scholarly journals Microbial Ecology of Greek Wheat Sourdoughs, Identified by a Culture-Dependent and a Culture-Independent Approach

Foods ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 1603
Author(s):  
Maria K. Syrokou ◽  
Christina Themeli ◽  
Spiros Paramithiotis ◽  
Marios Mataragas ◽  
Loulouda Bosnea ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Microbial Ecology ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Random Amplified Polymorphic Dna ◽  
Titratable Acidity ◽  
Rrna Gene ◽  
Pichia Membranifaciens ◽  
Of Greece ◽  
Culture Dependent

The aim of the present study was to assess the microecosystem of 13 homemade spontaneously fermented wheat sourdoughs from different regions of Greece, through the combined use of culture-dependent (classical approach; clustering by Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) and identification by PCR species-specific for Lactiplantibacillus plantarum, and sequencing of the 16S-rRNA and 26S-rRNA gene, for Lactic Acid Bacteria (LAB) and yeasts, respectively) and independent approaches [DNA- and RNA-based PCR-Denaturing Gradient Gel Electrophoresis (DGGE)]. The pH and Total Titratable Acidity (TTA) values ranged from 3.64–5.05 and from 0.50–1.59% lactic acid, respectively. Yeast and lactic acid bacteria populations ranged within 4.60–6.32 and 6.28–9.20 log CFU/g, respectively. The yeast: LAB ratio varied from 1:23–1:10,000. A total of 207 bacterial and 195 yeast isolates were obtained and a culture-dependent assessment of their taxonomic affiliation revealed dominance of Lb. plantarum in three sourdoughs, Levilactobacillus brevis in four sourdoughs and co-dominance of these species in two sourdoughs. In addition, Companilactobacillusparalimentarius dominated in two sourdoughs and Fructilactobacillussanfranciscensis and Latilactobacillus sakei in one sourdough each. Lactococcus lactis, Lb. curvatus, Leuconostoc citreum, Ln. mesenteroides and Lb. zymae were also recovered from some samples. Regarding the yeast microbiota, it was dominated by Saccharomyces cerevisiae in 11 sourdoughs and Pichia membranifaciens and P. fermentans in one sourdough each. Wickerhamomyces anomalus and Kazachstania humilis were also recovered from one sample. RNA-based PCR-DGGE provided with nearly identical results with DNA-based one; in only one sample the latter provided an additional band. In general, the limitations of this approach, namely co-migration of amplicons from different species to the same electrophoretic position and multiband profile of specific isolates, greatly reduced resolution capacity, which resulted in only partial verification of the microbial ecology detected by culture-dependent approach in the majority of sourdough samples. Our knowledge regarding the microecosystem of spontaneously fermented Greek wheat-based sourdoughs was expanded, through the study of sourdoughs originating from regions of Greece that were not previously assessed.

scholarly journals Microbial Ecology Dynamics during Rye and Wheat Sourdough Preparation

2013 ◽  
Vol 79 (24) ◽  
pp. 7827-7836 ◽  
Author(s):  
Danilo Ercolini ◽  
Erica Pontonio ◽  
Francesca De Filippis ◽  
Fabio Minervini ◽  
Antonietta La Storia ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Microbial Ecology ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Diversity Indices ◽  
Rrna Gene ◽  
Content Type ◽  
Gradient Gel Electrophoresis ◽  
Plate Counts ◽  
Relative Abundances

ABSTRACTThe bacterial ecology during rye and wheat sourdough preparation was described by 16S rRNA gene pyrosequencing. Viable plate counts of presumptive lactic acid bacteria, the ratio between lactic acid bacteria and yeasts, the rate of acidification, a permutation analysis based on biochemical and microbial features, the number of operational taxonomic units (OTUs), and diversity indices all together demonstrated the maturity of the sourdoughs during 5 to 7 days of propagation. Flours were mainly contaminated by metabolically active genera (Acinetobacter,Pantoea,Pseudomonas,Comamonas,Enterobacter,Erwinia, andSphingomonas) belonging to the phylumProteobacteriaorBacteroidetes(genusChryseobacterium). Their relative abundances varied with the flour. Soon after 1 day of propagation, this population was almost completely inhibited except for theEnterobacteriaceae. Although members of the phylumFirmicuteswere present at very low or intermediate relative abundances in the flours, they became dominant soon after 1 day of propagation. Lactic acid bacteria were almost exclusively representative of theFirmicutesby this time.Weissellaspp. were already dominant in rye flour and stably persisted, though they were later flanked by theLactobacillus sakeigroup. There was a succession of species during 10 days of propagation of wheat sourdoughs. The fluctuation between dominating and subdominating populations ofL. sakeigroup,Leuconostocspp.,Weissellaspp., andLactococcus lactiswas demonstrated. Other subdominant species such asLactobacillus plantarumwere detectable throughout propagation. As shown by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analysis,Saccharomyces cerevisiaedominated throughout the sourdough propagation. Notwithstanding variations due to environmental and technology determinants, the results of this study represent a clear example of how the microbial ecology evolves during sourdough preparation.

