ITRM20130257A1 - NEW BOWL OF LACTOBACILLUS - Google Patents

Description

"NEW STRAIN OF LACTOBACILLUS"

DESCRIPTION

The present invention refers to a new strain of lactic bacteria afferent to the Lactobacillus plantarum species and its uses. The strain of the present invention is capable of over-producing riboflavin and can be used in the production of food and feed, fermented and non-fermented, with significant increases in vitamin B2 levels and as a probiotic in the human and animal gastro-intestinal tract.

STATE OF THE PRIOR ART

The enrichment or fortification of food is a process that improves its nutritional quality through an increase in the content of specific macronutrients and / or micronutrients. Vitamins are essential micronutrients for the metabolism of living beings. Vitamin enrichment is of great interest in the food industry, as the vitamin content of foods can be depleted following the cooking, processing and conservation processes, and the refinement of raw materials. The spread of new lifestyles and eating patterns also contributes to determining deficiency states in the population. Thus, the need arises to fortify foods with â € ̃synthesisâ € ™ vitamins. Even in animal nutrition there is a need for â € fortifiedâ € ™ feeds. B vitamins are water-soluble and play an important role in cellular metabolism. Riboflavin (vitamin B2) belongs to group B. It is a heterocyclic compound consisting of flavin, a water-soluble compound formed by a substituted isoalloxazine, to which ribitol, an alcohol derived from ribose by reduction of its carbonyl group, is bound. Riboflavin is stable at high temperatures and oxygen, but is unstable when exposed to acids, alkalis and light. Riboflavin is the precursor of the coenzymes FMN (flavinadeninmononucleotide) and FAD (flavin-adenindinucleotide), which act as a â € œcarrierâ € of electrons in the biological oxidation-reduction reactions and play a fundamental role for hundreds of other proteins (FMN or FAD- dependent) called flavoproteins which act, in turn, as hydrogen transporters in biological redox reactions. Flavoproteins are also essential for the metabolism of amino acids, for the production of energy and for the activation of folate and pyridoxine in their coenzymatic forms. The daily requirement of vitamin B2 established in the United States is 1.1 mg and 1.3 mg, respectively for women and men, while the requirement established by the European Union is 1.6 mg per day (EU RDA).

There are basically two approaches for the production of riboflavin for the â € ̃exogenousâ € ™ fortification of foods: chemical and biotechnological. The chemical approach is based on the reaction of 3,4-xylidine with D-ribose in methanol (US4355158 A). The biotechnological approach mainly involves the use of microorganisms: Ashbya gossypii, Candida famata and Bacillus subtilis (Burgess et al., 2009. Bacterial vitamin B2, B11 and B12 overproduction: An overview. Int. J. Food Microbiol .133, 1-7). In the case of B. subtilis, high levels of riboflavin production were obtained following exposure of vitamin B2 producing strains to the toxic analogue of riboflavin: roseoflavin (EP0405370 B2). Exposure to this toxic analogue allows the selection of natural mutants of the producing strains, bearing point mutations on the regulatory element of the rib operon, which lead to the over-production of vitamin B2.

Lactic Acid Bacteria (LAB) are widely used as probiotics and as starter cultures for the production of fermented foods by virtue of their fermentation properties and QPS (Qualified Presumption of Safety, European Food Safety Authority) (EFSA) status. , 2012). There are strains of lactic bacteria that possess the molecular basis for the production of riboflavin (rib operon). Strains afferent to Lactobacillus plantarum species were obtained by exposure to roseoflavin (Burgess et al., 2006. A general method for selection of riboflavin-overproducing food grade micro-organisms. Microb. Cell Fact.5, 24; Capozzi et al. , 2011. Biotechnological production of vitamin B2-enriched bread and pasta. J. Agric. Food Chem. 59, 8013-20) and Leuconostoc mesenteroides (Burgess et al., 2006) capable of producing vitamin B2. The use of over-producing riboflavin lactic bacteria for food enrichment represents a convenient alternative to the chemical and biotechnological approaches described above. These strains of lactic bacteria, if compatible with the food production process, allow for enrichment in vitamin B2 in situ, ie directly in the food matrix.

There was therefore a great need to isolate new strains of lactic acid bacteria to be used for the enrichment of food or feed.

SUMMARY OF THE INVENTION

The inventors have succeeded in isolating a new bacterial strain belonging to the Lactobacillus plantarum (L. plantarum) species deposited at the CECT (Spanish Type Culture Collection) with the CECT filing number 8328 on 22 April 2013 from a durum wheat flour.

