ES2955258A1 - MUTANT STRAINS OF WEISSELLA CIBARIA FOR OVERPRODUCTION OF RIBOFLAVIN AND DEXTRAN (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Mutant strains of Weissella cibaria for overproduction of riboflavin and dextran. The present invention relates to mutant strains belonging to the Weissella cibaria species capable of overproducing dextran and riboflavin, as well as their use in the production of fermented food products enriched with dextran and riboflavin. (Machine-translation by Google Translate, not legally binding)

Description

DESCRIPTION

Mutant strains of Weissella cibaria for overproduction of riboflav ine and dextran

The present invention belongs to the technical field of food biotechnology, particularly to the development of lactic acid bacteria for the production of fermented food products. In particular, the present invention refers to mutant bacterial strains belonging to the species Weissella cibaria capable of overproducing dextran and riboflavin, as well as their use in the production of fermented foods.

BACKGROUND OF THE INVENTION

Vitamins belonging to group B are essential for human beings, since they cannot synthesize them, at least in the required quantities, and they obtain them mostly from exogenous sources. Among them is vitamin B2 or riboflavin, which is a precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), cofactors of oxido-reductase enzymes. There are no riboflavin deposits in man, so this vitamin has to be synthesized by his microbiota or consumed daily, the excess of ingestion being eliminated in the urine.

Deficiency of this vitamin, called arriboflavinosis, can cause health problems, such as liver and skin damage, changes in the brain and glucose metabolism. Furthermore, riboflavin appears to be involved in the prevention of migraine, anemia, cancer, hyperglycemia, hypertension, diabetes mellitus and, directly or indirectly, in the reduction of oxidative stress ( Thakur et al, 2017. Crit Rev Food Sci Nutr 57:3650 -3660).

The amount of riboflavin that is required to be ingested daily depends on various factors, such as age, sex and physiological state, with the recommended dose for a healthy adult being between 0.9 and 1.6 mg per day. Specifically, according to the “ European Food Information Council”, the recommended daily amount of riboflavin is 1.6 mg ( Turck et al, 2017. EFSA Journal 15: e04919). These doses can be achieved in developed countries in individuals with a balanced diet, as vitamin B2 is present in the green leaves of cereals, seeds (cereals and nuts) and in foods of natural origin. animal such as eggs, meat, milk and dairy products.

However, arriboflavinosis is a problem in underdeveloped countries and can occur in certain population groups in developed countries, including adolescents, pregnant women, the elderly, alcoholics, people with liver problems, vegetarians and vegans ( Mazur-Bialy et al , 2015. J Physiol Pharmacol 66:793-802). In addition to deficiencies of B vitamins due to unbalanced diets, the processing and cooking of vegetables increases the loss of these vitamins, including riboflavin ( Titcomb et al, 2019. Compr. Rev Food Sci Food Saf 18:1968- 1984).

Therefore, the food industry is required to develop foods fortified with B vitamins using overproducing bacteria isolated from different ecological niches. Lactic acid bacteria (LAB) can synthesize or use riboflavin during food fermentation, and the use of bacteria that overproduce this vitamin is of industrial interest. Thus, overproducing LAB strains belonging to the species Lactococcus lactis, Lactiplantibacillus plantarum (previously Lactobacillus plantarum), Limosilactobacillus fermentum (previously Lactobacillus fermentum) and Leuconostoc mesenteroides have been proposed to fortify fermented foods through in situ production using, among others, dairy matrices or based on cereals ( Thakur et al., 2016. Microb Biotechnol, 2016, 9:441-451).

On the other hand, LAB are capable of producing exopolysaccharides (EPS), among which dextrans stand out because they currently have a wide range of industrial use that includes their use as: cryoprotectants, humectants, blood plasma expanders or column matrices. chromatographic. Many LAB belonging to different genera produce dextran: Leuconostoc ( Zarouret al., 2017. In Microbial Production of Food Ingredients and Additives, eds AM Holban, and AM Grumezescu, 89-124. Cambridge: Academic press), Weissella ( Besrour-Aouam et al. al., 2021, Carbohy Polym 253:117254), Lactobacillus ( Nácher-Vázquez et al., 2017. Front Microbiol 8:2281) and Oenococcus ( Dimopoulou et al., 2018. Front Microbiol 9:1276).

The high molecular weight dextran produced by LAB ( Leuconostoc mesenteroides and lactobacilli), as well as by Saccharomyces cerevisiae, obtained the “food grade ” labeling issued by the EFSA in 2000. For this reason, they are used as additives for increase palatability in the baking, pastry and pastry industry, and in the production of ice cream, smoothies and other drinks. Studies carried out by different authors have shown the improvement of the qualities of bread, as the fermented dough is enriched with dextrans ( Wang et al., 2018. Food Hydrocholl 84:396-405). These biopolymers have a practically linear structure made up of glucopyranosides linked in their main chain by -(1^6) bonds, and a high molecular weight. Also, the addition of enzymatically produced dextran improved the volume and texture of white bread and bread containing 20% rye flour ( Chen et al., 2016. Int J Food Microbiol 239:95-102). Thus, dextrans are considered one of the new hydrocolloids that can be used as food ingredients. It has also been shown that high molecular weight dextrans produced by LAB act as immunostimulants in vitro and appear to have anti-inflammatory properties ( Zarour et al., 2017. Carbohy Polym 174:646-657), supporting their usefulness to improve the quality and the functionality of different products, including the production of functional fermented foods.

Strains belonging to the species Weissella cibaria capable of producing dextran have been previously described ( Besrour-Aouam et al., 2021. Carbohy Polym 253:117254), and EP3105339 A1 describes the method to produce dextran from a strain of W cibaria.

According to what was previously described, it would be useful for the food industry to obtain new LAB strains capable of overproducing both dextran and vitamins such as riboflavin, particularly during the production of fermented products with functional capacities.

DESCRIPTION OF THE INVENTION

The present invention relates to mutant strains belonging to the species Weissella cibaria capable of overproducing dextran and riboflavin, which comprise at least one mutation in the regulatory region of the riboflavin synthesis operon (also referred to throughout the invention as " riboswitch FMN"), where said regulatory region comprises or consists of the nucleotide sequence of SEQ ID NO: 6.

From parental strains of Weissella cibaria capable of producing concentrations elevated dextran, isolated from Type 1 baking sourdoughs (generated from spontaneous fermentation) of rye flour, preferably from the parental strains with deposit number CECT 30583 (BAL3C-5), CECT 30585 (BAL3C-7) and CECT 30584 (BAL3C-22) the inventors have obtained mutant strains (BAL3C-5 B2, BAL3C-7 B2 and BAL3C-22 B2, respectively) that maintain the dextran production capacity of the parental strains (preferably > 2 mg/ mL) and that, in addition, are overproducers of riboflavin (preferably > 0.8 μg/mL) (Table 1 and Figure 1).

Furthermore, the inventors have characterized the mutations responsible for the overproduction of riboflavin. The results obtained showed that the BAL3C-5 B2, BAL3C-7 B2 and BAL3C-22 B2 strains presented mutations in the regulatory region of the riboflavin synthesis operon (called “riboswitch FMN”) (SEQ ID NO: 6), particularly in the conserved sensor domain (aptamer), modifying its conformation (Figure 2).

