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FIELD OF THE INVENTION

The invention relates to a microbiological process for the production of a protein fraction with immuno-regulatory activity from cereal doughs.

Also described are a protein fraction obtainable by the process of the invention and its uses for the preparation of a nutraceutical and pharmaceutical composition for the treatment of coeliac disease.

STATE OF THE ART

Oral tolerance is a physiological condition characterized by the induction of a nonimmune response to food antigens and bacterial antigens belonging to the intestinal microbiota. Multiple cellular and molecular mechanisms are involved in the regulation of this fundamental property of the intestinal immune system. Many studies have been inspired by the knowledge of these events with the aim of stopping the beginning and/or progression of adverse immune conditions as evaluated in experimental models. The main obstacle to the use of such approaches is the lack of knowledge of the pathogenic antigens. Therefore, from this point of view, coeliac disease or celiac disease (CD), due to the fact that the pathogenic antigen (gluten) is known, represents a suitable model in order to establish prophylactic/therapeutic protocols capable of acting selectively on T cells with the aim of restoring a tolerance which is transferable also to other conditions. Having regard to the specific problem of CD, an approach of re-inducing tolerance to this antigen may provide an alternative treatment to the current gluten-free diet, currently the only available therapy.

From this point of view, different immunomodulatory strategies have been explored so far. The use of NexVax2 ^® , a desensitizing vaccine consisting of three dominant gluten peptides and administered by subcutaneous route, is current under investigation (ClinicalTrials.gov Identifier: NCT00879749).

With a completely different approach, the potential of cyclic or dimeric substituted analog peptides to be used as a blocker of the binding of native gliadin to HLA molecule was explored in vitro (Xia J et al, J Am Chem Soc 2006;128:1859; Huan J et al, Mucosal Immunol 201 1 ;4:1 12). In line with these results, we have recently found that enzymatically modified gliadin (transamidates) influences the immune response in the intestinal T cells (Gianfrani C et al, Gastroenterology 2007;133:780; patent WO 2008/053310) and in intestinal biopsies from coeliac patients (Lombardi E et al, J Leuk Biol 2013;93:479) drastically reducing the production of IFN-γ.

Taken together, these results suggest that masking of gliadin immunodominant epitopes can promote an alternative binding to HLA by low-affinity antigenic determinants, that is potentially capable of restoring oral tolerance. This concept can be extended also to other immune-based eating disorders such as food allergies.

So far, however, the only studies conducted to date in humans to prevent the onset of CD have made use of native and unmodified gluten molecules, and have obtained unsatisfactory results. In particular, a first intervention double-blind randomized study (PreventCD), in which children with genetic risk of developing the CD (HLA-DQ positive) (n = 944) received a small dose of gluten or a placebo per day in the age of 4-6 months, showed no differences between groups in the prevalence of CD at the age of 3 years (Vriezinga SL et al, N Engl J Med 2014; 371 ,1304-1315). The second study (CELIPREV) has shown that introduction of gluten at the age of 12 months (compared to 6 months) in infants genetically predisposed to develop CD (n = 832) can delay the onset of the disease, but does not change its incidence at the age of 5 (Lionetti E et al, N Engl J Med 2014;371 , 1295-1303).

Moreover, there are preparations of probiotic bacteria currently available on the market that have the aim of mitigating the symptoms associated with the CD, but they have no specific therapeutic effect.

The object of the present invention is therefore to provide a simple and economically advantageous method for the production of nutraceutical compositions for the treatment of immune and allergy based food intolerances, which do not present the disadvantages reported above for the known immunomodulatory molecules for the same application. SUMMARY OF THE INVENTION

The invention therefore relates to a process for the production of a soluble protein fraction (SPF) from cereal doughs, having the steps of:

a. culturing a S. mobarensis strain under conditions which allow aerobic growth, to obtain a bacterial medium containing specific bacterial metabolites;

b. centrifuging the bacterial medium containing specific bacterial metabolites of step a. to obtain a pellet and a supernatant;

c. collecting the supernatant of step b.;

d. contacting the supernatant of step c. with a dough of at least one cereal and with a lysine ethyl ester and incubating under conditions which allow formation of a suspension;

e. centrifuging the suspension of step d. thus obtaining a pellet and a supernatant; f. recovering the protein soluble fraction by precipitation of the supernatant of step e.

The invention also relates to a soluble protein fraction obtainable from cereal doughs according to the method comprising the steps of:

a. culturing a S. mobarensis strain under conditions which allow aerobic growth to obtain a bacterial medium containing specific bacterial metabolites;

b. centrifuging the bacterial medium containing specific bacterial metabolites of step a. to obtain a pellet and a supernatant;

c. collecting the supernatant of step b.;

d. contacting the supernatant of step c. with a dough of at least one cereal and with a lysine ethyl ester and incubating under conditions which allow formation of a suspension;

e. centrifuging the suspension of step d. thus obtaining a pellet and a supernatant; f. recovering the protein soluble fraction by precipitation of the supernatant of step e.

