A Species-Specific qPCR Method for Enumeration of Lactobacillus sanfranciscensis, Lactobacillus brevis, and Lactobacillus curvatus During Cocultivation in Sourdough

ABSTRACTThe structure and stability of the dominant lactic acid bacterium population were assessed during wheat flour sourdough type I propagation by using singly nine strains ofLactobacillus sanfranciscensis. Under back-slopping propagation with wheat flour type 0 F114, cell numbers of presumptive lactic acid bacteria varied slightly between and within starters. As determined by randomly amplified polymorphic DNA-PCR and restriction endonuclease analysis-pulsed-field gel electrophoresis analyses, only three (LS8, LS14, and LS44) starters dominated throughout 10 days of propagation. The others progressively decreased to less than 3 log CFU g−1. Partial sequence analysis of the 16S rRNA andrecAgenes and PCR-denaturating gradient gel electrophoresis analysis using therpoBgene allowed identification ofWeissella confusa,Lactobacillus sanfranciscensis,Lactobacillus plantarum,Lactobacillus rossiae,Lactobacillus brevis,Lactococcus lactissubsp.lactis,Pediococcus pentosaceus, andLactobacillusspp. as the dominant species of the raw wheat flour. At the end of propagation, one autochthonous strain ofL. sanfranciscensiswas found in all the sourdoughs. Except forL. brevis, strains of the above species were variously found in the mature sourdoughs. Persistent starters were found in association with other biotypes ofL. sanfranciscensisand withW. confusaorL. plantarum. Sourdoughs were characterized for acidification, quotient of fermentation, free amino acids, and community-level catabolic profiles by USING Biolog 96-well Eco microplates. In particular, catabolic profiles of sourdoughs containing persistent starters behaved similarly and were clearly differentiated from the others. The three persistent starters were further used for the production of sourdoughs and propagated by using another wheat flour whose lactic acid bacterium population in part differed from the previous one. Also, in this case all three starter strains persisted during propagation.

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Microbiota “Fingerprint” of Greek Feta Cheese through Ripening

Applied Sciences ◽

10.3390/app11125631 ◽

2021 ◽

Vol 11 (12) ◽

pp. 5631

Author(s):

Athina Tzora ◽

Aikaterini Nelli ◽

Chrysoula Voidarou ◽

George Fthenakis ◽

Georgios Rozos ◽

...

Keyword(s):

Lactobacillus Rhamnosus ◽

Small Ruminants ◽

Lactobacillus Brevis ◽

Lactobacillus Curvatus ◽

Flight Mass Spectrometry ◽

Protected Designation Of Origin ◽

White Cheese ◽

Lactobacillus Paraplantarum ◽

Feta Cheese ◽

Post Production

Feta is a Greek protected designation of origin (PDO) brined curd white cheese made from small ruminants’ milk. In the present research, Greek Feta cheese bacterial diversity was evaluated via matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Analysis of 23 cheese samples, produced in different regions of the country, was performed in two ripening times (three or six months post-production). The identified microbiota were primarily constituted of lactic acid bacteria. A total of 13 different genera were obtained. The dominant species in both ripening times were Lactobacillus plantarum (100.0% and 87.0%, at three or six months post-production, respectively), Lactobacillus brevis (56.5% and 73.9%), Lactobacillus paracasei (56.5% and 39.1%), Lactobacillus rhamnosus (13.0% and 17.4%), Lactobacillus paraplantarum (4.3% and 26.1%), Lactobacillus curvatus (8.7% and 8.7%). Other species included Enterococcus faecalis (47.8% and 43.5%), Enterococcus faecium (34.8% and 17.4%), Enterococcus durans (13.0% and 17.4%), Enterococcus malodoratus (4.3% and 4.3%), and Streptococcus salivarius subsp. thermophilus (21.7% and 30.4%). The increased ripening time was found to be correlated to decreased total solids (r = 0.616; p = 0.002), protein (r = 0.683; p < 0.001), and PH (r = 0.780; p < 0.001). The results of this study contribute to a better understanding of the core microbiota of Feta cheese.

