GABA-enriched food products with health benefits

Abstract

The present invention relates to a natural extract, preferably a yeast extract, which is rich in gamma-aminobutyric acid (GABA), to a product comprising such an extract and to the use thereof for preparing food products with health and well-being benefits intended for humans or animals. Such foodstuffs are in particular biscuits, treats or pâtés, or bakery products such as bread.

Description

[0001] GABA-ENRICHED FOOD PRODUCTS THAT ARE BENEFICIAL TO HEALTH

[0002] technical field

[0003] The present invention falls within the fields of agri-food and health, seeking natural solutions to improve the nutritional and medicinal qualities of ingested products. The solution proposed in this application is the provision of a natural extract rich in GABA, whose beneficial health properties are well-established (Pydi et al., J Biol Chem., 2014, 289(36): 25054-66; Sasaki et al., J Biol Macromol., 2014, 14(1): 43-57). For example, incorporating such an extract during a bread-making process can produce bread that is "healthy" for humans.

[0004] Prior state of the art

[0005] The non-proteinogenic amino acid GABA (gamma-aminobutyric acid) is naturally present in microorganisms, plants, and animals, where this bioactive compound plays various roles. GABA is thought to be involved in mammalian behavior by regulating stress and anxiety, modulating cognitive and brain functions, promoting sleep, and improving mood (Rashmi et al., 2018, Studies in Natural Products Chemistry, 57: 413-452).

[0006] In practice, positive effects of GABA administration in the context of certain chronic diseases have been observed in clinical trials:

[0007] Inoue et al. (2003, European Journal of Clinical Nutrition, 57: 490-495) conducted a randomized, single-blind, placebo-controlled trial in 39 hypertensive patients to evaluate the effect of daily intake of 100 ml of fermented milk containing 10 to 12 mg of GABA for 12 weeks. They observed a decrease in blood pressure in these patients and concluded that daily intake of fermented milk containing GABA may be useful for controlling blood pressure levels in mildly hypertensive patients.

[0008] Okada et al. (2000, Journal of the Japanese Society for Food Science and Technology, 47(8): 596-603), in a double-blind trial, identified a potential treatment for patients suffering from neurological disorders (insomnia, depression, and autonomic dysfunction) with the intake of sprouted rice enriched with GABA (26.4 mg / day). The recommended intake and toxic dose of GABA have not yet been established, but clinical studies based on the administration of a high dose of this compound (300 mg per day) revealed no serious adverse effects after four weeks of consumption (Byun et al., 2018, J Clin Neurol., 14(3): 291-295).

[0009] The human body is capable of producing its own reserves of GABA, but in small quantities, which can be further inhibited by a lack of estrogen, zinc, or vitamins, or by an excess of salicylic acid and food additives. Moreover, the typical daily human diet is relatively low in GABA, certainly insufficient to produce significant biological effects.

[0010] Therefore, it has been considered to provide exogenous GABA to humans to maintain or even improve their health.

[0011] Today, most GABA products are available as dietary supplements (tablets and capsules), while only a few GABA-containing foods and beverages are commercially available. For example, Kittibunchakul et al. (2021, Journal of Functional Foods, 86, 1047140) proposed a probiotic beverage based on brown rice, naturally rich in GABA, fermented using the lactic acid bacteria Lactobacillus penlosiis, which also produces GABA.

[0012] However, there is a clear need to develop new GABA-rich foods that are advantageously of natural origin, including foods that are universally and routinely consumed.

[0013] The aim of the present invention is to provide a natural extract containing gamma-aminobutyric acid (GABA) in a complex matrix (peptides, amino acids, RNA, etc.). Thus, the incorporation of such an extract into an ingestible product makes it possible to provide a sufficient quantity of GABA to obtain a beneficial effect on human or animal health, in a natural context also rich in other potentially useful compounds (in terms of nutrition, health, taste, etc.).

[0014] For example, document W02006 / 064791 describes a bacteria / yeast co-culture in a medium containing glutamic acid (e.g., soy) to obtain GABA for incorporation into food. Document JP4838628 describes the use of lactic acid bacteria to produce health-promoting GABA from asparagus. Document JP2001352940 describes the use of lactic acid bacteria to convert glutamic acid present in food materials (rice, wheat, etc.) into GABA. Document JP2022113666 describes fermented products containing GABA, and document W02010 / 027117 describes fermented algae containing GABA.

[0015] Detailed description of the invention

[0016] Thus, according to a first aspect, the present invention relates to a natural extract rich in gamma-aminobutyric acid (GABA).

[0017] For the purposes of this invention, a "GABA-rich extract" is defined as an extract containing at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%, or even at least 11, 12, 13, 14, or even at least 15% by weight of GABA, the percentage by weight being expressed relative to the dry weight of the extract. Advantageously, the extract contains at least 5% by weight of GABA. A content between 5 and 15% is particularly suitable for the purposes of this invention.

[0018] In the context of the invention, a natural extract is defined as one obtained from an element of nature, in particular from plant matter or from a microorganism such as a yeast or a bacterium. According to a particular embodiment, the extract according to the invention is obtained from a yeast, hereinafter referred to as "yeast extract" ("YE" for Yeast Extract).