It is also an object of the invention the use of the soluble protein fraction for the preparation of a nutraceutical product.

The invention also relates to a soluble protein fraction obtainable from cereal doughs, for medical use. Under a further aspect, the invention relates to the use of the soluble protein fraction obtainable from cereal doughs, for the treatment of coeliac disease.

Under yet another aspect, the invention relates to a pharmaceutical composition comprising the soluble protein fraction obtainable from cereal doughs, and a pharmacologically acceptable carrier.

In a further embodiment, the present invention relates to a nutraceutical composition comprising the soluble protein fraction obtainable from cereal doughs and other food matrices.

These and other objects as well as features and advantages of the present invention will be better understood from the following detailed description.

DESCRIPTION OF THE DRAWINGS

The invention will now be described in detail and with reference to the accompanying Figures in which:

Figure 1 shows a graph including the ability evaluation results of different culture media, recovered after exhaustive culture with S. mobarensis (see table 1 ), to produce SPF from wheat flour and in comparison to high doses of microbial transglutaminase (imTG).

Figure 2. shows a graph including the results of various cereal flours incubation with culture medium E to analyze the ability to extract proteins from the dough, as described in Example 2.

Figure 3. Experiments of co-culture of murine dendritic cells presenting gliadin (Figures 3A and Figure 3B) or SPF (Figures 3C and Figure 3D) with murine splenic antigen-specific CD4+ T cells, as described in Example 3.

Figure 4 shows a graph including the results of the ability real time PCR assessment of SPF to induce transcription of imRNA for IL-10 in intestinal biopsies of coeliac patients with manifest disease, as described in Example 4.

DETAILED DESCRIPTION OF THE INVENTION

The invention therefore relates to a process for the production of a soluble protein fraction (SPF) from cereal doughs, having the steps of:

a. culturing a S. mobarensis strain under conditions which allow aerobic growth, to obtain a bacterial medium containing specific bacterial metabolites; b. centrifuging the bacterial medium containing specific bacterial metabolites of step a. to obtain a pellet and a supernatant;

c. collecting the supernatant of step b.;

d. contacting the supernatant of step c. with a dough of at least one cereal and with a lysine ethyl ester and incubating under conditions which allow formation of a suspension;

e. centrifuging the suspension of step d. thus obtaining a pellet and a supernatant; f. recovering the protein soluble fraction by precipitation of the supernatant of step e.

The present method has the advantage that a soluble protein fraction with nutraceutical activity can be produced with low cost directly from a culture of S. mobarensis.

In the present invention when using the term:

- "soluble protein fraction" or "SPF" it is meant to include the cereal protein fractions resulting from catalytic activity of the culture supernatant;

- "S.mobarensis", "S.mobaraensis" or " Streptomyces mobarensis" what is meant is a species of gram-positive aerobic bacteria, of the genus of food grade Streptomycetes used for the production of antibiotics (penicillins) and microbial transglutaminase (imTG); and

- "nutraceutical" what is meant is an active principle obtained from a natural food or following a treatment which has a beneficial function, as it combines nutritional components to healing properties. Nutraceuticals may be administered either in the form of food "as such" or as enriched food of a specific active principle, or also in the form of food supplements in liquid formulations, tablets or capsules.

In one embodiment, the process according to the present invention is a process in which aerobic culturing of step a. is carried out under stirring at a temperature in a range of 25 °C to 30 °C, preferably employing an Erlenmeyer flask for a time ranging from 24 to 120 hours.

In a preferred embodiment, the bacterial growth is carried out at 28 °C, at 250 rpm for 96 hours, using orbital shakers.

After 96 hours of culturing the bacterial mycelium in a specific culture medium, its supernatant is capable of extracting a soluble protein fraction (SPF) from cereal doughs (wheat, corn, barley) in the presence of lysine ethyl ester, with greater efficiency than the same purified imTG.

This culture medium comprises at least glucose, potassium hydrogen phosphate, magnesium sulfate heptahydrate, magnesium chloride and starch; preferably, also casein peptone, meat peptone, vegetable peptone, soy peptone and cow's milk serum.

In a preferred embodiment, the culture medium comprises at least 1 % glucose, 0.2% potassium hydrogen phosphate, 0.1 % magnesium sulfate heptahydrate, 0.1 M magnesium chloride and 1 % starch. In addition, 1 .5% casein peptone, 1 .5% meat peptone, 1 .5% vegetable peptone, 1 .5% soy peptone, and 1 1 % bovine milk serum can also be added to the culture medium.

