Process for the preparation of gamma-aminobutyric acid using lactic acid bacteria

Abstract

The present invention relates to a process for the preparation of γ-aminobutyric acid (GABA) using lactic acid bacteria (LAB) on vegetal matrices. In particular, the invention relates to the use of selected lactic acid bacteria for the production of γ-aminobutyric acid (GABA) on vegetal matrices such as legume flours for beneficial use in food, pharmaceuticals or cosmetics.

Description

[0001] "PROCESS FOR THE PREPARATION OF GAMMA-AMINOBUTYRIC ACID

[0002] USING LACTIC ACID BACTERIA"

[0003] Cross-Reference to Related Applications

[0004] This Patent Application claims priority from Italian Patent Application No. 102023000013167 filed on June 26, 2023, the entire disclosure of which is incorporated herein by reference.

[0005] Technical Field

[0006] The present invention relates to a process for the preparation of y-aminobutyric acid (GABA) using lactic acid bacteria (LAB) on vegetal matrices. In particular, the invention relates to the use of selected lactic acid bacteria for the production of Y-amin°butyric acid (GABA) on vegetal matrices such as legume flours for beneficial use in food, nutraceuticals, pharmaceuticals or cosmetics.

[0007] Background of the Invention

[0008] Y~aminobutyric acid (GABA) is a four-carbon-atom amino acid made from L-glutamic acid through the activity of the enzyme glutamate decarboxylase (GAD). This molecule is widely present in microorganisms, plants and animals.

[0009] GABA is known to be a bioactive component with multiple physiological functions. Indeed, it acts as an important inhibitory neurotransmitter that sends chemical messages into the central nervous system of mammals. In addition, GABA also plays an important role in behaviour, cognition and the body's response to stress. GABA as a food supplement can improve states of insomnia and depression, strengthen the immune system, alleviate anxiety and menopausal syndrome, regulate blood pressure, combat obesity, and improve visual cortical function.

[0010] There are many foods containing GABA, but few of them can actually modulate human physiological functions due to their low GABA content.

[0011] Microorganisms are an important source of GABA. So far, it has been confirmed that many types of microorganisms can synthesise GABA, including yeasts, fungi and bacteria. Microorganisms grow faster than plants, do not need much space for cultivation and their use is sustainable and accepted by public opinion. Furthermore, it is easy to control the production of microorganisms through ad hoc biotechnological processes. Therefore, their potential use as a source of GABA has attracted considerable interest.

[0012] Lactic Acid Bacteria (LAB) have been widely used in the food industry, particularly in the production of fermented foods for many centuries. Due to the Generally Recognised as Safe (GRAS) status of LAB (recognised by EFSA and FDA) and their high application potential in the fermentation industry, GABA-producing LAB have received much attention in recent years. A large number of GABA-producing LAB have been isolated from fermented foods and used in the production of GABA-enriched foods.

[0013] A number of LAB strains with GABA production capacity have been isolated from traditional fermented foods such as cheese, kimchi, paocai, yoghurt and fermented soy beans.

[0014] The Lactobacillus genus, now reclassified into numerous subgenera, has abundant GABA-producing species, including Lactobacillus brevis, Lactobacillus buchnerirLactobacillus delbrueckii subsp. bulgaricus, Lactobacillus fermentum, Lactobacillus helveticus , Lactobacillus paracasei, and Lactobacillus plantarum. In addition, certain strains of Streptococcus thermophilus and Lactococcus lactis, species typically associated with the dairy area, show good GABA production capacities and are the best candidates for the production of GABA-rich dairy products. In recent years, it has been discovered that certain species of the genera EnterococcusrLeuconostocrPediococcusrPropionibacterium and Weissella are capable of producing GABA.

[0015] Fermented foods rich in L-glutamate are important sources for isolating GABA-producing LAB as this compound is a precursor of GABA in microbial conversion. In general, an acidic environment is beneficial for the growth of GABA- producing LAB, as in Korean kimchi and Chinese paocai.

[0016] The GABA production capacity of different species is highly variable depending on the strain. Numerous studies have shown that Lb. brevis and Lb. plantarum (now reclassified as Lactiplantibacillus plantarum) can produce a higher yield of GABA than other LAB species (Cui, Y.; Miao, K.; Niyaphorn, S.; Qu, X. Production of Gamma-Aminobutyric Acid from Lactic Acid Bacteria: A Systematic Review. Int. J. Mol. Sci. 2020, 21(3), 995).

[0017] However, the search for strains suitable for industrial exploitation, e.g. capable of producing good yields on inexpensive substrates and especially of vegetal origin (with greater long-term sustainability than animal sources) is of great industrial interest, especially in view of the likely recognition of functional properties by food authorities .

[0018] The need is therefore felt in the art for new microorganisms and methods for the preparation of GABA that can overcome the disadvantages of the prior art.

[0019] Therefore, the aim of the present invention is to provide a new lactic acid bacterium for the production of GABA and methods for producing GABA.

[0020] Summary of the Invention

[0021] This object is achieved by a lactic acid bacterium according to claim 1, its use according to claim 2, a method for GABA production according to claim 4, and a food product according to claim 12.

[0022] Brief Description of the Drawings

[0023] The present invention will now be described in detail by reference to the accompanying drawings, in which:

[0024] - Figure 1 illustrates the process diagram for the production of a GABA-enriched product according to the present invention.

[0025] - Figure 2 illustrates the sensory profiles of yoghurt as determined by sensory analysis. Y-ct, yoghurt from semiskimmed milk; Y-SCd and Y-SCc, yoghurt supplemented with the GABA-enriched ingredient (in the forms SCd and See) in the percentages of 2% (w / v).

[0026] - Figure 3 illustrates the sensory profiles of vegetal yoghurt-like products as determined by sensory analysis. Y- ct, non-supplemented vegetal yoghurt-like products; YL-SCd and YL-SCc, vegetal yoghurt-like products supplemented with the GABA-enriched ingredient (in the forms SCd and See) in the percentages of 2% (w / v).

[0027] - Figure 4 illustrates the sensory profile of wheat bread. Control wheat bread (P-ct) and breads fortified with SCd (P-Scd) and SCc (P-Sc-c) at 2% w / w. - Figure 5 illustrates the sensory profile of gluten- free bread control (Pgf-ct) and gluten-free breads fortified with SCd (Pgf-Scd) and SCc (Pgf-SCc) at 2% w / w.

[0028] Description of Embodiments

[0029] The authors of the present invention have selected a lactic acid bacterium, in particular, Lactiplantibacillus plantarum CL3 DSM34115 (filed on 15thDecember 2021 with the DSMZ), suitable substrates for its growth, in particular, legume flour, and developed a process for producing GABA in much higher yields than can be obtained using the known technique.

[0030] Further advantages of the present invention are the possibility of using low-cost and natural fermentation substrates (e.g. legume flour), avoiding the use of commercial and complex culture media to promote the growth of microorganisms and the biosynthesis of GABA, increased synthesis of GABA on food-derived matrices compared to the concentrations reported in literature, and a fermentation process that enables the production of a product with high physiological value, naturally enriched with GABA, vitamins, minerals, polyphenols, and live and viable lactic acid bacteria potentially probiotic, which can be used as i) an ingredient for the fortification of foods of various types, including yoghurt and derivatives, beverages and derivatives, dairy products and derivatives, bakery products (bread, biscuits) and derivatives, bars and derivatives, pasta and derivatives, functional foods, sauces and derivatives or ii) used directly as a dietary supplement and PET food or iii) for the formulation of pharmaceutical and cosmetic products.

[0031] The GABA-producing lactic acid bacterium of the invention used for the fermentation process is a strain of the Lactiplantibacillus plantarum species (food-grade microorganism and classified as QPS - qualified presumption of safety - by EFSA), isolated from natural (non-GMO) vegetal matrix.

[0032] Lactiplantibacillus plantarum CL3 DSM34115 was selected on the basis of its ability to synthesise high concentrations of GABA in culture medium and for its high proteolytic activity.