[0019] In general, yeast extracts are products known to those skilled in the art. According to the invention, "yeast extract" (or "yeast hydrolysate" or "yeast peptone") refers to the soluble fraction obtained after thermal, mechanical (using known methods such as high-pressure homogenization, mechanical milling, mechanical lysis using glass beads, ultrasonic disintegration, repeated freeze-thaw cycles, or osmotic shock), or enzymatic lysis (using an enzyme exogenous or endogenous to the yeast)... The objective of such lysis is to release the internal macromolecules of said yeast in their native state, in particular the pool of free amino acids, including free glutamic acid. The resulting co-products correspond to the insoluble fraction called "yeast hulls" and can be used in other processes.The processes for obtaining yeast hulls and yeast extracts are well known in the art (see, for example, the reference work “Yeast Technology”, 2nd edition, 1991, G. Reed and TW Nogodawithana, published by Van Nostrand Reinhold, New York, ISBN 0-442-31892-8). The insoluble and / or soluble fractions can then be dried.

[0020] Thus, a yeast extract can be presented in dry form, preferably as a fine, water-soluble powder, in liquid form or even as a concentrated liquid, or as a paste. A yeast extract consists mainly of protein, preferably at least 55% protein.

[0021] The difficulty overcome by the present invention is to obtain a natural extract rich in GABA, without the addition of exogenous GABA. Indeed, some plants, such as broccoli or sweet potatoes, contain GABA but in very small quantities, unusable for the desired properties.

[0022] Advantageously, the natural extract used in the context of the present invention is an extract naturally rich in glutamic acid (or glutamate). It should be noted that, in the context of this invention, "glutamic acid" (or "glutamate") refers to the amino acid in its free form, not incorporated into a peptide or protein.

[0023] In particular, an extract is said to be "rich in glutamic acid" if it contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%, or even 11, 12, 13, 14, 15, 20, or 25% by weight of free glutamic acid, the percentage by weight being expressed relative to the dry weight of the extract. Advantageously, the extract comprises at least 10% by weight of free glutamic acid. A content between 5 and 25%, for example between 10 and 20%, is particularly suitable for the purposes of the present invention. Natural extracts meeting this definition include, for example, yeast extracts, bacterial extracts, or plant extracts, advantageously yeast extracts.

[0024] According to a particular embodiment, such an extract is obtained from an organism, especially a plant or microorganism such as yeast or bacteria, capable of synthesizing or even secreting glutamic acid, advantageously in large quantities. As is known to those skilled in the art, the organism, advantageously a yeast, may be naturally capable of synthesizing glutamic acid in large quantities, or genetically modified for this purpose. Alternatively, glutamic acid present in the medium may be transported into and by the organism, advantageously a yeast.

[0025] Thus, and according to a particular aspect, the present invention relates to a yeast extract containing gamma-aminobutyric acid (GABA), said GABA being obtained from glutamic acid present as a free amino acid in said yeast.

[0026] An extract according to the invention differs from synthetic or purified GABA in that it also contains the components of the natural extract, particularly yeast extract, thus constituting an enriched form. A yeast extract according to the invention can be obtained from any yeast, advantageously meeting the above definition. Preferably, the yeast strain used for preparing the extract according to the invention belongs to the genus Saccharomyces, Kluyveromyces, or Candida (also known as Pichia or I. indnera). Preferably, the yeast strain used for preparing the extract belongs to the genus Saccharomyces and, more particularly, to the species Saccharomyces cerevisiae.

[0027] Obtaining GABA from glutamic acid involves the activation of glutamate decarboxylase (GAD) activity capable of ensuring the following conversion:

[0028] L-Glutamate + H+ GABA + CO2

[0029] In another aspect, the invention relates to a process for obtaining an extract, advantageously a yeast extract as described above, and the extract thus obtained. Advantageously, such a process comprises the following steps:

[0030] - preparation of yeast extract from yeast containing glutamic acid in the form of a free amino acid;

[0031] - incubation of the extract in the presence of a glutamate decarboxylase (GAD) enzyme.

[0032] At the end of this step, the resulting extract is low in glutamic acid, or even free of glutamic acid. For the purposes of this invention, a "low-glutamic acid extract" is defined as an extract that advantageously comprises less than 10%, or even less than 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or even 0.1% by weight of glutamic acid in free form, the percentage by weight being expressed relative to the dry weight of the extract. A "glutamic acid-free or glutamic acid-deprived extract" is defined as an extract that does not contain glutamic acid in free form, corresponding to the case where all the free glutamic acid present in the extract has been converted to GABA.

[0033] As is known to those skilled in the art, a natural extract, advantageously a yeast extract, can be incubated in the presence of such an enzyme under conditions, particularly of temperature and pH, that ensure this conversion. These conditions vary depending on the origin of the GAD enzyme. For example, and in relation to the enzyme produced by Levilactobacillus brevis, these conditions are:

[0034] - an acidic pH, for example of 5; and

[0035] - a temperature between 30 and 35°C, for example equal to 33°C.

[0036] Alternatively, a natural extract, advantageously a GABA-rich yeast extract according to the invention, is obtained via a bacterium exhibiting suitable glutamate decarboxylase activity. Thus, the bacterium implemented within the framework of the invention exhibits glutamate decarboxylase (GAD) activity; in other words, it is capable of producing a glutamate decarboxylase (GAD) enzyme. The proteins involved in this metabolic pathway, in particular the GadA and / or GadB genes encoding the enzyme and the GadC gene encoding the substrate transporter, are extensively documented in the prior art; see, for example, the review by Yogeswara et al. (Microorganisms 2020, 8(12), 1923; https: / / doi.org / 10.3390 / microorganisms8121923) in relation to lactic acid bacteria.

