Christèle Humblot

Pyrosequencing of Tagged 16S rRNA Gene Amplicons for Rapid Deciphering of the Microbiomes of Fermented Foods Such as Pearl Millet Slurries

ABSTRACT Pearl millet slurries, mixed with groundnuts or not, were chosen as a model to investigate the feasibility of obtaining a rapid overview of community structure and population dynamics of fermented foods using pyrosequencing of tagged 16S rRNA gene amplicons. From 14 fermented samples collected either in a traditional small-scale processing unit in Burkina Faso or at laboratory scale, 137,469 sequences of bacterial 16S rRNA gene amplicons were characterized. Except for a few Proteobacteria, almost all the bacterial sequences were attributed to cultivable bacteria. This approach enabled 80.7% of the sequences to be attributed to a family and 70% to a genus but did not enable identification to the species level. The bacterial sequences were assigned to four phyla, with Firmicutes representing the highest diversity, followed by Proteobacteria, Actinobacteria, and Bacteroidetes, which were found only in the slurries prepared in traditional production units. Most of the Firmicutes were lactic acid bacteria, mainly represented by members of the Lactobacillus, Pediococcus, Leuconostoc, and Weissella genera, whose ratio varied from the onset to the end of the fermentation. The other bacteria present at the beginning of fermentation were generally no longer detected at the end, which is consistent with already-known patterns in the microbial ecology of fermented foods. In conclusion, this method seems very promising for rapid and preliminary microbial characterization in many samples of an unknown food sample, by determining numerous nucleic sequences simultaneously without the need for cloning and cultivation-dependent methods.

Genetic Screening of Functional Properties of Lactic Acid Bacteria in a Fermented Pearl Millet Slurry and in the Metagenome of Fermented Starchy Foods

ABSTRACT Lactic acid bacteria (LAB) (n = 152) in African pearl millet slurries and in the metagenomes of amylaceous fermented foods were investigated by screening 33 genes involved in probiotic and nutritional functions. All isolates belonged to six species of the genera Pediococcus and Lactobacillus, and Lactobacillus fermentum was the dominant species. We screened the isolates for the abilities to survive passage through the gastrointestinal tract and to synthesize folate and riboflavin. The isolates were also tested in vitro for their abilities to survive exposure to bile salts and to survive at pH 2. Because the ability to hydrolyze starch confers an ecological advantage on LAB that grow in starchy matrixes as well as improving the nutritional properties of the gruels, we screened for genes involved in starch metabolism. The results showed that genes with the potential ability to survive passage through the gastrointestinal tract were widely distributed among isolates and metagenomes, whereas in vitro tests showed that only a limited set of isolates, mainly those belonging to L. fermentum, could tolerate a low pH. In contrast, the wide distribution of genes associated with bile salt tolerance, in particular bsh, is consistent with the high frequency of tolerance to bile salts observed. Genetic screening revealed a potential for folate and riboflavin synthesis in both isolates and metagenomes, as well as high variability among genes related to starch metabolism. Genetic screening of isolates and metagenomes from fermented foods is thus a promising approach for assessing the functional potential of food microbiotas.