[0033] Lactiplantibacillus plantarum CL3 DSM34115 is used as a starter in a process for the production of GABA. In the context of the present invention, "starter" means one or more microorganisms used in a live and viable state for the inoculation of food biomass for transformation by fermentation into products, e.g. ingredients or food or beverages, for food, nutraceutical, pharmaceutical or cosmetic use. The term "starter" also refers to the preparation in liquid or solid form, fresh or frozen or freeze dried, containing a high cell density of the aforesaid microorganisms in a live and viable form.

[0034] Further subject matter of the present invention is the use of Lactiplantibacillus plantarum CL3 DSM34115 for the production of y-aminobutyric acid or for the preparation of a product enriched with Y-amin°butyric acid.

[0035] The invention also relates to a process for the production of Y-amin°butyric acid or the preparation of a product enriched with Y-amin°butyric acid comprising the following steps: a) preparing a substrate for the cultivation of Lactiplantibacillus plantarum CL3 DSM34115 comprising a vegetal matrix and water and having a pH between 5 and 6.5; b) inoculating said substrate with

[0036] Lactiplantibacillus plantarum CL3 DSM34115; c) fermenting said substrate for between 18 and 72 hours at a temperature between 20 and 40°C to obtain a product enriched with Y-amin°butyric acid; d) optionally extracting y-aminobutyric acid.

[0037] In more detail, the procedural steps according to the present invention will be illustrated below.

[0038] 1. Substrate preparation

[0039] The substrate used for the production of Y-aminobutyricacid is an aqueous substrate suitable for the cultivation of the selected Lactiplantibacillus plantarum CL3 producing microorganism. For example, the substrate can be produced with food-grade components that therefore comply with the requirements for the use of vegetal ingredients in food sector (Regulation (EC) No 852 / 2004, Regulation (EC) No 2073 / 2005, Regulation (EC) No 1881 / 2006) regarding the contamination of microorganisms and mycotoxins.

[0040] In particular, the substrate can be obtained from the mixture of:

[0041] - drinking water, preferably between 50 and 95% of the final volume, more preferably 90%; and

[0042] - a vegetal matrix.

[0043] In the following, the term "vegetal matrix" refers to an ingredient of edible plant origin, i.e. free from substances of any kind that are harmful to humans and of suitable microbiological and hygienic quality with the following indicative composition: o Moisture: 6-14%, preferably 10% oFat: 2-5%, preferably 3.5% o Proteins: 15-25%, preferably 20% o Complex carbohydrates: 55-70%, preferably 60%

[0044] • of which Fibre: 10-17%, preferably 13.5% o Ash: 1.2-2.0%, preferably 1.6%

[0045] In one embodiment, the vegetal matrix is a legume flour comprising a legume flour selected from the group consisting of bean, Phaseolus vulgaris L.; pea, Pisum sativum L.; broadbean, Vicia faba L.; lupin, Lupinus albus; chickpea, Cicer arietinum L.; pigeon pea, Cajanus indicus; peanut, Arachis hypogaea L.; soy bean, Glycine max; lentil, Lens culinaris; grass pea, Lathyrus sativus; carob, Ceratonia siliqua; and mixtures thereof. The legume flour may also be in the form of legume grains, possibly sprouted.

[0046] In one embodiment, the legume flour is preferably a flour selected from the group consisting of chickpeas, lentils, and broad beans and mixtures thereof, more preferably a chickpea and lentil flour. In one embodiment, chickpea and lentil flour is presented as a mixture in a 1:1 ratio, preferably not previously heat-treated.

[0047] Preferably, the vegetal matrix is present in the substrate in an amount between 5 and 30% by weight of the final volume of the substrate, preferably 10%.

[0048] To enable the correct cultivation of Lactiplantibacillus plantarum CL3, the substrate must have a pH between 5 and 6.5 and a titration acidity (TTA) of 2.0-3.5. The preferred pH for the process is 5.5. 2. Enrichment

[0049] The substrate may also comprise a sugar matrix.

[0050] In the following, the term "sugar matrix" refers to a vegetal matrix rich in fermentable sugars with the following indicative composition: o Moisture: 5-14%, preferably 6% oFat: 0.2-1%, preferably 0.5% o Proteins: 3.5-7.5%, preferably 5% o Carbohydrates: 78-92%, preferably 88%

[0051] • Of which Fibre: 15-40%, preferably 37% o Ash: 1.5-5.0%, preferably 2.5%

[0052] In particular, the substrate can be enriched with sugar matrices such as grape must or carob pulp, preferably carob pulp. In particular, the following components can be used for enrichment:

[0053] - White or black grape must, preferably black grape, which previously underwent mutage (preferably by concentration), not containing sulphur dioxide and preferably not rectified. Grape must is used in percentages that guarantee a concentration between 0.1 and 10% sugar in the final substrate, preferably 1% (weight / volume);

[0054] - Carob pulp for food use, preferably in the form of dehydrated flour.

[0055] Carob pulp in the form of dehydrated flour can be used in percentages that ensure the concentration of between 1 and 10% carbohydrates (except fibres) in the final substrate, preferably 1% (weight / volume).

[0056] The enriched substrate has a pH between 5 and 6.5 and titration acidity (TTA) of 2.5-4.5. The preferred pH for the process is 5.5. The pH can be corrected by adding acidity regulators in the manner and in the doses prescribed for food use (e.g. sodium bicarbonate - E500 II - or citric acid - E330).

[0057] To remove precipitates and suspended particulates, the substrate can be filtered (porosity between 10 and 50 pm, preferably 20 pm) or subjected to centrifugation (5000-12000 rpm, preferably 10000 rpm) with removal of the pellet.

[0058] 3. Inoculum

[0059] Viable cells of the microorganism L. plantarum DSM34115 can be inoculated into the liquid substrate at a cell density between 105and 109UFC / mL, preferably 107UFC / mL.

[0060] The cells can be inoculated as a suspension in drinking water or saline (NaCl 9 g / L) at room temperature (18-25°C).

[0061] In one embodiment, the cells are derived from fresh culture in culture medium after collection by centrifugation or filtration and washing in drinking water or phosphate buffer (50mM at pH 7), preferably phosphate buffer. The cells prior to inoculation can be stored after dehydration by freeze-drying or frozen in the range of -20°C to -80°C, but used after verification of viability.

[0062] The elective medium for cultivation of the microorganism is general edible medium (GEM) with the following composition: 40 g / L glucose, 30 g / L soy peptone, 7 g / L freeze-dried yeast extract, 1 g / L magnesium sulphate heptahydrate in lOmM potassium phosphate buffer at pH 6.3 (Saarela, M., Rantala, M., Hallamaa, K., Nohynek, L., Virkajarvi, I., & Mattd, J. (2004). Stationary-phase acid and heat treatments for improvement of the viability of probiotic lactobacilli and bifidobacteria. Journal of Applied Microbiology, 96(6), 1205-1214). 4. Fermentation

[0063] Fermentation of the inoculated liquid substrate of the microorganism takes place at temperatures between 20 and 40°C, preferably between 25 and 35°C, more preferably 30°C, preferably under agitation between 50 and 300 rpm, more preferably at 150 rpm; preferably under non-strict anaerobic conditions. Fermentation lasts between 18 and 72 hours, preferably for 48 hours.

[0064] The fermented preparation, which can be used in liquid form (10% dry matter (d.m.)) or dehydrated form (90% d.m.), has a final GABA concentration of 200 mg / 1 in liquid form or 2500 mg / kg in dehydrated form.

[0065] 5. Fermented product stabilisation

[0066] The substrate obtained by fermentation comprises at least one legume flour, water and Lactiplantibacillus plantarum DSM 34115. It may further comprise a sugar matrix, preferably selected from the group consisting of grape must and carob pulp.

[0067] It can be used immediately or rapidly cooled in the range of 0-12°C within 2-6 hours, preferably 2 hours, and stored prior to freezing or dehydration for no longer than 12 hours in these refrigerated conditions.

[0068] The substrate can be separated from the cells by filtration (porosity between 0.22 and 50 pm, preferably 0.22 pm) or centrifugation (4000-12000 rpm, preferably 10000 rpm) with removal of the pellet.