scholarly journals Biodiversity andγ-Aminobutyric Acid Production by Lactic Acid Bacteria Isolated from Traditional Alpine Raw Cow’s Milk Cheeses

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Elena Franciosi ◽  
Ilaria Carafa ◽  
Tiziana Nardin ◽  
Silvia Schiavon ◽  
Elisa Poznanski ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Random Amplified Polymorphic Dna ◽  
Streptococcus Thermophilus ◽  
Lactobacillus Paracasei ◽  
Aminobutyric Acid ◽  
Cow’S Milk ◽  
Rrna Gene ◽  
Cow's Milk ◽  
Raw Cow’S Milk

“Nostrano-cheeses” are traditional alpine cheeses made from raw cow’s milk in Trentino-Alto Adige, Italy. This study identified lactic acid bacteria (LAB) developing during maturation of “Nostrano-cheeses” and evaluated their potential to produceγ-aminobutyric acid (GABA), an immunologically active compound and neurotransmitter. Cheese samples were collected on six cheese-making days, in three dairy factories located in different areas of Trentino and at different stages of cheese ripening (24 h, 15 days, and 1, 2, 3, 6, and 8 months). A total of 1,059 LAB isolates were screened using Random Amplified Polymorphic DNA-PCR (RAPD-PCR) and differentiated into 583 clusters. LAB strains from dominant clusters (n=97) were genetically identified to species level by partial 16S rRNA gene sequencing. LAB species most frequently isolated wereLactobacillus paracasei,Streptococcus thermophilus, andLeuconostoc mesenteroides. The 97 dominant clusters were also characterized for their ability in producing GABA by high-performance liquid chromatography (HPLC). About 71% of the dominant bacteria clusters evolving during cheeses ripening were able to produce GABA. Most GABA producers wereLactobacillus paracaseibut other GABA producing species includedLactococcus lactis,Lactobacillus plantarum,Lactobacillus rhamnosus,Pediococcus pentosaceus, andStreptococcus thermophilus. NoEnterococcus faecalisorSc. macedonicusisolates produced GABA. The isolate producing the highest amount of GABA (80.0±2.7 mg/kg) was aSc. thermophilus.

scholarly journals Dynamics and Biodiversity of Populations of Lactic Acid Bacteria and Acetic Acid Bacteria Involved in Spontaneous Heap Fermentation of Cocoa Beans in Ghana

2007 ◽  
Vol 73 (6) ◽  
pp. 1809-1824 ◽  
Author(s):  
Nicholas Camu ◽  
Tom De Winter ◽  
Kristof Verbrugghe ◽  
Ilse Cleenwerck ◽  
Peter Vandamme ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Fermentation Process ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Acetic Acid Bacteria ◽  
Cocoa Bean ◽  
Rrna Gene ◽  
Cocoa Bean Fermentation ◽  
Leuconostoc Pseudomesenteroides

