SOURDOUGH AND COMPOSITION THEREOF FOR PREPARING PLANT-BASED FOOD

PRODUCTS Technical field

[01] The present invention relates to a sourdough and a composition thereof for preparing plant-based food products, in particular a sourdough and a composition thereof suitable for decreasing the gluten content in plant-based food products.

Prior art

[02] The need to produce foods with a low gluten content has long been known, as celiac disease is now considered the most frequent food intolerance and affects about 1 % of the world population.

[03] Furthermore, recent studies carried out by the University of Copenhagen have shown that a diet low in gluten and high in vegetable fibers induces changes in the structure and function of the intestinal bacterial ecosystem, reducing the production of hydrogen and decreasing intestinal bloating.

[04] These reasons push an ever-growing audience of non-gluten intolerant consumers to choose a low-gluten diet, despite the fact that there are no medical needs related to this choice.

[05] Therefore, there are countless foods with high digestibility and low gluten content on the market, able to satisfy the nutritional needs of both the public subject to celiac disease and the even wider public who are looking for a light diet that promotes digestive functions.

[06] These foods include naturally gluten-free cereal-based foods such as rice, corn, buckwheat, millet, amaranth, quinoa, cassava, teff, sorghum.

[07] The need, in particular by consumers who are more attentive to their diet, to reduce the daily consumption of meat, considered potentially harmful both at an environmental and ecological level and for the health of the person is also known.

[08] Therefore, some types of meat substitute foods have been developed, capable of maintaining taste and consistency similar to those of meat, in order to preserve the taste of a varied diet in the consumer, simultaneously reducing health and environmental risks associated with meat consumption.

[09] The meat substitute food products currently on the market are products based on proteins derived from legumes such as soy or grasses such as wheat.

[10] In particular, wheat-based meat substitute food products generally consist of a gluten dough. A typical example is seitan, a product of oriental origin that is obtained by extracting gluten from wheat flour and then boiling the mixture obtained in water containing soy sauce, kombu seaweed and other flavors. [11] With the increased awareness of the potentially negative effects of meat consumption and the spread of vegetarian, vegan and macrobiotic diets, the demand for meat replacement products has increased exponentially in the last decade.

[12] The market, thus far, is plenty of gluten-based food products as an alternative to seitan.

[13] The patent EP 0 821 882 B1 in the name of Green Live Vegetable Meat S.r.l. discloses a method for producing a plant-based meat substitute product consisting of a mixture of gluten and preferably organic, cereal and/or legume flours, which is amalgamated with vegetable broth or water, possibly flavored and spiced, kneaded, molded and then boiled. The final product can be commercialized as a substitute for steaks, stews, sausages, minced meat, hamburgers, ham, bresaola, mortadella, pizzaiola and bacon. In particular, the mixture described by the patent consists of a percentage by weight between 45% and 99% of gluten and between 0.1% and 55% of a vegetable flour derived from cereals and/or legumes.

[14] The meat substitutes on the market generally have the disadvantage of containing a high percentage by weight of gluten, a protein complex that is hydrolyzed into peptides by the transglutaminase enzyme during digestion in the intestinal tract.

[15] A problem encountered in the use of these products concerns people predisposed to celiac disease, who can develop anti-tranglutaminase antibodies causing an inflammatory process that leads the T lymphocytes to "attack" the intestinal villous cells.

[16] In particular, gliadin, a protein present in gluten, activated by the transglutaminase enzyme, activates the T lymphocytes present in the lamina propria of the intestinal mucosa, which migrate to the subepithelial site and produce cytokines causing apoptosis of the intestinal villous cells.

[17] The apoptosis of the villous cells causes a flattening of the intestinal mucosa which leads to intestinal malabsorption with consequent pathological changes in the health of the person concerned.

[18] Therefore, the consumption of gluten-based food substitutes for meat is highly not recommended for people predisposed to celiac disease, which is currently considered the most frequent food intolerance affecting about 1% of the population.

[19] Furthermore, the consumption of such products is poorly sought after by the consumers pursuing a more digestible low-gluten diet.

[20] Patent application WO 2006/097949 A1 in the name of Actial Farmaceutica Lda et Al. describes a sourdough and a leavening composition comprising a mixture composed of at least six species of lactic bacteria and/or Bifidobacteria. The sourdough or leavening composition is used in the preparation of a baked product, which in turn includes the probiotic mixture present in the sourdough or leavening composition. The document also describes the process for preparing the baked product, which includes adding the sourdough or leavening composition to the other ingredients.

Disclosure

[21] The aim of the present invention is to solve the aforementioned problems, devising a sourdough and a composition thereof for preparing plant-based food products capable of carrying out proteolytic activity on gluten proteins and decreasing the gluten content of the finished product.

[22] Within this aim, a further object of the present invention is a sourdough and a composition thereof for preparing plant-based food products capable of operating synergistically to favor the fermentation processes inside the sourdough, enhancing the proteolytic activity on gluten proteins.