As shown by the experimental data reported here, this specific strain of Lactobacillus plantarum is characterized by a high capacity to produce riboflavin, about 3 times higher than the strains previously identified in the state of the art, and by a significant probiotic potential, which confers added value to the invention. It should in fact be emphasized that the strain of the present invention is the first of the Lactobacillus plantarum species which, at the same time, overproduces riboflavin and exhibits probiotic properties.

The high ability to produce riboflavin and the probiotic potential make it particularly useful in the riboflavin enrichment of food matrices, such as bread, pasta and yogurt.

The Lactobacillus plantarum species also has the status of QPS (Qualified Presumption of Safety, European Food Safety Authority) (EFSA, 2012) which indicates the safety of this microorganism in food. The strain object of the present invention is therefore particularly preferred for use as a probiotic producing vitamin B2 in the human and animal gastro-intestinal tract.

Therefore, the subject of the present invention is an isolated bacterial strain belonging to the Lactobacillus plantarum species, deposited at CECT, with the access number CECT 8328 and the compositions comprising it. Another object is their use in the treatment of gastrointestinal pathologies that present an alteration of the balance of the intestinal flora and / or associated with a deficiency of vitamin B2 and related compositions. A further object of the invention is a food or food supplement, comprising the bacterial strain described above and procedures for their preparation.

The advantages, characteristics and methods of use of the present invention will become evident from the following detailed description of some embodiments, presented by way of non-limiting example.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Concentration of riboflavin in chemically defined uninoculated medium (thesis 1), inoculated with Lactobacillus plantarum WCFS1 (non-riboflavin producing strain) (thesis 2), inoculated with the L. plantarum strain of the present invention (thesis 3). The reported values are means and standard deviation of a repeated experiment with 3 different independent cultures for each bacterial strain, and with duplicate repetition of the measurements.

Figure 2 reports the survival of Lactobacillus plantarum WCFS1 and the L. plantarum strain of the present invention in an in vitro system mimicking the conditions of the human gastrointestinal tract. The matrix is represented by physiological solution. The in vitro system has been reproduced in accordance with what described by Bove, et al. 2012 (Probiotic features of Lactobacillus plantarum mutant strains. Appl. Microbiol. Biotechnol. 96, 431â € “441). The reported values are means and standard deviation of a repeated experiment with 3 different independent cultures for each bacterial strain, and with duplicate repetition of the measurements.

Figure 3 reports the survival of L. plantarum WCFS1 and the L. plantarum strain of the present invention in an in vitro system mimicking the conditions of the human gastrointestinal tract. The matrix is represented by yogurt. The in vitro system has been reproduced in accordance with what described by Bove et al. (2012). The reported values are means and standard deviation of a repeated experiment with 3 different independent cultures for each bacterial strain, and with duplicate repetition of the measurements.

Figure 4 reports the survival of Lactobacillus plantarum WCFS1 and the L. plantarum strain of the present invention in an in vitro system mimicking the conditions of the human gastrointestinal tract. The matrix is represented by milk. The in vitro system has been reproduced in accordance with what described by Bove et al. (2012). The reported values are means and standard deviation of a repeated experiment with 3 different independent cultures for each bacterial strain, and with duplicate repetition of the measurements.

In Figure 5, adhesion capacity of L. plantarum WCFS1 and the L. plantarum strain of the present invention in vitro on colon adenocarcinoma cell lines (Caco-2). The method has been reproduced in accordance with what described by Bove et al. (2012). The reported values are means and standard deviation of a repeated experiment with 3 different independent cultures for each bacterial strain, and with duplicate repetition of the measurements.

In Figure 6, a measure of the inhibitory / antagonist power (radius of the inhibition zone) of L. plantarum WCFS1 and of the L. plantarum strain of the present invention on a panel of three pathogenic microorganisms (Listeria monocytogenes, Escherichia coli O157: H7 and Salmonella enterica). The method has been reproduced in accordance with what described by Gaudana et al. 2010 (Probiotic attributes of Lactobacillus strains isolated from food and of human origin. Br. J. Nutr. 103, 1620â € “1628). The reported values are means and standard deviation of a repeated experiment with 3 different independent cultures for each bacterial strain, and with duplicate repetition of the measurements.