ABSTRACT The Ghanaian cocoa bean heap fermentation process was studied through a multiphasic approach, encompassing both microbiological and metabolite target analyses. A culture-dependent (plating and incubation, followed by repetitive-sequence-based PCR analyses of picked-up colonies) and culture-independent (denaturing gradient gel electrophoresis [DGGE] of 16S rRNA gene amplicons, PCR-DGGE) approach revealed a limited biodiversity and targeted population dynamics of both lactic acid bacteria (LAB) and acetic acid bacteria (AAB) during fermentation. Four main clusters were identified among the LAB isolated: Lactobacillus plantarum, Lactobacillus fermentum, Leuconostoc pseudomesenteroides, and Enterococcus casseliflavus. Other taxa encompassed, for instance, Weissella. Only four clusters were found among the AAB identified: Acetobacter pasteurianus, Acetobacter syzygii-like bacteria, and two small clusters of Acetobacter tropicalis-like bacteria. Particular strains of L. plantarum, L. fermentum, and A. pasteurianus, originating from the environment, were well adapted to the environmental conditions prevailing during Ghanaian cocoa bean heap fermentation and apparently played a significant role in the cocoa bean fermentation process. Yeasts produced ethanol from sugars, and LAB produced lactic acid, acetic acid, ethanol, and mannitol from sugars and/or citrate. Whereas L. plantarum strains were abundant in the beginning of the fermentation, L. fermentum strains converted fructose into mannitol upon prolonged fermentation. A. pasteurianus grew on ethanol, mannitol, and lactate and converted ethanol into acetic acid. A newly proposed Weissella sp., referred to as “Weissella ghanaensis,” was detected through PCR-DGGE analysis in some of the fermentations and was only occasionally picked up through culture-based isolation. Two new species of Acetobacter were found as well, namely, the species tentatively named“ Acetobacter senegalensis” (A. tropicalis-like) and “Acetobacter ghanaensis” (A. syzygii-like).

scholarly journals Tracking of Intentionally Inoculated Lactic Acid Bacteria Strains in Yogurt and Probiotic Powder

2019 ◽  
Vol 8 (1) ◽  
pp. 5 ◽  
Author(s):  
Anshul Sharma ◽  
Jasmine Kaur ◽  
Sulhee Lee ◽  
Young-Seo Park
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Gene Sequence ◽  
Random Amplified Polymorphic Dna ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Bacterial Strains ◽  
Sequence Alignments ◽  
Gene Sequence Analysis ◽  
Rrna Gene Sequencing

The present work aimed at tracking intentionally inoculated lactic acid bacteria (LAB) strains in yogurt and probiotic powder. Leuconostoc (Leu.) mesenteroides (11251), Lactobacillus (L.) brevis (B151), and Lactobacillus plantarum (LB41K) strains were tracked in yogurt, and L. plantarum (LB41P) was tracked in a commercial probiotic powder. The yogurt was intentionally inoculated with the selected bacterial strains. Two types of yogurt with known and unknown bacterial pools were utilized. The standard 16S rRNA gene sequencing was used to evaluate the initial screening. The molecular typing tools, random amplified polymorphic DNA (RAPD), repetitive element palindromic PCR (rep-PCR), and comparative gene sequence analysis of selected housekeeping loci were used to track the inoculated dubious strains. Out of 30 random selections for each inoculation, the developed method identified seven (11251), nine (B151), and five (LB41K) colonies in the yogurt. The validation was performed by identifying 7 colonies (LB41P) out of 30 in the probiotic powder. The DNA banding profiles and the gene sequence alignments led to the identification of the correct inoculated strains. Overall, the study summarizes the use of molecular tools to identify the deliberately inoculated LAB strains. In conclusion, the proposed polyphasic approach effectively tracked the intentionally inoculated strains: Leu. mesenteroides, L. brevis, and L. plantarum (LB41K) in yogurt and L. plantarum (LB41P) in probiotic powder. The study demonstrates how to track industrially relevant misused LAB strains in marketable food products.

Antibiotic Resistance of Coagulase-Negative Staphylococci and Lactic Acid Bacteria Isolated from Naturally Fermented Chinese Cured Beef

2018 ◽  
Vol 81 (12) ◽  
pp. 2054-2063 ◽  
Author(s):  
JING WANG ◽  
MINGYUE LI ◽  
JING WANG ◽  
MIAOMIAO LIU ◽  
KUN YANG ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Random Amplified Polymorphic Dna ◽  
Selective Medium ◽  
Dilution Method ◽  
Agar Dilution Method ◽  
Rrna Gene ◽  
M Gene ◽  
Coagulase Negative Staphylococci