In this context, the BAL3C-5 B2 strain, which exhibits the phenotype of greater riboflavin overproduction, presents a change of T instead of G in the nucleotide in position 35 of the “FMN riboswitch” (G35T in SEQ ID NO: 6 , giving rise to the sequence SEQ ID NO: 7), which would destabilize the formation of the P2 helix of the aptamer. The BAL3C-22 B2 strain, with the phenotype of lower riboflavin overproduction, has cytosine C at position 43 replaced by T (C43T in SEQ ID NO: 6, giving rise to the sequence SEQ ID NO: 8). This mutation would also affect the stability of the P2 helix. For its part, the BAL3C-7 B2 strain has an A at position 129, instead of a G (G129A in SEQ ID NO: 6, giving rise to the sequence SEQ ID NO: 9), a mutation located in a position adjacent to helix P6. Consequently, the localization of mutations in the aptamer of the “riboswitch FMN” (SEQ ID NO: 6), of the rib operon (SEQ ID NO: 1) of the strains BAL3C-5 B2, BAL3C-7 B2 and BAL3C-22 B2 , together with the absence of mutations in the genes that encode the enzymes involved in riboflavin biosynthesis, indicates that these changes are directly responsible for the riboflavin overproduction phenotype of these strains, maintaining the ability to overproduce dextran.

On the other hand, the inventors generated fermented flour doughs enriched in riboflavin and dextran through in situ production with the Weissella cibaría BAL3C-5 B2 strain in co-culture with a yeast strain (Q1 of the species Saccharomyces cerevisiae isolated from a sourdough quinoa, IBFG collection). The results obtained indicated that flour doughs fermented with BAL3C-5 B2 showed an increase in the levels of both dextran and riboflavin (Tables 2 and 3).

Finally, the inventors have evaluated the content of riboflavin and dextran during the production of bread with the strains BAL3C-5 B2, BAL3C-7 B2, BAL3C-22 B2 of W. cibaria, without addition of yeast, in comparison with their parental strains. The results obtained, collected in Table 4, showed that both in the fermented flour doughs and in pilot breads (fermentation was carried out at 30 °C for 16 h; the bread was obtained after cooking it in an electric oven at 210 °C for 15 min) high levels of dextran were maintained (>2.5 mg/g) and the effect of the riboflavin-overproducing strains BAL3C-5 B2, BAL3C-7 B2 and BAL3C-22 B2 was observed, causing an increase of 1 .7-2.4 times the vitamin levels detected in the control sample (fermented without the addition of yeast or LAB and baked), with the riboflavin values detected in the breads made with each of the mutant strains being similar (4 .3 4.8 μg/g bread).

These bacteria will therefore be useful for the production, by fermentation, of functional foods enriched in situ in vitamin B2 (riboflavin) and dextrans, which can improve the texture and organoleptic conditions of the products and may also have potential as agents immunostimulants and anti-inflammatory. Among the possible applications of these mutant strains is their use for vitamin B2 fortification of fermented doughs made with white wheat flour and the production of enriched functional baking products (such as, but not limited to, breads, cookies, pasta). with vitamin B2, as well as products with better textures thanks to dextran, favoring a lower presence of added additives for certain sectors of the population (for example, people with celiac disease, non-celiac gluten intolerances, the elderly, diets poor in vitamins, etc.).

Thus, in a first aspect the present invention relates to a strain of parental Weissella cibaria that comprises at least one mutation in the regulatory region of the riboflavin synthesis operon ( "riboswitch FMN"), where said regulatory region comprises or consists of the nucleotide sequence SEQ ID NO: 6, and where said mutated strain is an overproducer of riboflavin and a producer of dextran, hereinafter “the strain of the invention”.

" Weissella cibaria strain" is understood as any bacterial strain that belongs to the species of Weissella cibaria, a species of Gram-positive lactic acid bacteria (LAB), located within the Leuconostocaceae family. Preferably, the present invention relates to strains of food-grade Weissella cibaria that are capable of producing dextran and riboflavin (parental strains), specifically producing high concentrations of dextran, but in the absence of overproduction of riboflavin. Examples of parental strains of Weissella cibaria include strains with deposit number, without limiting to: CECT 30583 (BAL3C-5), CECT 30585 (BAL3C-7) and CECT 30584 (BAL3C-22).

In a particular embodiment of the present invention, the parental Weissella cibaria strain is selected from among the strains with deposit number: CECT 30583, CECT 30585 and CECT 30584.

The Weissella cibaria BAL3C-5 strain was deposited on March 22, 2022 under the Treaty of Budapest in the Spanish Type Culture Collection as an International Depository Authority (based in Building 3 CUE, Parc Científic Universitat de Valencia, C/ Catedrático Agustín Escardino, 9, 46980 Paterna (Valencia) SPAIN). The deposit number assigned was CECT 30583. The depositor of the strain was the Higher Council for Scientific Research (CSIC).

The Weissella cibaria BAL3C-7 strain was deposited on March 22, 2022 under the Treaty of Budapest in the Spanish Type Culture Collection as an International Depository Authority (based in Building 3 CUE, Parc Científic Universitat de Valencia, C/ Catedrático Agustín Escardino, 9, 46980 Paterna (Valencia) SPAIN). The deposit number assigned was CECT 30585. The depositor of the strain was the Higher Council for Scientific Research (CSIC).

The Weissella cibaria BAL3C-22 strain was deposited on March 22, 2022 under the Treaty of Budapest in the Spanish Type Culture Collection as an International Deposit Authority (based in Building 3 CUE, Parc Científic Universitat de Valencia, C/ Catedrático Agustín Escardino, 9, 46980 Paterna (Valencia) SPAIN). The deposit number assigned was CECT 30584. The depositor of the strain was the Higher Council for Scientific Research (CSIC).

The present invention relates to strains of Weissella cibaria overproducing dextran, that is, they are strains that present an active dextran sucrase that synthesizes dextran, and that also comprise at least one mutation (in at least a single nucleotide) in the riboflavin synthesis operon ( rib operon), particularly in the region regulator of the riboflavin synthesis operon, also called “FMN riboswitch”. These mutant strains have an improved riboflavin production capacity and are therefore capable of overproducing both dextran and riboflavin.

As used herein, the terms "mutant", "mutant strain", "derived" or "derived strain" are equivalent and refer to any microorganism, particularly a bacterium of the Weissella cibaria species, that results from the mutation or change in the DNA of one (or several) genes of a strain of Weissella cibaria that overproduces dextran and has an improved riboflavin production capacity, as described in the present invention. The derived or mutant strain can be produced naturally or intentionally, by mutagenesis methods known in the state of the art, such as, for example, the growth of the original microorganism in the presence of mutagenic or stress-causing agents, or through genetic engineering aimed at modifying specific genes.

Strains with “overproduction of riboflavin and dextran” are understood to be those mutant strains that produce more riboflavin and a similar amount of dextran than the parental strain of Weissella cibaria from which they come, after the same or similar period of fermentation time. under the same or similar fermentation conditions (temperature, pH, anaerobiosis, without or with aeration, medium volume, saturation, optical density, etc.) and in the presence of a culture medium comprising a carbon or carbohydrate source thereof. or similar composition. Thus, the mutants give rise to an overproduction of riboflavin, compared to the yield obtained when the parental strain of Weissella cibaria is used, due to a deregulated expression of the rib operon.

Particularly, in the present invention, overproduction of dextran is understood to be a concentration or production equal to or greater than 2 mg/mL or 2.8 mg/g, and overproduction of riboflavin is understood to be a production equal to or greater than 0.8 μg/g. mL or 4 μg/g.

