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Like in many developing countries, the traditional Ethiopian diet relies mainly on starchy staple foods and often lacks sufficient animal-sourced foods which are crucial for cobalamin intake. Furthermore, the concentration of folate in traditionally prepared injera, an Ethiopian cereal-based fermented staple food, is highly variable and injera contains biologically inactive corrinoids. This study aimed to improve the cobalamin and folate content of injera by using cobalamin-producing Propionibacterium freudenreichii and folate-producing Lactiplantibacillus plantarum strains, both individually and combined. Since injera is fermented using backslopping, we also assessed the ability of these strains to produce cobalamin and folate consistently across successive fermentation batches. Changes in the microbial ecosystem were monitored using real-time PCR. The theoretical contribution of the injera prepared using the selected strains to the cobalamin and folate intake of children and women of reproductive age was also calculated. Results showed that using the selected bacterial strains individually increased cobalamin (up to 19.2 μg/100 g of dry matter) and folate (up to 180.2 μg/100 g of dry matter) levels in the injera dough over several backslopping fermentation batches. Regular consumption of the injera with enhanced vitamin content produced using each strain alone would be capable of fulfilling the entire recommended nutrient intake for cobalamin and up to 29 % of the recommended intake for folate for children and women of reproductive age. However, when the strains were used together, the production of both vitamins was reduced. The presence of certain common endogenous bacterial species and genera exhibited significant variability, highlighting the complex response of the native microbiota to the different inoculation strategies employed. Future experiments should consider selecting a microbial consortium comprising non-competing microorganisms to ensure the simultaneous production of cobalamin and folate in fermented foods., Competing Interests: Declaration of competing interest None., (Copyright © 2024. Published by Elsevier B.V.)

Worldwide, fermented foods (FF) are recognized as healthy and safe. Despite the rapid increase of research papers, there is a lack of systematic evaluation of the health benefits and risks of FF. The COST Action CA20128 "Promoting innovation of fermented foods" (PIMENTO) aims to provide a comprehensive assessment on the available evidence by compiling a set of 16 reviews. Seven reviews will cover clinical and biological endpoints associated with major health indicators across several organ systems, including the cardiovascular, gastrointestinal, neurological, immune, and skeletal systems. Nine reviews will address broader biological questions associated with FF including bioactive compounds and vitamin production, nutrient bioavailability and bioaccessibility, the role of FF in healthy diets and personalized nutrition, food safety, regulatory practices, and finally, the health properties of novel and ethnic FF. For each outcome assessed in the reviews, an innovative approach will be adopted based on EFSA's published guidance for health claim submissions. In particular, each review will be composed of three parts: (1) a systematic review of available human studies; (2) a non-systematic review of the mechanism of action related to the clinical endpoints measured by the human studies identified in part 1; and (3) a non-systematic review of the characterization of the FF investigated in the human studies identified in part 1. The evidence and research gaps derived from the reviews will be summarized and published in the form of a strategic road map that will pave the way for future research on FF., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Todorovic, Akpinar, Assunção, Bär, Bavaro, Berkel Kasikci, Domínguez-Soberanes, Capozzi, Cotter, Doo, Gündüz Ergün, Guzel, Harsa, Hastaoglu, Humblot, Hyseni, Hosoglu, Issa, Karakaş-Budak, Karakaya, Kesenkas, Keyvan, Künili, Kütt, Laranjo, Louis, Mantzouridou, Matalas, Mayo, Mojsova, Mukherjee, Nikolaou, Ortakci, Paveljšek, Perrone, Pertziger, Santa, Sar, Savary-Auzeloux, Schwab, Starowicz, Stojanović, Syrpas, Tamang, Yerlikaya, Yilmaz, Malagon-Rojas, Salminen, Frias, Chassard and Vergères.)