[23] A still further object of the present invention is to provide a sourdough and a composition thereof for preparing plant-based food products capable of degrading the allergenic fragments of gluten proteins, achieving a significant qualitative and quantitative reduction of these fragments.

[24] A further object of the present invention is to provide a sourdough and a composition thereof for preparing plant-based food products capable of giving the finished food product a taste, consistency and aroma similar to those of meat.

[25] A still further object of the present invention is to provide a sourdough and a composition thereof for preparing plant-based food products capable of giving the finished food product a consistency such that the sliceability of the product is improved maintaining the integrity of the product in the packaging step.

[26] A further object of the present invention is to provide a sourdough and a composition thereof for preparing plant-based food products capable of improving the shelf life of the food by increasing the shelf-life of the finished product.

[27] A further object of the present invention is to provide a plant-based food product with a reduced gluten content.

[28] A further object of the invention is to provide a plant-based food product capable of replacing meat and its derivatives, which can be digestible and not harmful to consumers with a predisposition to celiac disease.

[29] Another object of the invention is to provide an entirely vegetable-based food product capable of replacing meat and its derivatives while allowing the consumer a varied diet.

[30] A still further object of the invention is to provide a plant-based food product containing exclusively plant-based proteins.

[31] An even further object of the invention is to provide a plant-based food product with high nutritional properties and of high quality, but relatively low cost.

[32] The aforementioned objects are achieved, according to the present invention, by the sourdough for preparing plant-based food products of claim 1; by the composition for formulating a sourdough according to claim 4; by the plant-based food product according to claim 9; by the process for producing plant-based food products with low gluten content of claim 13 and by the use of a sourdough for preparing plant-based food products of claim 15.

[33] The sourdough for preparing plant-based food products object of the invention is composed of a mixture of flour and water acidified by a complex of lactic bacteria and yeast capable of starting a natural fermentation.

[34] Advantageously, the fraction of said lactic bacteria contained within said sourdough is composed of lactic bacteria of the Lactobacillus plantarum species, while the fraction of said yeasts contained within said sourdough consists of yeasts of the Saccharomyces cerevisiae species.

[35] Advantageously, the basic dough of said sourdough consists of a mixture of water and flours selected from gluten, cereal and legume flour.

[36] Preferably, the amount of water required to obtain said basic dough of said sourdough varies from 300g to 500g per an overall 1Kg of finished product.

[37] Advantageously, said sourdough, object of the invention, has shown a particular ability to digest gluten proteins.

[38] A composition for formulating said sourdough suitable for decreasing the gluten content in plant-based food products comprises at least one strain of Lactobacillus plantarum and at least one strain of Saccharomyces cerevisiae.

[39] Said at least one strain of Lactobacillus plantarum has shown a particular proteolytic capacity of gluten proteins, in particular in the degradation of allergenic fragments of said proteins, leading to a significant qualitative and quantitative reduction of these fragments.

[40] The proteolytic activity of said at least one strain of Lactobacillus plantarum is promoted by the presence within the composition of said at least one strain of Saccharomyces cerevisiae, which assists the fermentation processes of the bacterium by hydrolyzing the carbohydrates present in the substrate into simple molecules such as glucose and fructose, more usable by the bacterium, consequently increasing its cellular yield and enhancing its proteolytic activity.

[41] Said sourdough is used for formulating and producing plant-based food products, in particular for plant products with a low gluten content.

[42] Advantageously, said plant-based food products added with said sourdough show a low gluten content and high digestibility even for those predisposed to celiac disease.

[43] Preferably, said plant-based food products added with said sourdough are composed of gluten in a percentage by weight of from 20% to 40%.

[44] Preferably, said plant-based food products added with said sourdough are composed of at least one type of cereal flour in a percentage by weight from 5% to 15% and/or at least one type of legume flour in a percentage by weight of 10 % to 30%, or with a mixture of at least one type of cereal flour and at least one type of legume flour in a percentage by weight from 10% to 30%.

[45] Advantageously, said cereal flours are selected from hard wheat flours and soft wheat flours.

[46] Said cereal flours can be bran, wholemeal, fine, extra-fine flours.

[47] Preferably, said cereal flours are organic flours.

[48] Advantageously, said legume flours are selected from chickpea, pea, lupine, lentil flours.

[49] Preferably, said legume flours are organic flours.

[50] Said flour mixtures so obtained are added with said sourdough.

[51] Said mixture of gluten, flours and sourdough is preferably flavored by means of a mixture of spices.

[52] Advantageously, said spices present in said mixture are selected from a group comprising pepper, oregano, nutmeg, ginger, chilli and others.

[53] Said mixture of cereal and/or legume flours added with said mixture of spices is mixed with water and/or vegetable broth.

[54] Preferably the quantity of said required water and/or said vegetable broth varies from 200 g to 600 g for each kg of finished product obtained from said mixture of cereal and/or legume flours used.