[0037] According to a particular embodiment, a bacterium of interest carries a GadA and / or GadB gene encoding the GAD enzyme.

[0038] According to another embodiment, the bacterium also carries a GadC gene encoding a glutamate and GABA transporter.

[0039] These can be endogenous or exogenous genes, single or multicopy, chromosomally integrated or carried by a plasmid, and placed under the control of regulatory elements allowing their expression, such as a promoter. In a particular embodiment, they are called endogenous genes.

[0040] It has been shown that lactic acid bacteria, particularly those of the Lactobacillaceae family, are especially well-suited for implementation within the scope of the present invention. The bacteria listed below can be used alone or in combination.

[0041] Bacteria carrying at least one GadA and / or GadB gene are, for example, chosen from the group including: Lactobacillus plantarum.

[0042] Des baccières porteuses d'au moins un gène GadA et / ou GadB et d'au moins un gène GadC sont par exemple choisi dans le groupe comprendre les genres suivants : Levilactobacilus, I.enlilaclobacilus. Lactococcus, I. aclicaseibacillus. Furfurilactobacillus, I. euconosloc. Lactiplantibacillus, Bifidobacterium, Pediococcus, Enterococcus and Limosilactobacillus.

[0043] For example, it may be: Levilactobacillus brevis, Lentilactobacillus buchneri, Lactococcus lactis, Lacticaseibacillus paracasei, Furfurilactobacillus rossiae, Enterococcus faecium, Leuconostoc suionicum, Lactobacillus amylovorus, Lactiplantibacillus plantarum, Bifidobacterium dentium, Bifidobacterium adolescentis, Bifidobacterium angulatum, Pediococcus pentosaceus, Enterococcus casseliflavus, Limosilactobacillus reuteri, Enterococcus gallinarum, Limosilactobacillus oris, Pediococcus acidilactici, I mosi lactobacillus fermentum and Enterococcus hermanniensis.

[0044] Preferably, the bacterium, used alone or in mixture, belongs to a species chosen from the following group: Levilactobacilus brevis, Lentilactobacilus buchneri, and Lactococcus lactis, advantageously Levilactobacilus brevis.

[0045] As is known to those skilled in the art, the bacterium is cultured under conditions favorable to the growth of bacteria of the Lactobacillaceae family, for example, in an MRS (Man Rogosa, Sharpe) type culture medium. The culture conditions, particularly those relating to pH, temperature, and aerobic or anaerobic conditions (partial or total), depend on the strain chosen. As is known and as described in the examples, a strain of Levilactobacillus brevis is advantageously cultured under partially anaerobic conditions, at a temperature between 30 and 35°C and at a slightly acidic pH, for example, 6.2.

[0046] Advantageously, the bacterium is further cultured under conditions that promote the expression of genes in the pathway, including GadA, GadB and / or GadC.

[0047] At the end of growth and for the conversion stage, the culture thus obtained can be used as is, or in dried or even freeze-dried form, provided that these treatments do not affect the enzymatic activity.

[0048] By using a bacterium equipped with the GadC-encoded transporter, whole cells can be implemented to ensure the conversion of glutamic acid from the natural extract into GABA.

[0049] Alternatively, and especially when the bacteria do not possess such a transporter, the bacteria are subjected to lysis, then possibly centrifuged, and it is the lysate containing the enzyme of interest, possibly purified, that is used.

[0050] The extract, advantageously of yeast, is then incubated with the bacterial culture, possibly in the form of a bacterial lysate.

[0051] In one embodiment, the free glutamic acid content of the extract, particularly yeast extract, is adjusted. In practice, the extract is advantageously used in this incubation step at a concentration of at least 100, 150, 200, 250, or even 300 g / L, for example, between 150 g / L and 250 g / L. In another embodiment, the conversion is achieved by contacting the extract, advantageously yeast extract, as detailed above, with the bacteria, namely the bacterial must or bacterial lysate, as described above.

[0052] Incubation is advantageously carried out under conditions adapted to the bacterium used or to the enzyme in the case of a bacterial lysate.

[0053] Thus, as is well known to those skilled in the art, incubation conditions are adjusted according to the bacterial strain and the enzyme present to ensure optimal conversion activity. In this sense, the medium and incubation conditions are conducive to enzymatic activity and are not intended to promote bacterial growth.

[0054] It should also be noted that in this stage, there can be no growth of the yeast since it is in extract form and therefore in an inactivated form.

[0055] According to one embodiment, the incubation medium is not a medium for bacterial culture and consists solely of yeast extract and bacterial culture, optionally lysed.

[0056] Other conditions suitable for this conversion stage are:

[0057] - a temperature between 30 and 40°C,

[0058] - a pH between 5 and 7;

[0059] - an incubation time of approximately 15 to 70 hours, preferably between 24 and 70 hours.

[0060] The conversion step is carried out until the desired amount of free glutamic acid is consumed. For the purposes of this application concerning the use of the final extract for health, and assuming no GABA toxicity, the conversion to GABA must be optimal, advantageously exceeding 90%. As previously stated, the final extract advantageously comprises less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%, or even 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or even 0.1% by weight of free glutamic acid, the weight percentage being expressed relative to the dry weight of the extract. Even more advantageously, all of the glutamic acid in the extract is consumed.