In a particular embodiment of the present invention, the overproduction of dextran is equivalent to a concentration equal to or greater than 2 mg/mL, preferably equal to or greater than 2.5 mg/mL, more preferably equal to or greater than 3 mg/mL, more preferably equal to or greater than 4 mg/mL.

In another more particular embodiment of the present invention, the overproduction of dextran is equivalent to a concentration equal to or greater than 5 mg/mL, preferably equal to or greater than 5.5 mg/mL.

In another more particular embodiment of the present invention, the overproduction of dextran is equivalent to a concentration equal to or greater than 2.8 mg/g, preferably equal to or greater than 3 mg/g.

In a particular embodiment of the present invention, the overproduction of riboflavin is equivalent to a concentration equal to or greater than 0.8 μg/mL or 0.8 μg/g, preferably equal to or greater than 1 μg/mL or 1 μg/g, more preferably equal to or greater than 2 μg/mL or 1.25 μg/g.

In another more particular embodiment of the present invention, the overproduction of riboflavin is equivalent to a concentration equal to or greater than 2.5 μg/mL or 4.0 μg/g, preferably equal to or greater than 3 μg/mL or 4.3 μg. /g, more preferably equal to or greater than 3.4 μg/mL or 4.8 μg/g.

Overproduction of dextran and riboflavin can be determined by a variety of methods known in the art. Example methods for determining overproduction of dextran and riboflavin include, but are not limited to: measurement of the concentration (weight/volume) of dextran and riboflavin in the fermentation medium, the yield (weight/weight) of dextran and riboflavin per amount of substrate or the ratio of dextran and riboflavin formation (weight/volume/time).

It is routine practice for one skilled in the art to use methods and techniques to determine riboflavin and dextran levels. Examples of such methods include, but are not limited to, HPLC, mass spectrometry, immunoassay or activity assays, particularly fluorescence spectrometry, sulfuric phenol method, dextranase hydrolysis and quantification of the product by gas chromatography and mass spectrometry, measurement of refractive index.

In lactic acid bacteria (LAB), as in the rest of the Gram-positive bacteria, the Enzymes involved in riboflavin biosynthesis are encoded by genes that are grouped in the rib operon ( ribbG , ribB, ribA and ribH), whose expression is regulated by the element called “riboswitch FMN” located in the 5' untranslated region of its mRNA, and which code, respectively, for proteins:

RibG: diaminohydroxyphosphoribosylaminopyrimidine deaminase/5-amino-6-(5-phosphoribosylamino) uracil reductase, with 341 amino acids (aa) and 36.63 kDa, which has the amino acid sequence SEQ ID NO: 2;

RibB: riboflavin synthase with 195 aa and 21.16 kDa, which has the amino acid sequence SEQ ID NO: 3;

RibA: 3,4-dihydroxy-2-butanone 4-phosphate synthase with 393 aa and 42.9 kDa, which has the amino acid sequence SEQ ID NO: 4 and

RibH: 6,7-dimethyl-8-ribitillumacine synthase with 195 aa and 16.07 kDa, which has the amino acid sequence SEQ ID NO: 5.

The rib operon comprises the nucleotide sequence SEQ ID NO: 1, where said rib operon includes the “FMN riboswitch” (SEQ ID NO: 6). Said operon includes the genes encoding the Rib proteins described above (SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5).

The “FMN riboswitch” consists of a conserved sensor domain (aptamer), capable of binding the FMN effector, and a regulatory domain (expression platform), which can exhibit alternative conformations of transcriptional terminator (OFF state) or antiterminator (OFF state). ON). Said regulatory region or “FMN riboswitch” comprises the nucleotide sequence SEQ ID NO: 6.

SEQ ID NO: 6

Nucleotide sequence of the “FMN riboswitch” of W. cibaria strains CECT 30583, CECT 30585 and CECT 30584:

Particularly, the mutations responsible for the overproduction of riboflavin are arranged in the aptamer, modifying its conformation. The aptamer consists of a pentafoliate structure, made up of 5 hairpins (formed by stems P2 to P6) that interact at a distance to form the FMN binding site, closed by the regulatory helix (P1) that transmits the binding signal from the effector to the expression platform . The binding of FMN to the aptamer stabilizes the P1 helix and leads to the formation of a transcriptional terminator in the regulatory domain (OFF state), which prevents the expression of the rib operon. In the absence of the effector, on the contrary, an antiterminator structure is formed that allows transcription of the operon (ON state).

The BAL3C-5 B2, BAL3C-7 B2 and BAL3C-22 B2 strains present single nucleotide mutations within the regulatory region of the riboflavin synthesis operon (called “FMN riboswitch”) (SEQ ID NO: 6), specifically in the conserved sensor domain (aptamer), modifying its conformation. Particularly the mutations are found at position 35, 43 and/or 129 of the “FMN riboswitch” (positions underlined and marked in bold in SEQ ID NO: 6 described above) and present a thymine (T) instead of guanine (G ) at the nucleotide at position 35 (G35T), a thymine (T) instead of a cytosine (C) at position 43 (C43T), or an adenine (A) at position 129, instead of a guanine (G ) (G129A).

Thus, in another particular embodiment of the strain of the invention, the mutation in the regulatory region of the riboflavin synthesis operon is selected from:

(a) T instead of G at position 35 of SEQ ID NO: 6 (giving rise to the regulatory region of the riboflavin synthesis operon with sequence SEQ ID NO: 7);

(b) T instead of C at position 43 of SEQ ID NO: 6 (giving rise to the regulatory region of the riboflavin synthesis operon with sequence SEQ ID NO: 8); either

(c) A instead of G at position 129 of SEQ ID NO: 6 (giving rise to the regulatory region of the riboflavin synthesis operon with sequence SEQ ID NO: 9).

Thus, the mutated strains of the invention have a regulatory region of the riboflavin synthesis operon that comprises or consists of the nucleotide sequence of SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, where the SEQ ID NO: 7 corresponds to the sequence SEQ ID NO: 6 that comprises the G35T mutation, SEQ ID NO: 8 corresponds to the sequence SEQ ID NO: 6 that comprises the C43T mutation and SEQ ID NO: 9 corresponds to the sequence SEQ ID NO: 6 comprising the G129A mutation.

Particularly, the BAL3C-5 B2 strain (mutant strain obtained from the parental strain with deposit number CECT 30583), which exhibits the phenotype of greater riboflavin overproduction, presents a change of T instead of G in the nucleotide in position 35 of the “FMN riboswitch” (G35T), which would destabilize the formation of the P2 helix of the aptamer.

The BAL3C-22 B2 strain (mutant strain obtained from the parental strain with CECT deposit number 30584), with the phenotype of lower riboflavin overproduction, has cytosine C at position 43 replaced by T (C43T). This mutation would also affect the stability of the P2 helix.

For its part, the BAL3C-7 B2 strain (mutant strain obtained from the parental strain with CECT deposit number 30585) has an A at position 129, instead of a G (G129A). This mutation would affect the sequence spacing between helices P5 and P6.

Thus, in another particular embodiment, the strain of the invention is the parental strain Weissella cibaria with deposit number CECT 30583, where said strain comprises a regulatory region of the riboflavin synthesis operon that comprises or consists of the nucleotide sequence of the SEQ ID NO: 7.