Faecal microbiota plays a critical role in human health, but its relationship with nutritional status among schoolchildren remains under-explored. Here, in a double-blinded cluster-randomized controlled trial on 380 Cambodian schoolchildren, we characterize the impact of six months consumption of two types of rice fortified with different levels of vitamins and minerals on pre-specified outcomes. We investigate the association between the faecal microbiota (16SrRNA sequencing) and age, sex, nutritional status (underweight, stunting), micronutrient status (iron, zinc and vitamin A deficiencies, anaemia, iron deficient anaemia, hemoglobinopathy), inflammation (systemic, gut), and parasitic infection. We show that the faecal microbiota is characterised by a surprisingly high proportion of Lactobacillaceae. We discover that deficiencies in specific micronutrients, such as iron and vitamin A, correlate with particular microbiota profiles, whereas zinc deficiency shows no such association. The nutritional intervention with the two rice treatments impacts both the composition and functions predicted from compositional analysis in different ways. (ClinicalTrials.gov (Identifier: NCT01706419))., (© 2024. The Author(s).)

The microbiota significantly impacts digestive epithelium functionality, especially in nutrient processing. Given the importance of iron for both the host and the microbiota, we hypothesized that host-microbiota interactions fluctuate with dietary iron levels. We compared germ-free (GF) and conventional mice (SPF) fed iron-containing (65 mg/Kg) or iron-depleted (<6 mg/Kg) diets. The efficacy of iron privation was validated by iron blood parameters. Ferritin and Dmt1, which represent cellular iron storage and transport respectively, were studied in tissues where they are abundant: the duodenum, liver and lung. When the mice were fed an iron-rich diet, the microbiota increased blood hemoglobin and hepcidin and the intestinal ferritin levels, suggesting that the microbiota helps iron storage. When iron was limiting, the microbiota inhibited the expression of the intestinal Dmt1 transporter, likely via the pathway triggered by Hif-2α. The microbiota assists the host in storing intestinal iron when it is abundant and competes with the host by inhibiting Dmt1 in conditions of iron scarcity. Comparison between duodenum, liver and lung indicates organ-specific responses to microbiota and iron availability. Iron depletion induced temporal changes in microbiota composition and activity, reduced α-diversity of microbiota, and led to Lactobacillaceae becoming particularly more abundant after 60 days of privation. By inoculating GF mice with a simplified bacterial mixture, we show that the iron-depleted host favors the gut fitness of Bifidobacterium longum .

Folate (vitamin B9) and cobalamin (vitamin B12) deficiencies potentially affect millions of people worldwide, leading to different pathologies. In Ethiopia, the diet is characterized by high consumption of fermented cereal-based foods such as injera, a good source of folate but not of cobalamin, which is only found in foods of animal origin that are rarely consumed. Some of the bacteria responsible for the fermentation of cereals can synthesize cobalamin, but whether or not fermented cereal food products contain cobalamin remains underexplored. The objective of this study was to assess the folate and cobalamin content of injera collected from various households in Ethiopia at different stages of production. Global (16S rRNA gene sequencing) and specific (real-time PCR quantification of bacteria known for folate or cobalamin production) bacterial composition of these samples was assessed. UPLC-PDA was used to identify the cobalamin to see whether the active or inactive form was present. Surprisingly, teff flour contained 0.8 μg/100 g of cobalamin, most probably due to microbial contamination from the environment and the harvesting process. While fermentation increased the folate and cobalamin content in some households, their levels decreased in others. Conversely, cooking consistently reduced the level of the vitamins. Fresh injera contained, on average, 21.2 μg/100 g of folate and 2.1 μg/100 g of cobalamin, which is high, but with marked variation depending on the sample. However, the form of cobalamin was a corrinoid that is biologically inactive in humans. Injera fermentation was dominated by lactic acid bacteria, with significant correlations observed between certain bacterial species and folate and cobalamin levels. For example, a high proportion of Fructilactobacillus sanfranciscensis, a known folate consumer, was negatively correlated with the folate content of injera. On the contrary, Lactobacillus coryniformis, known for its cobalamin synthesis ability was present in high proportion in the cobalamin-rich samples. These findings highlight the complex interrelationship between microorganisms and suggest the involvement of specific bacteria in the production of folate and cobalamin during injera fermentation. Controlled fermentation using vitamin-producing bacteria is thus a promising tool to promote folate and cobalamin production in fermented food., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Micronutrient deficiencies or "hidden hunger" remains a serious public health problem in most low- and middle-income countries, with severe consequences for child development. Traditional methods of treatment and prevention, such as supplementation and fortification, have not always proven to be effective and may have undesirable side-effects (i.e., digestive troubles with iron supplementation). Commensal bacteria in the gut may increase bioavailability of specific micronutrients (i.e., minerals), notably by removing anti-nutritional compounds, such as phytates and polyphenols, or by the synthesis of vitamins. Together with the gastrointestinal mucosa, gut microbiota is also the first line of protection against pathogens. It contributes to the reinforcement of the integrity of the intestinal epithelium and to a better absorption of micronutrients. However, its role in micronutrient malnutrition is still poorly understood. Moreover, the bacterial metabolism is also dependent of micronutrients acquired from the gut environment and resident bacteria may compete or collaborate to maintain micronutrient homeostasis. Gut microbiota composition can therefore be modulated by micronutrient availability. This review brings together current knowledge on this two-way relationship between micronutrients and gut microbiota bacteria, with a focus on iron, zinc, vitamin A and folate (vitamin B9), as these deficiencies are public health concerns in a global context.

