Bioinformatics and metabolic flux analysis highlight a new mechanism involved in lactate oxidation in Clostridium tyrobutyricum / La bioinformática y el análisis del flujo metabólico destacan un nuevo mecanismo implicado en la oxidación del lactato en Clostridium tyrobutyricum

Climate 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 CO2 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 CO2 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.(AU)

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

Climate 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 CO2 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 CO2 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.

Green strategies to control redox potential in the fermented food industry.

Lactic acid bacteria (LAB) are important microorganisms in the food industry as functional starters for the manufacture of fermented food products and as probiotics. Redox potential (Eh) is a parameter of the physicochemical environment of foods that influences key oxidation-reduction reactions involved in process performances and product quality. Eh can be modified by different methods, using redox molecules, catalytic activity of enzymes or LAB themselves, technological treatments like electroreduction or heating, and finally gases. Nowadays new applications for food manufacture must undertake green process innovation. This paper presents the strategies for Eh modification in a sustainable manner for production of LAB biomass (starters, probiotics) and fermented food products (fermented milks, cheeses and others). While the use of chemical or enzymes may be subject to controversy, the use of gases offers new opportunities, in combination with LAB. Protection against food-borne microorganisms, an increasing growth and viability of LAB, and a positive impact on food flavour are expected.

Whole-Genome Sequencing and Annotation of Clostridium tyrobutyricum Strain Cirm BIA 2237, Isolated from Silage.

Clostridium tyrobutyricum is the main bacterial species leading to the late blowing defect, a major cause of spoilage in semihard and hard cheeses. This study reports the complete genome sequencing, assembly, and annotation of C. tyrobutyricum strain Cirm BIA 2237, formerly called CNRZ 608, isolated from silage.

A simple micro-batch ion-exchange resin extraction method coupled with reverse-phase HPLC (MBRE-HPLC) to quantify lactoferrin in raw and heat-treated bovine milk.

Lactoferrin is an iron-binding cationic glycoprotein (pI = 8.7) beneficial for mammal health, especially udder and milk preservation. A new simple two-step method of quantification was developed. Lactoferrin in 1 mL of bovine skim milk was first adsorbed onto 100 mg of macroporous sulfonated-resin at pH 6.8 by rotary stirring for 90 min at 20-25 °C. After washing the resin, lactoferrin was desorbed using 1 mL of 2 M NaCl containing phenylalanine as a dilution marker, then fully resolved and quantified by RP-HPLC at 220 nm using a wide-bore C4 silica column. This robust, inexpensive and flexible method improves selectivity (no protein interference) and sensitivity compared to previous HPLC methods. In-laboratory validation demonstrated its linearity (25 to 514 µg Lf mL-1), accuracy (110 to 98% recovery), and precision (<4%), which were comparable to immuno-based methods. The results for individual raw cow's milk were strongly correlated with results using an ELISA test.

The Microbiology of Traditional Hard and Semihard Cooked Mountain Cheeses.

Traditional cheeses originate from complex systems that confer on them specific sensory characteristics. These characteristics are linked to various factors of biodiversity such as animal feed, the use of raw milk and its indigenous microflora, the cheese technology, and the ripening conditions, all in conjunction with the knowledge of the cheesemaker and affineur. In Europe, particularly in France, the preservation of traditional cheesemaking processes, some of which have protected designation of origin, is vital for the farming and food industry in certain regions. Among these cheeses, some are made in the Alps or Jura Mountains, including Comté, Beaufort, Abondance, and Emmental, which are made from raw milk. The principle of hard or semihard cooked cheese, produced in the Alps and Jura Mountains, was to make a product during the summer-a period during which the animals feed more and milk production is high-with a shelf life of several months that could be consumed in winter. Today, these traditional cheeses are produced according to a specific approach combining science and tradition in order to better understand and preserve the elements that contribute to the distinctiveness of these cheeses. To address this complex problem, a global approach to the role of the raw milk microflora in the final quality of cheeses was initially chosen. The modifications resulting from the elimination of the raw milk microflora, either by pasteurization or by microfiltration, to the biochemistry of the ripening process and ultimately the sensory quality of the cheeses were evaluated. This approach was achieved mainly with experimental hard cooked cheeses. Other types of traditional cheese made with raw and pasteurized milk are also considered when necessary. Besides the native raw milk microflora, traditional lactic starters (natural or wild starters) also participate in the development of the characteristics of traditional hard and semihard cooked mountain cheeses. After an initial description, their roles are described, mainly for Comté.

Effect of mesophilic lactobacilli and enterococci adjunct cultures on the final characteristics of a microfiltered milk Swiss-type cheese.

The effect of four associations of adjunct cultures composed of mesophilic lactobacilli and enterococci, either solely or combined, on the microbiological, biochemical and sensory characteristics of Swiss-type cheese made using microfiltered cows' milk and supplemented with propionibacteria was studied. The global pattern of growth was similar to that generally observed in raw milk cheese and interactions between microflora were highlighted during ripening. Enterococci, which negatively affected the survival of streptococci starters, seemed to play a limited role in the formation of volatile compounds, probably due to their low levels throughout ripening. On the contrary, mesophilic lactobacilli, which affected the evolution of propionibacteria, enterococci and L. delbrueckii subsp. lactis starter counts, modified free amino acid content, production of volatile compounds and organoleptic properties of mature cheese. This population appeared to be of major importance in the formation of cheese flavor as it was positively related to numerous potential flavor compounds such as alcohols and their corresponding esters, acetaldehyde and 4-methyl-4-heptanone. The original mesophilic lactobacilli present in milk could play an important role in the sensorial diversity of raw milk Swiss-type cheeses such as Comte.

Milk acidification by Lactococcus lactis is improved by decreasing the level of dissolved oxygen rather than decreasing redox potential in the milk prior to inoculation.

Although redox potential is very rarely taken into account in food fermentation it could be as influential as pH on bacterial activities. Lactococcus lactis is already known to exhibit a powerful reducing activity in milk but its reduction activity was shown to occur prior to its acidification activity with a potential interaction between these two lactococcal activities. Therefore, acidification lag-type phase could be shortened by decreasing the redox potential of milk before inoculation. As the redox potential is highly dependent on the dissolved oxygen level, our objective was to study their separate and combined influences on acidification and growth kinetics of pure L. lactis strains in milk. Results showed that high level of dissolved oxygen is significantly more influential on growth, and even more on acidification kinetics, than initial decreased redox potential of milk. Reduction of milk was drastic and mostly due to bacterial activity. The redox potential of milk only dropped when dissolved oxygen was entirely consumed. When there was no dissolved oxygen from the beginning, L. lactis immediately decreased the redox potential of milk and acidified afterwards. When the level of dissolved oxygen was initially high, acidification and reduction of milk occurred at the same time. Acidification kinetics was then biphasic with a slower rate during the aerobic stage and a faster rate during the anaerobic stage. The seven strains tested demonstrated diversity in both their acidification kinetics and their adaptation to high level of dissolved oxygen, independent of their growth kinetics. To conclude, we have shown that the level of dissolved oxygen in milk has a dramatic influence on acidification kinetics and could be used to control acidification kinetics in dairy industries.