Thus, in another particular embodiment, the strain of the invention is the parental strain of Weissella cibaria with deposit number CECT 30584, where said strain comprises a regulatory region of the riboflavin synthesis operon that comprises or consists of the nucleotide sequence of the SEQ ID NO: 8.

Thus, in another particular embodiment, the strain of the invention is the parental strain Weissella cibaria with deposit number CECT 30585, where said strain comprises a regulatory region of the riboflavin synthesis operon that comprises or consists of the nucleotide sequence of SEQ ID NO: 9.

As one skilled in the art knows, the strain of the invention or the derived or mutant strain may be comprised in a bacterial population. Therefore, another aspect of the present invention relates to a bacterial population comprising the strain of the invention, hereinafter referred to as "bacterial population of the invention."

The term “bacterial population” refers to a set of microorganisms where there is at least one cell of the strain of the invention, in any phase of the development stage. and in any phase of growth.

As understood by those skilled in the art, the strain of the invention or the population of the invention may be contained within a composition.

Thus, in another aspect, the present invention relates to a composition comprising the strain of the invention or the population of the invention, as well as any combination thereof, hereinafter the "composition of the invention."

The term “composition”, generally defined, is a set of components that is formed by at least the strain of the invention in any concentration or the population of the strain of the invention, or a combination thereof. Particularly, the composition of the invention may be a food composition.

The term "food composition" of the present invention refers to a food or a composition necessary for the preparation or production of a food that, regardless of providing nutrients to the subject who takes it, beneficially affects one or more functions of the organism, so that it provides a better state of health and well-being. As a consequence, said food composition can be used for the prevention and/or treatment of a disease or the factor causing a disease.

In another more particular embodiment, the food is selected from the list that includes: dairy product, baking product, vegetable product, meat product, snack, chocolate, beverage or baby food.

Optionally, the composition according to any of those defined above may further comprise at least one additive. Thus, in a particular embodiment, the composition of the invention also comprises at least one additive.

The term "additive" refers to a compound, substance, solution, product that can improve, enhance or complement the activity of a strain of the Weissella cibaria species, in order to improve or preserve the flavor, appearance, freshness and quality, maintaining its chemical and organoleptic properties. Examples of additives include, without limitation, plants or plant extracts or components (polyphenols, argan oil, fining proteins), and chemicals or enzymes (sulfiting agents, lysozyme, glycosidases, pectinases, hemicellulases) or food additives.

In another particular embodiment of the composition of the invention, the additive is a food additive.

Examples of food additives include, but are not limited to, substances that prevent biological chemical alterations (antioxidants, synergists of antioxidants and preservatives), substances that stabilize physical characteristics (emulsifiers, thickeners, gelling agents, antifoams, anti-caking agents, anti-caking agents, humectants, moisture regulators). pH), substances that correct plastic qualities (baking improvers, winemaking correctors, ripening regulators) or substances that modify organoleptic characteristics (colors, flavor enhancers, artificial sweeteners, aromas) and probiotics or prebiotics.

It is routine practice for one skilled in the art to calculate the amount of strain of the invention, or in the bacterial population or in the composition of the invention, used for the production of dextran and riboflavin in the present invention.

In a particular embodiment of the present invention, the concentration of the strain of the invention is between 107 and 1010 colony forming units (CFU) per gram or milliliter of final composition.

In another particular embodiment, the concentration of the strain of the invention is between 108 and 109 CFU/g or mL of the final composition.

The strain, bacterial population or composition of the invention are capable of overproducing dextran and riboflavin and, in addition, would be useful for the production of functional foods enriched in situ in vitamin B2 (riboflavin) and dextrans, which can improve texture and food organoleptics.

Thus, another aspect of the present invention is the use of the strain, bacterial population or composition of the invention for the production of dextran and riboflavin.

Furthermore, another aspect of the present invention is the use of the strain, bacterial population or composition of the invention for the production of experimental fermented foods. enriched in dextran and riboflavin.

The term "fermented foods", as used in the present invention, refers to foods or food compositions that have been transformed or fermented by the action of bacteria and/or yeasts on the carbohydrates they contain, altering their biochemical characteristics. During In this process, the sugars are converted into acids, gas or alcohol, which act as natural preservatives. Examples of fermented foods include but are not limited to: fermented flour dough or bread, cheese, sausages, alcoholic beverages (beer or wine), yogurt, kefir , raw sauerkraut and traditional kimchi.

Another particular embodiment of the present invention is the use of the strain, bacterial population or composition of the invention for the production of fermented flour doughs.

The bacterial strains of the present invention are useful for the production of dextran and riboflavin. Thus, another aspect of the present invention is a method for the production of dextran and riboflavin, hereinafter the "first method of the invention", which comprises:

(i) cultivating the Weissella cibaria strain, the bacterial population or the composition of the invention in a culture medium comprising sucrose supplementation, and (ii) recovering the dextran and riboflavin obtained in (i) from the culture medium.

The terms " Weissella cibaria strain", "bacterial population" and "composition" have been defined in previous paragraphs of this document, and apply equally to this aspect of the invention, as well as to all its particular embodiments (alone or in combination).

As understood by those skilled in the art, the growth or cultivation of a microorganism is carried out in a culture medium that includes the necessary components for said microorganism to proliferate. Thus, in this document, the term "culture medium" refers to a substance, a compound, a mixture, a solution that comprises the carbon source necessary for the growth and metabolism of the microorganism, where said culture medium can be solid, semisolid, semiliquid or liquid.

"Culture media" suitable for use in the present invention are all those known in the state of the art as appropriate for the growth, fermentation activity and maintenance in culture of Weissella cibaria strains. The culture medium may also comprise: salts and/or buffers. Examples of these culture media are, but are not limited to, MRS (Man, Rogosa and Sharpe medium, Condalab), RAM (Riboflavin Assay Medium, Difco) or RAMS (Riboflavin Assay Medium with saccharose).

The culture medium is further supplemented with a carbon source, which may comprise, for example, but not limited to, glucose, sucrose, maltose, fructose, xylose, arabinose, or a mixture of both types of sugars, preferably with saccharose.

The cultivation of Weisella cibaria bacteria has to be carried out under the right conditions so that the bacteria can grow and synthesize dextran and riboflavin from the carbon source. The appropriate conditions of pH, temperature, humidity, etc., necessary to cultivate Weissella cibaria are widely known in the state of the art. In general, the growth of these bacteria requires a temperature above 15°C and below 45°C, and a time between 12 to 48 hours.

Thus, in a particular embodiment of the first method of the invention, step (i) is carried out for 15 to 35 hours and a temperature between 20 °C and 37 °C.

In another particular embodiment of the first method of the invention, step (i) is carried out at a temperature of between 25 °C and 35 °C, preferably between 28 °C and 32 °C.

In another even more particular embodiment, step (i) of the method of the invention is carried out at a temperature of 30 °C.

Thus, in a particular embodiment of the first method of the invention, step (i) is carried out for a time between 16 to 25 hours, preferably for 23 hours.

Once the Weissella cibaria strains have been cultured during step (i) of the first method of the invention, the dextran and riboflavin produced are recovered from the culture medium.

The term "recovery", as used in step (ii) of the first method for the Production of dextran and riboflavin of the invention refers to the collection of said compounds obtained after step (i). Said recovery can be carried out by any procedure known in the art, including mechanical and/or manual methods, such as centrifugation of the bacterial culture, collection of the supernatant and precipitation with ethanol or concentration by lyophilization.