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Direct Molecular Approach to Monitoring Bacterial Colonization on Vacuum-Packaged Beef

Applied and Environmental Microbiology ◽

10.1128/aem.00029-06 ◽

2006 ◽

Vol 72 (8) ◽

pp. 5618-5622 ◽

Cited By ~ 36

Author(s):

Cecilia Fontana ◽

Pier Sandro Cocconcelli ◽

Graciela Vignolo

Keyword(s):

Bacterial Communities ◽

Denaturing Gradient Gel Electrophoresis ◽

Bacterial Colonization ◽

Lactobacillus Sakei ◽

Lactobacillus Curvatus ◽

Specific Primers ◽

Specific Pcr ◽

Gradient Gel Electrophoresis ◽

Packaged Beef ◽

Species Specific

ABSTRACT Denaturing gradient gel electrophoresis allowed us to monitor total bacterial communities and to establish a pattern of succession between species in vacuum-packaged beef stored at 2 and 8°C for 9 weeks and 14 days. Species-specific PCR was used to confirm the presence of Lactobacillus sakei and Lactobacillus curvatus. Multiplex PCRs using 16S rRNA-specific primers allowed differentiation between Leuconostoc species. These methods provided the desired information about microbial diversity by detecting the main microorganisms capable of colonizing this ecological niche.

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Arginine Catabolism by Sourdough Lactic Acid Bacteria: Purification and Characterization of the Arginine Deiminase Pathway Enzymes from Lactobacillus sanfranciscensis CB1

Applied and Environmental Microbiology ◽

10.1128/aem.68.12.6193-6201.2002 ◽

2002 ◽

Vol 68 (12) ◽

pp. 6193-6201 ◽

Cited By ~ 81

Author(s):

Maria De Angelis ◽

Liberato Mariotti ◽

Jone Rossi ◽

Maurizio Servili ◽

Patrick F. Fox ◽

...

Keyword(s):

Lactic Acid ◽

Lactic Acid Bacteria ◽

Lactobacillus Brevis ◽

Inhibitory Effect ◽

Cell Extract ◽

Arginine Deiminase ◽

Lactobacillus Sanfranciscensis ◽

Arginine Catabolism ◽

Carbamate Kinase ◽

Ornithine Transcarbamoylase

ABSTRACT The cytoplasmic extracts of 70 strains of the most frequently isolated sourdough lactic acid bacteria were screened initially for arginine deiminase (ADI), ornithine transcarbamoylase (OTC), and carbamate kinase (CK) activities, which comprise the ADI (or arginine dihydrolase) pathway. Only obligately heterofermentative strains such as Lactobacillus sanfranciscensis CB1; Lactobacillus brevis AM1, AM8, and 10A; Lactobacillus hilgardii 51B; and Lactobacillus fructivorans DD3 and DA106 showed all three enzyme activities. Lactobacillus plantarum B14 did not show CK activity. L. sanfranciscensis CB1 showed the highest activities, and the three enzymes were purified from this microorganism to homogeneity by several chromatographic steps. ADI, OTC, and CK had apparent molecular masses of ca. 46, 39, and 37 kDa, respectively, and the pIs were in the range of 5.07 to 5.2. The OTCs, CKs, and especially ADIs were well adapted to pH (acidic, pH 3.5 to 4.5) and temperature (30 to 37°C) conditions which are usually found during sourdough fermentation. Internal peptide sequences of the three enzymes had the highest level of homology with ADI, OTC, and CK of Lactobacillus sakei. L. sanfranciscensis CB1 expressed the ADI pathway either on MAM broth containing 17 mM arginine or during sourdough fermentation with 1 to 43 mM added arginine. Two-dimensional electrophoresis showed that ADI, OTC, and CK were induced by factors of ca. 10, 4, and 2 in the whole-cell extract of cells grown in MAM broth containing 17 mM arginine compared to cells cultivated without arginine. Arginine catabolism in L. sanfranciscensis CB1 depended on the presence of a carbon source and arginine; glucose at up to ca. 54 mM did not exert an inhibitory effect, and the pH was not relevant for induction. The pH of sourdoughs fermented by L. sanfranciscensis CB1 was dependent on the amount of arginine added to the dough. A low supply of arginine (6 mM) during sourdough fermentation by L. sanfranciscensis CB1 enhanced cell growth, cell survival during storage at 7°C, and tolerance to acid environmental stress and favored the production of ornithine, which is an important precursor of crust aroma compounds.

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Detection of Human Intestinal Catalase-Negative, Gram-Positive Cocci by rRNA-Targeted Reverse Transcription-PCR

Applied and Environmental Microbiology ◽

10.1128/aem.03132-09 ◽

2010 ◽

Vol 76 (16) ◽

pp. 5440-5451 ◽

Cited By ~ 46

Author(s):

Hiroyuki Kubota ◽

Hirokazu Tsuji ◽

Kazunori Matsuda ◽

Takashi Kurakawa ◽

Takashi Asahara ◽

...