Lactobacillaceae and Cell Adhesion: Genomic and Functional Screening

The analysis of collections of lactic acid bacteria (LAB) from traditional fermented plant foods in tropical countries may enable the detection of LAB with interesting properties. Binding capacity is often the main criterion used to investigate the probiotic characteristics of bacteria. In this study, we focused on a collection of 163 Lactobacillaceace comprising 156 bacteria isolated from traditional amylaceous fermented foods and seven strains taken from a collection and used as controls. The collection had a series of analyses to assess binding potential for the selection of new probiotic candidates. The presence/absence of 14 genes involved in binding to the gastrointestinal tract was assessed. This enabled the detection of all the housekeeping genes (ef-Tu, eno, gap, groEl and srtA) in the entire collection, of some of the other genes (apf, cnb, fpbA, mapA, mub) in 86% to 100% of LAB, and of the other genes (cbsA, gtf, msa, slpA) in 0% to 8% of LAB. Most of the bacteria isolated from traditional fermented foods exhibited a genetic profile favorable for their binding to the gastrointestinal tract. We selected 30 strains with different genetic profiles to test their binding ability to non-mucus (HT29) and mucus secreting (HT29-MTX) cell lines as well as their ability to degrade mucus. Assays on both lines revealed high variability in binding properties among the LAB, depending on the cell model used. Finally, we investigated if their binding ability was linked to tighter cross-talk between bacteria and eukaryotic cells by measuring the expression of bacterial genes and of the eukaryotic MUC2 gene. Results showed that wild LAB from tropical amylaceous fermented food had a much higher binding capacity than the two LAB currently known to be probiotics. However their adhesion was not linked to any particular genetic equipment.

Brussels sprouts, inulin and fermented milk alter the faecal microbiota of human microbiota-associated rats as shown by PCR-temporal temperature gradient gel electrophoresis using universal, Lactobacillus and Bifidobacterium 16S rRNA gene primers

We investigated the effect of Brussels sprouts, inulin and a fermented milk on the faecal microbiota diversity of human microbiota-associated (HMA) rats by PCR-temporal temperature gradient gel electrophoresis (PCR-TTGE) using universal and group-specific 16S rRNA gene primers. The HMA rats were submitted to a control diet for 10 d (initial time), then switched to the experimental diets for 4 weeks (final time). Using universal primers, the mean degree of similarity between all faecal samples at initial time was 80.8 %. In the group consuming the control diet throughout the experiment, the mean degree of similarity between the PCR-TTGE profiles at initial v. final time was 76.8 %, reflecting a spontaneous temporal variation. The mean degree of similarity between control and experimental groups at final time was lower, 72.4 %, 74.4 % and 75.6 % for inulin, Brussels sprouts and fermented milk, respectively, indicating a dietary effect on the predominant populations. Using specific primers, bifidobacteria could be detected only in those rats that had consumed inulin, showing a specific increasing effect of this dietary compound. The Lactobacillus population was very heterogeneous at initial time but tended to homogenize within each dietary group. At final time, caecal contents were collected for analysis of SCFA and beta-glucuronidase activity. Inulin and Brussels sprouts increased the butyrate and acetate proportion, respectively, while the fermented milk did not modify the caecal biochemistry. This experiment shows for the first time that cruciferous vegetables are able to alter the diversity and the metabolic activities of the digestive microbiota in HMA rats.

1H nuclear magnetic resonance spectroscopy-based studies of the metabolism of food-borne carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline by human intestinal microbiota.

ABSTRACT 2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) is a mutagenic/carcinogenic compound formed from meat and fish during cooking. Following ingestion, IQ is metabolized mainly by liver xenobiotic-metabolizing enzymes, but intestinal bacteria may also contribute to its biotransformation. The aim of this study was to investigate the metabolism of IQ by the human intestinal microbiota. Following incubation of IQ (200 μM) under anoxic conditions with 100-fold dilutions of stools freshly collected from three healthy volunteers, we quantified residual IQ by high-pressure liquid chromatography (HPLC) analysis and characterized the production of IQ metabolites by in situ 1H nuclear magnetic resonance (1H-NMR) spectroscopic analysis of crude incubation media. In addition, we looked for IQ-degrading bacteria by screening collection strains and by isolating new strains from the cecal contents of human-microbiota-associated rats gavaged with IQ on a regular basis. HPLC and 1H-NMR analyses showed that the three human microbiota degraded IQ with different efficiencies (range, 50 to 91% after 72 h of incubation) and converted it into a unique derivative, namely, 7-hydroxy-IQ. We found 10 bacterial strains that were able to perform this reaction: Bacteroides thetaiotaomicron (n = 2), Clostridium clostridiiforme (n = 3), Clostridium perfringens (n = 1), and Escherichia coli (n = 4). On the whole, our results indicate that bacteria belonging to the predominant communities of the human intestine are able to produce 7-hydroxy-IQ from IQ. They also suggest interindividual differences in the ability to perform this reaction. Whether it is a metabolic activation is still a matter of debate, since 7-hydroxy-IQ has been shown to be a direct-acting mutagen in the Ames assay but not carcinogenic in laboratory rodents.

