Jerome COMBRISSON Ph.D

Microbial analyses of fermented milk products require selective
methods to discriminate between close species simultaneously present in
high amounts. A culture-based method combining novel chromogenic agar
media and appropriate incubation conditions was developed to enumerate
lactic acid bacteria (LAB) strains in fermented milk. M1 agar, containing
two chromogenic substrates, allowed selective enumeration of
Lactobacillus rhamnosus, two strains of Lactobacillus paracasei… Voir plus

Microbial analyses of fermented milk products require selective
methods to discriminate between close species simultaneously present in
high amounts. A culture-based method combining novel chromogenic agar
media and appropriate incubation conditions was developed to enumerate
lactic acid bacteria (LAB) strains in fermented milk. M1 agar, containing
two chromogenic substrates, allowed selective enumeration of
Lactobacillus rhamnosus, two strains of Lactobacillus paracasei subsp.
paracasei and Streptococcus salivarius subsp. thermophilus based on
differential β-galactosidase and β-glucosidase activities. Depending on
the presence of some or all of the above strains, M1 agar was
supplemented with L-rhamnose or vancomycin and incubations were carried
out at 37°C or 44°C to increase selectivity. A second agar medium, M2,
containing one chromogenic substrates was used to selectively enumerate
β-galactosidase producing L. delbrueckii subsp. bulgaricus at 47°C. By
contrast with the use of usual culture media, the chromogenic method
allowed unambiguous enumeration of each species, including discrimination
between the two L. paracasei, up to 109 CFU/g of fermented milk. In
addition, the relevance of the method was approved by enumerating
reference ATCC strains in pure cultures and fermented milk product. The
method could also be used for enumerations on non-Danone commercial
fermented milk products containing strains different from those used in
this study, showing versatility of the method. To our knowledge, this is
the first description of a chromogenic culture method applied to
selective enumeration of LAB. Voir moins

The International Dairy Federation (IDF) is a science-based, non-profit private sector organisation which represents the interests of various stakeholders in dairy (including dairy farmers, dairy processing industries, dairy suppliers, academics and governments/food control authorities) at the international level. IDF aims to identify, elaborate and disseminate best practice at the international level to guide the dairy sector and to harmonise the work of its members on a variety of issues… Voir plus

The International Dairy Federation (IDF) is a science-based, non-profit private sector organisation which represents the interests of various stakeholders in dairy (including dairy farmers, dairy processing industries, dairy suppliers, academics and governments/food control authorities) at the international level. IDF aims to identify, elaborate and disseminate best practice at the international level to guide the dairy sector and to harmonise the work of its members on a variety of issues, including standards for methods of analysis and sampling (MAS). Voir moins

A rapid and specific analytical method to enumerate viable Bifidobacteria in dairy products containing a mix of lactic acid bacteria was developed to proof of concept. A polyclonal antibody against Bifidobacterium animalis ssp. lactis (B. lactis) was used for specific detection by flow cytometry (FCM). The antibody, which targeted the cell wall of B. lactis, had a high labelling efficiency of 95.5±1.9% of the population. A viability probe, 5, 6-carboxyfluorescein diacetate, was combined with… Voir plus

A rapid and specific analytical method to enumerate viable Bifidobacteria in dairy products containing a mix of lactic acid bacteria was developed to proof of concept. A polyclonal antibody against Bifidobacterium animalis ssp. lactis (B. lactis) was used for specific detection by flow cytometry (FCM). The antibody, which targeted the cell wall of B. lactis, had a high labelling efficiency of 95.5±1.9% of the population. A viability probe, 5, 6-carboxyfluorescein diacetate, was combined with the antibody for real-time quantification of viable B. lactis. Enumeration results from FCM on four different commercial dairy products that contain B. lactis showed a good correlation with the standard culture-based method. Moreover, it provided a species-specific method to enumerate viable B. lactis within 2 h; the standard method counts the total cultivable Bifidobacteria genus on plates after 72 h. This study demonstrated that FCM could be a rapid solution for probiotic analysis in dairy products.

Keywords
Bifidobacteria; Flow cytometry; Antibody Voir moins

Traditionally, culture-based methods have been used to enumerate microbial populations in dairy products. Recent developments in molecular methods now enable faster and more sensitive analyses than classical microbiology procedures. These molecular tools allow a detailed characterization of cell physiological states and bacterial fitness and thus, offer new perspectives to integration of microbial physiology monitoring to improve industrial processes. This review summarizes the methods… Voir plus

Traditionally, culture-based methods have been used to enumerate microbial populations in dairy products. Recent developments in molecular methods now enable faster and more sensitive analyses than classical microbiology procedures. These molecular tools allow a detailed characterization of cell physiological states and bacterial fitness and thus, offer new perspectives to integration of microbial physiology monitoring to improve industrial processes. This review summarizes the methods described to enumerate and characterize physiological states of technological microbiota in dairy products, and discusses the current deficiencies in relation to the industry’s needs. Recent studies show that PCR-based methods can successfully be applied to quantify fermenting microbes and probiotics in dairy products. Flow cytometry and omics technologies also show interesting analytical potentialities. However, they still suffer from a lack of validation and standardization for quality control analyses, as reflected by the absence of performance studies and official international standards. Voir moins

