Prospects for Food Fermentation in South-East Asia, Topics From the Tropical Fermentation and Biotechnology Network at the End of the AsiFood Erasmus+Project

Fermentation has been used for centuries to produce food in South-East Asia and some foods of this region are famous in the whole world. However, in the twenty first century, issues like food safety and quality must be addressed in a world changing from local business to globalization. In Western countries, the answer to these questions has been made through hygienisation, generalization of the use of starters, specialization of agriculture and use of long-distance transportation. This may have resulted in a loss in the taste and typicity of the products, in an extensive use of antibiotics and other chemicals and eventually, in a loss in the confidence of consumers to the products. The challenges awaiting fermentation in South-East Asia are thus to improve safety and quality in a sustainable system producing tasty and typical fermented products and valorising by-products. At the end of the “AsiFood Erasmus+ project” (www.asifood.org), the goal of this paper is to present and discuss these challenges as addressed by the Tropical Fermentation Network, a group of researchers from universities, research centers and companies in Asia and Europe. This paper presents current actions and prospects on hygienic, environmental, sensorial and nutritional qualities of traditional fermented food including screening of functional bacteria and starters, food safety strategies, research for new antimicrobial compounds, development of more sustainable fermentations and valorisation of by-products. A specificity of this network is also the multidisciplinary approach dealing with microbiology, food, chemical, sensorial, and genetic analyses, biotechnology, food supply chain, consumers and ethnology

New Genes Involved in Mild Stress Response Identified by Transposon Mutagenesis in Lactobacillus paracasei

International audienceLactic acid bacteria (LAB) are associated with various plant, animal, and human niches and are also present in many fermented foods and beverages. Thus, they are subjected to several stress conditions and have developed advanced response mechanisms to resist, adapt, and grow. This work aimed to identify the genes involved in some stress adaptation mechanisms in LAB. For this purpose, global reverse genetics was applied by screening a library of 1287 Lactobacillus paracasei transposon mutants for mild monofactorial stresses. This library was submitted independently to heat (52 degrees C, 30 min), ethanol (170 g.L-1, 30 min), salt (NaCl 0.8 M, 24 h), acid (pH 4.5, 24 h), and oxidative (2 mM H2O2, 24 h) perturbations which trigger mild monofactorial stresses compatible with bacterial adaptation. Stress sensitivity of mutants was determined either by evaluating viability using propidium iodide (PI) staining, or by following growth inhibition through turbidity measurement. The screening for heat and ethanol stresses lead respectively to the identification of 63 and 27 genes/putative promoters whose disruption lead to an increased sensitivity. Among them, 14 genes or putative promoters were common for both stresses. For salt, acid and oxidative stresses, respectively 8, 6, and 9 genes or putative promoters were identified as essential for adaptation to these unfavorable conditions, with only three genes common to at least two stresses. Then, RT-qPCR was performed on selected stress response genes identified by mutant screenings in order to evaluate if their expression was modified in response to stresses in the parental strain. Eleven genes (membrane, transposase, chaperone, nucleotide and carbohydrate metabolism, and hypothetical protein genes) were upregulated during stress adaptation for at least two stresses. Seven genes, encoding membrane functions, were upregulated in response to a specific stress and thus could represent potential transcriptomic biomarkers. The results highlights that most of the genes identified by global reverse genetics are specifically required in response to one stress and that they are not differentially transcribed during stress in the parental strain. Most of these genes have not been characterized as stress response genes and provide new insights into the adaptation of lactic acid bacteria to their environment

Chapter 17 - Relationship between fermented food, oral microbiota, and taste perception

International audienceThe oral cavity hosts an important amount of microorganisms characterized by an important diversity. This diversity is partly due to the presence of various ecological niches in the oral cavity with different physicochemical and biochemical properties. The totality of these microorganisms constitutes the oral microbiota. The oral microbiota has been studied for years, especially for its implication in various oral pathologies including dental caries and periodontal disease. However, its implication in other physiological functions has started to be studied recently. Among them, we can mention taste perception. The implication of the oral microbiota in this perception appears highly credible since the surface of the tongue, which is at the interface between the oral cavity and the taste receptors, is covered by a rich and diverse microbial biofilm. Similarly, saliva, which is the fluid where sapid compounds are diluted before reaching taste receptors, is also a medium where planktonic bacteria are strongly represented. Besides the potential role of this oral microbiota, the implication of food microbiota and particularly fermented foods microbiota to taste perception is poorly known although the consumption of these products leads to the introduction of billions of exogenous microorganisms in the oral cavity. The aim of this chapter is then, in the first part, to give an overview of the characteristics of the oral microbiota. A second part will focus on the fermented food microbiota and the products of their metabolism having a potential impact on taste perception. Finally, the last part will review the potential implications of the oral microbiota in taste perception