Determination of expression and activity of genes involved in starch metabolism in Lactobacillus plantarum A6 during fermentation of a cereal-based gruel

Traditional fermented gruels prepared from cereals are widely used for complementary feeding of young children in Africa and usually have a low energy density. The amylase activity of some lactic acid bacteria (LAB) helps increase the energy content of gruels through partial hydrolysis of starch, thus enabling the incorporation of more starchy material while conserving the desired semi-liquid consistency for young children. Even if this ability is mainly related to the production of alpha-amylase (E.C. 3.2.1.1), in a recent molecular screening, genes coding for enzymes involved in starch metabolism were detected in the efficient amylolytic LAB Lactobacillus plantarum A6: alpha-glucosidase (E.C. 3.2.1.20), neopullulanase (E.C. 3.2.1.135), amylopectin phosphorylase (E.C. 2.4.1.1) and maltose phosphorylase (E.C. 2.4.1.8). The objective of this study was to investigate the expression of these genes in a model of starchy fermented food made from pearl millet (Pennisetum glaucum). Transcriptional and enzymatic analyses were performed during the 18-h fermentation period. Liquefaction was mainly caused by an extracellular alpha amylase encoded by the amyA gene specific to the A6 strain among L. plantarum species and found in both Lactobacillus amylovorus and Lactobacillus manihotivorans. The second most active enzyme was neopullulanase. Other starch metabolizing enzymes were less often detected. The dynamic detection of transcripts of gene during starch fermentation in pearl millet porridge suggests that the set of genes we investigated was not expressed continuously but transiently.

Behavior of lactobacilli isolated from fermented slurry (ben-saalga) in gnotobiotic rats.

Most bacterial strains, which have been studied so far for their probiotic functions, are extensively used by manufacturers in developed countries. In our work, we sought to study a mix (called BSL) comprising three strains belonging to Lactobacillus fermentum, L. paraplantarum and L. salivarius, that were isolated from a traditional African pearl millet based fermented slurry. Our objective was to study this BSL cocktail in gnotobiotic rats, to evaluate their survival and their behavior in the digestive tract conditions. After a single oral inoculation of germfree rats with BSL, the species established stably in the digestive tract with the following hierarchy of abundance: L. salivarius> L. plantarum> L. fermentum. BSL cocktail was metabolically active since it produced 50 mM lactate and it expressed genes involved in binding mechanism in the caecum. Furthermore, the global morphology of the colon epithelium was not disturbed by the BSL cocktail. BSL cocktail did not modify mucus content and host mucus-related genes (MUC1, MUC2, MUC3 or resistin-like molecule β). The cocktail of lactobacilli enhanced the proliferating cell nuclear antigen (PCNA) at a level comparable to what was observed in conventional rats. PCNA was involved in proliferation and DNA repair, but the presence of the cocktail did not provoke proliferative events (with Ki67 as indicator), so we suppose BSL may help gut preservation. This work is the first step towards the selection of strains that are derived from traditional fermented food to formulate new probiotic mixture.

Lactic acid fermentation as a tool for increasing the folate content of foods.

ABSTRACT Folate is an essential micronutrient involved in numerous vital biological reactions. The dietary consumption of naturally occurring vitamin B9 is often inadequate in many countries, and supplementation or fortification programs (using synthetic folic acid) are implemented to alleviate folate deficiency. Other food-based alternatives are possible, such as the use of lactic acid bacteria (LAB) to synthesize folate during fermentation. Many studies have been conducted on this topic, and promising results were reported for some fermented dairy products. However, in other studies, folate consumption by LAB or rather low folate production were observed, resulting in fermented foods that may not significantly contribute to the recommended B9 intake. In addition, the optimum conditions for folate biosynthesis by LAB are still not clear. The aim of this review was thus to (i) clarify the ability of LAB to produce folate in food products, (ii) check if the production of folate by LAB in various fermented foods is sufficient to meet human vitamin B9 requirements and (iii) suggest ways to optimize folate production by LAB in fermented food products.