[55] In order to give greater elasticity to the dough a percentage varying between 5% and 20% of at least one vegetable oil is advantageously added to the mixture so obtained.

[56] Preferably said vegetable oil is selected from extra virgin olive oil and sunflower oil.

[57] It is possible to vary the consistency, taste and aroma of the food products obtained by combining water and/or vegetable broth in different proportions with the various types of cereal and legume flours, the different types of spices, vegetable oils and gluten.

[58] The process for producing plant-based food products with low gluten content includes the steps of: a) mixing said gluten with at least one said type of cereal flour, at least one said type of legume flour or one said mixture of at least one said type of cereal flour and at least one said type of legume flour; b) adding said mixture obtained with said sourdough; c) flavoring said mixture of gluten, flours and sourdough with a mixture of spices; d) adding water and/or vegetable broth to said mixture and kneading until a homogeneous mixture is obtained; e) stuffing said mixture into a substantially cylindrical or possibly flattened shape, using suitable shapes, with typical sausage casings (fibrous, cellulose, etc.); f) boiling the sausage in water and/or vegetable broth and let it cool. [59] Advantageously, at least one vegetable oil is added to said mixture to give greater elasticity to said mixture.

[60] Preferably, said molded dough is boiled in water and/or vegetable broth for 60-90 minutes and left to cool overnight to obtain a consistency such as to improve the sliceability of the product allowing to maintain the integrity of the product during the packaging step.

Description of drawings

[61] The details of the invention will become more evident from the detailed description of the composition of the sourdough for producing plant-based food products and low-gluten plant-based food product.

[62] The accompanying drawings illustrate the data obtained from the studies conducted to refine the sourdough as claimed and to investigate the proteolytic activity of the claimed sourdough on gluten proteins.

Figure 1 shows the result of the ITS amplification of the yeasts extracted from the first yeast sample analyzed;

Figure 2 shows the result of the ITS amplification of the lactic bacteria extracted from the first yeast sample analyzed;

Figure 3 represents an example of development of the MM6 strain on Gluten Media;

Figure 4 shows the results of the electrophoretic run of the yeast strains extracted from the second sample of yeast analyzed and of the clone MW1 obtained from the isolation of the first sample of the sourdough;

Figure 5 shows the results of the electrophoretic runs of the strains of lactic bacteria extracted from the second sample of yeast analyzed and of the LP1 clone obtained from the isolation of the first sample of the sourdough;

Figures 6-10 are chromatograms representing the results of the mass analysis of the sample called "Delicate Yeast" using 70% Ethanol for protein extraction;

Figures 11-14 are chromatograms representing the results of the mass analysis of the sample called "Delicate Yeast" using DMF 1.5M for protein extraction;

Figures 15, 16 are chromatograms representing the results of the mass analysis of the sample called "Delicate No Yeast" using 70% Ethanol for protein extraction;

Figures 17-19 are chromatograms representing the results of the mass analysis of the sample called "Delicate No Yeast" using DMF 1 5M for protein extraction;

Figures 20-22 show the separations on gel electrophoresis with optimized conditions of the samples of soft wheat flour, hard wheat flour, gluten, flour mixture, Delicate after TTS and Veghiamo® with the analysis obtained on the densitometer on the side;

Figures 23-27 are HPLC chromatograms representing the results of the mass analysis of the samples of gluten extracted with 70% Ethanol, LG4 flour mixture, LG8 flour mixture, Veghiamo® extracted with 70% Ethanol, Veghiamo® after gel extraction electrophoresis; Figures 28-57 are chromatograms representing the results of the mass analysis of the samples of gluten, flour mixture, Delicate after TTS, Veghiamo®.

Description of embodiments of the invention

[63] All technical and scientific terms mentioned in this document have meanings that can be commonly understood by the person skilled in the art, unless otherwise defined.

[64] The sourdough for preparing plant-based food products object of the invention is composed of a mixture of flour and water acidified by a complex of lactic bacteria and yeast capable of starting a natural fermentation.

[65] In particular, the fraction of lactic bacteria contained within the sourdough is composed of lactic bacteria of the Lactobacillus plantarum species, while the fraction of yeast contained within the sourdough consists of yeasts of the Saccharomyces cerevisiae species.

[66] The basic dough of the sourdough consists of a mixture of water and flours selected from gluten, cereal and legume flour.

[67] The amount of water required to obtain the basic dough of the mother yeast varies from 300g to 500g per overall 1Kg of yeast.

[68] The sourdough object of the invention has shown a particular ability to digest gluten proteins.

[69] The analyzes carried out for the characterization of the components of the sourdough and the investigations on the proteolytic activity of the sourdough on gluten proteins (a-gliadin, b-gliadin and glutein) are reported below.

1. Analysis of a first sample of sourdough

[70] Initially, a sample of sourdough of interest for formulating plant-based food products was analyzed. The sample, dated 26.04.2017 was called PH407 and was subjected to microbiological analysis for the research and counting of the microbiological strains of interest.