[0069] The fermented product can be stored until use in frozen form (in the range of -20 to -80°C, preferably -20°C) or dehydrated by vacuum evaporation, centrifugal vacuum evaporation, spray drying, fluidised bed, freeze drying, drum drying, at temperatures not exceeding 75°C and not below 55°C.

[0070] In the following, the present invention will be illustrated by means of a few examples, which are not intended to be considered as limiting the scope of the invention.

[0071] Example 1.

[0072] Production of GABA on vegetal matrix with Lactiplantibacillus plantarum CL3 strain

[0073] 1.1 Microorganisms

[0074] The following lactic acid bacteria previously selected for use as starters for the fermentation of vegetal matrices were included in the experiment. All the strains belong to the Crop Collection of the Department of Soil, Plant and Food Sciences of the University of Bari and were previously selected for protechnological aptitudes such as adaptation to vegetal matrices, growth and acidification capacity, proteolytic activity, ability to synthesise GABA and release of compounds with antioxidant activity. Below is the list of microorganisms and some representative bibliographical references. It is emphasised that the inclusion of microorganisms already identified for optimal technological performance was carried out to highlight the exceptional characteristics of the strain of the invention, Lactiplantibacillus plantarum DSM34115, with reference to proteolytic activity and the ability to synthesise GABA.

[0075] 1. Lactiplantibacillus plantarum H64 (Verni et al., 2022, Journal of Applied Microbiology, 00:1-15);

[0076] 2. Lactiplantibacillus plantarum PU1 (Verni et al., 2020, Frontiers in Microbiology, 11:1831; Pontonio et al., 2019 Frontiers in Microbiology, 10, 1550; Schettino et al., 2021, Antioxidants 10 (5), 742; Rizzello et al., 2008,

[0077] Journal of Agricultural and Food Chemistry, 56:6936-43);

[0078] 3. Lactiplantibacillus plantarum C48 (Curiel et al., 2015, International Journal of Food Microbiology, 196:51-61; Rizzello et al., 2008, Journal of Agricultural and Food Chemistry, 56:6936-43; Coda et al., 2010, International J. Food Microbiol., 137: 236-245; Verni et al 2022, Journal of Applied Microbiology, 00:1-15);

[0079] 4. Pediococcus pentosaceus F01 (Verni et al., 2017, Frontiers in Microbiology, 8: 2461; Verni et al. 2022, Journal of Applied Microbiology, 00:1-15)

[0080] 5. Levilactobacillus brevis MRS4 (Verni et al. 2022, Journal of Applied Microbiology, 00:1-15);

[0081] 6. Lactiplantibacillus plantarum C2 (Rizzello et al., 2013. Microbial Cell Factories, 12: 44);

[0082] 7. Lactiplantibacillus plantarum LB1 (Rizzello et al., 2010 Food Chemistry, 119:1079-1089);

[0083] 8. Lactiplantibacillus plantarum H48 (Verni et al., 2020, Frontiers in Microbiology, 11:1831);

[0084] 9. Lactiplantibacillus plantarum T6B4 (Rizzello et al., 2017. International Journal of Food Microbiology, 241, 252-261);

[0085] 10. Lactiplantibacillus plantarum T0A10 (Torreggiani et al., 2023 Antioxidants 12(8), 1521);

[0086] 11. Lactiplantibacillus plantarum T6C16 (Rizzello et al., 2017. International Journal of Food Microbiology, 241, 252-261);

[0087] 12. Lactiplantibacillus plantarum 18S9 (Montemurro et al., 2023, Foods 12(3):485); 13. Lactiplantibacillus plantarum MRS1 (De Pasquale et al., 2020, International Journal of Food Microbiology, 316,108426);

[0088] 14. Lactiplantibacillus plantarum 1A7 (Coda et al., 2011, Applied And Environmental Microbiology, 77: 3484-3492;

[0089] 15. Lactiplantibacillus plantarum PRO17 (Verni et al., 2020, Frontiers in Microbiology, 11:1831);

[0090] 16. Furfurilactobacillus rossiae LB5 (Rizzello et al., 2010 Food Chemistry, 119:1079-1089);

[0091] 17. Furfurilactobacillus rossiae T0A16 (Rizzello et al., 2017. International Journal of Food Microbiology, 241, 252-261; Rizzello et al., 2016, Food Microbiology 56, 1-13);

[0092] 18. Levilactobacillus brevis MRS4 (De Pasquale et al., 2020, International Journal of Food Microbiology, 316, 108426);

[0093] 19. Levilactobacillus brevis AM7 (Verni et al., 2023, International Journal of Food Microbiology 407, 110403);

[0094] 20. Pediococcus pentosaceus Hll (Verni, et al., 2020 Frontiers in Microbiology, 11:29);

[0095] 21. Pediococcus pentosaceus T1A13 (Rizzello et al., 2017. International Journal of Food Microbiology, 241, 252- 261);

[0096] 22. Pediococcus pentosaceus 176 (Verni et al., 2017, Frontiers in Microbiology, 8: 2461; Verni, et al., 2020 Frontiers in Microbiology, 11:29);

[0097] 23. Pediococcus pentosaceus 1214 (Verni et al., 2017, Frontiers in Microbiology, 8: 2461; Verni, et al., 2020 Frontiers in Microbiology, 11:29) 24. Pediococcus pentosaceus 102 (Verni et al., 2017, Frontiers in Microbiology, 8: 2461; Verni, et al., 2020 Frontiers in Microbiology, 11:29)

[0098] 25. Pediococcus pentosaceus 1014 (Verni et al., 2017, Frontiers in Microbiology, 8: 2461; Verni, et al., 2020 Frontiers in Microbiology, 11:29)

[0099] 26. Pediococcus pentosaceus F01 (Verni et al., 2017, Frontiers in Microbiology, 8: 2461; Verni, et al., 2020 Frontiers in Microbiology, 11:29);

[0100] 27. Pediococcus pentosaceus OA1 (Verni et al., 2017, Frontiers in Microbiology, 8: 2461; Verni, et al., 2020 Frontiers in Microbiology, 11:29);

[0101] 28. Pediococcus pentosaceus S3N3 (Verni et al., 2017, Frontiers in Microbiology, 8: 2461; Verni, et al., 2020 Frontiers in Microbiology, 11:29);

[0102] 29. Pediococcus pentosaceus BAR4 (Verni et al., 2017, Frontiers in Microbiology, 8: 2461; Verni, et al., 2020 Frontiers in Microbiology, 11:29);

[0103] 30. Pediococcus acidilactici 10MM0 (Verni et al., 2017, Frontiers in Microbiology, 8: 2461; Verni, et al., 2020 Frontiers in Microbiology, 11:29);

[0104] 31. Pediococcus sp. 156 (Verni et al., 2017, Frontiers in Microbiology, 8: 2461; Verni, et al., 2020 Frontiers in Microbiology, 11:29);

[0105] 32. Leuconostoc mesenteroides 12MM1 (Pontonio et al., 2020, Antioxidants, 9, 1258);

[0106] 33. Leuconostoc mesenteroides 157 (Verni et al., 2017, Frontiers in Microbiology, 8: 2461);

[0107] 34. Weissella confusa KAS3 (Montemurro et al., 2020, Food Microbiology, 103491); 35. Welssella confusa NEY6 (Pontonio et al., 2015 Food Microbiology, 47: 99-110);

[0108] 36. Lactlplantlbaclllus plantarum DSM34115, isolated from vegetal matrices and identified on a molecular basis (16S rDNA gene sequencing, according to the method described in Pontonio, et al., 2015. Food microbiology, 47, 99-110.) was included in the test.