Scope: Fighting obesity and associated comorbidities through dieting is not always sustained and results in a subsequent weight gain, a phenomenon referred to as weight cycling. Diet is among the most important factors in modifying the composition of gut microbiota. The objective of this work is to determine whether weight cycling affects the composition and the predicted function of mouse fecal bacteria on a long-term basis., Methods and Results: Mice fed for 40 weeks with either high fat (HF), low fat (LF), or cycled diets (starting and ending by one of the two diets, and the reverse) exhibit a bacterial profile specific to each of the four groups. A higher proportion of Firmicutes and Bacteroidota phyla are observed in mice on Hf and LF diet, respectively. The proportion of functions dedicated to amino acid metabolism is higher in mice on HF or LF/HF diets, whereas the mice on LF or HF/LF diets have a higher proportion of functions involve in carbohydrate metabolism and vitamin B biosynthesis., Conclusion: Compared to continuous HF or LF diets, cyclic diet specifically alters the composition and function of the mouse fecal microbiota, suggesting that fight against weight gain should be considered on a long-term basis., (© 2022 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH GmbH.)

Folate deficiencies are widespread in Africa due to predominantly cereal-based diets. The objective of this work was to test the feasibility of using folate-producing microorganisms to increase folate content of tef injera , a traditional Ethiopian fermented staple food. To this end, a strain of Lactobacillus plantarum previously isolated from fermented tef batter and a commercial Saccharomyces cerevisiae were used alone and in combination to prepare injera . Ten successive fermentations using backslopping from the fermented batter prepared with L. plantarum inoculation were performed to mimic the traditional backslopping. The highest folate content was obtained with S. cerevisiae (53.5 μg/100 ​g fresh material). All the combinations were efficient and could cover up to 22 % of the recommended nutrient intakes. All injera prepared with selected inoculums were preferred by sensory panelists to the traditional one. This work demonstrates the possibility to increase folate intake using folate-producing microorganisms in the conditions normally encountered in households., Competing Interests: The authors declare no conflict of interest., (© 2022 The Authors.)

The consumption of rice bran has been shown to have a positive effect on nutritional status and prevention of chronic diseases related to hundreds of metabolites with bioactivity. Consumption after fermentation can lead to specific beneficial effects, yet is lacking complete characterization when fermented with diverse strains. The objective of this study was to examine the effect of fermentation on the rice bran metabolite profile. Bacterial probiotics ( Bifidobacterium longum, Limosilactobacillus fermentum, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus and, Escherichia coli ) were used to ferment rice bran alone or after incubation with yeast probiotic Saccharomyces boulardii . Fermented rice bran was methanol extracted and analyzed by UPLC-MS/MS. The metabolome of the two fermentation types was deeply modified when compared with non-fermented rice bran. The two-step fermentation provided alternative substrate to the bacteria in a few cases. Key metabolites of high nutritional value (essential amino acids, vitamins) and gut health (arabinose, maltotriose) were identified., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Seyoum, Humblot, Baxter, Nealon, Weber and Ryan.)