Bioinformatics and metabolic flux analysis highlight a new mechanism involved in lactate oxidation in Clostridium tyrobutyricum

International audienceClimate change and environmental issues compel us to find alternatives to the production of molecules of interest from petrochemistry. This study aims at understanding the production of butyrate, hydrogen, and CO 2 from the oxidation of lactate with acetate in Clostridium tyrobutyricum and thus proposes an alternative carbon source to glucose. This specie is known to produce more butyrate than the other butyrate-producing clostridia species due to a lack of solvent genesis phase. The recent discoveries on flavin-based electron bifurcation and confurcation mechanism as a mode of energy conservation led us to suggest a new metabolic scheme for the formation of butyrate from lactate-acetate co-metabolism. While searching for genes encoding for EtfAB complexes and neighboring genes in the genome of C. tyrobutyricum , we identified a cluster of genes involved in butyrate formation and another cluster involved in lactate oxidation homologous to Acetobacterium woodii . A phylogenetic approach encompassing other butyrate-producing and/or lactate-oxidizing species based on EtfAB complexes confirmed these results. A metabolic scheme on the production of butyrate, hydrogen, and CO 2 from the lactate-acetate co-metabolism in C. tyrobutyricum was constructed and then confirmed with data of steady-state continuous culture. This in silico metabolic carbon flux analysis model showed the coherence of the scheme from the carbon recovery, the cofactor ratio, and the ATP yield. This study improves our understanding of the lactate oxidation metabolic pathways and the role of acetate and intracellular redox balance, and paves the way for the production of molecules of interest as butyrate and hydrogen with C. tyrobutyricum

Does the lingual film intervene in the perception of taste?

International audienceThe biological film at the surface of the tongue, composed of saliva and microorganims, is an important interface between exogenous taste molecules and receptors involved in taste perception. However, the relationships between the composition of the lingual film and the perception of taste are not fully described. Thus, the aim of the present work was to study the differences in the biochemical (protein concentration, metabolite profiles) and microbiological compositions of the tongue films from 20 adult subjects, and to explore the possible relationships between composition and taste sensitivity assessed by means of a rapid at-home test. Preliminary results showed that intra-subject variability in film composition was smaller than inter-subject variability. The pH of the lingual film varied between 6.40 and 7.40. For one given subject, the tongue film pH was always higher than the saliva pH. The range of protein concentration was 0.4-1.0 mg/g of film and the mean number of bacteria was around 3×108 CFU/g of film sample. qPCR will also be performed to complete these results using phyla-specific primers (Firmicutes, Bacteroidetes, Fusobacteria, Actinobacteria, β-Proteobacteria, γ-Proteobacteria) and genus-specific primers (Veillonella, Streptococcus, and Prevotella). In conclusion, lingual film composition reveals variability between subjects that could be linked to variability in taste perception

Table2.XLSX

<p>Lactic acid bacteria (LAB) are associated with various plant, animal, and human niches and are also present in many fermented foods and beverages. Thus, they are subjected to several stress conditions and have developed advanced response mechanisms to resist, adapt, and grow. This work aimed to identify the genes involved in some stress adaptation mechanisms in LAB. For this purpose, global reverse genetics was applied by screening a library of 1287 Lactobacillus paracasei transposon mutants for mild monofactorial stresses. This library was submitted independently to heat (52°C, 30 min), ethanol (170 g.L⁻¹, 30 min), salt (NaCl 0.8 M, 24 h), acid (pH 4.5, 24 h), and oxidative (2 mM H₂O₂, 24 h) perturbations which trigger mild monofactorial stresses compatible with bacterial adaptation. Stress sensitivity of mutants was determined either by evaluating viability using propidium iodide (PI) staining, or by following growth inhibition through turbidity measurement. The screening for heat and ethanol stresses lead respectively to the identification of 63 and 27 genes/putative promoters whose disruption lead to an increased sensitivity. Among them, 14 genes or putative promoters were common for both stresses. For salt, acid and oxidative stresses, respectively 8, 6, and 9 genes or putative promoters were identified as essential for adaptation to these unfavorable conditions, with only three genes common to at least two stresses. Then, RT-qPCR was performed on selected stress response genes identified by mutant screenings in order to evaluate if their expression was modified in response to stresses in the parental strain. Eleven genes (membrane, transposase, chaperone, nucleotide and carbohydrate metabolism, and hypothetical protein genes) were upregulated during stress adaptation for at least two stresses. Seven genes, encoding membrane functions, were upregulated in response to a specific stress and thus could represent potential transcriptomic biomarkers. The results highlights that most of the genes identified by global reverse genetics are specifically required in response to one stress and that they are not differentially transcribed during stress in the parental strain. Most of these genes have not been characterized as stress response genes and provide new insights into the adaptation of lactic acid bacteria to their environment.</p