[0109] 1.2 Fermentation substrates

[0110] The microorganisms listed in paragraph 1.1. were inoculated into the following substrates: a) chickpea flour (10% w / w) in water b) lentil flour (10% w / w) in water c) wheat flour (10% w / w) in water

[0111] The composition of the flour used is shown in Table 1. Table 1

[0112] 1.3 Fermentation conditions

[0113] All the strains were propagated continuously in De Man, Rogosa and Sharpe (MRS, Oxoid Basingstoke, Hampshire, UK) medium at 30 °C every 24 hours. When used for fermentation, the microorganism was cultivated until the exponential growth phase was reached (approximately 16 hours). Subsequently, the cells were recovered by centrifugation at 9.000 x g at 4 °C for 10 min, washed twice in 50 mM phosphate buffer (4 °C, pH 7.0), resuspended in running water (final cell density of approximately 8 loglO CFU / mL) and used as a starter for the fermentation of the substrate mentioned in point 1.2. Specifically, the substrate was inoculated at a cell density of approximately 7 loglO CFU / mL, and fermentation was continued for 24 hours at 30 °C under mechanical agitation (100 rpm) . At the end of the fermentation process, the substrate was refrigerated to 10 °C and processed within 4 hours of reaching this temperature.

[0114] For analysis, the fermented samples were dehydrated in a ventilated oven at 65 °C.

[0115] 1.4 Determination of proteolytic activity

[0116] At the end of the fermentation process described above, an aqueous extract was obtained from each substrate fermented with the individual strains according to the method described below.

[0117] The aqueous extracts were prepared according to the method initially described by Osborne in 1907 and later modified by Weiss (Weiss et al., 1993, Electrophoresis, 14, 805-816). Specifically, 10 grams of sample were suspended in 8 mL of 50 mM Tris-HCl (pH 8.8). The suspension was kept at 4 °C for one hour, vortexed at 15-minute intervals and centrifuged at 12000 rpm for 20 minutes. The supernatant was then recovered and analysed.

[0118] The aqueous extract was analysed to define the concentration in peptides and total free amino acids of the samples. Peptides and total free amino acids are derivatives of proteolysis (products of protein degradation) and therefore their concentrations used as indices of the degree of proteolysis. Proteolysis is considered an important requirement because it leads to the release of the GABA precursor, i.e. glutamic acid, the concentration of which was also determined, as described below. The presence of abundant free glutamic acid allows lactic acid bacteria to produce GABA (through gad enzyme activity) without substrate limitation. Proteolytic activity, and in particular the ability to release glutamic acid from native proteins in a leguminous substrate, was therefore considered as an important criterion for the selection and identification of a new microbial starter for GABA production.

[0119] The peptides were analysed by OPA assay. Aqueous extracts, obtained as described above, were pre-treated with 5 pL / mL of 10% TFA. The contents of the eppendorfs were vortexed and subjected to a 10-minute centrifuge at 10000 rpm. At the end, the supernatant was recovered and analysed. Specifically, 730 pL of reaction mixture (Church et al., 1983, Journal of Dairy Science, 66, 1219-1227.) and 18.25 pL of extract were mixed and spectrophotometrically read at 340 nm. A triptone solution (0.10 - 2 mg / mL) was used for the calibration curve.

[0120] Instead, total free amino acids and glutamic acid concentration were determined in the samples after protein precipitation using 5-sulphosalicylic acid dihydrate (0.5 % w / v) (Sigma-Aldrich, Darmstadt, Germany), centrifugation and 0.22 pm filtration, using a Biochrom 30+ series Amino Acid Analyzer (Biochrom Ltd, Cambridge Science Park, England) with a cation exchange column (20 with an inner diameter of 0.46 cm), for post-column derivatisation. The result was expressed as mg / kg dry matter.

[0121] Two types of controls were added to the analyses, for each substrate, which were then used as references: uninoculated and unfermented substrates (i.e. chickpea flour (10% w / w) in water; lentil flour (10% w / w) in water; wheat flour (10% w / w) in water; substrates not inoculated but incubated under the same conditions as the fermented products (to highlight the activity of endogenous enzymes and indigenous microorganisms) .

[0122] 1.5 Determination of the GABA concentration

[0123] GABA was analysed in the samples after protein precipitation using 5-sulphosalicylic acid dihydrate (0.5 % w / v) (Sigma-Aldrich, Darmstadt, Germany), centrifugation and 0.22 pm filtration, using a Biochrom 30+ series Amino Acid Analyzer (Biochrom Ltd., Cambridge Science Park, England) with a cation exchange column (20 with an inner diameter of 0.46 cm), for post-column derivatisation. The result was expressed as mg / kg dry matter.

[0124] 1.6 Statistical analysis

[0125] The collected data were treated by one-way ANOVA and compared by Tukey's procedure (P<0.05).

[0126] 1.7 Identification of the selected starter and comparison with other GABA-producing starters

[0127] 1.7.1 Proteolytic activity and release of glutamic acid from native proteins

[0128] The production of GABA, as described above, is the result of several competing factors. If the basis of production is the need to use a microbial strain with a specific enzymatic activity, there is also a need for this to be expressed intensively under the process conditions and for the substrate to contain the precursor from which the functional compound is derived by enzymatic conversion, glutamic acid.

[0129] Glutamic acid is present within the polymeric structure of proteins, bound by peptide bonds to other amino acids, and its availability as a free amino acid (a necessary condition for conversion to GABA) is rare in nature or in any case requires rather costly technological (chemicalphysical) and synthetic steps.

[0130] The present invention has identified a microbial strain that is capable of producing high amounts of GABA but simultaneously has a marked ability to hydrolyse native substrate proteins (by means of pronounced proteolytic activity) leading to a natural and abundant release of free amino acids and glutamate, the precursor of the functional compound. The abundant release of glutamic acid underlies the improved synthesis of GABA compared to other previously identified microbial strains, as this is often present in limited amounts in natural substrates.

[0131] To quantify and compare the proteolytic activity of the different microbial strains examined, several analytical parameters were examined. The first indicator of proteolytic activity is the concentration of peptides. Peptides are protein fragments, generally consisting of linear amino acid sequences that are generated by an initial hydrolysis of proteins as a result of the activity of proteinases and endopeptidases. The release of peptides by Lactiplantibacillus plantarum DSM34115 was significantly higher than by the other microorganisms, in all matrices considered (Table 2). It is emphasised that the cluster of comparison microorganisms includes microorganisms previously selected for their protechnological properties, highlighting the exceptional nature of the newly identified strain (Table 2) .

[0132] In particular, Table 2 shows the peptide concentration (expressed as g / kg dry matter) in chickpea, lentil and wheat flour fermented with single-inoculum lactic acid bacteria, initial cell density 7 loglO CFU / mL).

[0133] Table 2 a~eDifferent letters within the column indicate significantly (P<0.05) different data

[0134] Together with peptides, proteolysis leads to the release of free amino acids, which are released from the peptide themselves, as a result of the activity of cytoplasmic exopeptidases. The concentration in free amino acids is considered the most important indicator of the proteolytic activity of a microbial strain. Also for this parameter, as shown for peptide release, the exceptionality of Lactiplantibacillus plantarum DSM34115 is confirmed, which shows, in all matrices considered, significantly (and markedly) higher concentrations than those found in samples inoculated with previously selected microorganisms included in the comparison (Table 3). In particular, Table 3 shows the total free amino acid concentration (expressed as mg / kg dry matter) in chickpea, lentil and wheat flours fermented with single-inoculum lactic acid bacteria, initial cell density 7 loglO CFU / mL).

[0135] Table 3

[0136] a~eDifferent letters within the column indicate significantly (P<0.05) different data

[0137] Analysis of the residual glutamic acid at the end of fermentation shows that proteolytic activity leads to a release of abundant substrate for GABA conversion in the medium. The concentrations found in samples inoculated and fermented with Lactiplantibacillus plantarum DSM34115 were significantly (P<0.05) and far higher than those found for the other selected microorganisms (Table 4). It is emphasised that this capacity of Lactiplantibacillus plantarum DSM34115 ensures abundant GABA production even under conditions of limited precursor availability in the fermentation substrate.