In Figure 7, relative variation of adhesion on colon adenocarcinoma cell lines (Caco-2) by E. coli O157: H7, when placed in antagonism with L. plantarum WCFS1 and with the L. plantarum strain of the present invention . The method has been reproduced in accordance with what described by Koo et al. 2012 (Recombinant probiotic expressing Listeria adhesion protein attenuates Listeria monocytogenes virulence in vitro. PLoS ONE 7, e29277). The reported values are means and standard deviation of a repeated experiment with 3 different independent cultures for each bacterial strain, and with duplicate repetition of the measurements.

In Figure 8, relative variation of adhesion on colon adenocarcinoma cell lines (Caco-2) by E. coli O157: H7, when placed in antagonism with L. plantarum WCFS1 and with the L. plantarum strain of the present invention . The method has been reproduced in accordance with what described by Koo et al. (2012). The reported values are means and standard deviation of a repeated experiment with 3 different independent cultures for each bacterial strain, and with duplicate repetition of the measurements.

Figure 9. Concentration of riboflavin in acid slurry produced starting from uninoculated slurry (thesis 1), from slurry inoculated with Lactobacillus plantarum WCFS1 (strain not producing riboflavin) (thesis 2), from slurry inoculated with the L. plantarum strain of the present invention (thesis 3). Values reported are means and standard deviation of experimental duplicates.

Figure 10. Concentration of riboflavin in bread produced starting from uninoculated dough (thesis 1), from a mixture inoculated with Lactobacillus plantarum WCFS1 (strain not producing riboflavin) (thesis 2), from a mixture inoculated with the L. plantarum strain of present invention (thesis 3). Values reported are means and standard deviation of experimental duplicates.

Figure 11. Concentration of riboflavin in paste produced without the addition of lactic bacteria (thesis 1), ii) with the addition of Lactobacillus plantarum WCFS1 (thesis 2), iii) with the addition of the L. plantarum strain of the present invention (thesis 3). Values reported are means and standard deviation of experimental duplicates.

Figure 12. Concentration of riboflavin in pizza produced starting from uninoculated dough (thesis 1), from dough inoculated with Lactobacillus plantarum WCFS1 (strain not producing riboflavin) (thesis 2), from dough inoculated with the L. plantarum strain of present invention (thesis 3). Values reported are means and standard deviation of experimental duplicates.

Figure 13. Concentration of riboflavin in yogurt produced from uninoculated milk (thesis 1), milk inoculated with Lactobacillus plantarum WCFS1 (non-riboflavin producing strain) (thesis 2), milk inoculated with the L. plantarum strain of the present invention (thesis 3). Values reported are means and standard deviation of experimental duplicates.

In the figures shown (fig. 1-8) the strain of L. plantarum object of the present invention is indicated as L. plantarum B2, in figures 9-13 it is indicated as thesis 3.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a new bacterial strain belonging to the Lactobacillus plantarum (L. plantarum) species characterized by a high capacity to produce riboflavin and by a high probiotic activity.

The bacterial strain according to the present description can be used in any form, for example in suspension, freeze-dried, encapsulated, frozen. Freeze-drying and freezing are intended as a means of temporary inactivation of bacteria, which does not prevent the restoration of cell viability. The bacteria in the aforesaid forms can be prepared in any of the ways known to the person skilled in the art detailed in the laboratory manuals. Other forms of temporary bacterial inactivation, other than lyophilization and freezing, are included in the embodiments of the invention.

In recent years, the alteration of the balance of the intestinal bacterial flora has been associated with the onset or even the presence of various intestinal and extra intestinal pathologies. Therefore, the probiotic strain described here can be validly used for the prevention and / or treatment of gastrointestinal pathologies that show an alteration of the balance of the intestinal flora, or of gastrointestinal pathologies presenting such alteration among the most evident symptoms. By way of non-limiting example, these diseases / disorders include: diverticulosis, SIBO (bacterial overgrowth in the small intestine), IBD (inflammatory bowel disease), IBS (irritable bowel syndrome), diarrhea (for example diarrhea of the traveler), various dyspeptic forms, intestinal swelling, meteorism, diarrhea caused by taking antibiotics, food intolerances, alterations in the defenses of the immune system. The strain of the present invention, given its high production of vitamin B2, can also be used profitably in the prevention and / or for the treatment of gastrointestinal pathologies associated with a deficiency of vitamin B2.

Also subject of the present invention are compositions comprising the bacterial strain described here (CECT 8328). Such compositions may be for example in the form of solution, suspension, emulsion, capsule, tablet, powder, granulate and contain one or more excipients such as for example binders, diluents, absorbents, etc. According to one embodiment, such compositions are pharmaceutical compositions which will therefore comprise pharmaceutical grade carriers and / or excipients. These pharmaceutical compositions will preferably be for use in the treatment and / or prevention of gastrointestinal pathologies which show an alteration of the balance of the intestinal flora, or of gastrointestinal pathologies presenting such alteration among the most evident symptoms.