[71] In particular, the analysis focused on detecting the presence of lactic bacteria and yeasts, known to be the microorganisms most involved in the fermentation processes useful in the context of food biotransformations.

[72] In addition, the generic and generically polluting flora present within the sample was analyzed.

[73] Table 1 shows the numbers obtained:

Table 11

[74] The generic and generically polluting flora in Plate Count Agar is considered of little relevance for the production process of the plant-based food product, therefore it was not considered necessary to isolate the individual strains.

[75] The colonies obtained on the higher diluted agar plate were analyzed, representing the dominant flora with respect to the composition of the sourdough. The colonies were analyzed from the morphological point of view and through microscopic observation.

[76] To proceed with the pure isolation of the strains of interest, a number equal to the square root of the number of colonies similar in morphology and microscopic observation is chosen. Seven colonies were isolated from the selective yeast medium (WL Nutrient Agar) and 5 colonies from the selective lactic acid medium (MRS Agar).

[77] The strains isolated in purity were subjected to DNA extraction and PCR amplification.

[78] Initially, an initial screening was carried out for both lactic bacteria and yeasts using ITS (Internal Transcribed Spacers) in order to evaluate the presence of different species (results in Figure 1 and Figure 2).

[79] Subsequently, the amplification of the 26S rDNA was performed for the yeasts and the amplification of the 16S rDNA for the lactic bacteria. Subsequently, the samples and the species they belonged to were sequenced.

[80] The lactic acid bacteria isolated, as emerged from the data obtained, all belong to the Lactobacillus plantarum species, having a high proteolytic activity. [81] The isolated yeasts all belong to the Saccharomyces cerevisiae species.

2. Investigation of the proteolytic activity of 6 Lactobacillus plantarum strains [82] Subsequently, a qualitative research of the proteolytic activity of the six strains of Lactobacillus plantarum was carried out, present within the sample of the sourdough called PH407 dated 26.04.2017 previously analyzed.

[83] Lactic acid bacteria were inoculated on Gluten Media, an agar medium containing gluten as the sole source of nitrogen. [84] The six strains analyzed were first developed in MRS culture medium and placed in an incubator at 37°C for 48 hours under anaerobic conditions.

[85] Subsequently, the cells were centrifuged and washed with Ringer's solution and two drops with a diameter of 1 cm for each culture were deposited on a plate of Gluten Media (Figure 3 shows an example of development of the MM6 strain on Gluten Media).

[86] The plates were incubated at 37° C for 48 hours under anaerobic conditions.

[87] The growth of the strains tested on gluten-containing medium was evaluated and the proteolytic activity of the strains was evaluated as a function of the diameter of the precipitation zone formed around the colony.

[88] All strains except number 2 grew on Gluten Media, but none of the strains tested showed a precipitation halo around the colony. This does not necessarily indicate the absence of proteolytic activity in the tested strains, but may be indicative of a proteolytic activity of the strains below the detection limit of the technique described above.

3. Analysis of a second sample of sourdough

[89] An investigation was carried out on a second sample of sourdough dated 15.12.2017. The sample was subjected to microbiological analysis for the research and selective counting of each group of interest.

[90] In particular, the analysis focused on detecting the presence of lactic bacteria and yeasts, known to be the microorganisms most involved in the fermentation processes useful in the context of food biotransformations.

[91] In addition, the generic and generically polluting flora present within the sample was analyzed.

[92] Table 2 shows the numbers obtained

Table 22

[93] The colonies obtained on the higher diluted agar plate were analyzed, representing the dominant flora with respect to the composition of the sourdough. The colonies were analyzed from the morphological point of view and through microscopic observation.

[94] To proceed with the pure isolation of the strains of interest, a number equal to the square root of the number of colonies similar in morphology and microscopic observation is chosen.

[95] In order to proceed with the identification of lactic bacteria and dominant yeasts, 3 colonies from the selective medium for yeasts (called F1, F2 and F3) and 3 colonies from the selective medium for lactic bacteria (called F5, F6 and F7) were purely isolated.

[96] The strains isolated in purity were then subjected to DNA extraction and PCR amplification in order to characterize the isolated clones.

[97] In particular, Multilocus Sequence Typing was implemented for yeasts, a method aimed at the amplification of microsatellites, while Random Amplification of Polymorphic DNA (RAPD-PCR) was implemented for lactic bacteria.

[98] It was possible to verify, by inserting in the molecular analysis two clones obtained from the isolation carried out on the first sample of sourdough (in which the lactic bacterium is called LP1 and the yeast MW1), that the clones isolated in the second sample of the sourdough were the same clones isolated from in the first analyzed sample. This step also indirectly confirms the species of the obtained isolated clones.

[99] As for the yeasts, as shown in Figure 4, the three isolated clones of the second sample show a profile identical to the clone MW1 purified during the first investigation and are confirmed to belong to the Saccharomyces cerevisiae species.