Table3.XLSX

<p>Lactic acid bacteria (LAB) are associated with various plant, animal, and human niches and are also present in many fermented foods and beverages. Thus, they are subjected to several stress conditions and have developed advanced response mechanisms to resist, adapt, and grow. This work aimed to identify the genes involved in some stress adaptation mechanisms in LAB. For this purpose, global reverse genetics was applied by screening a library of 1287 Lactobacillus paracasei transposon mutants for mild monofactorial stresses. This library was submitted independently to heat (52°C, 30 min), ethanol (170 g.L⁻¹, 30 min), salt (NaCl 0.8 M, 24 h), acid (pH 4.5, 24 h), and oxidative (2 mM H₂O₂, 24 h) perturbations which trigger mild monofactorial stresses compatible with bacterial adaptation. Stress sensitivity of mutants was determined either by evaluating viability using propidium iodide (PI) staining, or by following growth inhibition through turbidity measurement. The screening for heat and ethanol stresses lead respectively to the identification of 63 and 27 genes/putative promoters whose disruption lead to an increased sensitivity. Among them, 14 genes or putative promoters were common for both stresses. For salt, acid and oxidative stresses, respectively 8, 6, and 9 genes or putative promoters were identified as essential for adaptation to these unfavorable conditions, with only three genes common to at least two stresses. Then, RT-qPCR was performed on selected stress response genes identified by mutant screenings in order to evaluate if their expression was modified in response to stresses in the parental strain. Eleven genes (membrane, transposase, chaperone, nucleotide and carbohydrate metabolism, and hypothetical protein genes) were upregulated during stress adaptation for at least two stresses. Seven genes, encoding membrane functions, were upregulated in response to a specific stress and thus could represent potential transcriptomic biomarkers. The results highlights that most of the genes identified by global reverse genetics are specifically required in response to one stress and that they are not differentially transcribed during stress in the parental strain. Most of these genes have not been characterized as stress response genes and provide new insights into the adaptation of lactic acid bacteria to their environment.</p

Table1.XLSX

<p>Lactic acid bacteria (LAB) are associated with various plant, animal, and human niches and are also present in many fermented foods and beverages. Thus, they are subjected to several stress conditions and have developed advanced response mechanisms to resist, adapt, and grow. This work aimed to identify the genes involved in some stress adaptation mechanisms in LAB. For this purpose, global reverse genetics was applied by screening a library of 1287 Lactobacillus paracasei transposon mutants for mild monofactorial stresses. This library was submitted independently to heat (52°C, 30 min), ethanol (170 g.L⁻¹, 30 min), salt (NaCl 0.8 M, 24 h), acid (pH 4.5, 24 h), and oxidative (2 mM H₂O₂, 24 h) perturbations which trigger mild monofactorial stresses compatible with bacterial adaptation. Stress sensitivity of mutants was determined either by evaluating viability using propidium iodide (PI) staining, or by following growth inhibition through turbidity measurement. The screening for heat and ethanol stresses lead respectively to the identification of 63 and 27 genes/putative promoters whose disruption lead to an increased sensitivity. Among them, 14 genes or putative promoters were common for both stresses. For salt, acid and oxidative stresses, respectively 8, 6, and 9 genes or putative promoters were identified as essential for adaptation to these unfavorable conditions, with only three genes common to at least two stresses. Then, RT-qPCR was performed on selected stress response genes identified by mutant screenings in order to evaluate if their expression was modified in response to stresses in the parental strain. Eleven genes (membrane, transposase, chaperone, nucleotide and carbohydrate metabolism, and hypothetical protein genes) were upregulated during stress adaptation for at least two stresses. Seven genes, encoding membrane functions, were upregulated in response to a specific stress and thus could represent potential transcriptomic biomarkers. The results highlights that most of the genes identified by global reverse genetics are specifically required in response to one stress and that they are not differentially transcribed during stress in the parental strain. Most of these genes have not been characterized as stress response genes and provide new insights into the adaptation of lactic acid bacteria to their environment.</p