In one embodiment, the compositions may be a food or dietary supplement comprising the bacterial strain described here (CECT 8328) in any of the above forms. The quantity of bacteria to be included in the food can be any value deemed suitable by the technician in the sector, for example a quantity between 10 <7> and 10 <11> CFU (colony forming units) per gram of food / matrix-vehicle.

The foods or food supplements may include one or more suitable food ingredients and / or preservatives and / or acidifiers and / or antioxidants and / or immunostimulants and / or dyes and / or further bacterial strains, preferably probiotics and / or yeasts, preferably belonging to the Saccharomyces cerevisae species. The food or dietary supplement can conveniently be, by way of non-limiting example, in the form of liquid, thickened liquid, semi-solid, lyophilisate, powder, granules, emulsion, pasta, pizza, dough , suspension, pulp, chewable bars, yoghurt, cheese, fruit juice or compote, beverage, dairy food product, gelatine, syrup, elixir, milk, condensed milk, milk powder, flavored powder, artificial milk.

In the present description, the term food refers to both a food for humans and animals, therefore a feed for animals such as, for example, cattle, pigs, horses, sheep, primates, canids, felines, goats, rodents or other mammals from breeding in general.

The preparation of foods and supplements can be carried out, for example, by lyophilization of bacterial cultures of the strain described above, by mixing lyophilized or suspended cultures with water or with other suitable excipients such as preservatives, flavorings, dyes. These can be grown directly or added at any stage of food preparation.

The object of the present invention is therefore also a process for the preparation of a food or dietary supplement, for example according to any of the forms described above, comprising the step of adding the strain belonging to the Lactobacillus plantarum species deposited with the CECT access number 8328 in any of the stages of preparation of said food or dietary supplement. Preferably, the amount of strain added is between 10 <7> and 10 <11> CFU per gram. The procedure may also include the addition of additional bacterial strains such as a single strain or more prebiotic strains belonging to the species Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus delbrueckii, Lactobacillus helveticus, Lactobacillus plantarum, Lacillus plantarum, Lacillus plantar Enterococcus faecium, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum and / or yeasts such as Saccharomyces cerevisae.

In one embodiment of the process, the riboflavin overproducing bacterial strain object of the present invention can therefore be advantageously mixed with other strains. These mixtures may also contain yeasts (eg S. cerevisiae), depending on the composition of the microbial starters used for the specific food production. The inoculation of the food matrix can take place for example with a concentration of viable microorganisms ranging from about 10 <7> CFU / g (colony forming units per gram) to around 10 <11> CFU / g.

According to an embodiment, in the case of unfermented foods, the process may include a â € ̃pre-fermentationâ € ™ step in which all or part of the raw materials are inoculated with the over-producing microorganisms. The duration of the pre-fermentation step is variable and can, for example, last from a few minutes to about 16 hours, depending on compatibility with the technological process of food production. In the case of fermented foods, the bacterial biomass is added as a microbial starter, or to supplement the microbial initiator used for the specific production.

The foods (or feeds) and food supplements of the present disclosure will be foods enriched in riboflavin and capable of acting as probiotic foods in the human or animal gastrointestinal tract.

Examples are reported below which have the purpose of better illustrating the present invention and some specific embodiments, these examples are in no way to be considered as a limitation of the previous description and subsequent claims.

EXAMPLES

Example 1

1.1 Isolation and selection of the Lactobacillus plantarum strain (CECT 8328)

The strain of Lactobacillus plantarum (CECT 8328) was isolated starting from a commercial durum wheat flour supplied by the “Azienda Agricola Vito Roberto” (via Nazionale 9, 83030 Savignano Irpino, AV). From the semolina an acid mixture was obtained by mixing with water and periodic â € ̃rinfreschiâ € ™ for 5 days. A sample of this acid slurry was subjected to decimal dilutions in sterile physiological solution (NaCl, 8.5 g / L). The different dilutions were plated in de De Man, Rogosa et Sharpe (as described in De Man et al., 1960) a culture medium suitable for lactobacilli. The Lactobacillus plantarum strain was isolated from one of the plates.