Iron deficiency is the most frequent nutritional deficiency in the world with an estimated 1.4 billion people affected. The usual way to fight iron deficiency is iron fortification, but this approach is not always effective and can have undesirable side effects including an increase in the growth and virulence of gut bacterial pathogens responsible for diarrhea and gut inflammation. Iron is mainly absorbed in the duodenum and is tightly regulated in mammals. Unabsorbed iron enters the colonic lumen where many microorganisms, referred to as gut microbiota, reside. Iron is essential for these bacteria, and its availability consequently affects this microbial ecosystem. The aim of this review is to provide further insights into the complex relationship between iron and gut microbiota. Given that overcoming anemia caused by iron deficiency is still a challenge today, gut microbiota could help identify more efficient ways to tackle this public health problem.

Rapid physical growth and the onset of menstruation during adolescence can increase the risk of iron deficiency (ID) and related adverse effects. However, little is known about the risk of anemia and ID among adolescent girls in Ethiopia. Therefore, we aimed to determine the prevalence of ID, low iron stores, and anemia and characterize selected risk factors in Huruta, Arsi Zone, Oromia Region, Ethiopia. A cross-sectional study was conducted among non-pregnant adolescent girls (15-19 years of age; n = 257). Data on household socio-demographic characteristics, anthropometric measurements, and women's dietary diversity score (WDDS) were collected. Hemoglobin (Hb) and serum ferritin (SF), C-reactive protein (CRP), and α-1-acid-glycoprotein (AGP) concentrations were measured. Diets were predominantly plant-based, with a low consumption of animal source foods, fruits, and dark-green leafy vegetables. Only 4% of the adolescent girls had adequate dietary diversity (WDDS ≥5), and 35% were underweight. The prevalence of anemia (Hb <11 g/dL, 8.7%) and clinical ID (SF <15 µg/L, 8.7%) was low, but 41% had marginal iron stores (SF <50 µg/L). The low prevalence of ID, despite a predominantly plant-based diet is atypical and calls for adapted strategies to address low iron stores in this and other similar settings of Ethiopia.

Folate deficiencies are widespread around the world. Promoting consumption of folate-rich foods could be a sustainable option to alleviate this problem. However, these foods are not always available. Cereals, being a staple food, could contribute to folate intake. They are fermented prior to consumption in many African countries, and fermentation can modify the folate content. In Ethiopia, injera is a widely consumed fermented flat bread. The main drivers of its fermentation are lactic acid bacteria (LAB). The aim of this work was to isolate and identify folate-producing LAB from injera fermented dough and to evaluate their ability to increase folate status after depletion in a rat model. Among the 162 strains isolated from 60 different fermentations, 19 were able to grow on a folate-free culture medium and produced 1 to 43 µg/L (24 h, 30 °C incubation). The four highest folate producers belonged to the Lactobacillus plantarum species. The most productive strain was able to enhance folate status after depletion in a rat model, despite the relatively low folate content of the feed supplemented with the strain. Folate-producing L. plantarum strain has potential use as a commercial starter in injera production.

Folate deficiency can cause a number of diseases including neural tube defects and megaloblastic anemia, and still occurs in both developed and developing countries. Cereal-based food products are staple foods in many countries, and may therefore be useful sources of folate. The production of folate by microorganisms has been demonstrated in some cereal-based fermented foods, but has never been studied in a traditional African cereal based food spontaneously fermented. The microbiota of ben-saalga, a pearl-millet based fermented porridge frequently consumed in Burkina Faso, has a good genetic potential for the synthesis of folate, but the folate content of ben-saalga is rather low, suggesting that folate is lost during the different processing steps. The aim of this study was therefore to monitor changes in folate content during the different steps of preparing ben-saalga, from pearl-millet grains to porridge. Traditional processing involves seven different steps: washing, soaking, grinding, kneading, sieving, (spontaneous) fermentation, and cooking. Two type of porridge were prepared, one using a process adapted from the traditional process, the other a modified process based on fermentation by backslopping. Dry matter and total folate contents were measured at each step, and a mass balance assessment was performed to follow folate losses and gains. Folate production was observed during the soaking of pearl-millet grains (+26% to +79%), but the folate content of sieved batters (2.5 to 3.4μg/100g fresh weight) was drastically lower than that of milled soaked grains (17.3 to 19.4μg/100g FW). The final folate content of the porridges was very low (1.5 to 2.4μg/100g FW). The fermentation had no significant impact on folate content, whatever the duration and the process used. This study led to a better understanding of the impact on folate of the different processing steps involved in the preparation of ben-saalga., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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.