[0138] Table 4 shows the residual glutamic acid concentration (expressed as mg / kg dry matter) in chickpea, lentil and wheat flours fermented with single-inoculum lactic acid bacteria (initial cell density of 7 loglO CFU / mL), in comparison with the non-inoculated controls 1 and 2. Table 4

[0139] a~eDifferent letters within the column indicate significantly (P<0.05) different data

[0140] 1.7.2 GABA production

[0141] The concentrations of GABA in the fermented products were in only a few cases significantly higher than in the respective non-fermented matrices. All the strains used are lactic acid bacteria already identified as elective starters for the fermentation of vegetal matrices. In particular, some of them, such as Lactiplantibacillus plantarum H64, Lactiplantibacillus plantarum PU1, Lactiplantibacillus plantarum C48 and Lactiplantibacillus plantarum PR017 are already reported in literature as efficient GABA producers (Verni et al., 2022, Journal of Applied Microbiology, Volume 133, Issue 1, 1stJuly 2022, Pages 76-90; Siragusa et al., 2007, Applied and Environmental Microbiology, Vol. 73, No. 22). Other application examples where the strains included in the comparison have been reported as efficient GABA producers are provided below:

[0142] L. plantarum PU1 (previously classified as Lactococcus lactis PU1) has previously been selected as a producer of GABA in various matrices (Rizzello et al., 2008 Journal of Agricultural and Food Chemistry, 56:6936-43; Coda et al., 2010, International J. Food Microbiol., 137: 236- 245);

[0143] L. plantarum C48 was also previously selected as a starter for GABA production in cereals, pseudocereals and legumes by Coda et al., 2010 (International J. Food Microbiol., 137: 236-245).

[0144] L. plantarum H64 was recently selected as a starter for GABA production in a substrate obtained from waste bread by Verni et al., 2022 (Journal of Applied Microbiology, 00:1- 15)

[0145] The microorganism Lactiplantibacillus plantarum DSM34115 proved, on all substrates and under the same fermentation conditions considered, to be the microorganism capable of synthesising the highest quantities of functional amino acid. The specific results, in terms of y-aminobutyric acid concentration, expressed as mg / kg dry matter, are shown in Table 5.

[0146] Table 5

[0147] a~cData in the same column marked with different letters are significantly different.

[0148] Example 2.

[0149] Production of a fermented product enriched with GABA from legumes supplemented with carob pulp

[0150] 2.1 Substrate preparation

[0151] The substrate for the biotechnological production of GABA was obtained from the mixture of: Drinking water 90% w / v;

[0152] Mixture of chickpea and lentil flours (1:1 ratio), 10% w / v. The composition of the legume flour mixture had the following nutritional label: o Moisture: 12.2 % o Fat: 3.5 % o Proteins: 23.4 % o Carbohydrates: 60.8 %

[0153] ■ of which Fibre 15.1 % o Ash 0.8 % Carob pulp flour with the following composition: moisture 6 %; proteins 5 %; fat 0.5 % dry matter (dry matter); carbohydrates 51.6 % of d.m. (of which 31.7 sugars); fibre 37% of d.m., total polyphenols 3.5% of d.m., ash 2.5% of d.m.

[0154] The legume substrate with unfermented carob pulp supplement (SC) was mixed with a mechanical stirrer (150 rpm) for 12 minutes until a homogeneous suspension was obtained, prior to inoculation, at room temperature. The substrate had pH 5.210.4 and titration acidity (TTA) of 4.5 ±0.2.

[0155] 2.2 Inoculation and fermentation

[0156] The L. plantarum strain DSM34115 was propagated continuously in De Man, Rogosa and Sharpe medium (MRS, Oxoid Basingstoke, Hampshire, UK) at 30 °C every 24 hours. When used for fermentation, the microorganism was cultivated until the exponential growth phase was reached (approximately 16 hours). Subsequently, the cells were recovered by centrifugation at 9.000 x g at 4 °C for 10 min, washed twice in 50 mM phosphate buffer (4 °C, pH 7.0), resuspended in running water (final cell density of approximately 8 loglO CFU / mL) and used as a starter for the fermentation of the substrate mentioned in point 2.1. Specifically, the substrate was inoculated at a cell density of approximately 7 loglO CFU / mL, and fermentation was continued for 24 hours at 30 °C under mechanical agitation (100 rpm). At the end of the fermentation process, the substrate was refrigerated to 10 °C and processed within 4 hours of reaching this temperature.

[0157] 2.3 Fermented product stabilisation

[0158] The fermented substrate (SCr), suitably refrigerated to stop microbial activities, was intended for three different technological routes:

[0159] Subjected to freezing (-20°C).

[0160] Subjected to dehydration in ventilated desiccator at 70 °C after being arranged in trays (1 cm liquid thickness) (SCd).

[0161] Subjected to centrifugation for separation of microbial cells and insoluble particulate matter derived from the flours and then dehydrated in a ventilated desiccator at 70°C after being arranged in trays (1 cm liquid thickness) (SCc).

[0162] 2.4 Characterisation

[0163] The pH of the substrate was determined with the pH- meter M.507 (Crision, Milan, Italy) equipped with a food probe. The total titratable acidity (TTA) was determined on 10 g of sample homogenised with 90 mL of distilled water, and is expressed as the amount in mL of 0.1 M NaOH to reach pH 8.3.

[0164] The cell density of lactic acid bacteria was determined on 10 mL of each sample, resuspended in 90 mL of a sterile sodium chloride solution (0.9 % w / v) and homogenised at room temperature in a Bag Mixer 400 P (Interscience, St Nom, France) . The decimal dilutions were then seeded in MRS medium (Oxoid, Basingstoke, Hampshire, UK) supplemented with cycloheximide (0.1 g / L) and incubated for 48 hours at 30 °C.

[0165] GABA was analysed in the samples after protein precipitation using 5-sulphosalicylic acid dihydrate (0.5 % w / v) (Sigma-Aldrich, Darmstadt, Germany), centrifugation and 0.22 pm filtration, using a Biochrom 30+ series Amino Acid Analyzer (Biochrom Ltd., Cambridge Science Park, England) with a cation exchange column (20 with an inner diameter of 0.46 cm), for post-column derivatisation.

[0166] Protein (total nitrogen x 5.7), lipids, moisture, total fibre and ash content were determined as described by methods 46-11A, 30-10.01, 44-15A, 32-05.01 and 08-01.01 of the American Association of Cereal Chemists (AACC, 2010). Carbohydrates were calculated as the difference [100— (protein+lipids+ash+fibre)].

[0167] All the analyses were replicated three times on three different independently produced samples. The collected data were treated by one-way ANOVA and compared by Tukey's procedure (P<0.05). The detailed results are shown in Table 6 (unfermented substrate (SC), fermented product (SCr), fermented product after dehydration (SCd) and fermented product after dehydration preceded by centrifugation with removal of the insoluble precipitate (SCc)).

[0168] Table 6 a~dData in the same row marked with different letters are significantly different. As can be seen from the data in Table 3, the unfermented substrate contains negligible concentrations of GABA. Substantial concentrations are reached at the end of the fermentation process due to the synthesis activity of the selected producing microorganism L. plantarum DSM34115 (corresponding to 200mg / L). Dehydration of the liquid substrate leads to a GABA (non-volatile compound) concentration of just over 10 times. If the insoluble fraction is removed by centrifugation (as in SCc), the dehydrated preparation obtained (in pulverulent form) has less dietary fibre (insoluble and separated as pellets by centrifugation), while most of the other water-soluble substances not removed by centrifugation undergo a significant, though not statistically significant, increase, including GABA, which in SCc reaches 2500 mg / kg, concentrations slightly lower but still above 2400 mg / kg are found in Scd.

[0169] Example 3.