[100] As regards lactic bacteria, as shown in Figure 5, the three isolated clones of the second sample show identical profile to the MM1 clone purified during the first investigation and are confirmed to belong to the Lactobacillus plantarum species.

4. Investigation into the proteolytic activity of the sourdough

[101] 4.1 - In a first analysis two samples were analyzed to investigate the proteolytic activity of the sourdough on gluten proteins (a-gliadin, b-gliadin and glutein): the first sample was called "Delicate No Yeast", while the second sample was called "Delicate Yeast".

[102] The samples underwent separate extraction procedures. In particular, the extraction took place both by means of 70% ethanol useful for the extraction of the gliadin protein present in the samples, and by means of a 1.5M DMF solution useful for the extraction of peptide fragments derived from the hydrolysis of gliadin.

[103] Therefore, in total 4 extracts were analyzed, which were then centrifuged, separated and concentrated:

RDM1106: Delicate Yeast, extraction with 70% Ethanol;

RDM1107: Delicate Yeast, extraction with DMF 1 5M;

RDM 1108: Delicate No Yeast, extraction with 70% Ethanol;

RDM1109: Delicate No Yeast, extraction with DMF 1.5M.

[104] During the extraction process the samples have a tendency to decompose, therefore it is possible to detect the presence of peptides slightly different from those expected, in particular during the hydrolysis process glutamine (Gin, Q) is transformed into glutamic acid (Glu, E ).

[105] Due to the instrumentation used, it was not possible to identify the weight of the whole protein (a-gliadin, b-gliadin and glutein), but the allergenic peptide sequences deriving from the proteins were identified.

[106] The mass analysis of the sample called "Delicate Yeast" using 70% Ethanol for extraction are shown in the chromatograms in Figure 6-10.

[107] The molecular weights indicated in the chromatograms indicate:

[108] P.M. 3909.019 represents the repetitive unit of gliadin-a (33-Mer). Sequence: LQLQPFPQPQLPYPQPQLYPQPQLPYPQPQPF;

[109] P.M. 1506. 13 fragment of gliadin-a in which glutamic acid Glu (E) is substituted for glutamine Gin (Q). Sequence: PEPELPYPPEPELP;

[110] P.M. 1458.678. 12 gliadin-y fragment. Sequence: FSQPQQQFPQPQ;

[111] P.M. 1455. 12 fragment representing the hydrolysis product of a-gliadin. In mass, M/4 = 365;

[112] P.M. 3147. 26 active gliadin-y fragment. Sequence:

FLQPQQPFPQQPQQPYPQQPQQPFPQ;

[113] P.M. 2407. 20 gliadin-g fragment in which 6 Glu replace 6 Gin. Sequence:

LEPEEPFPEEPEQPYPQQPQ;

[114] P.M. 1669.846. 14 fragment of gliadin-a in which glutamine Q is transformed into glutamic acid E. Sequence: PEPELPYPEPELPY.

[115] Mass analysis of the sample called "Delicate Yeast" using DMF 1.5M for extraction are shown in the chromatograms in Figure 11-14.

[116] The molecular weights indicated in the chromatograms indicate:

[117] P.M. 1526.74. 13 particularly immunogenic a-gliadin fragment. Q transformed into E for a total of 4E, P.M. 1530.74. Sequence: LGEEEPFPPEEPY;

[118] P.M. 3795.935. 32 33-Mer a-gliadin fragment active with E instead of Q for a total of 8E. Sequence: ELEPFPEPELPYPEPELPYPEPELPYPQPQPF;

[119] P.M. 1085.544. 9 fragment of inactive 33-Mer. Sequence: LPYPQPQPF;

[120] P.M. 3909.019. 33 fragment of 33-Mer a-gliadin. Sequence:

LQLQPFPQPQLPYPQPQLPYQPQLPYPQPQLPYPQPQLPYPQPQPF;

[121] P.M. 2401.17. 20 g-gliadin fragment. Sequence: LQPQQPFPQQPQQPYPQQPQ;

[122] P.M. 1992.0. 17 a-gliadin fragment. Sequence: QLQPFPQPQLPYPQPQS;

[123] P.M. 1501.78. 13 a-gliadin fragment. Sequence: PQPQLPYPQPQLP.

[124] Mass analysis of the sample called "Delicate No Yeast" using 70% Ethanol for extraction are shown in the chromatograms in Figure 15, 16.

[125] The molecular weights indicated in the chromatograms indicate:

[126] P.M. 3909.019. 33 fragment of 33-Mer a-gliadin. Sequence:

LQLQPFPQPQLPYPQPQLPYQPQLPYPQPQLPYPQPQLPYPQPQPF;

[127] P.M. 1526.74. 13 particularly immunogenic a-gliadin fragment. Q transformed into E for a total of 4E. P.M.: 1530.74. Sequence: LGEEEPFPPEEPY; [128] P.M.: 1404.766. 12 inactive 33-Mer fragment. Sequence: LQLQPFPQPQLP.