With the aim of selecting starter cultures with interesting probiotic potential and with the ability to produce folate in a food matrix, yeast strains isolated from fermented cereal-based African foods were investigated. A total of 93 yeast strains were screened for their tolerance to pH 2 and 0.3% of bile salts. Pichia kudriavzevii isolates gave the best results. Selected P. kudriavzevii strains were tested for survival to the simulated human digestion and for adhesion to Caco-2 cells. Moreover, presence of folate biosynthesis genes was verified and production of extra and intra-cellular folate determined during growth in culture medium. 31% of yeast strains could tolerate pH 2, while 99% bile salts. Survival rate after simulated digestion ranged between 11 and 45%, while adhesion rate between 12 and 40%. Folate production was mainly intracellular, maximum after 24 h of growth. To be closer to traditional cereal-based fermentations, a P. kudriavzevii strain with good probiotic potential was co-inoculated with Lactobacillus fermentum strains in a pearl millet gruel. This resulted in in situ folate production that peaked after 4 h. The use of strains with both probiotic and nutritional enrichment properties may have a greater impact for the consumers., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Cereals are staple foods in most African countries, and many African cereal-based foods are spontaneously fermented. The nutritional quality of cereal products can be enhanced through fermentation, and traditional cereal-based fermented foods (CBFFs) are possible sources of lactic acid bacteria (LAB) with useful nutritional properties. The nutritional properties of LAB vary depending on the species and even on the strain, and the microbial composition of traditional CBFFs varies from one traditional production unit (TPU) to another. The nutritional quality of traditional CBFFs may thus vary depending on their microbial composition. As the isolation of potentially useful LAB from traditional CBFFs can be very time consuming, the aim of this study was to use PCR to assess the nutritional potential of LAB directly on the metagenomes of pearl-millet based fermented porridges (ben-saalga) from Burkina Faso. Genes encoding enzymes involved in different nutritional activities were screened in 50 metagenomes extracted from samples collected in 10 TPUs in Ouagadougou. The variability of the genetic potential was recorded. Certain genes were never detected in the metagenomes (genes involved in carotenoid synthesis) while others were frequently detected (genes involved in folate and riboflavin production, starch hydrolysis, polyphenol degradation). Highly variable microbial composition - assessed by real-time PCR - was observed among samples collected in different TPUs, but also among samples from the same TPU. The high frequency of the presence of genes did not necessarily correlate with in situ measurements of the expected products. Indeed, no significant correlation was found between the microbial variability and the variability of the genetic potential. In spite of the high rate of detection (80%) of both genes folP and folK, encoding enzymes involved in folate synthesis, the folate content in ben-saalga was rather low (median: 0.5μg/100g fresh weight basis). This work highlighted the limit of evaluating the nutritional potential of the microbiota of traditional fermented foods by the only screening of genes in metagenomes, and suggests that such a screening should be completed by a functional analysis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Lactic acid bacteria (LAB) synthesize a wide variety of biochemical compounds during food fermentation. Carotenoids provide important biological functions for bacteria, and their consumption by humans has many beneficial effects. In this study, the presence of several genes involved in the production of carotenoids was determined by BLAST analysis and PCR in a collection of 156 LAB isolated from traditional amylaceous African fermented foods. Only the crtE gene and the crtNM operon were present and detected in Lactobacillaceae. Most of the strains with positive PCR detection of the operon crtNM produced carotenoid-like compounds when grown in MRS broth. The carotenoids produced differed from compounds previously identified in other LAB except for one peak, which was closely related to 4,4'-diaponeurosporene already reported in the literature in Lactobacillus plantarum species. Most producing strains belonged to Lactobacillus fermentum and L. plantarum species but a few Pediococcus acidilactici were also producers. Furthermore, the most efficient L. plantarum was able to synthesize carotenoids in a cereal fermented food. Genetic screening was shown to be efficient since, in all cases, it eliminated the need for biochemical analysis of strains in which no amplicons of the operon crtNM were obtained., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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.