[0061] According to a particular embodiment, an extract according to the invention further has the following characteristics, expressed as a percentage by weight relative to the dry weight of the extract:

[0062] - a nitrogen content of 0 to 20%, for example 7 to 20%; and / or

[0063] - a free amino acid content of 0 to 30%, for example 10 to 20%; and / or a total amino acid content of 20 to 50%, for example 30 to 40%.

[0064] After the conversion process has stopped, several processing schemes for the resulting extract can be considered:

[0065] According to a first embodiment, and at least in the case of incubation with live bacteria, it is subjected to thermal inactivation, for example at a temperature of 90°C. In this case, the product obtained contains inactivated bacteria.

[0066] According to another embodiment, after this thermal inactivation step, the bacteria are removed from the extract by any technique known to those skilled in the art, for example by tangential membrane filtration (microfiltration or ultrafiltration), by centrifugation, or by a combination of these different techniques.

[0067] These steps may prove unnecessary in the case of the implementation of a bacterial lysate.

[0068] Thus, at this stage, the extract may contain inactivated bacteria or be free (or essentially devoid) of bacteria.

[0069] According to another characteristic, an extract according to the invention is water-soluble.

[0070] According to a particular embodiment, the extract thus obtained is dried, advantageously by spray drying, for example using a spray dryer. In the context of this application, it has been shown that the GABA present in the extract withstands these heat treatments and remains stable.

[0071] In one particular embodiment, the extract according to the invention is in the form of a dry extract. Alternatively, it may be in liquid or powder form. In another particular embodiment, the extract according to the invention may be diluted in a physiologically acceptable carrier or excipient. A physiologically acceptable carrier or excipient is one that is aromatically neutral and suitable for administration in humans or animals. Examples of physiologically acceptable carriers or excipients include maltodextrins, triacetin, propylene glycol, vegetable glycerin, glycerol, soluble fibers, yeast derivatives such as yeast extracts, barks, and autolysates, and fats such as palm oil.

[0072] An extract according to the invention possesses health-beneficial properties of interest for any product intended for oral administration, that is, for ingestion. One aspect of the invention therefore relates to the use of such an extract in this context. As such, it can be defined as a postbiotic, namely a preparation of inanimate microorganisms and / or their components that confers a health benefit to its host.

[0073] Thus, the invention relates to a food product containing an extract as described above and the use of such an extract to obtain a food product beneficial to health, both in humans and animals.

[0074] The beneficial effect on health may include, for example, an improvement in the general condition of humans, an improvement in the general behavior of animals, a reduction in stress and / or anxiety and / or fear.

[0075] According to a particular embodiment, the invention relates to the extract according to the invention or food product comprising this extract for its use to improve the state of well-being and health, and / or to reduce the risk of disease in humans or animals.

[0076] In other words, the invention may relate to a use of the extract according to the invention or of the food product containing this extract, to improve the state of well-being and health, and / or to reduce the risk of disease in humans or animals.

[0077] In other words, the invention may relate to the use of the extract according to the invention or of the food product containing this extract for the preparation of a medicinal product to improve well-being and health, and / or to reduce the risk of disease in humans or animals. Preferably, the animal is a companion animal or an animal in captivity, preferably a companion animal chosen from among dogs, cats, rabbits, guinea pigs, hamsters, ferrets, horses, ponies, and donkeys, and even more advantageously a companion animal chosen from among dogs or cats.

[0078] A food product, as defined in the present invention, can be considered any substance providing nutrition and / or supplementation to a living being, particularly an animal or a human being. The food product can therefore be in either solid or liquid form. For example, in the context of human nutrition, a food product might be a prepared meal, jam, a fermented beverage such as wine or beer, an energy drink, or a protein sports drink. In the context of animal nutrition, it might include treats, kibble, or pâté, including kibble or treats such as biscuits.In practice, a food enriched with an extract according to the invention, in particular enriched in GAB A and capable of influencing a wide variety of functions involved in the state of well-being and health, or the reduction of the risk of diseases, corresponds to a functional food, also called a nutraceutical or medicinal food.

[0079] Alternatively, it may be a food supplement, the main ingredient of which is the extract according to the invention. By definition, a food supplement (or additive or supplement) is a foodstuff intended to supplement the normal diet and which constitutes a concentrated source of nutrients or other substances having a nutritional or physiological effect. With regard to animals, such a supplement may be added to drinking water or to feed such as, for example, cereals and / or legumes.

[0080] Within the scope of the invention, a prime food product is bread. Indeed, bread is a staple food consumed by 80% of global consumers with high frequency, and the increasing demand for healthier options is a dominant trend in the bread market.

[0081] Note that the bread is obtained by mixing several ingredients including flour and yeast which may contain traces of GABA but in insufficient quantity for the desired effect.

[0082] Thus, the raw materials used in breadmaking, mainly flour and yeast, generally have glutamic acid and GABA levels between 0 and 50 ppm.

[0083] Within the framework of this application, it has been demonstrated that the integration of an extract according to the invention was compatible with breadmaking processes and made it possible to obtain a bread rich in GABA, in addition to the favorable effects of the yeast extract in particular on the volume of the bread, without degrading its olfactory qualities.