[0170] Production of a fermented product enriched with GABA obtained on a legume substrate enriched with grape must

[0171] 3.1 Substrate preparation

[0172] The substrate for the biotechnological production of GABA was obtained from the mixture of:

[0173] Drinking water 90% w / v;

[0174] Mixture of chickpea and lentil flours (1:1 ratio), 10% w / v. The composition of the legume flour mixture had the following nutritional label: o Moisture: 12.2 % o Fat: 3.5 % o Proteins: 23.4 % o Carbohydrates: 60.8 %

[0175] • Of which Fibre 15.1 % o Ash 0.8 %

[0176] Concentrated black grape must (whose fermentation has been stopped by dehydration, without addition of sulphur dioxide, having a fermentable sugar concentration of 6111% w / v), 1.65% (v / v)

[0177] The legume substrate supplemented with unfermented grape must (SM) was mixed with a mechanical stirrer (150 rpm) for 12 minutes until a homogeneous suspension was obtained, prior to inoculation, at room temperature. The substrate had pH 5.210.2 and titration acidity (TTA) of 4.5 10.2.

[0178] 3.2 Inoculation and fermentation

[0179] The L. plantarum strain DSM34115 was propagated continuously in De Man, Rogosa and Sharpe medium (MRS, Oxoid Basingstoke, Hampshire, UK) at 30 °C every 24 hours. When used for fermentation, the microorganism was cultivated until the exponential growth phase was reached (approximately 16 hours). Subsequently, the cells were recovered by centrifugation at 9.000 x g at 4 °C for 10 min, washed twice in 50 mM phosphate buffer (4 °C, pH 7.0), resuspended in running water (final cell density of approximately 8 loglO CFU / mL) and used as a starter for the fermentation of the substrate mentioned in point 3.1. Specifically, the substrate was inoculated at a cell density of approximately 7 loglO CFU / mL, and fermentation was continued for 24 hours at 30 °C under mechanical agitation (100 rpm). At the end of the fermentation process, the substrate was refrigerated to 10 °C and processed within 4 hours of reaching this temperature.

[0180] 3.3 Fermented product stabilisation

[0181] The refrigerated fermented substrate (SMr) was intended for three different technological routes:

[0182] Subjected to freezing (-20°C). Subjected to dehydration in ventilated desiccator at 70 °C after being arranged in trays (1 cm liquid thickness) (SMd).

[0183] Subjected to centrifugation for separation of microbial cells and insoluble particulate matter derived from the flours and then dehydrated in a ventilated desiccator at 70 °C after being arranged in trays (1 cm liquid thickness) (SMc).

[0184] 3.4 Characterisation

[0185] The pH of the substrate was determined with the pH- meter M.507 (Crision, Milan, Italy) equipped with a food probe. The total titratable acidity (TTA) was determined on 10 g of sample homogenised with 90 mL of distilled water, and is expressed as the amount in mL of 0.1 M NaOH to reach pH 8.3.

[0186] The cell density of lactic acid bacteria was determined on 10 mL of each sample, resuspended in 90 mL of a sterile sodium chloride solution (0.9 % w / v) and homogenised at room temperature in a Bag Mixer 400 P (Interscience, St Nom, France) . The decimal dilutions were then seeded in MRS medium (Oxoid, Basingstoke, Hampshire, UK) supplemented with cycloheximide (0.1 g / L) and incubated for 48 hours at 30 °C.

[0187] GABA was analysed in the samples after protein precipitation using 5-sulphosalicylic acid dihydrate (0.5 % w / v) (Sigma-Aldrich, Darmstadt, Germany), centrifugation and 0.22 pm filtration, using a Biochrom 30+ series Amino Acid Analyzer (Biochrom Ltd., Cambridge Science Park, England) with a cation exchange column (20 with an inner diameter of 0.46 cm), for post-column derivatisation.

[0188] Protein (total nitrogen x 5.7), lipids, moisture, total fibre and ash content were determined as described by methods 46-11A, 30-10.01, 44-15A, 32-05.01 and 08-01.01 of the

[0189] American Association of Cereal Chemists (AACC, 2010). Carbohydrates were calculated as the difference [100— (protein+lipids+ash+fibre)].

[0190] All the analyses were replicated three times on three different independently produced samples. The collected data were treated by one-way ANOVA and compared by Tukey's procedure (P<0.05). The detailed results are shown in Table 7 (unfermented substrate (SM), fermented substrate (SMr), fermented substrate after dehydration (SMd) and fermented substrate after dehydration preceded by centrifugation with removal of the insoluble precipitate (SMc)). Table 7 adData in the same row marked with different letters are significantly different.

[0191] As can be seen from the data in Table 4, even in the case of the substrate supplemented with grape must, the unfermented matrix contains negligible concentrations of GABA, which, however, reach substantial concentrations at the end of the fermentation process due to the synthesis activity of the selected producing microorganism L. plantarum DSM34115 (corresponding to approximately 188 mg / L) . Dehydration of the liquid substrate leads to a GABA (non-volatile compound) concentration of just over 10 times. If the insoluble fraction is removed by centrifugation (as in SMc), the dehydrated preparation obtained (in pulverulent form) has less dietary fibre (insoluble and separated as pellets by centrifugation), while most of the other water- soluble substances not removed by centrifugation undergo a significant, though not statistically significant, increase, including GABA, which in SMc reaches 2500 mg / kg, concentrations slightly lower but still above 2400 mg / kg are found in SMd. There is no substantial difference in the final result in terms of GABA concentration when comparing the substrate supplemented with carob pulp (previous example) with must supplementation.

[0192] Example 4.

[0193] Production of yoghurt from semi-skimmed cow’s milk with high GABA content

[0194] - Yoghurt production

[0195] Commercial pasteurised semi-skimmed cow's milk with the following composition was used for the preparation of a yoghurt: water 88.50 proteins 3.3% carbohydrates 4.8%, of which fibre 0 fat 1.98% ash 0.55%

[0196] The milk was inoculated with Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus in a freeze-dried phase in a 1:1 ratio (log inoculum density 7.00 CFU / mL each). The milk was incubated at a temperature of 41°C in agitation for 8h.

[0197] The product enriched with GABA in the SCd and SCc forms of Example 2 was added to the yoghurt (pH 4.3± 0.2) at the rate of 2% (w / v) (Y-SCd and Y-SCc). The preparation was mixed with mechanical stirring and curd breaking, refrigerated at 4°C and analysed during the subsequent 24 h storage period.

[0198] An unsupplemented sample was used as a control (Y-ct).

[0199] - Yoghurt characterisation

[0200] The product was characterised nutritionally (moisture, proteins, dietary fibre, fat, ash, carbohydrates, and energy value). The methods used for the determination of moisture, fibre, protein, fat and ash were ISO 5534:2004, AOAC 985.29, ISO 8968-1:2014, ISO 1211:2010, ISO / CD 9877, respectively. The carbohydrates were calculated by difference as stated in Reg. No. 77 of 16thFebruary 1993 while the energy value as stated in Reg. EU 1169 / 2011. In addition, pH, TTA, GABA concentration were determined as described above. The viscosity was also determined using an A & D SV-10 viscometer (A & D Company Ltd., Japan). The characterization results obtained are shown in the following Table 8.

[0201] Table 8 a~cData in the same row marked with different letters are significantly different.

[0202] Sensory analysis The sensory analysis was conducted by a panel of 10 trained tasters (5 men and 5 women, average age: 32 years old, range: 20-48 years) at the Department of Soil, Plant and Food Sciences, University of Bari. Prior to sensory analysis, the drinks were stored under refrigerated conditions for approx. 24 hours and then poured into glasses for tasting. Each taster received one glass per sample. The sensory attributes selected for describing the organoleptic profile of the drinks are shown in Table 9. A scale from 0 to 10, with 10 being the maximum value, was used to describe the individual attributes. The sensory attributes were selected and discussed with the tasters in an introductory session .

[0203] Table 9

[0204]

[0205] The results of the sensory analysis are illustrated in Figure 2.

[0206] Conclusions

[0207] As can be seen from the results of the study, many of the analytical parameters are not affected by the addition of the fermented product obtained by the method of the invention in its dehydrated form. It is important to emphasise that the pH values are not altered, nor is the survival of the starter lactic acid bacteria, whose viability for this type of product (conventional yoghurt) must necessarily remain above a threshold regulated by law. However, a significant increase in carbohydrates is possible. The concentration of GABA in fortified products reaches 60mg / 100ml (600 mg / 1) or more. The colourimetric coordinates of the product vary significantly, especially for the brightness parameters (L), which decreases in fortified products, and red index "a" (which increases in fortified products). From a sensory point of view, it is clear that the use of the dehydrated fermented product not separated from the solid fraction in suspension (SCd) markedly influences the aftertaste of the product, unlike the SCc formulation.