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.

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.

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.

Lactic acid bacteria and more particularly lactobacilli have been used for the production of fermented foods for centuries. Several lactobacilli have been recognized as probiotics due to their wide range of health-promoting effects in humans. However, little is known about the molecular mechanisms underpinning their probiotic functions. Here we reviewed the main beneficial effects of lactobacilli and discussed, when the information is available, the molecular machinery involved in their probiotic function. Among the beneficial effects, lactobacilli can improve digestion, absorption and availability of nutrients. As an example, some strains are able to degrade carbohydrates such as lactose or α-galactosides that may cause abdominal pain. Furthermore, they can hydrolyze compounds that limit the bioavailability of minerals like tannin and phytate due to tannin acylhydrolase and phytase activities. In addition, it was shown that some lactobacilli strains can improve mineral absorption in Caco-2 cells. Lactobacilli can also contribute to improve the nutritional status of the host by producing B group vitamins. More recently, the role of lactobacilli in energy homeostasis, particularly in obese patients, is the object of an increased interest. Lactobacilli are also involved in the prevention of diseases. They have potential to prevent carcinogenesis through the modulation of enzymes involved in the xenobiotic pathway, and may prevent cardiovascular diseases such as hypertension through the production of a bioactive peptide that may have angiotensin converting enzyme inhibitor activity. Lactobacilli are increasingly studied for the treatment of inflammatory bowel diseases and exhibit interesting potential in the reduction of pain perception. The ability of some strains to bind to intestinal cells, their pathogen-associated molecular patterns and the metabolites they produce confer interesting immunomodulatory effects. Finally, pathogenic fungi, virus or bacteria can be inhibited by probiotics. They can reinforce the intestinal barrier, simply occupying the ecological niche, or they can have an active role by synthesizing various metabolites inhibiting pathogen development. Lactobacilli have a long standing history with foods and humans but comparatively, their history as probiotics is recent. Their effects are investigated in in vitro and in vivo models leading sometimes to contradictory or controversial results that make necessary final demonstrations through clinical trials. Researches on molecular mechanisms involved in the beneficial effects reviewed here are necessary for a better understanding of these effects, but could also lead to the development of molecular tools to help the screening of the probiotic potential of lactobacilli that are common inhabitants of numerous fermented foods around the world., (Copyright © 2010 Elsevier B.V. All rights reserved.)

To assess the ability of lactic acid bacteria to improve some nutritional characteristics of the pearl millet-soybean slurry to prepare complementary foods for young children in African countries, inoculation was performed using strains previously selected for their ability to hydrolyse starch, phytate, or alpha-galactooligosaccharides (alpha-GOS). For the sake of comparison with the action of a natural microflora, fermentation was also performed by back slopping inoculation, that is, with a sample obtained from spontaneously fermented traditional pearl millet slurry obtained from a small scale processing unit in Burkina Faso (Ouagadougou). Starter cultures thrived on the slurry as shown by counts on MRS agar, TTGE fingerprints, and fermentation patterns. The fermentation of precooked slurries inoculated by back slopping or with mixed cultures containing the amylolytic strain Lb. plantarum A6 enabled partial starch hydrolysis. Corresponding gruels had a suitable consistency for young child feeding at high dry matter content, and a high energy density: 88.7 +/- 4.2 and 75.8 +/- 5.1 kcal/100 g of sweetened gruel, for the gruels inoculated by back slopping or with Lb. plantarum A6, respectively. Unexpectedly, no decrease in phytates was observed in any of the experiments, suggesting the presence of one or more inhibitory compounds in soybean. Furthermore, preprocessing conditions before fermentation affect the carbohydrate composition of slurry and have a more profound effect than fermentation on the reduction of the alpha-GOS content.