[0084] However, a number of technical prejudices explained why this solution had never been considered: Indeed, breadmaking is characterized by a series of steps that can reduce the amount of GABA or affect its integrity, particularly during fermentation, as yeast can consume glutamate and GABA, and then during baking, as GABA participates in Maillard reactions involving reducing sugars and amino acids. By promoting these reactions, there was a fear that its addition would disrupt the bread's flavor. From another perspective, the present invention therefore relates to the use of a natural extract as described above in a baking process, specifically for producing bread enriched with GABA.

[0085] For the purposes of this application, "bread making" refers to all the operations involved in transforming flour into bread. The extract according to the invention is intended to be used in any type of bread making, whether on a small or large scale.

[0086] More generally, an extract according to the invention can be used in the preparation of any bakery product including bread, pastries and cakes.

[0087] As is well known, bread making requires flour, water, salt, and a raising agent, typically baker's yeast and / or sourdough starter. The flour provides fermentable sugars used by the yeast and proteins, especially gluten, which gives bread dough its viscoelastic texture. The fermenting microflora, provided by the baker's yeast or sourdough starter, ferments the carbohydrates into carbon dioxide (CO2) and ethanol. The gas fills the alveoli of the gluten network and makes the dough rise, while the ethanol evaporates during baking.

[0088] The yeasts used for breadmaking, also called "baker's yeasts," are living cells generally obtained from different strains of the genus Saccharomyces, in particular Saccharomyces cerevisiae, Saccharomyces chevalieri, Saccharomyces bayanus, or Saccharomyces boulardii, advantageously from the species Saccharomyces cerevisiae.

[0089] Yeasts can be found in several forms: yeast cream, pressed yeast, dry yeast, or frozen yeast. Fresh yeasts are characterized by a higher water content compared to dry yeasts. Fresh yeasts include both yeast creams and pressed yeasts. Yeast creams, also called "liquid yeasts," are aqueous suspensions of yeast cells with a cream-like viscosity. These aqueous suspensions of live yeast cells generally have a dry matter content of at least 12% by weight, particularly from 12 to 40% by weight. A yeast cream, for example, might have a dry matter content between 12 and 25% by weight, preferably between 14 and 22% by weight. Pressed yeasts include both compressed block yeasts and crumbled compressed yeasts.Pressed yeast in compact blocks, also called "yeast loaves," is characterized by a dry matter content between 26% and 35%. Crumbled pressed yeast has a water content between 21% and 35%. Dry yeast is characterized by a dry matter content exceeding approximately 92%. Frozen yeast is characterized by a dry matter content between 74% and 80%.

[0090] Unlike a "baker's" yeast, a natural extract, advantageously a yeast extract according to the invention, does not have fermentative power, thus not ensuring fermentation during breadmaking.

[0091] In the present invention, "sourdough" refers to a bread-making starter resulting from a mixture of flour and water in which the metabolic activity of a heterogeneous population of lactic acid bacteria and yeasts takes place, either through spontaneous fermentation (so-called "natural" sourdough) or through fermentation initiated by a starter culture, with or without feeding. This term also encompasses industrially produced sourdoughs such as live active sourdough or starter sourdough.

[0092] Traditional bread-making processes generally include the following steps:

[0093] - Kneading: mixing the ingredients that make up the dough, called bread dough

[0094] - Division: separating the dough (according to weight)

[0095] - Shaping: shaping the dough into loaves

[0096] Proofing: fermentation, generally at a temperature between 15 and 45°C, for one to several hours

[0097] Cooking: for example at 230°C for 30 minutes.

[0098] The breadmaking processes covered by this application can be either of the direct scheme type (“NO-TIME DOUGH”) or of the indirect scheme type (“SPONGE and DOUGH”).

[0099] A direct scheme involves virtually no first fermentation between intensive kneading and the division of the dough, the resulting dough pieces being fermented in a mold between 35°C and 40°C, then baked.

[0100] A Sponge and Dough type bread-making process is described, for example, in the reference book "Bakers Handbook" by EJ Pyler, published by Sosland Publishing Co. A "SPONGE and DOUGH" scheme is a bread-making process comprising two fermentation stages:

[0101] - a first stage, called "SPONGE", which corresponds to the fermentation of a dough comprising 50 to 70% of the total flour used, part of the water and all or part of the yeast, for several hours, generally between 3 and 6 hours, and more particularly between 3 and 4 hours; and - a second stage, called "DOUGH", in which the SPONGE obtained after the above fermentation is combined with the rest of the flour, the rest of the water and the other ingredients of the dough, the mixture thus formed is kneaded, divided, put in a mold and fermented, then baked, this second fermentation in the mold corresponding to the proofing.

[0102] In the present invention, "dough" or "baking dough" refers to the mixture of ingredients used in preparing the final bread product. Typically, such dough comprises flour, water, salt, and a raising agent such as yeast.

[0103] Typically, yeast represents between 0.5 and 10% by weight, and preferably from 0.5 to 3% by weight relative to the total weight of the composition, namely the dough.

[0104] The extract according to the invention can be incorporated at any time during the preparation process before the cooking stage. Preferably, the natural extract, advantageously yeast extract, is added during the kneading stage, along with the previously mentioned ingredients.

[0105] According to a particular aspect, the present invention relates to a bread dough comprising an extract as defined above and the bread or bakery product obtained from said dough.