Identification and transcriptional profile of Lactobacillus paracasei genes involved in the response to desiccation and rehydration

International audienceLactobacillus paracasei is able to persist in a variety of natural and technological environments despite physico-chemical perturbations, in particular alternations between desiccation and rehydration. However, the way in which it adapts to hydric fluctuations and the genetic determinants involved are not clearly understood. To identify the genes involved in adaptation to desiccation, an annotated library of L. paracasei random transposon mutants was screened for viability after desiccation (25% relative humidity, 25 °C). We found 16 genes that have not been described as being involved in this response. Most of them are linked to either the transport of molecules or to cell wall structure and function. Our screening also identified genes encoding DNA related enzymes and an alarmone necessary for L. paracasei survival. Subsequently, the expression of the identified genes was measured at five stages of the dehydration-rehydration process to decipher the chronology of genetic mechanisms. They were classified into four different transcriptional profiles: genes upregulated during both desiccation and rehydration phases, genes upregulated during the desiccation phase only, genes downregulated during both desiccation and rehydration and genes downregulated only during the rehydration stage. Thus, genetic response to hydric fluctuations seems to occur during desiccation and can continue or not during rehydration. The genes identified should contribute to improve the stabilization of Lactobacillus starters in dry state

Maitrise de la fermentation butyrique par la microflore lactique réductrice

International audienceClostridium tyrobutyricum, bactérie anaérobie stricte, endosporulée, est à l’origine de l’apparition du défaut appelé «gonflement tardif » lors de l’étape d’affinage de nombreux fromages à pâtes pressées non cuites et cuites. Selon d’Incecco et al. (2018), elle passerait de l’état spore à un état de cellule végétative à la fin de l’étape d’acidification. Par ailleurs dans le projet ANR FoodREDOX, il a été montré qu’un ensemencement dans le lait d’une microflore abaissant le potentiel d’oxydoréduction, ralentirait la consommation de lactates et la production de butyrate par C. tyrobutyricum lors de l’affinage des fromages. Ainsi, connaître et comprendre les mécanismes de germination, sporulation et le métabolisme de cette bactérie, jusque-là encore peu connus ou incomplets, sont nécessaires pour comprendre l’impact d’une microflore réductrice sur ces mécanismes et de trouver un moyen de lutte à l’aide de cette dernière.Dans l’étude bio-informatique que nous avons réalisée, les séquences génomiques et/ou protéiques de protéines clés des différents mécanismes de la souche C. tyrobutyricum KCTC 5387 (ATCC 25755T ) ont été comparées à celles d’autres espèces ou genres bactériens par le biais du logiciel Blast proposé par le site Pubmed. Les résultats ont confirmé la présence des principaux facteurs sigmas et Spo0A du mécanisme de sporulation et tendent vers un mécanisme de germination se rapprochant à celui du genre Bacillus. Pour la fermentation butyrique à partir du lactate, les schémas actuels ne sont pas en totale corrélation avec l’analyse du génome. En effet, certainesenzymes présentent généralement dans les schémas ne le sont pas dans ce dernier, engendrant ainsi undéséquilibre redox. Cependant de nouveaux mécanismes, appelés « Flavin-Based Electron Bifurcation » retrouvés chez C. tyrobutyricum, seraient les éléments manquants pour équilibrer et comprendre le métabolisme chez ce dernier.En conclusion, les facteurs principaux du schéma général de sporulation chez le genre Clostridium sont retrouvés chez C. tyrobutyricum mais les mécanismes intermédiaires restent encore incertains. Le mécanisme de germination tend vers celui du genre Bacillus mais son déclenchement demeure encore inconnu. Enfin, le nouveau schéma métabolique obtenu, après les différentes comparaisons génomiques ou protéiques, doit être validé avec des données de flux métaboliques