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.

2-amino-3-methylimidazo[4,5-f]quinoline (IQ) is a genotoxic/carcinogenic compound formed in meat and fish during cooking. Following absorption in the upper part of the gastrointestinal tract, IQ is mainly metabolized in the liver by xenobiotic-metabolizing enzymes. Among them, UDP-glucuronosyl transferases lead to harmless glucuronidated derivatives that are partly excreted via the bile into the digestive lumen, where they come into contact with the resident microbiota. The purpose of this study is to investigate if microbial beta-glucuronidase could contribute to IQ genotoxicity by releasing reactive intermediates from IQ glucuronides. We constructed a beta-glucuronidase-deficient isogenic mutant from a wild-type Escherichia coli strain carrying the gene uidA encoding this enzyme and compared the genotoxicity of IQ in gnotobiotic rats monoassociated with the wild-type or the mutant strain. The Comet assay performed on colonocytes and hepatocytes showed that the presence of beta-glucuronidase in the digestive lumen dramatically increased (3-fold) the genotoxicity of IQ in the colon. This deleterious effect was paralleled by slight modifications of the pharmacokinetics of IQ. The urinary and faecal excretion of the parent compound and its conjugated derivatives reached a maximum 24-48 h after gavage in rats harbouring the beta-glucuronidase-deficient strain. In rats associated with the wild-type strain, the kinetics of urinary excretion showed a biphasic curve with a second, smaller peak after 144 h. This is the first in vivo demonstration that bacterial beta-glucuronidase plays a pivotal role in the genotoxicity of a common food-borne carcinogen.

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 microM) 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 (1)H nuclear magnetic resonance ((1)H-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 (1)H-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.

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.

We investigated the chemoprotective effects of four common constituents of the human diet, i.e. a fermented milk, inulin, oligofructose and Brussels sprouts, towards 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-induced genotoxicity in male Fischer 344 rats harbouring a human intestinal microflora. We found that the four dietary components significantly reduced IQ-induced DNA damage in hepatocytes (reduction ranged from 74% with inulin to 39% with Brussels sprouts) and colonocytes (reduction ranged from 68% with inulin to 56% with Brussels sprouts). This chemoprotective effect correlated with the induction of hepatic UDP-glucuronosyl transferase following Brussels sprouts consumption, and with alterations of bacterial metabolism in the distal gut (acidification, increase of butyrate proportion, decrease of beta-glucuronidase activity) following inulin consumption.

Cruciferous vegetables, such as Brassica, which contain substantial quantities of glucosinolates, have been suggested to possess anticarcinogenic activity. Cutting and chewing of cruciferous vegetables releases the thioglucosidase enzyme myrosinase, which degrades glucosinolates to isothiocyanates and other minor metabolites. Cooking of cruciferous vegetables inactivates the myrosinase enzyme, allowing intact glucosinolates to reach the large intestine, where they can be degraded by the indigenous microflora into isothiocyanates. This local release of isothiocyanates may explain the protective effect of cruciferous vegetables on the colon epithelium. However, little is known about the amounts and identities of glucosinolate metabolites produced by the human microflora. The production of allyl isothiocyanate from sinigrin was investigated in a dynamic in vitro large-intestinal model, after inoculation with a complex microflora of human origin. Sinigrin and allyl isothiocyanate concentrations were analysed in the lumen and dialysis fluid of the model. Peak levels of allyl isothiocyanate were observed between 9 and 12 h after the addition of sinigrin. The model was first set up with a pooled and cultured human microflora, in which 1 and 4% of, respectively, 1 and 15 mM sinigrin, was converted into AITC. However, the conversion rate was remarkably higher if different individual human microflora were used. Between 10% and 30% (mean 19%) of the sinigrin was converted into allyl isothiocyanate. The results of this study suggest that allyl isothiocyanate is converted further into other, yet unknown, metabolites.