Recent developments in molecular methods aid in detecting and quantifying food microorganisms through faster, more sensitive and more specific procedures than classical microbiology techniques. Molecular tools were widely used to detect pathogens in food but yet remain insufficient to directly quantify them in food products. Nevertheless, recent studies show that quantitative PCR (qPCR)-based methods are applied with success in enumerating fermenting microbes and health promoting bacteria in… Voir plus

Recent developments in molecular methods aid in detecting and quantifying food microorganisms through faster, more sensitive and more specific procedures than classical microbiology techniques. Molecular tools were widely used to detect pathogens in food but yet remain insufficient to directly quantify them in food products. Nevertheless, recent studies show that quantitative PCR (qPCR)-based methods are applied with success in enumerating fermenting microbes and health promoting bacteria in dairy products. The increasing number of sequenced genomes is now a crucial support to designing specific primers and probes. Technological advances in qPCR –based methods including viable quantitative PCR are also highlighted and could be promising strategies for quantifying merely viable cells, detecting multiple microorganisms in a single reaction and constructing high throughput PCR workflows. Application of real-time PCR in the food industry has proven its reliability nevertheless its widespread use in the dairy industry will need technical recommendations and standardisation methods. Voir moins

Molecular methods are being increasingly applied to detect, quantify and study microbial populations in food or during food processes. Among these methods, PCR-based techniques have been the subject of considerable focus and ISO guidelines have been established for the detection of food-borne pathogens. More particularly, real-time quantitative PCR (qPCR) is considered as a method of choice for the detection and quantification of microorganisms. One of its major advantages is to be faster than… Voir plus

Molecular methods are being increasingly applied to detect, quantify and study microbial populations in food or during food processes. Among these methods, PCR-based techniques have been the subject of considerable focus and ISO guidelines have been established for the detection of food-borne pathogens. More particularly, real-time quantitative PCR (qPCR) is considered as a method of choice for the detection and quantification of microorganisms. One of its major advantages is to be faster than conventional culture-based methods. It is also highly sensitive, specific and enables simultaneous detection of different microorganisms. Application of reverse-transcription-qPCR (RT-qPCR) to study population dynamics and activities through quantification of gene expression in food, by contrast with the use of qPCR, is just beginning. Provided that appropriate controls are included in the analyses, qPCR and RT-qPCR appear to be highly accurate and reliable for quantification of genes and gene expression. This review addresses some important technical aspects to be considered when using these techniques. Recent applications of qPCR and RT-qPCR in food microbiology are given. Some interesting applications such as risk analysis or studying the influence of industrial processes on gene expression and microbial activity are reported. Voir moins

In a previous study using fusion of the deregulated lactose promoter lacTp* and reporter genes, we suggested that Lactobacillus casei could initiate de novo protein synthesis during intestinal transit. In order to confirm this finding and extend it to other promoters, we adopted a reverse transcriptase quantitative PCR (RT-QPCR) approach combined with a transcriptional fusion system consisting of luciferase genes under the control of four promoters (ccpA, dlt, ldh, and lacT*) from L. casei… Voir plus

In a previous study using fusion of the deregulated lactose promoter lacTp* and reporter genes, we suggested that Lactobacillus casei could initiate de novo protein synthesis during intestinal transit. In order to confirm this finding and extend it to other promoters, we adopted a reverse transcriptase quantitative PCR (RT-QPCR) approach combined with a transcriptional fusion system consisting of luciferase genes under the control of four promoters (ccpA, dlt, ldh, and lacT*) from L. casei DN-114 001. Promoter expression was monitored during cell growth, and variable luciferase activities were detected. In 3-day cultures, all the genetically modified strains survived but without exhibiting luciferase activity. Luciferase mRNA levels determined by RT-QPCR analysis (RNA/CFU) were not significant. The cultures were administered to human-microbiota-associated mice, and the feces were collected 6 h later. L. casei promoters lacTp* and ldhp initiated mRNA synthesis during gastrointestinal transit. The promoters, ccpAp and dltp, exhibited no luciferase activity, nor was de novo-synthesized luciferase mRNA detected in the feces. L. casei seems to adapt its physiology to the gastrointestinal tract environment by modulating promoter activities. The approach (fecal transcriptional analysis) described herein may, moreover, be of value in studying gene expression of transiting bacteria in human fecal specimens.
ACKNOWLEDGMENTS :
We are grateful to Donald White for correcting the English of the manuscript and to Jerome Combrisson, Denis Mater, and Olivier Goniak for their helpful advice. Voir moins

