The late blowing in cheese: a new molecular approach based on PCR and DGGE to study the microbial ecology of the alteration process

Cocolin, Luca Simone; Innocente, Nadia; Biasutti, M.; Comi, Giuseppe

doi:10.1016/s0168-1605(03)00296-4

Cited by 119 publications

(64 citation statements)

References 16 publications

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“…With a nonspecific primer for the Clostridium genus, the sensitivity of such a method was estimated to be 10 4 CFU/g. In this case, an enrichment period of cheese samples before DNA extraction is needed (7). Based on in vitro tests on pure cultures, we expect clostridial species to be detected even when they constitute only 10% of the clostridial population.…”

Section: Discussionmentioning

confidence: 99%

“…Consequently, a set of specific primers must be available to identify all the agents responsible for alteration. Denaturing gradient gel electrophoresis (DGGE) (10), temperature gradient gel electrophoresis (28), or temporal temperature gradient gel electrophoresis (TTGE) (37) is suitable for the analysis of mixed microbial communities (7,24) and can also be focused on the numerically subdominant bacteria by specific amplification of 16S rRNA genes (14). This tool leads to the separation of DNA fragments of the same length but with different base-pair sequences.…”

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Development and Validation of PCR Primers To Assess the Diversity of Clostridium spp. in Cheese by Temporal Temperature Gradient Gel Electrophoresis

Bourhis

Saunier

Doré

et al. 2005

Appl Environ Microbiol

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A nested-PCR temporal temperature gradient gel electrophoresis (TTGE) approach was developed for the detection of bacteria belonging to phylogenetic cluster I of the genus Clostridium (the largest clostridial group, which represents 25% of the currently cultured clostridial species) in cheese suspected of late blowing. Primers were designed based on the 16S rRNA gene sequence, and the specificity was confirmed in PCRs performed with DNAs from cluster I and non-cluster I species as the templates. TTGE profiles of the PCR products, comprising the V5-V6 region of the 16S rRNA gene, allowed us to distinguish the majority of cluster I species. PCR-TTGE was applied to analyze commercial cheeses with defects. All cheeses gave a signal after nested PCR, and on the basis of band comigration with TTGE profiles of reference strains, all the bands could be assigned to a clostridial species. The direct identification of Clostridium spp. was confirmed by sequencing of excised bands. C. tyrobutyricum and C. beijerinckii contaminated 15 and 14 of the 20 cheese samples tested, respectively, and C. butyricum and C. sporogenes were detected in one cheese sample. Most-probable-number counts and volatile fatty acid were determined for comparison purposes. Results obtained were in agreement, but only two species, C. tyrobutyricum and C. sporogenes, could be isolated by the plating method. In all cheeses with a high amount of butyric acid (>100 mg/100 g), the presence of C. tyrobutyricum DNA was confirmed by PCR-TTGE, suggesting the involvement of this species in butyric acid fermentation. These results demonstrated the efficacy of the PCR-TTGE method to identify Clostridium in cheeses. The sensitivity of the method was estimated to be 100 CFU/g.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Development and Validation of PCR Primers To Assess the Diversity of Clostridium spp. in Cheese by Temporal Temperature Gradient Gel Electrophoresis

Bourhis

Saunier

Doré

et al. 2005

Appl Environ Microbiol

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“…After the enrichment, 5 mL of the cultures were transferred into conical tubes and centrifuged at 14,000g at 4 C for 5 min. The settled bacterial cells were collected and subjected to DNA extraction by the method of Cocolin et al (2004). PCR was used to amplify a variable region (V3 region) of the bacterial 16S rRNA gene, with the GC-clamp attached to the forward primer 341F-GC (5 0 -CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGG GGGGCCTACGGGAGGCAGCAG-3 0 ) and the reverse primer 534R (5 0 -ATTACCGCGGCTGCTGG-3 0 ) (Muyzer et al 1993).…”