By using the Weissella cibaria strains BAL3C-5 B2, BAL3C-7 B2 and BAL3C-22 B2, riboflavin and dextran can be obtained.

Thus, in another particular embodiment of the first method of the invention, the Weissella cibaria strain of step (i) is the parental strain with CECT deposit number 30583 (BAL3C-5), where said strain comprises a regulatory region of the operon of the synthesis of riboflavin comprising or consisting of the nucleotide sequence of SEQ ID NO: 7.

Thus, in another particular embodiment of the first method of the invention, the Weissella cibaria strain of step (i) is the parental strain with CECT deposit number 30584 (BAL3C-22) where said strain comprises a regulatory region of the operon of the riboflavin synthesis comprising or consisting of the nucleotide sequence of SEQ ID NO: 8.

Thus, in another particular embodiment of the first method of the invention, the Weissella cibaria strain of step (i) is the parental strain with CECT deposit number 30585 (BAL3C-7) where said strain comprises a regulatory region of the operon of the riboflavin synthesis comprising or consisting of the nucleotide sequence of SEQ ID NO: 9.

Particularly, the BAL3C-5 B2 strain (mutant strain obtained from the parental strain with deposit number CECT 30583), which presents a change of T instead of G in the nucleotide in position 35 of the “FMN riboswitch” (G35T ), exhibits the phenotype of greater riboflavin overproduction.

Thus, in another more particular embodiment of the first method of the invention, step (i) refers to cultivating the parental strain of Weissella cibaria with deposit number CECT 30583 where said strain comprises a regulatory region of the riboflavin synthesis operon that comprises or consists of the nucleotide sequence of SEQ ID NO: 7, that is, the mutant strain derived from BAL3C-5.

The bacterial strains of the present invention are useful for the production, by fermentation, of functional foods enriched in situ in vitamin B2 (riboflavin) and dextrans that can improve the texture and organoleptic conditions of the products. Among the possible applications of these mutant strains is their use for vitamin B2 fortification of white wheat flour and the production of functional baking products (breads, cookies, pasta) enriched with vitamin B2.

Thus, another aspect of the present invention refers to a method for producing fermented foods enriched in dextran and riboflavin, hereinafter the "second method of the invention" comprising:

(i) inoculate the strain, bacterial population or composition of the invention into a food dough or mixture, and

(ii) ferment, in the presence of sucrose, the food mass obtained in (i).

The terms " Weissella cibaria strain", "bacterial population", "composition" and "fermented foods" have been defined in previous paragraphs of this document, and apply equally to this aspect of the invention, as well as to all its aspects. particular realizations (alone or in combination).

In a particular embodiment of the second method of the invention, step (ii) is carried out at a temperature between 20 and 35 °C, for 6 to 24 hours.

In a particular embodiment of the second method of the invention, the food dough is a fermented flour dough.

In another particular embodiment of the second method of the invention, the bread dough comprises flour from cereal grains or herbs that are selected from: wheat, rice, corn, barley, oats, rye, buckwheat or buckwheat, quinoa and combinations. between them.

As a person skilled in the art knows, the baking doughs for fermented foods made with cereals or pseudocereals (for example, bread or pastries) may also comprise yeasts, to carry out the fermentation of the food product.

Thus, in another particular embodiment, the food dough also comprises at least one yeast.

The term "yeast" as used herein refers to microorganisms called fungi, capable of carrying out the decomposition by fermentation of various organic compounds, mainly sugars or carbohydrates. Examples of yeast species in the present invention include, but are not limited to, Saccharomyces cerevisiae.

It is routine practice for a person skilled in the art to calculate the amount of strain of the invention, or in the bacterial population or in the composition of the invention, or, where appropriate, the amount of yeast, used for the production of dextran and riboflavin. in the present invention.

In a particular embodiment of the second method of the present invention, the concentration of the Weissella cibaria strain is between 108 and 1010 and/or the concentration of the yeast is between 106 and 109 colony forming units (CFU) per gram or milliliter of food mass.

In another particular embodiment, the concentration of the strain of the invention is between 107 and 109 and of the yeast is between 107 and 108 CFU/g or mL of the final composition.

Particularly, by using the Weissella cibaria BAL3C-5 B2, BAL3C-7 B2 and BAL3C-22 B2 strains, fermented flour doughs enriched in riboflavin and dextran can be obtained, both in co-culture with yeast and without yeast.

Thus, in another particular embodiment of the second method of the invention, the Weissella cibaria strain of step (i) is the parental strain with CECT deposit number 30583 (BAL3C-5), where said strain comprises a regulatory region of the operon of the synthesis of riboflavin comprising or consisting of the nucleotide sequence of SEQ ID NO: 7.

Thus, in another particular embodiment of the second method of the invention, the Weissella cibaria strain of step (i) is the parental strain with CECT deposit number 30584 (BAL3C-22) where said strain comprises a regulatory region of the operon of the synthesis of riboflavin comprising or consisting of the nucleotide sequence of SEQ ID NO: 8.

Thus, in another particular embodiment of the second method of the invention, the Weissella cibaria strain of step (i) is the parental strain with CECT deposit number 30585 (BAL3C-7) where said strain comprises a regulatory region of the operon of the riboflavin synthesis comprising or consisting of the nucleotide sequence of SEQ ID NO: 9.

DESCRIPTION OF THE FIGURES

Figure 1. Detection of riboflavin production by riboflavin-overproducing W. cibaria strains (BAL3C-5 B2, BAL3C-7 B2 and BAL3C-22 B2) and their parental strains (BAL3C-5, BAL3C-7 and BAL3C- 22) vitamin producers. The bacteria were grown in medium containing glucose (RAM) or glucose plus sucrose (RAMS) and their growth (OD600 nm) and the fluorescence emitted by the riboflavin produced by them were measured in real time.

Figure 2. Schematic representation of the possible “ON” and OFF” conformations of the “FMN riboswitch”. The mechanism of action of the wild “FMN riboswitch” is based on the change, promoted by the binding of the effector, from the “ON” state. (antiterminator structure) to the "OFF” state (transcriptional terminator). Above the structure of the "OFF” state, the aptamer and the expression platform are indicated. The transcriptional terminator sequence is highlighted in red.

Figure 3. In situ detection of dextran sucrase (Dsr) activity in supernatants of cell-free BAL cultures. The indicated W. cibaria (BAL3C-5 B2, BAL3C-7 B2 and BAL3C-22 B2) were cultured in RAM or RAMS media and the culture supernatants were fractionated on SDS-polyacrylamide gels and, after renaturation of the proteins , were analyzed in situ to detect Dsr activity. S, “Precision Plus Protein Dual Color Standards” molecular weight standard (Bio-Rad).

EXAMPLES

Next, the invention will be illustrated through tests carried out by the inventors, which demonstrate the effectiveness of the product of the invention.

Example 1. Selection of riboflavin overproducing strains

The strains CECT 30583 (BAL3C-5), CECT 30585 (BAL3C-7) and CECT 30584 (BAL3C-22) belonging to the species W. cibaria and isolated from rye flour sourdoughs ( Llamas-Arriba et al., 2021 Foods 10:2004) were used to select mutant strains that overproduce vitamin B2 by treatment with roseoflavin.