[0106] Within the framework of the invention, a dough may be a dough without added sugar, a slightly sweetened dough or a sweetened dough, the sugar being most often sucrose.

[0107] In the present invention, "bread" means any product obtained by baking dough made by kneading a mixture comprising at least flour, water, and salt, and leavened with a raising agent, in particular yeast. More generally, "bread product" or "bakery product" means any product resulting from breadmaking, including pastries and viennoiseries.

[0108] According to another aspect, the present invention relates to a bread product, such as a loaf of bread, obtained from a dough comprising an extract according to the invention, in particular after fermentation and baking.

[0109] Advantageously, such a product is rich in GABA.

[0110] Within the framework of the invention and in connection with bread dough, bread product or more generally a food product, "GABA-rich product" means that the product includes at least 0.01, 0.015, 0.02, 0.03, 0.04, 0.05%, or even 0.1 or 0.5% by weight of GABA, the percentage by weight being expressed in relation to the weight of the product or dough. In other words, a food product, a bakery product or a bakery dough according to the invention advantageously comprises at least 100, 150, 200, 300, 400, 500, or even 1000 or even 3000 ppm of GABA, preferably an amount between 100 ppm and 3000 ppm, even more preferably between 150 ppm and 500 ppm, for example 150 ppm or 200 ppm.

[0111] Assuming that a man consumes approximately 80g of bread per day, the daily intake of GABA via such a product could therefore be at least 1mg, 5mg, 10mg or even 50mg or even 100mg per day.

[0112] According to another particular embodiment of the present invention, the food product provides nutrition and / or supplementation and is in the form of kibble or treats for use in animals, preferably dogs, to improve the animal's general behavior and reduce stress, anxiety, and / or fear. Preferably, the kibble or treats contain between 50 ppm and 500 ppm, for example, between 150 and 300 ppm, or approximately 150 ppm, of the extract according to the invention.

[0113] According to another particular embodiment of the present invention, the food product is in the form of kibble, treats or pâtés, and preferably comprises between 50 ppm and 500 ppm of the extract according to the invention for its use in animals, preferably dogs, to improve general behavior, reduce stress and / or anxiety and / or fear.

[0114] In other words, the invention may relate to the use of the food product in the form of kibble, treats or pâtés, and preferably comprising between 50 ppm and 500 ppm of the extract according to the invention in the animal, preferably the dog, to improve general behavior, reduce stress and / or anxiety and / or fear.

[0115] In other words, the invention may relate to a use of the food product for the preparation of kibble, treats or pâtés, preferably comprising between 50 ppm and 500 ppm of the extract according to the invention in the animal, preferably the dog, to improve general behavior, reduce stress and / or anxiety and / or fear.

[0116] As demonstrated in the examples below, and remarkably so, the extract according to the invention is compatible with food production processes, particularly breadmaking, and gives the resulting product a GABA content compatible with health benefits, without impairing the appearance and taste of said product, particularly bread. FIGURES

[0117] Figure 1: Average specific volume (in cm³) 3 / g) loaves obtained from different formulations:

[0118] Test 1; a "control" formulation corresponding to a dough without yeast extract

[0119] Test 2; the "control" formulation, in which a commercial yeast extract (Lesaffre - Springer Reveal 292 PW) was added

[0120] Test 3; the "control" formulation, to which pure GABA was added

[0121] Test 4; the "control" formulation, in which a GABA-rich yeast extract according to the invention has been added

[0122] Figure 2: Spiderweb chart - main descriptors (White flour odor and aroma; Fermented odor and aroma; Cracker odor and aroma; Malty odor and aroma; Umami; Salty; Cracker crust aroma) and their scores (from 0 to 10)

[0123] + : significant difference (ANOVA, 5%)

[0124] EXAMPLES OF ACHIEVEMENTS

[0125] The invention and its resulting advantages will become clearer from the following embodiments, supported by the accompanying figures. These examples, however, are not intended to be exhaustive.

[0126] 1 / of the asset according to the invention

[0127] 1 / Preparation of yeast extract

[0128] A yeast extract of Saccharomyces cerevisiae was prepared and used at a concentration of between 30g / L and 300g / L.

[0129] 2 / Preparation of the bacterium carrying the glutamate decarboxylase activity

[0130] The bacterium Levilactobacillus brevis was cultured under suitable conditions on an MRS (Man Rogosa, Sharpe) type medium.

[0131] The culture thus obtained is used as is or subjected to freeze-drying and then used in the step of converting glutamic acid to GABA.

[0132] 3 / Conversion of glutamic acid to GABA

[0133] The conversion is carried out at a temperature between 30 and 40°C and a pH between 5 and 7. Inoculation of the bacterial extract with the bacterial wort initiates the conversion. The conversion time is approximately 24 to 70 hours. The reaction is stopped when the conversion to GABA is greater than 90%, ideally complete. The resulting GABA-rich extract is then subjected to thermal inactivation and subsequently centrifuged.

[0134] The composition of the final extract obtained, in dry form, is given in the table below:

[0135] Table 1

[0136] II / Use of the active ingredient according to the invention in a process of

[0137] 1 / Preparation of the different bread doughs:

[0138] The experiments were carried out on a "crusted bread" type formulation called "bastard", with a dough comprising water, T55 flour, salt, an improver containing ascorbic acid and baking enzymes, and "Blue Swallow" pressed yeast.