The genomic and transcriptomic basis of the potential of Lactobacillus plantarum A6 to improve the nutritional quality of a cereal based fermented food

The objective of this work was to investigate the nutritional potential of Lactobacillus plantarum A6 in a food matrix using next generation sequencing. To this end, we characterized the genome of the A6 strain for a complete overview of its potential. We then compared its transcriptome when grown in a food matrix made from pearl millet to and its transcriptome when cultivated in a laboratory medium. Genomic comparison of the strain L. plantarum A6 with the strains WCFS1, ST-III, JDM1 and ATCC14917 led to the identification of five regions of genomic plasticity. More specifically, 362 coding sequences, mostly annotated as coding for proteins of unknown functions, were specific to L. plantarum A6. A total of 1201 genes were significantly differentially expressed in laboratory medium and food matrix. Among them, 821 genes were up-regulated in the food matrix compared to the laboratory medium, representing 23% of whole genomic objects. In the laboratory medium, the expression of 380 genes, representing 11% of the all genomic objects was at least double than in the food matrix. Genes encoding important functions for the nutritional quality of the food were identified. Considering its efficiency as an amylolytic strain, we investigated all genes involved in carbohydrate metabolism, paying particular attention to starch metabolism. An extracellular alpha amylase, a neopullulanase and maltodextrin transporters were identified, all of which were highly expressed in the food matrix. In addition, genes involved in alpha-galactoside metabolism were identified but only two of them were induced in food matrix than in laboratory medium. This may be because alpha galactosides were already eliminated during soaking. Different biosynthetic pathways involved in the synthesis of vitamin B (folate, riboflavin, and cobalamin) were identified. They allowed the identification of a potential of vitamin synthesis, which should be confirmed through biochemical analysis in further work. Surprisingly, some genes involved in metabolism and bioaccessibility of iron were identified. They were related directly to the use of transport of iron, or indirectly to metabolism of polyphenols, responsible of iron chelation in the food. The combination of genomics and transcriptomics not only revealed previously undocumented nutritional properties of L. plantarum A6, but also documented the behaviour of this bacterium in food.

Prevalence and Fate of Bacillus cereus in African Traditional Cereal-Based Foods Used as Infant Foods

The objective of the present work was to estimate the prevalence of Bacillus cereus group species in traditional cereal-based lactic acid-fermented slurries and nonfermented flours used to prepare infant foods in an African context. High counts on mannitol-egg yolk-polymixin agar medium were determined for the fermented slurries (median, 4.5 × 10(4) CFU/ml of slurry) compared with the nonfermented flours, most of whose counts were lower than 10(-1) CFU/g. Virulence genes were characterized in 60 isolates from 26 traditional cereal-based foods in Ouagadougou (Burkina Faso). Seventy-two and 38 % of isolates were positive for the complete set of genes coding for hemolysin BL and nonhemolytic enterotoxin, respectively, suggesting a high enterotoxigenic potential for these foodborne isolates. No potentially emetic toxin-producing strains were detected. Because of the high counts found for fermented slurries, survival tests with vegetative cells inoculated in fermented slurries were performed, which showed that growth of B. cereus was inhibited. This result suggests that fermentation in traditional production units is presumably not adequately controlled, enabling growth during any unit operations before fermentation, or even during the fermentation step, when the process was poorly controlled. However, adding nisin (0.1 mg/ml) enabled a 5-log reduction in the B. cereus population in 5 h, suggesting that the use of nisin could be a way to upgrade the hygienic quality of this type of food.