The 3 parental bacteria were grown independently in MRS medium (Man, Rogosa and Sharpe medium, Condalab) at 30 °C to an optical density at a wavelength of 600 nm (OD600 nm) of 1.5. Subsequently, the cultures were diluted 1:100 in RAM medium (riboflavin assay medium, Difco) lacking riboflavin supplemented with roseoflavin (10 μg/mL) and incubated under the same conditions until they reached the beginning of the stationary phase.

Subsequently, the bacteria were subjected to increasing concentrations of roseoflavin (50 μg/mL, 75 μg/mL, 100 μg/mL, 150 μg/mL, 200 μg/mL) through sequential dilution and subsequent growth in medium supplemented with said compound. The cultures of W. cibaria BAL3C-7 and BAL3C-22 showed growth even in the presence of a roseoflavin concentration of 200 μg/mL, while W. cibaria BAL3C-5 only showed tolerance to 150 μg/mL of the compound.

These cultures were sown in Petri dishes containing MRS supplemented with 1.6% agar, to isolate colonies of roseoflavin-resistant strains, after growth at 30 °C for 48 h. Next, the bacteria contained in 3 of the colonies (one for each parental strain) were recovered by growth in liquid MRS medium. Furthermore, these 3 isolates, once their resistance to roseoflavin was proven by growth in MRS in the presence of the compound, were preserved at -80°C in MRS medium without roseoflavin and supplemented with 20% glycerol.

Example 2. Evaluation of the riboflavin and dextran production capacity of lactic acid bacteria in culture medium

The three isolates described in example 1 were called W. cibaria BAL3C-5 B2, BAL3C-7 B2 and BAL3C-22 B2 and were analyzed together with their respective strains. parental W. cibaria CECT 30583 (BAL3C-5), CECT 30585 (BAL3C-7) and CECT 30584 (BAL3C-22). Dextran is produced extracellularly by LAB in a reaction catalyzed by dextran sucrases (Dsr) using sucrose as a substrate. Furthermore, riboflavin produced by bacteria can be secreted into the extracellular medium. Thus, the supernatants of bacterial cultures of the 6 strains grown in commercial RAM medium containing 2% glucose or in RAMS supplemented with 2% sucrose for 23 h at 30 °C were analyzed to determine riboflavin levels by fluorescence spectrometry. as previously described ( Mohedano et al., 2019. Front Microbio! 10:1748) and dextran by the sulfuric phenol method ( Besrour-Aouam et al., 2021. Carbohy Polym 253:117254). The cell biomass achieved was estimated by measuring the OD600 nm. The results obtained are shown in Table 1.

Table 1. Comparative analysis of riboflavin-overproducing W. cibaria strains and their corresponding parental strains. ( 1) Ratio of the production of the compounds by each of the riboflavin overproducing strains ( B2) with respect to its corresponding wild strain ( wt).

The supplementation of the medium with sucrose (RAMS versus RAM) led to the 6 strains analyzed reaching, after 23 h of growth, a higher biomass, as estimated by the OD600 nm values detected. Concerning dextran production, all 6 bacteria were able to produce the biopolymer at high levels (> 5 mg/mL) in the RAMS medium. Furthermore, no significant difference was observed between the EPS production of the riboflavin-overproducing mutant strains (designated B2) and their respective parental strains.

Regarding riboflavin production, B2 bacteria showed higher levels than their respective wild strains in the two growth media RAM (28-61-fold increase) and RAMS (9-19-fold increase), being higher the production of vitamin B2 by all strains in the medium supplemented with sucrose. Furthermore, the BAL3C-5 B2 strain was the one that showed the highest levels of riboflavin production (3.45 μg/mL in RAMS medium). Thus, the results obtained showed that the analysis of the supernatants of the LAB in the RAMS medium allowed the quantitative assessment of the dextran produced and the vitamin B2 secreted by them.

Furthermore, these results demonstrated that strains BAL3C-5 B2, BAL3C-7 B2 and BAL3C-22 B2 were overproducers of riboflavin and maintained the ability to overproduce dextran. The ability of the 6 lactic acid bacteria to produce riboflavin during their growth in RAMS and RAM medium in microtiter plates (Sterile 96-Well Optical White w/Lid Cell Culture, Thermo Fisher Scientific, Rochester, NY, United States) was also evaluated. on a Variouskan Flask System (Thermo Fisher Scientific, Waltham, MA, United States). To this end, simultaneous measurements were made, in real time, of the OD600 nm of the cultures and the fluorescence of vitamin B2 produced by them (excitation at a wavelength of 440 nm and emission detection at 520 nm). The results obtained (Figure 1) confirmed those described in Table 1 and revealed the overproduction of riboflavin by the strains BAL3C-5 B2, BAL3C-7 B2 and BAL3C-22 B2 during the exponential and stationary growth phases, although no differences between the growth of overproducing strains and their respective parental strains during exponential growth.

Example 3. Characterization of the mutations of riboflavin overproducing strains

The complete nucleotide sequence (SEQ ID NO: 1) of the rib operon (including the regulator 'riboswitch FMN') of the wild strains CECT 30583 (BAL3C-5), CECT 30585 (BAL3C-7) and CECT 30584 (BAL3C) was determined. -22) of W. cibaria and the corresponding isogenic strains overproducing riboflavin (BAL3C-5 B2, BAL3C-7 B2 and BAL3C-22 B2). The determination was carried out by automatic sequencing based on the Sanger method, with a strategy of "primer walking" and using PCR fragments that contained the complete sequence of the rib operon as template DNA. From the sequence of the rib genes, the amino acid sequence of the proteins RibG (SEQ ID NO: 2), RibB (SEQ ID NO: 3), RibA (SEQ ID NO: 4), and RibH (SEQ ID NO: 5) was inferred. with the help of the bioinformatics programs contained in the DNASTAR Lasergene 15 package.

The results obtained showed that the sequence was identical in the three wild strains, while each of the strains with the riboflavin overproducing phenotype presented a change in a single nucleotide within the "FMN riboswitch" aptamer.

In this context, the BAL3C-5 B2 strain, which exhibits the phenotype of greater riboflavin overproduction of the 3 mutant strains studied, presents a G ^ U change in the nucleotide in position 35 of the “FMN riboswitch” (numbered from the site predicted transcription initiation), which would destabilize the formation of the P2 helix of the aptamer. The BAL3C-22 B2 strain, with the phenotype of lower riboflavin overproduction, has C43 (which would pair with G35 in the wild aptamer) replaced by U. This mutation would also affect the stability of the P2 helix, although to a lesser extent. than the mutation found in the BAL3C-5 B2 strain, since a GU pair could still form. For its part, the BAL3C-7 B2 strain has an A at position 129, instead of a G. Previous crystallographic studies have shown that the G conserved in the same relative position of the aptamer is involved in the interaction with the FMN effector. Consequently, the localization of mutations in the FMN riboswitch aptamer of the rib operon of strains BAL3C-5 B2, BAL3C-7 B2 and BAL3C-22 B2 indicates that these changes are directly responsible for the riboflavin overproduction phenotype of these strains. .