[0139] The flour and yeast used in these examples contain glutamic acid and GABA, but in small quantities, as shown in the table below:

[0140] Table 2 Glutamic acid and GABA content in raw materials

[0141] Different samples were prepared, according to the quantities described in Table 3 below:

[0142] - a "control" formulation ("Control"; test 1) corresponding to a dough without yeast extract; the "control" formulation, in which a commercial yeast extract (Lesaffre - Springer Reveal 292 PW) has been added ("+ Springer Reveal 292 PW Yeast Extract"; test 2); the "control" formulation, in which pure GABA (Sigma Aldrich - Gamma Aminobutyric acid, BioXtra > 99%) has been added ("+ pure GABA"; test 3); the "control" formulation, in which a GABA-rich yeast extract according to the invention has been added ("+ GABA-rich Extract"; test 4).

[0143] Table 3 Tested Formulations

[0144]

[0145] 2 / Processing of different bread doughs:

[0146] The process used for the different types of pasta is the same and essentially comprises 3 steps: mixing of ingredients; raising of the pasta (fermentation) for 110 minutes; baking for 30 minutes at 230°C.

[0147] 3 / Analysis of bread products:

[0148] During the process, the level of CO2 production was measured by the Risograph system.

[0149] Bread volumes were measured using Volscan Profiler

[0150] For each sample, the GABA content of the final crumb was analyzed by HPLC.

[0151] 3-1 / CO2 Production

[0152] The results obtained with the Risograph, after 110 minutes of fermentation, are shown in the table below:

[0153] Pure GABA showed no impact on CO2 production, considering a fermentation time of 110 minutes. The addition of yeast extract had a positive impact on CO2 production, increasing it by approximately 10% at 110 minutes. The GABA-rich yeast extract according to the invention also had a positive impact on CO2 production at 110 minutes (+7.5%).

[0154] 3-2 / Bread volume

[0155] The results of the Volscan Profiler, allowing us to evaluate the average specific volume (in cm³) 3 / g), are shown in Figure 1. The results are expressed as % difference between trials 2, 3 and 4 and the control (trial 1).

[0156] These results are consistent with those concerning CO2 production: loaves made with commercial yeast extract have the highest volumes, but the incorporation of a yeast extract according to the invention also has a positive effect. 3-3 / Sensory evaluation of the loaves obtained

[0157] The sensory evaluation was carried out on fresh bread by a panel of experts:

[0158] - Test under white light

[0159] - Blind tasting, in a random order

[0160] - 6 expert panelists

[0161] - Quantitative (data analysis): rating of the intensity of each parameter on a scale of 10 points (0 to 10). The parameters are: White flour odor and aroma; Fermented odor and aroma; Cracker odor and aroma; Malty odor and aroma; Umami; Saltiness; Cracker crust aroma.

[0162] The results in Figure 2 reveal that:

[0163] - The GABA-rich yeast extract according to the invention is significantly more malty;

[0164] - The flavor profile of this bread is very different from the control. This solution provides significant flavors (malt and / or crackers) but no unpleasant taste.

[0165] 3-4 / GABA content

[0166] GABA content was measured in samples of crumb from the final loaf.

[0167] Free gamma-aminobutyric acid (GABA) was quantified by reversed-phase HPLC-UV at 260 nm after derivatization using the AccQ-Tag Ultra kit (Waters, USA). A 3 pL injection volume was injected into the chromatographic system equipped with a pre-column (XBridge B EH C18; 2.1 x 5.0 mm; 3.5 pm, Waters, USA) and a column (XBridge BEH C18; 3.0 x 150 mm; 3.5 pm, Waters, USA). The mobile phase consisted of four channels: Eluent A (A), Water / Eluent B 90 / 10 (v / v) (B), Ultrapure Water (C), and Eluent B (D).

[0168] The preparation of the different samples was carried out according to the following steps:

[0169] - Dilution of 5 g of sample in 20 ml of ultrapure water;

[0170] - Homogenization of the solution by Ultra-turrax;

[0171] Addition of 5 ml of internal standard;

[0172] Centrifugation and filtration.

[0173] The results are presented in the table below:

[0174] Table 5 GABA (free) content measured in the crumb and GABA (free) content calculated on the basis of the initial raw material input (theoretical)

[0175] As expected, the standard (control) condition resulted in a low level of GABA in the final loaf. The same was true for the loaf made with only Springer Reveal 292 PW yeast extract.

[0176] The theoretical calculated value (considering the GABA contribution of the different ingredients to the dough) and the actual GABA content, measured within the framework of this invention, are similar.

[0177] The GABA content remains stable during bread making.

[0178] In the present invention, the incorporation of pure GABA (Sigma Aldrich - Gamma Aminobutyric acid, BioXtra > 99%) or of yeast extract rich in GABA makes it possible to obtain a bread product comprising more than 0.015 (corresponding to 150 ppm) or even more than 0.02% (corresponding to 200 ppm) by weight of GABA, the percentage by weight being expressed in relation to the weight of the product.

[0179] In conclusion, an extract according to the invention offers a natural ingredient that combines the benefits of GABA (for health) and a yeast extract (for breadmaking), and is also fully compatible with breadmaking processes.

[0180] III / Use of the active ingredient according to the invention as a food product beneficial to the health of dogs

[0181] 1 / Purpose of the trial in dogs

[0182] The aim of this protocol is to explore the improvement of the state of well-being and health, and / or the reduction of the risk of disease in animals by using the active ingredient according to the invention as a supplement in animal feed.