Section: Denaturing Gradient Gel Electrophoresis (Dgge)mentioning

confidence: 99%

The effect of cultivar, wilting and storage period on fermentation and the clostridial community of alfalfa silage

Zheng

Niu

Zuo

et al. 2017

Italian Journal of Animal Science

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The objective of this study was to gain deeper insights into chemical transformations and the clostridial community dynamics during the ensiling of alfalfa. A factorial experiment was conducted with four alfalfa cultivars [Sanditi (A1), AC Caribou (A2), WL319HQ (A3) and 4030 (A4)] Â three wilting durations [0 h (direct-cut), 2 h and 4 h] Â three storage periods (14, 28 and 56 days). The clostridial community was examined using denaturing gradient gel electrophoresis. High butyric acid and ammonia nitrogen (NH 3 -N) contents and clostridia numbers were observed in direct-cut silage, with higher values observed in A4 than in the other three cultivars. However, butyric acid and NH 3 -N contents and clostridia numbers decreased with wilting regardless of the cultivar. Although Clostridium ghonii and Clostridium sartagoforme were common to all direct-cut and wilted silages, bands for these species were faint. Differences in the appearing time of these species were observed, bands for C. ghonii were found after 14 and 28 days of ensiling, while those for C. sartagoforme were only found after 56 days. In addition, in direct-cut silage, distinct bands for Clostridium perfringens were detected in A1, A2 and A3, while those for Clostridium sporogenes were detected in A4. The inactive spores of clostridia could be observed in 4-h wilted silage as no butyric acid was detected. It was concluded that the enhanced clostridial fermentation of direct-cut silage was attributed mainly to C. perfringens and C. sporogenes, while C. ghonii and C. sartagoforme were involved in the restricted clostridial fermentation of 2-h wilted silage. ARTICLE HISTORY

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“…Denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE) and temporal temperature gradient gel electrophoresis (TTGE) are widely used to study cheese microbial communities Ercolini et al, 2001;Ogier et al, 2002;Randazzo et al, 2002;Ercolini et al, 2003;Mauriello et al, 2003;Andrighetto et al, 2004;Cocolin et al, 2004;Ercolini et al, 2004;Henri-Dubernet et al, 2004;Lafarge et al, 2004;Ogier et al, 2004;Rantsiou et al, 2004;Le Bourhis et al, 2005;Flórez and Mayo, 2006;Randazzo et al, 2006;Cocolin et al, 2007;El-Baradei et al, 2007;Le Bourhis et al, 2007;Parayre et al, 2007;Abriouel et al, 2008;Bonetta et al, 2008;Ercolini et al, 2008;Gala et al, 2008;Henri-Dubernet et al, 2008;Nikolic et al, 2008;Rantsiou et al, 2008b;Van Hoorde et al, 2008;Alegría et al, 2009;Casalta et al, 2009;Dolci et al, 2009;Giannino et al, 2009;Serhan et al, 2009;Dolci et al, 2010;Fontana et al, 2010;Fuka et al, 2010;Randazzo et al, 2010;Van Hoorde et al, 2010;Masoud et al, 2011). Target sequences from rRNA or housek...…”

Section: Pcr-based Methods For Microbial Diversity Investigationmentioning

confidence: 99%

Application of PCR-Based Methods to Dairy Products and to Non-Dairy Probiotic Products

Monnet¹,

Matijašić²

2012

Polymerase Chain Reaction

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The late blowing in cheese: a new molecular approach based on PCR and DGGE to study the microbial ecology of the alteration process

Cited by 119 publications

References 16 publications

Development and Validation of PCR Primers To Assess the Diversity of Clostridium spp. in Cheese by Temporal Temperature Gradient Gel Electrophoresis

Development and Validation of PCR Primers To Assess the Diversity of Clostridium spp. in Cheese by Temporal Temperature Gradient Gel Electrophoresis

The effect of cultivar, wilting and storage period on fermentation and the clostridial community of alfalfa silage

Application of PCR-Based Methods to Dairy Products and to Non-Dairy Probiotic Products

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