Example 4. Identification of the determinants of dextran production

The nucleotide sequence of the dsr genes of the riboflavin overproducing strains (SEQ ID NO: 10) was determined as indicated in example 3. The genes of the 3 strains showed a length of 4,342 nt, were identical to each other and only showed 100% nucleotide identity with the dsr gene of W. cibaria CH ₂ , a strain isolated from cheese from the western Himalayas. The amino acid sequence of the Dsr was inferred from the nucleotide sequence of the gtf genes with the EditSeq 15 software program (DNASTAR R Lasergene 15) and showed that they encode a protein of 159,099 Da (SEQ ID NO: 11, which has at its amino-terminal end a signal peptide involved in processing and secretion in Gram-positive bacteria. The analysis of the sequence of amino acids of the protein with the SignalP-5.0 program allowed us to predict that the processed extracellular Dsr has a molecular mass of 156,411 Da. With the aim of detecting the active form of Dsr in situ and in gel, the supernatants of cultures of the strains BAL3C-5 B2, BAL3C-7 B2 and BAL3C-22 B2, grown in RAMS and RAM medium for 24 h, were analyzed. as described in example 2, by electrophoresis in an 8% SDS polyacrylamide gel at a constant voltage of 100 V.

Then, to detect Dsr activity, the previously described procedure was used ( Besrour-Aouam et al., 2021. Carbohy Polym 253:117254). This method involves: (i) the removal of SDS by washing, (ii) the synthesis of dextran by the Dsr for 17 h in the presence of sodium acetate buffer supplemented with 10% sucrose and (iii) the development of the activity by staining with periodic acid-Schiff. Subsequently, proteins were stained with 25% Coomassie blue and their molecular weight was estimated by reference to the Precision Plus Protein Dual Color Standards marker (Bio-Rad), which includes polypeptides in the range of 10-250 kDa.

Gel development (Figure 3) showed an intense band of activity approximately at the expected position (156 kDa) in the supernatants of the 3 W. cibaria strains grown in RAMS medium and a very faint band in the cultures grown in RAM medium. . Consequently, the results obtained revealed that Dsr production is induced when sucrose is present in the culture medium.

Example 5. Evaluation of the strains W. cibaria BAL3C-5 B2 and W. cibaria BAL3C-5 during the production of fermented flour doughs

During growth in culture medium, the BAL3C-5 B2 strain was, among the 6 bacteria studied, the one that showed the greatest capacity to overproduce riboflavin (Table 1 and Figure 1). Therefore, the capacity of said strain and that of its parent strain to produce vitamin B2 and dextran during flour fermentation was evaluated, both individually and co-inoculated with the yeast Saccharomyces cerevisiae Q1.

The flours used were organic and of high extraction (whole or semi-whole): breadmaking wheat of strength W200 ("Molinos del Duero y Compañía General de Harinas, SL" Carr. de Villalpando, 13, 49029 Zamora) and pseudocereal flours. buckwheat and quinoa supplied by Molendum Ingredients (Calle de la Milana S/N, 49530 Coreses, Zamora).

To generate the fermented flour doughs, the Q1 yeast was cultivated, on the one hand, from a saturated culture of 12-24 h in rich YEPD medium (yeast extract, peptone, 2% dextrose), inoculating yeast cells in 50 mL of fresh YEPD medium, at an initial OD600 nm of « 0.1 (1 unit of OD600 nm is equivalent to « 107 cells/mL). The cultures were incubated in 250 mL flasks with constant aeration, at 28 °C and orbital shaking at 230 rpm. The cells were collected by centrifugation at an OD600nm <1 in early-mid exponential phase, when the yeasts are actively dividing (budding) (5-6 h), and washed twice with sterile distilled water to prepare the corresponding inoculum (dilutions ) to ferment flour. On the other hand, LAB were cultured in MRSS medium (5% sucrose) with an initial pH adjusted to 7.0 at 30 °C and under constant aeration. The bacterial cells were collected by centrifugation after 2-3 h, washed twice with 0.9% saline solution and the appropriate dilutions were made to inoculate flour masses. The microorganisms contained in 13 mL of water with 5% sucrose [3x108 CFU/g of LAB mass (BAL3C-5 or BAL3C-5 B2) and/or 3x107 CFU/g of yeast mass Q1] and were mixed with 40 g of each flour (50% wheat quinoa or only buckwheat) and 80 mL of 5% sucrose (70% hydration), to induce in situ the synthesis of dextran by the LAB. The ingredients were mixed with elongated spatulas until forming a homogeneous mass and the tubes containing the masses were incubated in an oven at 28 °C for 24 h. Subsequently, the fermented masses were analyzed by evaluating the extracellular dextran produced by the BAL3C-5 B2 and BAL3C-5 strains, as well as the riboflavin secreted by said BAL and/or by the Q1 yeast. To do this, the masses generated were diluted 1:2 with water (final volume 3) and centrifuged at 8000 x g for 20 min. The supernatant obtained was used to fluorimetrically determine the levels of riboflavin as previously indicated and to determine the levels of exopolysaccharides (EPS) after 3 precipitations with 3 volumes of absolute ethanol and titration of neutral sugars by the sulfuric phenol method. To calculate the concentration of the vitamin or EPS, calibration curves of riboflavin or glucose dissolved in water were used, respectively.

Table 2 presents the values obtained for each of the samples analyzed, as well as the initial riboflavin contributions of the non-fermented doughs made with wheat and quinoa (TQ) or buckwheat (TS) flours. The results obtained indicated that the Spanish flours used to make the doughs have a high content of soluble riboflavin (0.28-0.34 jg/g dough). An increase in riboflavin levels was observed in all fermented doughs, even when they contained only the Q1 yeast (0.64-0.7 jg /g). Furthermore, it was observed that, with the two types of flour used, the fermentation carried out with Q1 and BAL3C-5 (0.69 jg /g, TQ2 and 0.83 jg /g, TS2) and, in a greater proportion, with BAL3C -5 B2 (0.78 |jg/g TQ3 and 1.25 jg /g TS3) generated higher levels of soluble vitamin B2.

Table 2. Determination of the concentration of soluble vitamin B ² present in fermented flour doughs.

Table 3 shows the results obtained by quantifying the dextran present in the masses. All doughs fermented with microorganisms showed levels of soluble EPS (1-1.9 mg/g; TQ1-TQ3 and TS1-TS3) much higher than those detected in unfermented doughs (0.22-0.26 mg/g). g; TQ and TS). Furthermore, cofermentation with LAB resulted in a higher increase in EPS content (up to 8.6 times), more pronounced in the case of doughs made with wheat and quinoa and, as expected, no major differences were observed between fermented flour doughs co-inoculated with BAL3C-5 B2 (TQ3 and TS3) and with CECT 30583 (BAL3C-5) (TQ2 and TS2).

Table 3. Determination of the concentration of soluble EPS present in the fermented flour doughs.

Example 6. Evaluation of the strains BAL3C-5 B2, BAL3C-7 B2, BAL3C-22 B2 of W. cibaria and its parent strains during bread making without the addition of yeast

The LAB were evaluated independently by carrying out a control fermentation without the addition of LAB, only with the indigenous microbiota, present in the flour doughs without inoculation. (control). The microorganisms were cultured as described in example 5.

The flour used in this case was organic wheat flour refined with strength W200, from Molinos del Duero (Cerecinos de Campos, Zamora). A dough was made with 400 g of flour, 300 mL of water with 5% sucrose and 0.64% salt, mixed well with a mixer and portions of 50 g of dough were divided, which were inoculated with 1x109 CFU of BAL/g. These pieces were kneaded and divided into 3 micro-loaves of 15 g each. Fermentation was carried out at 30 °C for 16 h. After fermentation, and before baking at 210 °C for 15 min, a CFU/g count was performed in MRS-agar medium to detect BAL and in YEPD medium to detect yeasts (Table 4). The data obtained are detailed in Table 4. In the dough not inoculated with bacteria, as expected, only low concentrations of LAB (4.37x106 CFU/g) present in the microbiota of the dough were detected. Furthermore, in all doughs fermented with LAB, values >1x109 CFU/g were detected. Also, in all the doughs analyzed, a practically negligible level of endogenous flour yeasts was observed (1.8-6.0x101 CFU/g).