ABSTRACT This study provided phenotypic and molecular analysis of the antibiotic resistance within coagulase-negative staphylococci and lactic acid bacteria isolated from naturally fermented Chinese cured beef. A total of 49 strains were isolated by selective medium and identified at the species level by 16S rRNA gene sequencing as follows: Staphylococcus carnosus (37), Lactobacillus plantarum (6), Weissella confusa (4), Lactobacillus sakei (1), and Weissella cibaria (1). All strains were typed by random amplified polymorphic DNA fingerprinting, and their antibiotic resistances profiles to 15 antibiotics were determined as the MIC by using the agar dilution method. All the tested strains were sensitive to ampicillin, and most of them were also sensitive to penicillin, gentamycin, neomycin, norfloxacin, and ciprofloxacin with low MICs. High resistance to streptomycin, vancomycin, erythromycin, roxithromycin, lincomycin, and kanamycin was widely observed, while the resistant levels to tetracycline, oxytetracycline, and chloramphenicol varied. The presence of corresponding resistance genes in resistant isolates was investigated by PCR, with the following genes detected: tet(M) gene in 9 S. carnosus strains and 1 W. confusa strain; erm(F) gene in 10 S. carnosus strains; ere(A) gene in 6 S. carnosus strains; ere(A) gene in 4 S. carnosus strains and 1 L. plantarum strain; and str(A) gene and str(B) gene in 3 S. carnosus strains. The results indicated that multiple antibiotic resistances were common in coagulase-negative staphylococci and lactic acid bacteria strains isolated from naturally fermented Chinese cured beef. Safety analysis and risk assessment should be performed for application in meat products.

Study on molecular identification of lactic acid bacteria from fermented milks and “Smen” (a traditional steppe butter) and their enzyme producing attributes

2019 ◽  
Vol 9 (3) ◽  
pp. 95-103
Author(s):  
Asmaa Labtar ◽  
Saliha Larouci ◽  
Amel Guermouche ◽  
Farid Bensalah
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Pcr Amplification ◽  
Titratable Acidity ◽  
Streptococcus Thermophilus ◽  
Fermented Milk ◽  
Rrna Gene ◽  
Bacterial Strains ◽  
Search Tool ◽  
Fermented Dairy

Streptococcus thermophilus and Lactobacillus belong to a widely-knowngroup of bacteria that have been frequently used as starter bacteria in fermented dairy products. The aim of this study was to isolate, characterize andidentify of lactic acid bacteria (LAB) from raw cow’s fermented milk and‘Smen’ (a traditional steppe butter) in steppic area of Algeria to study someof their important enzyme-producing attributes. All selected strains of lacticacid bacteria (LAB) were identified and typed by phenotypic and genotypiccriteria.Six strains were identified as cocci thermophilic LAB and two of themwere selected and identified by using polymerase chain reaction (PCR) amplification for proA gene. Six strains of lactobacilli were identified as Lactobacillus genus using 16S rRNA gene. Species identification was performed by 16SrRNA gene sequencing and sequences were analysed using Basic LengthAlignment Search Tool (BLAST) programs. A phylogenetic tree based on 16SrRNA genes was constructed by the neighbor-joinin method.S. thermophilusstrains screened for their ability to produce lactic acid, titratable acidity, andpH was measured at 2h intervals, proteolytic activity in milk was examined.In another part, the four selected Lactobacillus were able to hydrolyze X-Galby production of β-galactosidase enzyme. All strains of LAB exhibited production of diacetyl. Overall, this study provides bacterial strains having potentialfor dairy industry.

scholarly journals Polyphasic Approach to Bacterial Dynamics during the Ripening of Spanish Farmhouse Cheese, Using Culture-Dependent and -Independent Methods

2008 ◽  
Vol 74 (18) ◽  
pp. 5662-5673 ◽  
Author(s):  
Antonio M. Martín-Platero ◽  
Eva Valdivia ◽  
Mercedes Maqueda ◽  
Inés Martín-Sánchez ◽  
Manuel Martínez-Bueno
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Community Dynamics ◽  
16S Rrna Gene Sequencing ◽  
Starter Cultures ◽  
Molecular Techniques ◽  
Rrna Gene ◽  
Lactobacillus Curvatus ◽  
Ripening Period ◽  
Culture Dependent

ABSTRACT We studied the dynamics of the microbial population during ripening of Cueva de la Magahá cheese using a combination of classical and molecular techniques. Samples taken during ripening of this Spanish goat's milk cheese in which Lactococcus lactis and Streptococcus thermophilus were used as starter cultures were analyzed. All bacterial isolates were clustered by using randomly amplified polymorphic DNA (RAPD) and identified by 16S rRNA gene sequencing, species-specific PCR, and multiplex PCR. Our results indicate that the majority of the 225 strains isolated and enumerated on solid media during the ripening period were nonstarter lactic acid bacteria, and Lactobacillus paracasei was the most abundant species. Other Lactobacillus species, such as Lactobacillus plantarum and Lactobacillus parabuchneri, were also detected at the beginning and end of ripening, respectively. Non-lactic-acid bacteria, mainly Kocuria and Staphylococcus strains, were also detected at the end of the ripening period. Microbial community dynamics determined by temporal temperature gradient gel electrophoresis provided a more precise estimate of the distribution of bacteria and enabled us to detect Lactobacillus curvatus and the starter bacteria S. thermophilus and L. lactis, which were not isolated. Surprisingly, the bacterium most frequently found using culture-dependent analysis, L. paracasei, was scarcely detected by this molecular approach. Finally, we studied the composition of the lactobacilli and their evolution by using length heterogeneity PCR.

