promising resource for multiple industrial applications.

scholarly journals Handling of spurious sequences affects the outcome of high-throughput 16S rRNA gene amplicon profiling

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Sandra Reitmeier ◽  
Thomas C. A. Hitch ◽  
Nicole Treichel ◽  
Nikolaos Fikas ◽  
Bela Hausmann ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Diversity Analysis ◽  
Careful Attention ◽  
Operational Taxonomic Units ◽  
Basic Concepts ◽  
Gnotobiotic Mice ◽  
Mock Communities

Abstract16S rRNA gene amplicon sequencing is a popular approach for studying microbiomes. However, some basic concepts have still not been investigated comprehensively. We studied the occurrence of spurious sequences using defined microbial communities based on data either from the literature or generated in three sequencing facilities and analyzed via both operational taxonomic units (OTUs) and amplicon sequence variants (ASVs) approaches. OTU clustering and singleton removal, a commonly used approach, delivered approximately 50% (mock communities) to 80% (gnotobiotic mice) spurious taxa. The fraction of spurious taxa was generally lower based on ASV analysis, but varied depending on the gene region targeted and the barcoding system used. A relative abundance of 0.25% was found as an effective threshold below which the analysis of spurious taxa can be prevented to a large extent in both OTU- and ASV-based analysis approaches. Using this cutoff improved the reproducibility of analysis, i.e., variation in richness estimates was reduced by 38% compared with singleton filtering using six human fecal samples across seven sequencing runs. Beta-diversity analysis of human fecal communities was markedly affected by both the filtering strategy and the type of phylogenetic distances used for comparison, highlighting the importance of carefully analyzing data before drawing conclusions on microbiome changes. In summary, handling of artifact sequences during bioinformatic processing of 16S rRNA gene amplicon data requires careful attention to avoid the generation of misleading findings. We propose the concept of effective richness to facilitate the comparison of alpha-diversity across studies.

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