In a more preferred embodiment, the culture medium comprises 1 % glucose, 0.2% potassium hydrogen phosphate, 0.1 % magnesium sulfate heptahydrate, 0.1 M magnesium chloride, 1 % starch and 1 .5% vegetable peptone. In addition to being optimal for a catalytic activity of the culture supernatant, this culture medium also has the benefit of being cost effective.

In one embodiment, the process according to the present invention is a process wherein the centrifuging step b. is carried out for 15 minutes at 10000 rpm at room temperature.

In a further embodiment, after step c. of the presend process, a phase c'. is added in which freezing of the supernatant of step c. is carried out.

The supernatants may in fact be frozen and stored, for example at - 20 °C, to be used subsequently.

To verify the formation of the suspension of step d. the insoluble component remaining from the misture is monitored.

In yet a further embodiment, the dough of at least one cereal of step d. of the present process is prepared by resuspension of a flour in a 0.4M NaCI solution, centrifugation, for example at 1000 rpm for 10 min and washing of the pellet thus obtained.

In a preferred embodiment, the cereal flour is resuspended in a 0.4M NaCI solution and stirred for 10 min. The suspension it then centrifuged and the pellet (dough) washed several times with water. The dough it then finely dispersed in the culture supernatant of S. mobarensis and incubated at 30 °C for 2 hours in the presence of a lysine ethyl ester having a concentration of 20 imM. The suspension is then centrifuged and the supernatant is collected. Finally, the soluble protein fraction (SPF) is recovered (pellets) by precipitation with 10% TCA (trichloroacetic acid), it is washed with anhydrous acetone, dialysed, dried and preserved.

Advantageously, in one preferred embodiment, the flour being used is selected from the group consisting of wheat flour, barley flour, maize, kamut, rye, spelled and oats flour and all the cereal flour and semolina toxic for coeliacs.

The invention also relates to a soluble protein fraction obtainable from cereal doughs according to the method comprising the steps of:

a. culturing a S. mobarensis strain under conditions which allow aerobic growth, to obtain a bacterial medium containing specific bacterial metabolites;

b. centrifuging the bacterial medium containing specific bacterial metabolites of step a. to obtain a pellet and a supernatant;

c. collecting the supernatant of step b.;

d. contacting the supernatant of step c. with a dough of at least one cereal and with a lysine ethyl ester and incubating under conditions which allow formation of a suspension;

e. centrifuging the suspension of step d. thus obtaining a pellet and a supernatant; f. recovering the protein soluble fraction by precipitation of the supernatant of step e.

This protein fraction has the advantage of stimulating the production of the regulatory cytokine IL-10 in antigen-specific CD4+ T cells in the presence of dendritic cells (DCs).

Surprisingly, it has been seen that in biopsies from coeliac patients, the soluble protein fraction (SPF) induces an increased transcription of IL-10 compared to the stimulatory protein (gliadin).

The result so surprisingly obtained allows restoration, through exposure to SPF fraction, of a normal immune regulatory response state with respect to the gluten component, characterized by an increase in cytokine IL-10, eliminating the inflammatory response that is associated with the pathogenesis of CD. This result is of particular value as obtained not only in the animal model of CD but also in intestinal biopsies of coeliac patients.

It is also an object of the invention the use of the soluble protein fraction for the preparation of a nutraceutical product.

The nutraceutical product according to the present invention has antigen-specific immuno-regulatory properies, useful in immune based food intolerances and allergies.

This nutraceutical product, useful in the field of immune and allergy based food intolerances, is advantageously prepared by a methodology according to the present invention, which keeps costs low and gives a high product yield.

Advantageously, the soluble protein fraction fits well in the current international context of nutraceuticals, which is directed in particular to the prevention/mitigation/treatment theme of food immune based intolerance and allergies, as this is a product made from food grade material, which can have both a nutritional added value due to the presence of lysine, and a functional value due to the previously highlighted regulatory properties of the antigen(gluten)-specific immune function.

The invention also relates to a soluble protein fraction obtainable from cereal doughs, for medical use.

The immunological data obtained experimentally identify the potential of SPF of the present invention for inducing an immune-regulatory phenotype in the response of antigen-specific T cells, even with an inflammatory state in progress as, for example, that induced by a normal diet in patients with coeliac disease. Under a further aspect, the invention relates to the use of the soluble protein fraction obtainable from cereal doughs, for the treatment of coeliac disease.

The protein fraction according to the present invention has antigen-specific immuno-regulatory properties, useful for preventing, improving or treating food allergies and intolerances having an immune basis, such as the coeliac disease or coeliac disease.