Keyword(s):

Reverse Transcription ◽

Population Level ◽

23S Rrna ◽

Specific Primer ◽

Gram Positive ◽

Reverse Transcription Pcr ◽

Qpcr Method ◽

Primer Sets ◽

Species Specific ◽

Gram Positive Cocci

ABSTRACT An analytical system based on rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) for enumeration of catalase-negative, Gram-positive cocci was established. Subgroup- or species-specific primer sets targeting 16S or 23S rRNA from Enterococcus, Streptococcus, and Lactococcus were newly developed. The RT-qPCR method using these primers together with the previously reported primer sets specific for the Enterococcus genus, the Streptococcus genus, and several Streptococcus species was found to be able to quantify the target populations with detection limits of 103 to 104 cells per gram feces, which was more than 100 times as sensitive as the qPCR method (106 to 108 cells per gram feces). The RT-qPCR analysis of fecal samples from 24 healthy adult volunteers using the genus-specific primer sets revealed that Enterococcus and Streptococcus were present as intestinal commensals at population levels of log10 6.2 ± 1.4 and 7.5 ± 0.9 per gram feces (mean ± standard deviation [SD]), respectively. Detailed investigation using species- or subgroup-specific primer sets revealed that the volunteers harbored unique Enterococcus species, including the E. avium subgroup, the E. faecium subgroup, E. faecalis, the E. casseliflavus subgroup, and E. caccae, while the dominant human intestinal Streptococcus species was found to be S. salivarius. Various Lactococcus species, such as L. lactis subsp. lactis or L. lactis subsp. cremoris, L. garvieae, L. piscium, and L. plantarum, were also detected but at a lower population level (log10 4.6 ± 1.2 per gram feces) and prevalence (33%). These results suggest that the RT-qPCR method enables the accurate and sensitive enumeration of human intestinal subdominant but still important populations, such as Gram-positive cocci.

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Bacteriophage Ecology in a Commercial Cucumber Fermentation

Applied and Environmental Microbiology ◽

10.1128/aem.01914-12 ◽

2012 ◽

Vol 78 (24) ◽

pp. 8571-8578 ◽

Cited By ~ 19

Author(s):

Z. Lu ◽

I. M. Pérez-Díaz ◽

J. S. Hayes ◽

F. Breidt

Keyword(s):

Lactic Acid ◽

Starter Culture ◽

Lactobacillus Brevis ◽

Starter Cultures ◽

Broad Host Range ◽

Bacterial Isolates ◽

Content Type ◽

Phage Ecology ◽

Low Salt ◽

Species Specific

ABSTRACTTo reduce high-salt waste from cucumber fermentations, low-salt fermentations are under development. These fermentations may require the use of starter cultures to ensure normal fermentations. Because potential phage infection can cause starter culture failure, it is important to understand phage ecology in the fermentations. This study investigated the phage ecology in a commercial cucumber fermentation. Brine samples taken from a fermentation tank over a 90-day period were plated onto deMan-Rogosa-Sharpe agar plates. A total of 576 lactic acid bacterial isolates were randomly selected to serve as potential hosts for phage isolation. Filtered brine served as a phage source. Fifty-seven independent phage isolates were obtained, indicating that 10% of the bacterial isolates were sensitive to phage attack. Phage hosts includeLactobacillus brevis(67% of all hosts),Lactobacillus plantarum(21%),Weissella paramesenteroides,Weissella cibaria, andPediococcus ethanolidurans. Nearly 50% of phages were isolated on day 14, and the majority of them attackedL. brevis. Some phages had a broad host range and were capable of infecting multiple hosts in two genera. Other phages were species specific or strain specific. About 30% of phage isolates produced turbid pinpoint plaques or only caused reduced cell growth on the bacterial lawns. Six phages with distinct host ranges were characterized. The data from this study showed that abundant and diverse phages were present in the commercial cucumber fermentation, which could cause significant mortality to the lactic acid bacteria population. Therefore, a phage control strategy may be needed in low-salt cucumber fermentations.