Cultivation and molecular monitoring of halophilic microorganisms inhabiting an extreme environment presented by a salt-attacked monument

2009 ◽  
Vol 9 (1) ◽  
pp. 59-72 ◽  
Author(s):  
Jörg Ettenauer ◽  
Katja Sterflinger ◽  
Guadalupe Piñar
Keyword(s):  
Microbial Community ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Random Amplified Polymorphic Dna ◽  
Extreme Environment ◽  
Salt Crystallization ◽  
Porous Space ◽  
Rrna Gene ◽  
Bacterial Strains ◽  
Halophilic Microorganisms ◽  
Culture Dependent

AbstractIn the last few years several investigations, based on culture-dependent and -independent techniques, have shown that salt-attacked stone surfaces present a habitat for extremely salt tolerant and moderate halophilic microorganisms. The inner walls of the Chapel of St. Virgil in Vienna (Austria) are an example of this phenomenon. Salt crusts cover most of the wall surfaces and salt crystallization in the porous space of the stone is causing decohesion of material and destruction of the original medieval paintings. The salt, together with the oligotrophic conditions, creates a very special and extreme habitat for halotolerant and halophilic microorganisms.In this study we investigate and monitor the cultivable and non-cultivable members of the microbial community present on the stonework of the medieval Chapel of St. Virgil after several severe disturbances of the microbial environment caused by desalination and disinfection treatments. With this finality, a combination of culture-dependent and -independent techniques was selected. The genetic diversity of a total of 104 bacterial strains isolated from the stone samples was analysed by denaturing gradient gel electrophoresis (DGGE), random amplified polymorphic DNA (RAPD) analysis and 16S rRNA gene sequencing. Strains were distributed over 29 groups on the basis of their RAPD patterns. Only 19 groups were differentiated by DGGE. Comparative sequence analyses showed that the isolated strains belong to related species of the generaHalobacillus(47.1%),Bacillus(35.6%),Acinetobacter(4.8%),Halomonas(3.9%),Nesterenkonia(2.9%),Paucisalibacillus(2.9%),Paenibacillus(1%),Staphylococcus(1%) andExiguobacterium(1%).In addition, polymerase chain reaction DGGE fingerprints, in combination with the creation of clone libraries and sequencing analyses, were used to monitor and identifyArchaea, the non-cultivable fraction of the microbial community. The detected archaeal sequences were closely related to different uncultured archaeons as well as to the cultured generaHalococcusandHalalkalicoccusandHalobacterium.Cultivation and molecular analyses revealed the presence of highly specialized microorganisms that were able to thrive and survive after several desalination and disinfection treatments in the extreme environment presented by the salt-attacked Chapel of St. Virgil.

scholarly journals Case Study of the Distribution of Mucosa-Associated Bifidobacterium Species, Lactobacillus Species, and Other Lactic Acid Bacteria in the Human Colon

2003 ◽  
Vol 69 (12) ◽  
pp. 7545-7548 ◽  
Author(s):  
D. S. Nielsen ◽  
P. L. Møller ◽  
V. Rosenfeldt ◽  
A. Pærregaard ◽  
K. F. Michaelsen ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Sampling Site ◽  
Human Colon ◽  
Lactobacillus Species ◽  
Rrna Gene ◽  
Associated Bacteria ◽  
Gradient Gel Electrophoresis

ABSTRACT The distribution of mucosa-associated bacteria, bifidobacteria and lactobacilli and closely related lactic acid bacteria, in biopsy samples from the ascending, transverse, and descending parts of the colon from four individuals was investigated by denaturing gradient gel electrophoresis (DGGE). Bifidobacterial genus-specific, Lactobacillus group-specific, and universal bacterial primers were used in a nested PCR approach to amplify a fragment of the 16S rRNA gene. DGGE profiles of the bifidobacterial community were relatively simple, with one or two amplicons detected at most sampling sites in the colon. DGGE profiles obtained with Lactobacillus group-specific primers were complex and varied with host and sampling site in the colon. The overall bacterial community varied with host but not sampling site.

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