Under yet another aspect, the invention relates to a pharmaceutical composition comprising the soluble protein fraction obtainable from cereal doughs, and a pharmacologically acceptable carrier. In a further embodiment, the present invention relates to a nutraceutical composition comprising the soluble protein fraction obtainable from cereal doughs and other food matrices. Such nutraceutical composition may comprise the individual protein components of the soluble fraction. The use of a single well- characterized component may bring benefits in particular for the pharmacological application (eg. a better definition of the pharmacokinetics of the product, and therefore of the dosage, etc.).

Below are reported the exemplary embodiments of the present invention provided by way of illustrative examples.

EXAMPLES

Example 1 : Preparation of the S. mobarensis culture supernatant

Various culture media have been used for the growth of bacteria of Streptomyces mobarensis, indicated with letter A to G, depending on the components, in Table

1 .

Table 1 :

A 100 ml culture was obtained using a 250 ml Erienmeyer flask in orbital shaker at 28 °C, at 250 rpm, with the fermentation time in the range of 24h-120h, preferably 96 hours. After fermentation, the cultures were centrifuged for 15 minutes at 10000 rpm at room temperature.

After centrifugation the supernatants were collected, which could be used immediately or frozen at -20 °C.

Example 2: preparation of the soluble protein fraction (SPF) from wheat flour

10 g of cereal flour (for example wheat, corn or barley flour) is resuspended in 80 ml 0.4 M NaCI and stirred for 10 min. The suspension it then centrifuged and the pellet (dough) washed several times with water. The dough is then finely dispersed in 20 ml of S. mobarensis culture supernatant (obtained as described in Example 1 ) and incubated at 30 °C for 2h in the presence of 20 imM lysine ethyl ester (having a molecular formula C8H18N2O2). The suspension is then centrifuged and the supernatant is collected. Finally, the soluble protein fraction (SPF) is recovered by precipitation with 10% TCA of the supernatant obtained by centrifugation of the suspension. The precipitated soluble protein fraction is washed with anhydrous acetone, subjected to exhaustive dialysis, finally dried in a lyophilizer and stored at -20 °C.

The residual insoluble protein fraction is analyzed by extraction of the dough in 70% ethanol at 60 °C for 30 minutes followed by Bradford colorimetric analysis. Results:

Incubation of various S. mobarensis culture supernatants decreased in a differentiated way the recovery of prolamine from wheat flour, as shown in the data of Figure 1 . In particular, media A and E have given the highest productions of SPF, fully comparable to the yield obtained in parallel with 10U of purified imTG. The cheapest medium E was then selected for subsequent studies. The medium "E" was capable of extracting SPF with similar levels also from other cereal flours (Figure 2).

Example 3: In vitro tests

Splenocytes were isolated from mice parenterally immunized with the antigen (gliadin or SPF). CD4+ T cells were then purified by immuno-magnetic sorting based on the manufacturer's instructions. Antigen-specific CD4+ T cells were co- cultured with syngeneic dendritic cells developed from bone marrow (DC), according to a previously established protocol, pre-incubated with the specific antigen (cell T:DC ratio, 2:1 ) After 72 h, the supernatants were collected and analyzed for levels of IFN-γ and IL-10 by ELISA.

Results:

The immune properties of SPF produced from wheat flour have been specifically analysed in experiments of co-culture with dendritic cells from mice and gliadin- specific CD4+ T cells. An interesting result was that the gliadin-specific CD4+ T cells are able to produce IFN-γ even when stimulated with SPF containing DCs. Surprisingly, it was found that the gliadin-containing DCs induce significant secretion of IL-10 in SPF-specific CD4+ T cells (Figure 3, p = 0.006).

Example 4: Ex vivo test

Intestinal biopsies isolated from ten free-diet coeliac patients (age range 12-51 years; mean age, 24 years) were grown for 8 hours in the presence of a gliadin peptic-tryptic digest or SPF and analyzed for IFN-γ imRNA levels and IL-10 by real time PCR.

Results:

The ability of SPF to induce IL-10 in vitro in intestinal biopsies of coeliac patients with established disease (CD1 -10; age range 12-51 years; mean age, 24 years; Marsh degrees range: II- lllc) was also tested. Following a challenge with SPF, biopsies of five patients (CD2, -5, -6, -8 and -9) were found reactive; In these patients SPF was able to induce an increased transcription of the gene for IL-10 compared to the control (gliadin; Figure 4, p <0.05)

From the detailed description and from the Examples given above, the advantages achieved by the method of the present invention become apparent. In particular, this method has surprisingly and advantageously proved to be suitable for the production of a soluble protein fraction with nutraceutical properties, characterized by a re-induction of an antigen-specific immune regulatory response. At the same time, since this method is fast and easy to perform, it is also convenient from the economic point of view.