1.2 Protocol for the selection of riboflavin-producing lactic acid bacteria

To evaluate the ability of lactic acid bacteria to produce riboflavin, the chemically defined medium described by Terrade et al. Was used. 2009 (A new chemically defined medium for wine lactic acid bacteria. Food Res. Int. 42, 363-367; pag. 364),

modified by eliminating riboflavin and using D-glucose to replace D-

ribose (see Table 1).

Table 1 - Chemically defined medium described by Terrade et al. (2009) amended

eliminating riboflavin and using D-glucose to replace D-ribose.

Compounds Concentration (g / l) Glucose Carbohydrates 10

4-Aminobenzoic 1

Biotin 2

Choline chloride 2

Cyanocobalamin 1

Folic Acid 2

Vitamins Nicotinic Acid 2

Ca-D-pantotein 2

Pyridoxine HCl 2

Riboflavin - Thiamine - HCl 1

L-Alanine 0.2

L-Arginine 0.75

L-Asparagine 0.15

L-Aspartic Acid 0.35

L-Cysteine 0.2

L-Glutamic acid 0.5

L-Glutamine 0.2

L-Glycine 0.5

Amino acids L-histidine 0.5

L-Isoleucine 0.2

L-Leucine 0.2

L-Lysine x 2 HCl 0.25

L-Methionine 0.15

L-Phenylanine 0.2

L-Proline 0.5

L-Serine 0.4

L-Threonine 0.35

L-Tryptophan 0.2

L-Tyrosine 0.2

L-Valine 0.2

CuSO4x 5H2O 1.50

Salts FeSO4x 7H2O 2

ZnSO4x 7H2O 1.35

MnSO4x 4H2O 0.10

MgSO4x 7H2O 0.10

K2HPO41

CaCl20.44

1.3 Protocol for the selection of natural mutants on roseoflavin

The strain of Lactobacillus plantarum (CECT 8328) selected as being able to grow in a chemically defined medium free of riboflavin, has been subjected to treatment with a toxic analogue of riboflavin, roseoflavin. The strain was exposed to increasing concentrations of roseoflavin, according to the method reported by Burgess et al. (2006). The bacteria were exposed to four different concentrations of roseoflavin (10 mg / L, 50 mg / L, 100 mg / L and 200 mg / L). After exposure to the highest concentration of roseoflavin, a plate smear of the bacterial culture was performed. After a 48 hour incubation period, ten randomly collected colonies were isolated and inoculated in chemically defined medium free of riboflavin. Riboflavin was extracted from exponential-phase cultures by acid-enzymatic hydrolysis and subsequently quantified by high-performance liquid chromatography. Among the isolates analyzed, the strain of L. plantarum called B2 was selected, which showed the highest production of riboflavin.

Example 2: over-production of riboflavin

To demonstrate the efficacy of the Lactobacillus plantarum strain (CECT 8328) in the enrichment of chemically defined medium in riboflavin (formulation in accordance with the work of Terrade et al., 2009, modified by eliminating riboflavin and using D-glucose in substitution of D-ribose) three theses were set up: uninoculated medium (thesis 1), medium inoculated with Lactobacillus plantarum WCFS1 (strain not producing riboflavin) (thesis 2), medium inoculated with Lactobacillus plantarum B2 (thesis 3). The extraction of riboflavin and the HPLC analyzes, for the relative quantization of the vitamin, were carried out in accordance with the method of Jakobsen et al., 2008 (Optimization of the determination of thiamin, 2- (1 hydroxyethyl) thiamin, and riboflavin in food samples by use of HPLC. Food Chem. 106, 1209-1217). The results are shown in figure 1.

Below is a comparative table showing the higher riboflavin production of the strain of the present invention, compared to those related to previous research.

Comparative tables

ID. Strain Riboflavin production mg / L * L. plantarum WCSF1 0.000

L. plantarum B2 (CECT 8328) 1,596

L. plantarum CB301 (Burgess et al., 2006) 0,600 (approximately)

L. plantarum CB305 (Burgess et al., 2006) 0,600 (approximately)

L. plantarum UNIFGPL104 (Capozzi et al., 2011) 0,642

L. plantarum UNIFGPL 209 (Capozzi et al., 2011) 0.586

Example 3

Probiotic properties of the Lactobacillus plantarum strain (CECT 8328)

The invention envisages the possible use of Lactobacillus plantarum B2 (CECT 8328) as a probiotic that produces vitamin B2 in the gastrointestinal tract.