[129] Mass analysis of the sample called "Delicate No Yeast" using DMF 1 5M for extraction are shown in the chromatograms in Figure 17-19.

[130] The molecular weights indicated in the chromatograms indicate:

[131] P.M. 1992.0. 17 a-gliadin fragment. Sequence: QLQPFPQPQLPYPQPQS;

[132] P.M. 1532.74. 13 particularly immunogenic a-gliadin fragment. Q transformed into E for a total of 5E. Sequence: LGEEEPFPPEEPY;

[133] P.M. 1463.678. 12 y-gliadin fragment with 5E. Sequence: FSEPEEEFPEPQ;

[134] P.M. 2221.085. 19 a-gliadin fragment. Sequence: LGQQQPFPPQQPYPQPQPF.

[135] In conclusion, the presence of the particularly allergenic portion P.M. 1526 present in almost all samples is evident from the analyzed extracts. The presence of the 33Mer fragment of gliadin-a with molecular weight 3909.019 was observed. During the hydrolysis the transformation of glutamine (Gin, Q) into glutamic acid (Glu, E) takes place, the presence of these fragments was observed in the above analysis. Therefore, allergenic peptides are not attacked by yeast, while proteins are degraded. The latter are degraded into allergenic peptides in the digestive system.

[136] 4.2 - Subsequently, 6 different samples were analyzed:

1) Soft Wheat Flour Lot: FMV 201902210008

2) Hard Wheat Flour Lot: FMV 201903270003

3) Gluten Lot: FMV 201903080002

4) Mixture:

Soft Wheat Flour Hard Wheat Flour Chickpea flour Gluten

Lot: FMV 201902200004

5) Delicate after TTS 10/04/2019 Lot: 020070419

6) Veghiamo® Lot: 0600303-expiry 18/06/2019

[137] Proteins were initially extracted by gel electrophoresis. Subsequently, the gliadin bands with PM: 40KDa were cut and extracted to analyze the samples by HPLC-MS.

[138] Individual samples were subjected to extraction procedures. In particular, the gliadins were extracted with 70% EtOH. All the samples were quantified on 1 gr of material and the results obtained were reported in mg in the tables below.

Table 88

[140] The greatest quantity of gliadin, among the analyzed flours, is present in gluten 72.01 mg on 1 gr of analyzed sample.

[141] In subsequent samples, an inconsistency in the results was observed. The samples, Delicate after TTS and Veghiamo®, where the Delicate is the sample that underwent all the treatments and finally was subjected to cutting to obtain the final product called "Veghiamo®", from the quantification analysis the results were different. We can observe that the Delicate sample shows as a result of the quantification a value of gliadins equal to 33.99 mg while in the Veghiamo® the value was equal to 22.79 mg.

[142] The proteins contained in the samples were separated by gel electrophoresis. Several tests were made to obtain the best possible separation and optimal data were obtained using a 10% electrophoresis gel and MOPS as a buffer.

[143] Figures 20-22 show the separations of the samples on gel electrophoresis with optimized conditions with the analysis obtained by the densitometer at the side. The data have been reported through histograms: on the abscissa there are the ranges of molecular weights (PM) in KDa while on the ordinates the average value of the density read by the densitometer.

[144] The areas of interest for the gliadins are between 50-31 KDa. As we can see, the density of gliadins in flour is very high in all the histograms shown.

[145] The presence of 3 bands between 50-31 KDa with different average density values was also observed in the "Delicate after TTS" sample, while in the "Veghiamo®" sample, under the same conditions of separation in gel electrophoresis, only a single band of gliadin was observed in the same range 50-31 KDa. Results that confirm what was stated for the data obtained from the quantification.

[146] Subsequently, a degradation was performed with the trypsin enzyme and finally the peptides were extracted with a suitable extracting mixture and analyzed with HPLC-MS.

[147] Extraction procedure:

[148] Each single band was cut with a surgical scalpel.

[149] Since the bands were colored with Coomassie Blue for viewing on the densitometer, the bands were subjected to an initial decolorization step using a 100mM solution of ammonium bicarbonate/acetonitrile (1: 1, V / V), incubated at room temperature and vortexed every 30 minutes. More acetonitrile was added and left to incubate until the bands turned white and folded. The bands were stored at -20 ° C.

[150] The extraction had been carried out using trypsin in a buffer solution until each single band was completely covered. After 30 minutes, where deemed necessary, more trypsin was added to prevent the bands from drying out and being no longer in contact with the trypsin. Then, they were left to incubate overnight at 37° C.

[151] After one night the enzyme was removed and a 5% formic acid/acetonitrile solution was added to extract the peptides that underwent digestion by the enzyme. Samples have been stored at -20 ° C.