[0208] Example 5. Production of vegetal yoghurt-like products with high GABA content

[0209] - Production of vegetal yoghurt

[0210] A vegetal yoghurt-like drink was produced using rice flour with the following composition: Proteins 9.8% of d.m. Carbohydrates 86.4% of d.m. of which fibre 3.8% of d.m.. Fat 3.0 % of d.m.. Ash 0.7% of d.m..

[0211] The rice flour was resuspended in 20% drinking water, supplemented with 1% food grade glucose, and the suspension was heat-treated at 80°C for 15 minutes, as a bivalent intervention aimed at pasteurising the substrate and gelatinising the starch in the preparation. The microorganism Lactiplantibacillus plantarum DSM33326, was used as a starter for the fermentation process. The cells, in freeze dried form, were added to the preparation to obtain an initial cell density of log 7.00 CFU / mL each. The preparation was incubated at a temperature of 30°C under agitation for 16h.

[0212] At the end of fermentation (pH 4.3± 0.2), SCd and SCc were added at a rate of 2% (w / v) (YL-SCd and YL-SCc). The preparation was mixed with mechanical stirring and refrigerated at 4 °C and analysed during the following 24h storage period.

[0213] An unsupplemented sample was used as a control (YL-ct).

[0214] - Yoghurt characterisation

[0215] The product was characterised nutritionally (proteins, carbohydrates and dietary fibre, fat, ash and energy value).

[0216] The methods used for the determination of moisture, proteins, dietary fibre, fat and ash were respectively ISO 712: 2010, ISO 16634: 2016 (Part 2), AOAC 985.29, Ministerial Decree No. 4 of 23rdJuly 1994, ISO 2171: 2007. The carbohydrates were calculated by difference as stated in Reg. No. 77 of 16thFebruary 1993 while the energy value as stated in Reg. EU 1169 / 2011.

[0217] In addition, pH, TTA, GABA concentration were determined as described above. The viscosity was also determined using an A & D SV-10 viscometer (A & D Company Ltd., Japan).

[0218] As the samples consisted almost entirely of grains, the characterisation included the determination of the starch hydrolysis index (HI), an in vitro parameter determined as a predictive index of the glycaemic index in vivo. The determination of the starch hydrolysis index was performed by applying a protocol that mimics in vivo digestion processes as described by De Angelis et al. (De Angelis et al., 2009, European Food Research and Technology, 229(4), 593-601) .

[0219] First, the total starch content of the samples was determined using the Megazyme K-RSTAR kit (Megazyme International Ireland Limited, Bray, Ireland), with the aim of estimating the portion of the sample containing 1g of starch. Subsequently, aliquots containing 1 g starch were subjected to an enzymatic treatment after which the concentration of released glucose was determined with a suitable D-glucose assay kit (GOPOD Format, Megazyme) according to the manufacturer's instructions. The degree of starch digestion was expressed as the percentage of potentially available starch hydrolysed after 180 minutes. Soft wheat bread was used as a reference to estimate the hydrolysis index (HI=100).

[0220] All the analyses were replicated three times on three different independently produced samples. The collected data were treated by one-way ANOVA and compared by Tukey's procedure (P<0.05).

[0221] The characterisation data are shown in Table 10 below (YL-SCd=supplemented yoghurt-like product enriched with GABA SCd; YL-SCc=supplemented yoghurt-like product enriched with GABA See).

[0222] Table 10.

[0223] a~cData in the same row marked with different letters are significantly different.

[0224] Sensory analysis

[0225] The sensory analysis was conducted by a panel of 10 trained tasters (5 men and 5 women, average age: 32 years old, range: 20-48 years) at the Department of Soil, Plant and Food Sciences, University of Bari. Prior to sensory analysis, the drinks were stored under refrigerated conditions for about 24 hours and then poured into glasses for tasting. Each taster received one glass per sample. The sensory attributes selected for describing the organoleptic profile of the drinks are shown in Table 6 above. A scale from 0 to 10 was used to describe the individual attributes, with 10 being the maximum value. The sensory attributes were selected and discussed with the tasters in an introductory session. The results of the sensory analysis are shown in Figure 3.

[0226] Conclusions

[0227] Many of the analytical parameters are unaffected by the addition of the fermented product covered by the present invention, in its dehydrated form. It is important to underline that the pH is not altered, nor is the survival of the starter lactic acid bacteria. However, there is a significant increase in protein, fibre and polyphenolic compounds. The concentration of GABA in fortified products reaches 60mg / 100mL (600 mg / L) or more. The colourimetric coordinates of the product vary significantly, especially for the brightness parameters (L), which decreases in fortified products, and red index "a", which increases in fortified products. The starch hydrolysis index HI, which correlates with the in vivo glycaemic index, does not change significantly (fortified products vs. control). From a sensory point of view, it is clear that the use of the dehydrated ingredient not separated from the solid fraction in suspension (SCd) markedly influences the aftertaste of the product, unlike the SCc formulation. In addition, the SCd formulation leads to an increase in the perception of "particles" in the final product and consequently to a lower score for "uniformity".

[0228] The results of the sensory analysis are shown in Figure 3.

[0229] EXAMPLE 6.

[0230] Production of bread from wheat flour with high GABA content

[0231] Production of bread from wheat flour

[0232] A commercial common wheat flour with the following composition was used for the production of wheat bread: moisture, 14.5 % proteins, 10.0 % (11.75 % of d.m.) carbohydrates, 71 % (83.43% of d.m.) fibre, 2.5 % (2.94 % of d.m.) fat, 1.0 % (1.17 % of d.m.) ash, 0.6 % (0.70 % of d.m.)

[0233] The flour was mixed with drinking water (62.5% w / w flour in water, using a kneading machine (M 5-8 spiral kneading machine, Mecnosud, Flumeri)).

[0234] 2% w / w of the fermented product enriched with GABA was added to the dough in the SCd or SCc forms (in the breads coded as P-SCd and P-SCc, respectively).

[0235] 1.5% w / w commercial brewer's yeast in compressed form was added to the two doughs plus a third obtained without fortification for the production of a control bread (P-ct).

[0236] The doughs were incubated in a leavening cell at 28°C for 1.5 hours and then baked in an oven (Combo 3, Zucchelli, Verona) at a temperature of 200°C for about 45 minutes.

[0237] - Bread characterisation

[0238] The pH of the doughs before baking was determined using a FiveEasy Plus pH meter (Mettler-Toledo, Columbus, Ohio, USA); the total titratable acidity (TTA) was determined by titrating the acidity of 10g of sample with a 0.1 M NaOH solution to pH 8.4.

[0239] The nutritional label of the breads was determined using official methods. In detail, moisture, proteins, dietary fibre, fat and ash were determined, respectively, using ISO 712: 2010, ISO 16634: 2016 (Part 2), AOAC 985.29, Ministerial Decree No. 4 of 23rdJuly 1994, ISO 2171: 2007, while the carbohydrates were calculated by difference as indicated in Legislative Decree No. 77 of 16thFebruary 1993.

[0240] GABA was analysed in the breads after protein precipitation using 5-sulphosalicylic acid dihydrate (0.5 % w / v) (Sigma-Aldrich, Darmstadt, Germany), centrifugation and 0.22 pm filtration, using a Biochrom 30+ series Amino Acid Analyzer (Biochrom Ltd., Cambridge Science Park, England) with a cation exchange column (20 with an inner diameter of 0.46 cm), for post-column derivatisation.

[0241] The specific volume of the breads was measured by moving rape seeds in accordance with the AACC International method 10-05.01 (2001) and calculated as the ratio of volume to mass (cm3 / g).