The experiments to which figs. 2-8 help to outline the probiotic potential of the strain in question. The different experiments conducted in vitro, in fact, study various properties that are generally evaluated in a potential probiotic microorganism. Specifically, the following are considered: the ability to survive the gastro-intestinal environment and therefore reach the body district where probiotic activity is assumed to be maximum viable and with a significant degree (Fig. 2,3,4); the adhesive capacity of microbial cells on human intestinal epithelium reconstituted in vitro, which in turn is indicative of the colonizing capacity of the probiotic in the target body district (greater adhesion greater â € œtechmentâ € prolonged probiotic effect) (fig. 5); the ability to counteract the growth / proliferation of pathogenic microorganisms specific to the gastrointestinal tract (fig. 6) and to hinder their adhesion (and therefore their development / rooting within the target organ) on the epithelium human intestinal reconstituted in vitro (fig. 7-8).

Figures 2-4

The in vitro systems that simulate the human oro-gastro-intestinal tract (OGI) are widely used in the pre-screening phases of potential probiotics, as they test the ability of the microorganism to resist particularly adverse chemical-physical conditions (e.g. high acidity , digestive action of enzymes, emulsifying activity of bile, etc.) which it encounters when it is taken in the form of food and which can undermine its vitality inside the host. By estimating the viability of the ingested probiotic (with food), the OGI systems therefore provide useful information about its ability to reach vital (quantification of the vital title capable of reaching the target site) the digestive tract sector of maximum / effective probiotic activity: the middle-distal part of the intestine (ileum and colon). Development and use of such in vitro systems are encouraged by international bodies dealing with nutrition and health (FAO / WHO); moreover, in some cases, the validity of these systems, compared to the situation found in vivo, has been directly confirmed by clinical-scientific studies, as in the case of the L. plantarum species. The data shown in fig. 2-4 compare the survival capacity of L. plantarum WCFS1 to L. plantarum B2, conveyed by simple saline solution, or by food matrices commonly used for the formulation of probiotic food products (yogurt and milk). The data show that the L. plantarum B2 strain is able to withstand OGI stresses, including the most extreme ones related to the highly acidic environment of the gastric tract, maintaining a viability and a titre comparable to that already observed, under similar conditions. , using similar OGI systems for other commercial probiotics. Starting from an initial microbial titer of about 10 <9> CFU per mL of vehicle matrix, the titer is always detectable. The survival of the L. plantarum B2 strain is comparable, and in some cases better, than that of the reference strain L. plantarum WCFS1. The figures also show the important effect of the vehicle matrix on microbial vitality: due to the complexity of its composition, its richness in fermentable sugars, proteins and lipids, it can both support growth and microbial metabolism, and help protect the probiotic from OGI stress.

Figure 5

The data shown in fig. 5 indicate that the adhesive capacities of L. plantarum B2 and L. plantarum WCFS1 on monolayers of human intestinal epithelial cells are comparable and comparable to those observed also for other commercial probiotic strains. An appreciable power of adhesion on human intestinal epithelium reconstituted in vitro is another characteristic sought and evaluated in the screening phases of potential probiotics, since on the basis of this it is possible to reasonably estimate the ability of the microorganism to colonize the intestine. of the host in vivo and therefore persist in it in order to prolong the beneficial effect (eg production of vitamins and long-chain fatty acids); moreover, a close association between microbial cells and host cells favors phenomena such as immunomodulation and antagonism towards potential pathogens.

Figures 6-7-8

The figures refer to the antagonistic effect that the L. plantarum B2 strain can have with respect to intestinal pathogens carried by food. This aspect is also currently evaluated for the characterization of potentially beneficial microorganisms and represents a valid selection criterion for possible probiotics. The ability to counteract the growth of pathogens may in fact underlie the therapeutic properties typical of probiotics such as the restoration of the normal gastro-intestinal microflora, the treatment and prevention of enteric dysbiosis.

The data shown in fig. 6 indicate that the L. plantarum B2 strain is substantially capable of inhibiting the in vitro growth of the two pathogenic strains considered. The radius of the growth inhibition halo on the plate, in fact, is a measure of the power to hinder the proliferation of the pathogen. It is probable that this effect is attributable to the production of diffusible factors with antimicrobial activity and / or to mechanisms of competition for nutritional resources.