[152] Analysis HPLC-MS [153] The mass analysis of the samples are shown below. In detail, the following samples were analyzed:

• Gluten Lot: FMV 201903080002 (LG3-LG6)

• Flour mixture, Lot: FMV 201902200004 (LG4-LG8)

• Delicate after TTS 10/04/2019 Lot: 020070419 (LG5-LG7-LG15-LG17)

• Veghiamo® Lot: 0600303-expiry 18/06/2019 (LG11-LG19)

[154] The fractions searched in mass were the following:

^■ LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF 33-mer 3909.019 (other diagnostic weights: m/6 = 652.8 e m/4 = 979.6);

^■ FLQPQQPFPQQPQQPYPQQPQQPFPQ 26-mer 3145.53 and fragments;

^■ LQPQQPFPQQPQQPYPQQPQ 20-mer 2401.17 and fragments;

^■ LGQQQPFPPQQPYPQPQPF 19-mer 2221.085 and fragments;

^■ QLQPFPQPQLPYPQPQS 17-mer 1992.0 and fragments;

^■ PQPQLPYPQPQLPY 14-mer 1664.846 17-mer 1992.0 and fragments;

^■ LGQQQPFPPQQPY 13-mer 31-43 of a-gliadin, particularly immunogenic 1526.74 and fragments;

^■ FSQPQQQFPQPQ 12-mer range 1458.678 and fragments.

[155] The sequences may also be of diagnostic interest:

^■ SGQGSFQPSQQ fragment 206-216 1149.494;

^■ SSQGSFQPSQQN 1293.548;

^■ PQPQLPYPQPQLP 13-mer 1501.78;

^■ QLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF 32 fragment of 33-mer 3795.935;

^■ QPFPQPQLPYPQPQLPYPQPQLPYPQPQPF 29-mer fragment of 33-mer 3554.792;

^■ LQLQPFPQPQLP 12-mer inactive 33-mer fragment 1404.766;

^■ LQLQPFPQPQ 10-mer fragment of inactive 33-mer 1194.629;

^■ LPYPQPQPF 9-mer fragment of inactive 33-mer 1085.544;

^■ QPQPFPQQPYP 12-mer 1325.63.

[156] In addition, sequences were also sought that showed one or more deaminations of the glutamine (Q) residues that yielded glutamic acid (E).

[157] The first analysis concern the extraction of gliadins with EtOH before undergoing separation with gel electrophoresis.

[158] A column with the following dimensions was used: C18 XSelect Peptide CSH 4.6x100 mm, 130A, 3.5microm column. Solvents used: A = H20/ CH3CN 95:5+0.1%TFA, B = H20/ CH3CN 5:95+0.1 %TFA.

[159] From the HPLC chromatograms (Figures 23-27) it was possible to observe the clear division of the gliadins in relation to the elution time from the column.

[160] The a/b-gliadins elute from 7 min to 16 min. The g-gliadins from 16 min to 18 min. Finally the w-gliadins elute from 18 min to 30 min.

[161] Gluten has a high concentration of gliadins.

[162] LG4, LG8 and “Veghiamo®” are the samples after gel electrophoresis. The presence of high and narrow peaks can be observed due to lipophilic substances remaining in the column.

[163] On the basis of the results obtained, the column was replaced with a C18 110 A 100 * 3.0 mm, the solvents used H20 + 0.01% of HCOOH and CH3CN + 0.01% of HCOOH.

[164] All mass analyzes were analyzed based on the mass/charge ratio (m/z), where m is the molecular mass, z indicates the number of positive charges present on the molecule. On the basis of the data in the literature, these molecules take a maximum number of 10 charges, for this reason each individual mass was analyzed with z = 1,2,3.10.

[165] Both gluten and the mixture of flours have a large amount of fragments that have been associated with the sequences 33-mer, 12-mer, 13-mer, 14-mer, 17-mer, 19-mer and 29- mer. The same deaminated sequences are found, with glutamic acid instead of glutamine.

[166] The "Delicate" sample has a greater quantity of toxic fragments than the sample called "Veghiamo®", but the "Delicate" has only the 10-mer and 12-mer fragments, which are inactive fractions while the "Veghiamo® ’’Contains as inactive fractions the 9-mer, 10-mer and 12-mer fragment of 33-mer.

[167] In conclusion, the "Veghiamo®" sample shows a lower quantity of toxic gliadins than the "Delicate after TTS" sample, therefore the fresh yeast is able to further degrade the gluten proteins, resulting in a significant qualitative and quantitative reduction of the allergenic fragments.

[168] Figures 28-57 show all the mass analysis indicating the toxic and inactive fragments found.

[169] According to the results of the analysis carried out, the composition for formulating the sourdough suitable for decreasing the gluten content in plant-based food products object of the present invention comprises at least one strain of Lactobacillus plantarum and at least one strain of Saccharomyces cerevisiae.

[170] The at least one strain of Lactobacillus plantarum present in the composition object of the invention, has shown a particular proteolytic capacity of gluten proteins, in particular in the degradation of allergenic fragments of said proteins, leading to a significant qualitative and quantitative reduction of these fragments.