[0242] The structural characteristics of the breads were determined by Texture Profile Analysis (TPA), using a FRTS- 100N texture analyser (Imada, Toyohashi, Japan) equipped with an FR-HA-30J cylindrical probe. The analyses were conducted using the following parameters: test speed 1 mm / s, 30% sample deformation and two compression cycles. The indices measured were: hardness, cohesiveness and chewiness.

[0243] The colour coordinates of the crust (measured with a Minolta CR-10 colorimeter) were reported as L, a and b coordinates.

[0244] As the samples consisted almost entirely of grains, the characterisation included the determination of the starch hydrolysis index (HI) in vitro parameter determined as a predictive index of the glycaemic index in vivo. The determination of the starch hydrolysis index (HI) was carried out by applying the protocol described above.

[0245] The data collected in all described analyses were treated by one-way ANOVA and compared by Tukey's procedure (P<0.05).

[0246] The results of the characterisation of the breads are shown in Table 11 below.

[0247] Table 11

[0248]

[0249] Sensory analysis

[0250] The sensory analysis of the breads produced was conducted by 10 trained tasters (5 men and 5 women, average age: 32 years old, range: 20-48 years old). Prior to sensory analysis, the breads were cooled to room temperature for 5-6 hours and cut into 1.5 cm thick slices. Each taster received one slice per sample. The attributes used to describe the bread's organoleptic characteristics are shown in Table 9 and a scale from 0 to 10, with 10 being the maximum value, was used to describe the individual attributes. The sensory attributes were discussed with the tasters in an introductory session.

[0251] The data collected in all described analyses were treated by one-way ANOVA and compared by Tukey's procedure (P<0.05).

[0252] Table 12

[0253]

[0254] Conclusions

[0255] The addition of the GABA-rich ingredient of the present invention produces minimal effects on the main characteristics of the bakery product, especially when the SCc form, obtained after separation of the insoluble fraction by centrifugation, is used. A very slight change in the nutritional label includes the increase in carbohydrates, which, however, does not correspond to significant changes in either the energy value or the starch hydrolysis index (which is actually slightly lower in bread supplemented with Scd, which has a higher fibre content). The addition of SCd, unlike See, causes a slight but significant decrease in the specific volume of the bread and an increase in its hardness. Both forms of the ingredient cause an appreciable decrease in the brightness of the crust with changes in the colorimetric indices. Indeed, amino acids and added sugars promote the Maillard reaction during cooking. The GABA content is unaffected by processing and cooking and is greater than or equal to 50mg / 100g (500mg / kg) in both fortified formulations. Few variations were discernible in taste in the panel test; certainly noteworthy is that in the Scd form the ingredient causes an increase in the perception of the "sour" sensory attribute (Figure 4).

[0256] EXAMPLE 7.

[0257] Production of gluten-free bread with high GABA content

[0258] Production of gluten-free bread

[0259] For the production of gluten-free bread, a mixture of commercial maize flour and rice flour in a 1:1 ratio with the following composition was used: Maize flour moisture, 13.6 % proteins, 7.2 % (8.3 % of d.m.) carbohydrates, 73.0 % (84.5 % of d.m.) fibre, 4.5 % (5.2 % of d.m.) fat, 1.3 % (1.5 % of d.m.) ash, 0.4 % (0.5 % of d.m.)

[0260] Rice flour moisture, 11.2 % proteins, 7.4 % (8.3 % of d.m.) carbohydrates, 80.0 % (90.1 % of d.m.) fibre, 0.5 % (0.6 % of d.m.) fat, 0.6 % (0.7 % of d.m.) ash, 0.3 % (0.3 % of d.m.)

[0261] The flour was mixed with drinking water (62.5% w / w flour in water, using a kneading machine (M 5-8 spiral kneading machine, Mecnosud, Flumeri)).

[0262] The following were added to the dough:

[0263] - 2% w / w of the ingredient with a high GABA content in the SCd or SCc forms (in the breads coded as Pgf-SCd and Pgf-SCc, respectively).

[0264] - 1.5% w / w commercial brewer's yeast in tablet form;

[0265] - 1% w / w xanthan gum as a structuring agent.

[0266] A third dough without fortifications was produced to obtain a gluten-free bread to be used as a control (Pgf-ct).

[0267] The doughs were incubated in a leavening cell at 28°C for 2.0 h and then baked in a Combo 3 oven (Zucchelli, Verona) at a temperature of 200°C for approximately 45 minutes .

[0268] - Characterisation of the gluten-free bread

[0269] The characterisation of the breads, including sensory and statistical analysis, was carried out as previously described in section 4.2 for the characterisation of wheat breads .

[0270] The results of the characterisation of the breads are shown in Table 13 below.

[0271] Table 13

[0272]

[0273] - Sensory analysis The sensory profile of the gluten-free bread control (Pgf-ct) and gluten-free breads fortified with SCd (Pgf-Scd) and SCc (Pgf-SCc) at 2% w / w is shown in Figure 5.

[0274] Conclusions

[0275] The addition of the GABA-rich ingredient of the present invention produces minimal effects on the main characteristics of the bakery product, especially when the SCc form, obtained after separation of the insoluble fraction by centrifugation, is used. A very slight change in the nutritional label includes the increase in protein, which, however, does not correspond to significant changes in either the energy value or the starch hydrolysis index (which is actually slightly lower in fortified breads). The addition of SCd, and See increased the hardness of the breads, but had no effect on the specific volume of the products. Both forms of the ingredient cause an appreciable decrease in the brightness of the crust with changes in the colorimetric indices. Amino acids and added sugars promote the Maillard reaction during cooking. The GABA content is unaffected by processing and cooking and is greater than or equal to 52mg / 100g (520mg / kg) in both fortified formulations. Among the variations perceptible in the panel test, it should certainly be emphasised that in the Scd form the ingredient causes an increase in the perception of the "sour" sensory attribute (Figure 5) and that the SCc ingredient positively influences the chewability, aroma and elasticity of the gluten-free bread.

Claims

CLAIMS1.- Lactiplantibacillus plantarum CL3 DSM34115.2.- Use of Lactiplantibacillus plantarum CL3 DSM34115 for the preparation of Y-amin°butyric acid or of a product enriched with Y-amin°butyric acid.

3. - Use according to claim 2 characterized in that said product enriched with Y-amin°butyric acid is a food product, a nutraceutical product, a pharmaceutical product or a cosmetic product.4.- Method for the production of Y-amin°butyric acid or of a product enriched with Y-amin°butyric acid comprising the steps of: a) preparing a substrate for the cultivation of Lactiplantibacillus plantarum CL3 DSM34115 comprising a vegetal matrix and water and having a pH between 5 and 6.5; b) inoculate said substrate with Lactiplantibacillus plantarum CL3 DSM34115; c) ferment said substrate for between 18 and 72 hours at a temperature between 20 and 40°C to obtain a product enriched in Y-amin°butyric acid; d) optionally extract Y-amin°butyric acid.5.- Method according to claim 4, characterized in that said vegetal matrix is a legume flour.6.- Method according to claim 5, characterized in that said legume flour is a legume flour selected from the group consisting of bean, pea, broad-bean, lupin, chickpea, pigeon pea, peanut, soy bean, lentil, grass pea, carob, and mixtures thereof .7.- Method according to claim 5, characterized in that said legume flour is a mixture of chickpea flour and lentilflour.8.- Method according to claim 4, characterized in that said step (a) is conducted in the presence of a sugar matrix.9.- Method according to claim 8, characterized in that said sugar matrix is chosen from the group consisting of grape must and carob pulp.10.- Method according to claim 4, characterized in that said vegetal matrix is present in an amount between 5 and 30% by weight on the final volume of the substrate and said water is present in an amount between 50 and 95% by weight on the final volume of the substrate.11.- Method according to claim 4, characterized in that said inoculum is performed at a density between 105and 109CFU / ml.12.- Fermented product comprising at least one legume flour, water and Lactiplantibacillus plantarum DSM 34115.13.- Fermented product according to claim 12 further comprising a sugar matrix selected from the group consisting of grape must and carob pulp.14.- Food, nutraceutical, pharmaceutical or cosmetic product comprising the fermented product according to any of claims 12 and 13.

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