[0183] This study will span a period of 6 to 9 months, during which dogs will be observed to analyze the evolution of their anxious behavior with supplementation of the active ingredient according to the invention. Furthermore, there will also be idiosyncratic observations (concomitant behavioral changes), an assessment of the effects observed during weaning, and an evaluation of client satisfaction. Based on this data, it will be possible to make recommendations regarding the behavioral phenotypes that could benefit from supplementation with the active ingredient according to the invention.

[0184] 2 / Study design

[0185] Each dog will receive daily 150 ppm of GABA-rich yeast extract according to the invention in the form of 4 "biscuit" treats corresponding to 1.5 g of GABA-rich yeast extract per day and per 10 kg dog.

[0186] A case series, including the evaluation of dogs using baseline data for comparative purposes. Through the animal behavior reference clinic, approximately 20 dogs exhibiting various behavioral changes related to fear / anxiety exacerbated by stress are planned.

[0187] The dogs will generally be in good physical health (or with long-term conditions under effective management), but a subpopulation will be included in which behavioral problems appear to be associated with some form of stress-related gastrointestinal disturbance (directly or indirectly).

[0188] Previous treatments (e.g. antibiotics) will be noted, but only the simultaneous use of a hypoallergenic diet will be considered an exclusion criterion.

[0189] This profile is chosen as a potential indication for the active ingredient according to the invention, based on its proposed anxiolytic effect in preclinical models. All dogs will undergo a risk-benefit analysis related to the proposed intervention.

[0190] To do this, it is planned to consult at least 40 dogs to identify 20 suitable for supplementation.

[0191] The study will proceed as follows:

[0192] 3 / Results

[0193] Each dog will be assessed individually, with baseline data used as a reference. 3-1 / Behavior

[0194] Primary outcome: Overall measure of problem severity, in accordance with behavioral clinic protocols. Group-level effects will be adjusted based on the baseline value.

[0195] Secondary Outcomes: Behavioral Measures. The temperament assessment will conform to the screens currently used routinely at the behavioral clinic and will include the following:

[0196] 1. The primary affect (PANAS; Sheppard and Mills, 2002)

[0197] 2. frustration (CFQ; McPeake et al., 2019)

[0198] 3. possibly impulsivity (DIAS; Wright et al., 2011) 4. the Lincoln Canine Anxiety Scale (Mills et al., 2020).

[0199] 3-2 / Other criteria

[0200] The fecal score and the gut microbiota will also be analyzed.

Claims

DEMANDS 1. Yeast extract containing gamma-aminobutyric acid (GABA), said GABA being obtained from glutamic acid present as a free amino acid in said yeast.

2. Extract according to claim 1, characterized in that it contains at least 5% by weight of GABA, the percentage by weight being expressed in relation to the dry weight of the extract.

3. Extract according to any one of claims 1 to 2, characterized in that it contains less than 10% by weight of glutamic acid, advantageously less than 5%, even more advantageously less than 1%, the percentage by weight being expressed in relation to the dry weight of the extract.

4. Extract according to any one of claims 1 to 3, characterized in that it is a water-soluble extract, advantageously in dry form.

5. Extract according to any one of claims 1 to 4, characterized in that it is obtained from a yeast capable of synthesizing glutamic acid.

6. Extract according to claim 5, characterized in that it is obtained from a yeast of the species Saccharomyces cerevisiae.

7. Extract according to any one of claims 1 to 6, characterized in that the GABA is obtained using the following process: - preparation of yeast extract from yeast containing glutamic acid in the form of a free amino acid; - incubation of the extract in the presence of a glutamate decarboxylase (GAD) enzyme.

8. Extract according to claim 7, characterized in that the glutamate decarboxylase (GAD) enzyme is derived from a bacterium or a lysate of such a bacterium producing said enzyme, advantageously carrying the GadA and / or GadB or even GadC genes.

9. Extract according to claim 8, characterized in that the bacterium belongs to the family of Lactobacillaceae, advantageously chosen from the group consisting of: Levilactobacilus brevis, Lentilactobacilus buchneri and Lactococcus laclis, preferably the bacterium is Levilactobacilus brevis.

10. Extract according to any one of claims 8 to 9, characterized in that the incubation between the bacterium and the yeast extract is carried out under conditions promoting the conversion of glutamic acid to GABA.

11. Extract according to any one of the preceding claims, characterized in that it comprises inactivated bacteria or is free from bacteria.

12. Food product, in particular kibble, sweets, bread dough or bakery product, comprising an extract according to any one of claims 1 to 11, wherein the extract is advantageously present in an amount between 100 ppm and 3000 ppm, preferably between 150 ppm and 500 ppm, for example 150 ppm or 200 ppm.

13. Use of an extract according to any one of claims 1 to 11 or of a food product according to claim 12 to improve the state of well-being and health, and / or to reduce the risk of disease in humans or animals.

14. Use according to claim 13, wherein the food product is in the form of kibble, treats or pâté, and preferably comprises between 50 ppm and 500 ppm of the extract according to any one of claims 1 to 11, for use in animals, preferably dogs, to improve general behavior, reduce stress and / or anxiety and / or fear.

15. Use of an extract according to any one of claims 1 to 11 in a bread-making process, advantageously for the preparation of a bread enriched with GABA.

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