Subsequently, samples of the unbaked doughs and the breads obtained after baking were analyzed. To determine, from the same samples, the total concentration of riboflavin and dextran present in the breads, the following methodology was designed and used. 5 g of the unbaked fermented doughs and 5 g of the baked breads were analyzed. The samples were placed in 100 mL bottles, 25 mL of 0.1 M HCl were added and heated at 121 °C for 60 min. Subsequently, the suspension was neutralized by adding 4 M sodium acetate until reaching a pH of 6.5. Next, 0.5 mL of a solution of Chaetomium erraticum dextranase (Sigma-Aldrich-D0443, 0.6 g of the enzyme per sample) was added and the samples were incubated at 30 °C for 18 h. After incubation, the samples were brought to a volume of 50 mL by adding 0.01 M HCl and, to remove solid residues, they were centrifuged at 8,000 x g for 10 min. The supernatants were filtered through cheesecloth and subsequently through a 0.22 μm filter. Finally, the filtrate was aliquoted and kept frozen at −20 °C until further analysis.

Table 4. Analysis of the total content of riboflavin and dextran in doughs fermented for 16 h and in the breads generated by baking, as well as the dextran content in the breads.

1The content of riboflavin and dextran in flour processed as breads was 1.54±0.03 yg/g and 0.24±0.05 mg/g, respectively.

The aforementioned procedure involves the conversion of the flavins present in the sample into riboflavin, which was quantified by fluorescence spectrometry as described in example 2. The results obtained, collected in Table 4, showed that both in the fermented masses and In the breads, the effect of the 3 riboflavin-overproducing LAB, BAL3C-5 B2, BAL3C-7 B2, and BAL3C-22 B2, was observed, causing an increase of 1.7-2.4 times over the vitamin levels detected in the control sample. , the highest value being that detected with the BAL3C-5 B2 strain (4.25 jg /g bread).

On the other hand, the samples obtained from the breads were used to determine the concentration of dextran present in them. Treatment with C. erraticum dextranase, described previously in this example 6, specifically hydrolyzes dextran to isomaltose, the concentration of which was determined by analysis of 50 μL of the samples using gas chromatography and mass spectrometry. The results obtained (Table 4) revealed that the breads obtained through fermentations with the 6 LAB analyzed showed high levels of dextran (2.8-3.2 mg/g) much higher than those detected in the control bread (0.79 mg /g). Also, they showed that the BAL3C-5 B2 strain was the best producer of dextran during the fermentations, as had already been observed with the results of example 5. In addition, the methodology used proved to be suitable for generating suitable samples to assess total flavins and dextran. .

Claims (26)

1. A bacterial strain of parental Weissella cibaría characterized by comprising at least one mutation in the regulatory region of the riboflavin synthesis operon, where said regulatory region comprises or consists of the nucleotide sequence of SEQ ID NO: 6, and where said mutated strain is an overproducer of riboflavin and dextran. 2. The strain according to claim 1 wherein the parental Weissella cibaria strain is selected from among the strains with deposit number: CECT 30583, CECT 30584 and CECT 30585. 3. The strain according to claim 1 or 2 wherein the mutation in the regulatory region of the riboflavin synthesis operon is selected from: (a) T instead of G at position 35 of SEQ ID NO: 6; (b) T instead of C at position 43 of SEQ ID NO: 6; either (c) A instead of G at position 129 of SEQ ID NO: 6. 4. The strain according to any one of claims 1 to 3 wherein said strain is the parental Weissella cibaria strain with CECT deposit number 30583 characterized in that it comprises the mutation T instead of G at position 35 of SEQ ID NO: 6 . 5. The strain according to any one of claims 1 to 3 wherein said strain is the parental Weissella cibaria strain with CECT deposit number 30584 characterized in that it comprises the mutation T instead of C at position 43 of SEQ ID NO: 6 . 6. The strain according to any one of claims 1 to 3 wherein said strain is the parental Weissella cibaria strain with CECT deposit number 30585 characterized in that it comprises the mutation A instead of G at position 129 of SEQ ID NO: 6.7. 7. A bacterial population comprising the strain according to any one of the claims 1 to 6. 8. A composition comprising the strain according to any one of claims 1 to 6 or the bacterial population according to claim 7. 9. The composition according to claim 8 wherein said composition is a food composition. 10. The composition according to claim 8 or 9 further comprising at least one additive, preferably a food additive. 11. The composition according to any one of claims 8 to 10 wherein said composition comprises a concentration of the strain of between 107 and 1010 colony forming units (CFU) per gram or milliliter of final composition. 12. Use of the strain according to any one of claims 1 to 6, the bacterial population according to claim 7 or the composition according to any one of claims 8 to 11 for the production of dextran and riboflavin. 13. Use of the strain according to any one of claims 1 to 6, the bacterial population according to claim 7 or the composition according to any one of claims 8 to 11 for the production of fermented foods enriched in dextran and riboflavin, preferably flour doughs fermented. 14. Method for the production of dextran and riboflavin comprising: (i) cultivating the strain according to any one of claims 1 to 6, the bacterial population according to claim 7 or the composition according to any one of claims 8 to 11 in a culture medium comprising sucrose supplementation, and (ii) recover the dextran and riboflavin obtained in (i) from the culture medium. 15. Method according to claim 14 wherein step (i) is carried out for 12 to 48 hours and at a temperature between 15 °C and 45 °C. 16. Method according to claim 15 wherein step (i) is carried out for 15 to 35 hours, preferably for 23 hours. 17. Method according to claim 15 wherein step (i) is carried out at a temperature between 20 and 37 °C, preferably at 30 °C. 18. Method according to any one of claims 14 to 17 wherein the strain is the parental Weissella cibaria strain with CECT deposit number 30583 characterized in that it comprises the T mutation instead of G at position 35 of SEQ ID NO: 6. 19. Method for producing fermented foods enriched in dextran and riboflavin comprising: (i) inoculating the strain according to any one of claims 1 to 6, the bacterial population according to claim 7 or the composition according to any one of claims 8 to 11 into a food mass or mixture, and (ii) ferment, in the presence of sucrose, the dough or food mixture obtained in (i). 20. The method according to claim 19 wherein step (ii) is carried out for 6 to 24 hours and at a temperature between 20 and 35 °C. 21. The method according to claim 19 or 20 wherein the food dough is a fermented flour dough. 22. The method according to claim 21 wherein the flour dough comprises flour from cereal grains or herbal grains that are selected from: wheat, rice, corn, barley, oats, rye, buckwheat, quinoa and combinations from the same. 23. The method according to any one of claims 19 to 22 wherein the food dough also comprises at least one yeast. 24. The method according to claim 23 wherein the concentration of the yeast is between 107 and 108 colony forming units (CFU) per grams or milliliters of food mass. 25. The method according to any one of claims 19 to 24 wherein the concentration of the Weissella cibaria strain is between 107 and 109. 26. The method according to any one of claims 19 to 25 wherein the strain is the parental Weissella cibaria strain with CECT deposit number 30583 characterized in that it comprises the T mutation instead of G at position 35 of SEQ ID NO: 6.