Competitive adhesion experiments on monolayers of human intestinal epithelial cells, shown in Fig. 7-8, aim to clarify whether antagonism with the pathogen can also occur at the level of colonizing power on the intestinal epithelium. The antagonistic effect is observed in vitro both when the probiotic potential and the pathogen are co-incubated in contact with the epithelial monolayers (fig. 7), and when, and more markedly, the epithelial monolayer is first exposed to the probiotic and then treated with the pathogen (fig. 8). In the first case, the antagonism may depend on competition mechanisms for adhesion to the same receptors and binding sites present on the epithelial cells; in the second case, the rapid saturation of the adhesion sites by the probiotic, combined with the production of diffusible factors that limit the growth of the pathogen, can contribute to the net decrease of the colonizing power by the harmful strain. The data reported suggest that the L. plantarum B2 strain can significantly hinder the engraftment of pathogens even in vivo, at the level of the host mucosa.

Example 4

Production of sour dough

To demonstrate the efficacy of Lactobacillus plantarum B2 in the riboflavin enrichment of an acid slurry, we prepared three theses: uninoculated acid slurry (thesis 1), acid slurry inoculated with Lactobacillus plantarum WCFS1 (strain not producing riboflavin) (thesis 2 ), acid mixture inoculated with Lactobacillus plantarum B2 (thesis 3). The 'mother' sour dough was obtained by mixing 3 kg of soft wheat flour with 5 L of water, 30 g of table salt, 90 g of compressed brewer's yeast, i) without adding lactic bacteria (thesis 1), ii) with the addition of Lactobacillus plantarum WCFS1 (thesis 2), iii) with the addition of Lactobacillus plantarum B2 (thesis 3). Lactobacilli were added to the mixture to obtain a concentration equal to 2Ã — 10 <8> CFU / mL (colony forming units per milliliter). The dough was subjected to fermentation for 18 hours at 30 ° C. The biomass for inoculation was obtained by inoculating from a cryopreserved stock of the strain in purity, in a quantity of 1%, MRS medium (de De Man, Rogosa et Sharpe; De Man et al., 1960. A medium for the cultivation of lactobacilli. J. Appl. Microbiol. 23, 130-135), allowing the broth culture to grow at 30 ° C for 24 hours and subjecting the cells to two washes with physiological solution (7000 rpm for 15 min). Riboflavin extraction and HPLC analyzes were carried out in accordance with the Jakobsen method (2008).

Figure 9 reports the riboflavin content in the three experimental theses.

Example 5

Production of bread

To demonstrate the efficacy of Lactobacillus plantarum B2 in enriching bread with riboflavin, we set up, on a pilot plant, three theses: uninoculated dough (thesis 1), mixture inoculated with Lactobacillus plantarum WCFS1 (strain not producing riboflavin) (thesis 2), mixture inoculated with Lactobacillus plantarum B2 (thesis 3). The dough for bread making was obtained by mixing 2 kg of soft wheat flour with 400 mL of 3% compressed brewer's yeast solution, 400 mL of 6% sugar solution and 3% table salt. , dissolved animal fats in the ratio of 3%, water (determined following the evaluation of the farinographic absorption at 500 Brabender Units), i) without the addition of lactic bacteria (thesis 1), ii) with the addition of Lactobacillus plantarum WCFS1 (thesis 2), iii) with the addition of Lactobacillus plantarum B2 (thesis 3). Lactobacilli were added to the mixture to obtain a concentration equal to 2Ã — 10 <8> CFU / mL (colony forming units per milliliter). This was followed by dividing, first leavening (1 hour, 30 ° C), rolling, second leavening (35 min, 30 ° C), rolling, third leavening (45 min, 30 ° C), cooking in an electric oven at 220-230 ° C for 30 minutes. The biomass for inoculation was obtained by inoculating from a cryopreserved stock of the strain in purity, in a quantity of 1%, MRS medium (de De Man, Rogosa et Sharpe; De Man et al., 1960), leaving grow the broth culture at 30 ° C for 24 hours and subject the cells to two washes with physiological solution (7000 rpm for 15 min). Riboflavin extraction and HPLC analyzes were carried out in accordance with the Jakobsen method (2008).

Figure 10 reports the riboflavin content in the three experimental theses.

Example 6

Pasta production

To demonstrate the efficacy of Lactobacillus plantarum B2 in enriching pasta with riboflavin, we prepared three theses on a pilot plant. The re-milled durum wheat semolina was mixed with water until it reached a humidity of 16.5%, i) without the addition of lactic bacteria (thesis 1), ii) with the addition of Lactobacillus plantarum WCFS1 (thesis 2), iii) with the addition of Lactobacillus plantarum B2 (thesis 3). The contact time of the semolina with the microorganisms was 2 hours. The pasta making involved: kneading (about 10 min), extrusion (operating pressure between 9.1-12.1 MPa and vacuum of 700 torr), drying at low temperature (50 ° C for 18 h). The dough was processed into spaghetti with a diameter of 1.7 mm. Lactobacilli were added to the mixture to obtain a concentration equal to 2Ã — 10 <7> CFU / mL (colony forming units per milliliter). The biomass for the inoculation was obtained by inoculating from a cryopreserved stock of the strain in purity, in quantities of 1%, in MRS medium (de De Man, Rogosa et Sharpe; De Man et al., 1960 ), leaving the broth culture to grow at 30 ° C for 24 hours and subjecting the cells to two washes with physiological solution (7000 rpm for 15 min). Riboflavin extraction and HPLC analyzes were carried out in accordance with the Jakobsen method (2008).