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Microbial Quality and Direct PCR Identification of Lactic Acid Bacteria and Nonpathogenic Staphylococci from Artisanal Low-Acid Sausages

Applied and Environmental Microbiology ◽

10.1128/aem.69.8.4583-4594.2003 ◽

2003 ◽

Vol 69 (8) ◽

pp. 4583-4594 ◽

Cited By ~ 169

Author(s):

T. Aymerich ◽

B. Martín ◽

M. Garriga ◽

M. Hugas

Keyword(s):

Lactic Acid ◽

Lactic Acid Bacteria ◽

Point Of View ◽

Lactobacillus Curvatus ◽

Direct Pcr ◽

Specific Pcr ◽

Food Borne ◽

Fermented Sausages ◽

Species Specific ◽

Staphylococcus Simulans

ABSTRACT Detection of six species of lactic acid bacteria and six species of gram-positive catalase-positive cocci from low-acid fermented sausages (fuets and chorizos) was assessed by species-specific PCR. Without enrichment, Lactobacillus sakei and Lactobacillus curvatus were detected in 11.8% of the samples, and Lactobacillus plantarum and Staphylococcus xylosus were detected in 17.6%. Enriched samples allowed the detection of L. sakei and S. xylosus in all of the samples (100%) and of Enterococcus faecium in 11.8% of the sausages. The percentages of L. curvatus, L. plantarum, Staphylococcus carnosus, and Staphylococcus epidermidis varied depending on the sausage type. L. curvatus was detected in 80% of fuets and in 57% of chorizos. L. plantarum was found in 50% of fuets and 100% of chorizos. S. epidermidis was detected in only 11.8% of fuets, and S. carnosus was detected in only 5.9% of chorizos. Lactococcus lactis, Staphylococcus warneri, and Staphylococcus simulans were not detected in any sausage type. From a microbiological point of view, 70.6% of the samples could be considered of high quality, as they had low counts of Enterobacteriaceae and did not contain any of the food-borne pathogens assayed.

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Lactic Acid Bacteria Isolation from Spontaneous Sourdough and Their Characterization Including Antimicrobial and Antifungal Properties Evaluation

Microorganisms ◽

10.3390/microorganisms8010064 ◽

2019 ◽

Vol 8 (1) ◽

pp. 64 ◽

Cited By ~ 20

Author(s):

Elena Bartkiene ◽

Vita Lele ◽

Modestas Ruzauskas ◽

Konrad J. Domig ◽

Vytaute Starkute ◽

...

Keyword(s):

Lactic Acid ◽

Lactic Acid Bacteria ◽

Research Effort ◽

Lactobacillus Brevis ◽

Industrial Applications ◽

Starter Cultures ◽

Lactobacillus Curvatus ◽

Antifungal Properties ◽

Wide Range ◽

Food And Feed

This research effort aimed at isolating and phenotypically characterizing lactic acid bacteria (LAB) isolates from a spontaneous rye sourdough manufactured following traditional protocols, as well as at evaluating their antimicrobial and antifungal properties as key features for future industrial applications. Thirteen LAB strains of potential industrial interest were isolated and identified to species-level via PCR. Most of the sourdough isolates showed versatile carbohydrate metabolisms. The Leuconostoc mesenteroides No. 242 and Lactobacillus brevis No. 173 demonstrated to be gas producers; thus, revealing their heterofermenter or facultative homofermenter features. Viable counts higher than 7.0 log10 (CFU/mL) were observed for Lactobacillus paracasei No. 244, Lactobacillus casei No. 210, L. brevis No. 173, Lactobacillus farraginis No. 206, Pediococcus pentosaceus No. 183, Lactobacillus uvarum No. 245 and Lactobacillus plantarum No. 135 strains, after exposure at pH 2.5 for 2 h. Moreover, L. plantarum No. 122, L. casei No. 210, Lactobacillus curvatus No. 51, L. paracasei No. 244, and L. coryniformins No. 71 showed growth inhibition properties against all the tested fifteen pathogenic strains. Finally, all LAB isolates showed antifungal activities against Aspergillus nidulans, Penicillium funiculosum, and Fusarium poae. These results unveiled the exceptionality of spontaneous sourdough as a source of LAB with effective potential to be considered in the design of novel commercial microbial single/mixed starter cultures, intended for application in a wide range of agri-food industries, where the antimicrobial and antifungal properties are often sought and necessary. In addition, metabolites therefrom may also be considered as important functional and bioactive compounds with high potential to be employed in food and feed, as well as cosmetic and pharmaceutical applications.

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