[171] The proteolytic activity of the at least one strain of Lactobacillus plantarum is promoted by the presence within the composition of the at least one strain of Saccharomyces cerevisiae, which assists the fermentation processes of the bacterium by hydrolyzing the carbohydrates present in the substrate into simple molecules such as glucose and fructose, more usable by the bacterium, consequently increasing its cellular yield and enhancing its proteolytic activity.

[172] The composition of Lactobacillus plantarum and Saccharomyces cerevisiae for the formulation of a sourdough object of the invention has shown a particular ability to digest gluten proteins.

[173] Therefore, the sourdough containing this composition is used for the formulation and production of plant-based food products, in particular for plant products with a low gluten content.

[174] Plant-based food products added with said sourdough show a low gluten content and high digestibility even for those predisposed to celiac disease. The ability of the sourdough to degrade gluten proteins and to reduce allergenic fragments derived from the degradation of these proteins is demonstrated by the aforementioned analysis.

[175] The plant-based food products that are added with the sourdough are composed of gluten in a percentage by weight from 20% to 40%, at least one type of cereal flour in a percentage by weight from 5% to 15% and/or at least one type of legume flour in a percentage by weight from 10% to 30%, or with a mixture of at least one type of cereal flour and at least one type of legume flour in a percentage by weight from 10% to 30%.

[176] The cereal flours are selected from hard wheat and soft wheat flours.

[177] Said cereal flours can be bran, wholemeal, fine, extra-fine flours.

[178] Preferably, said cereal flours are organic flours.

[179] The legume flours are selected from chickpea, pea, lupine, lentil flours.

[180] Preferably, said legume flours are organic flours.

[181] The flour mixtures so obtained are added with the sourdough.

[182] The mixture of gluten, flours and sourdough is preferably flavored with a mixture of spices.

[183] The spices present in said mixture are selected from a group comprising pepper, oregano, nutmeg, ginger, chilli and others.

[184] The mixture of cereal and/or legume flours added with the mixture of spices is mixed with water and/or vegetable broth.

[185] The quantity of water and/or vegetable broth required varies from 200 g to 500 g for each kg of finished product.

[186] In order to give greater elasticity to the dough a percentage varying between 5% and 20% of at least one vegetable oil is advantageously added to the mixture so obtained.

[187] The vegetable oil is selected from extra virgin olive oil and sunflower oil.

[188] It is possible to vary the consistency, taste and aroma of the food products obtained by combining water and/or vegetable broth in different proportions with the various types of cereal and legume flours, the different types of spices, vegetable oils and gluten.

[189] Examples of plant-based food products obtained from the processing of the components described above are the vegetable cold sliced products of the Veghiamo® line.

[190] A first example is the lupine vegetable sliced product, which contains water, 25% lupine flour, 22% wheat gluten, sunflower oil, wheat starch, natural flavorings, salt, apple cider vinegar, a mixture of spices and aromatic plants, chickpea flour, durum wheat flour, acidity regulator: sodium bicarbonate, sourdough.

[191] A second example is the spiced vegetable sliced product, which contains water, 32% wheat gluten, 8% durum wheat, chickpea flour, extra virgin olive oil, natural flavors, soft wheat flour, salt, a mixture of spices and aromatic plants, sourdough, sunflower oil.

[192] A third example is the vegetable sliced product with turmeric and ginger, which contains water, 32% wheat gluten, 9% durum wheat, chickpea flour, sunflower oil, natural flavors, salt, 2% ginger powder, 0.4% turmeric powder, chili powder, sourdough, pepper powder, garlic powder.

[193] The products described by way of example, forming part of a wider range of plant-based products marketed by the Applicant, are susceptible of numerous modifications and variations according to the different requirements.

[194] In the practical implementation of the invention, the ingredients used, as well as the shape and size, can be changed according to requirements.

[195] The process for the production of plant-based food products with a low gluten content is easily understood from the above description.

[196] Gluten is mixed with at least one type of cereal flour, at least one type of legume flour or a mixture of at least one type of cereal flour and at least one type of legume flour.

[197] The mixture so obtained are added with the sourdough.

[198] The mixture of gluten, flours and sourdough is flavored with a mixture of spices.

[199] Water and/or vegetable broth are added and the ingredients are mixed until a homogeneous mixture is obtained.

[200] To give greater elasticity to the dough, at least one vegetable oil is added to the mixture thus obtained.

[201] The dough is modeled in a substantially cylindrical shape with rounded ends obtaining the typical shape of sausages such as bresaola or salami.

[202] The modeled dough is boiled in water and/or vegetable broth for 60-90 minutes and left to cool overnight until a consistency is obtained that improves the sliceability of the product maintaining the integrity of the product during the packaging step.

[203] The described procedure permits to obtain a plant-based food product with a low gluten content obtained thanks to the proteolytic activity of the sourdough on gluten proteins. Therefore, the finished product is digestible and not harmful for consumers with a