Figure 11 reports the riboflavin content in the three experimental theses.

Example 7

To demonstrate the effectiveness of Lactobacillus plantarum B2 in the enrichment of pizza riboflavin we have prepared three theses: uninoculated acid mixture (thesis 1), acid mixture inoculated with Lactobacillus plantarum WCFS1 (strain not producing riboflavin) (thesis 2), acid mixture inoculated with Lactobacillus plantarum B2 (thesis 3). The dough was obtained by mixing 3 kg of soft wheat flour with 1.8 L of water, 60 g of table salt, 75 g of compressed brewer's yeast, 10 mL of extra virgin olive oil, i) without addition of lactic bacteria (thesis 1), ii) with the addition of Lactobacillus plantarum WCFS1 (thesis 2), iii) with the addition of Lactobacillus plantarum B2 (thesis 3). Lactobacilli were added to the mixture to obtain a concentration equal to 2Ã — 10 <8> CFU / mL (colony forming units per milliliter). The dough was subjected to fermentation for 2 hours at 30 ° C. The preparation and filling is followed by cooking in an electric oven at 220-230 ° C for 30 minutes. The biomass for the inoculation was obtained by inoculating from a cryopreserved stock of the strain in purity, in a quantity of 1%, MRS medium (de De Man, Rogosa et Sharpe; De Man et al., 1960) , allowing the broth culture to grow at 30 ° C for 24 hours and subjecting the cells to two washes with physiological solution (7000 rpm for 15 min). The riboflavin extraction and HPLC analyzes were carried out in accordance with the Jakobsen method (2008).

Figure 12 reports the riboflavin content in the three experimental theses.

Claims (13)

CLAIMS 1. Isolated bacterial strain belonging to the Lactobacillus plantarum species, deposited at the CECT (Spanish Type Culture Collection), with the access number CECT 8328. 2. Bacterial strain according to claim 1 frozen, lyophilized, encapsulated or in suspension. 3. Bacterial strain according to claim 1 or 2 for use in the treatment of gastrointestinal pathologies which present an alteration of the balance of the intestinal flora and / or associated with a deficiency of vitamin B2. 4. Composition comprising the bacterial strain according to claim 1 or 2. 5. Composition according to claim 4 in the form of solution, suspension, emulsion, capsule, tablet, powder, granulate. 6. Food or dietary supplement, comprising the bacterial strain according to claim 1 or 2 and one or more food ingredients and / or preservatives and / or acidifiers and / or antioxidants and / or immunostimulants and / or dyes and / or further bacterial strains and / or yeasts. 7. Food or dietary supplement according to claim 6 wherein the quantity of said strain belonging to the Lactobacillus plantarum species is comprised between 10 <7> and 10 <11> CFU per gram of composition. 8. Food or dietary supplement according to claim 6 or 7 in the form of liquid, thickened liquid, semi-solid, lyophilisate, powder, granules, emulsion, pasta, pizza, dough, suspension, pulp , chewable bars, yoghurt, cheese, juice, fruit compote, beverage, dairy food product, gelatine, syrup, elixir, milk, condensed milk, milk powder, flavored powder , of artificial milk. 9. Process for the preparation of a food or dietary supplement according to any one of claims 6 to 8 comprising the step of adding the strain belonging to the Lactobacillus plantarum species deposited with the access number CECT 8328 in any of the preparation stages of said food or dietary supplement. 10. Process according to claim 9 wherein said strain is added in an amount comprised between 10 <7> and 10 <11> CFU per gram. 11. Process according to claim 9 or 10, in which in said step of adding further bacterial strains and, or even one or more yeasts, preferably belonging to the Saccharomyces cerevisae species, are also used. 12. Process according to any one of claims 9 to 11 wherein said addition step precedes a fermentation step. 13. A composition comprising the bacterial strain according to claim 3 and one or more vehicles and pharmaceutically acceptable additives.