The cell density and the genetic structure of bacterial subcommunities (further named pools)
present in the various microenvironments of a silt loam soil were investigated. The microenvironments
were isolated first using a procedure of soil washes that separated bacteria located outside
aggregates (outer part) from those located inside aggregates (inner part). A nondestructive physical
fractionation was then applied to the inner part in order to separate bacteria located inside… Voir plus

The cell density and the genetic structure of bacterial subcommunities (further named pools)
present in the various microenvironments of a silt loam soil were investigated. The microenvironments
were isolated first using a procedure of soil washes that separated bacteria located outside
aggregates (outer part) from those located inside aggregates (inner part). A nondestructive physical
fractionation was then applied to the inner part in order to separate bacteria located inside stable
aggregates of different size (size fractions, i.e., two macroaggregate fractions, two microaggregate
fractions, and the dispersible day fraction). Bacterial densities measured by acridine orange direct
counts (AODC) and viable heterotrophic (VH) cell enumerations showed the heterogeneous quantitative
distribution of cells in soil. Bacteria were preferentially located in the inner part with 87.6%
and 95.4% of the whole AODC and VH bacteria, respectively, and in the microaggregate and
dispersible clay fractions of this part with more than 70% and 80% of the whole AODC and VH
bacteria, respectively. The rRNA intergenic spacer analysis (RISA) was used to study the genetic
structure of the bacterial pools. Different fingerprints and consequently different genetic structures
were observed between the unfractionated soil and the microenvironments, and also among the
various microenvironments, giving evidence that some populations were specific to a given location
in addition to the common populations of all the microenvironments. Cluster and multivariate
analysis of RISA profiles showed the weak contribution of the pools located in the macroaggregate
fractions to the whole soil community structure, as well as the clear distinction between the pool
associated to the macroaggregate fractions and the pools associated to the microaggregate ones. Voir moins

We examined the mineralization of pentachlorophenol (PCP) in sterile and non-sterile soil with or without added bacteria (Mycobacterium chlorophenolicum PCP-1). The soil used had no history of PCP contamination. Microcosms (30 g dry weight of soil) were incubated with labelled PCP (6.76% 13C, a non-radioactive stable isotope, 22 mg kg-1 dry weight) for 60 days. M. chlorophenolicum PCP-1 (7.8 x 10(6) cells g-1 dry weight) was added to some samples. 50% of the PCP was mineralized in non-sterile… Voir plus

We examined the mineralization of pentachlorophenol (PCP) in sterile and non-sterile soil with or without added bacteria (Mycobacterium chlorophenolicum PCP-1). The soil used had no history of PCP contamination. Microcosms (30 g dry weight of soil) were incubated with labelled PCP (6.76% 13C, a non-radioactive stable isotope, 22 mg kg-1 dry weight) for 60 days. M. chlorophenolicum PCP-1 (7.8 x 10(6) cells g-1 dry weight) was added to some samples. 50% of the PCP was mineralized in non-sterile soil with or without the exogenous bacteria. Only 5% of the PCP was mineralized in sterile soil with or without bacteria. These data suggest that the PCP was not accessible to M. chlorophenolicum and that the indigenous soil microflora can mineralize PCP Voir moins

A single DNA procedure to recover bacterial DNA from various soil microenvironments which differ in their physical, chemical and structural properties was developed. These microenvironments, obtained by a combination of soil washes and physical fractionation, were the outer part or macroporosity (outside and surface of aggregates), the inner part or microporosity (inside of aggregates) and various size and stability classes of soil aggregates and particles. The DNA extraction method involved… Voir plus

A single DNA procedure to recover bacterial DNA from various soil microenvironments which differ in their physical, chemical and structural properties was developed. These microenvironments, obtained by a combination of soil washes and physical fractionation, were the outer part or macroporosity (outside and surface of aggregates), the inner part or microporosity (inside of aggregates) and various size and stability classes of soil aggregates and particles. The DNA extraction method involved sample homogenization and cell disruption by grinding in liquid nitrogen, followed by enzymatic lysis with lysozyme and proteinase K. High yields of high molecular weight DNA (≥ 23 kb) were obtained for all microenvironments. Crude DNA yields for the various soil microenvironments were between 0.7 and 51.4 μg DNA·g−1 soil sample and were positively correlated with bacterial cell abundance (r = 0.91). Further purification steps allowed to recover at least 60 % of the DNA extracted from the various microenvironments. The suitability of the extracted DNA to undergo enzymatic amplification reactions and the effectiveness of the extraction procedure in recovering DNA from various native bacterial groups was tested using primers for archaebacterial 16S rDNAs, universal and group-specific eubacterial 16S rDNAs primers (β- and γ-proteobacteria, High G+C Gram-positive bacteria, and Bacillus species and relatives). Successful amplification of less ubiquitous genes was also obtained with primers targeting nitrogen fixation (nifH) and mercury resistance (merRTΔP) genes. Voir moins