Novel GABA-rich masking product

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 masking flavours, in particular bitter and acid flavours, undesirable notes, in particular of sweeteners or proteins, and metallic notes, or trigeminal sensations such as astringency, in a product, advantageously a food, pharmaceutical or nutraceutical product, or an aromatic preparation.

Description

[0001] NEW GABA-RICH MASK PRODUCT

[0002] technical field

[0003] The present invention falls within the fields of agri-food and health, seeking natural solutions to mask undesirable tastes in ingested products. The solution proposed in this application is the provision of a natural extract rich in GABA, whose blocking properties, particularly for bitterness, are known (Pydi et al., J Biol Chem., 2014, 289(36): 25054-66; Sasaki et al., J Biol Macromol., 2014, 14(1): 43-57).

[0004] Prior state of the art

[0005] Today, more and more consumers are looking for healthier and more environmentally friendly food.

[0006] To meet this growing demand, food manufacturers have developed new solutions in recent years focused on plant-based products (development of meat and dairy analogues using non-animal proteins), nutrition (protein drinks or drinks fortified with various compounds such as vitamins, amino acids, minerals, etc.) and the reduction of sugar, fat and salt in existing products.

[0007] The development of these new solutions is accompanied by many challenges, particularly around taste, which remains one, if not the main, criterion for purchasing agri-food products worldwide.

[0008] One of the main challenges is bitterness, which is present in many "new" ingredients used in these products: in sweeteners, in some salt substitutes (KC1 for example), in many vegetable proteins (pea, soy, potato etc.), in many plant extracts, in food supplements (vitamins, minerals, amino acids) etc.

[0009] Other undesirable notes also need to be removed, blocked or masked: sour taste, astringency, metallic notes, green vegetal notes, rancid notes, etc.

[0010] Certain ingredients are therefore developed to help solve these flavor profile problems and improve the organoleptic quality of consumed products. Some of these ingredients contain "bitter blockers," that is, compounds capable of "blocking" bitter taste receptors, in order to prevent the perception of the bitterness initially present in the consumed product. No perfect solution exists, and the development of new ingredients, added to solutions already available on the market, makes it possible to create customized solutions that combine these different technologies (for example, masking agents for pea protein, masking agents for rebaudioside A (Reb A), masking agents for KC1, etc.).

[0011] 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 integration of such an extract into an ingestible product makes it possible to provide a sufficient quantity of GABA to achieve the desired masking effect in a natural context, i.e., without impact on human or animal health, but also rich in other potentially useful compounds (in terms of nutrition, taste, etc.).

[0012] Document JP2004275098 describes a kelp extract enriched with GABA for seasoning food.

[0013] Detailed description of the invention

[0014] Thus, according to a first aspect, the present invention relates to a natural extract rich in gamma-aminobutyric acid (GABA).

[0015] 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.

[0016] 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).

[0017] 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.

[0018] 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.

[0019] 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, unsuitable for the desired masking properties.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] An extract according to the invention differs from synthetic or purified GABA in that it also contains the components of the natural extract, in particular yeast extract, thus constituting an enriched form.

[0025] 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.

[0026] Obtaining GABA from glutamic acid involves the activation of glutamate decarboxylase (GAD) activity capable of ensuring the following conversion:

[0027] L-Glutamate + H+ - GABA + CO2

[0028] 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:

[0029] - preparation of yeast extract from yeast containing glutamic acid in the form of a free amino acid;

[0030] - incubation of the extract in the presence of a glutamate decarboxylase (GAD) enzyme.

[0031] Following this process, 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 advantageously containing 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.

[0032] 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:

[0033] - an acidic pH, for example of 5; and

[0034] - a temperature between 30 and 35°C, for example equal to 33°C.

[0035] Alternatively, a natural extract, advantageously a GABA-rich yeast extract according to the invention, is obtained via a bacterium exhibiting suitable glutamate decarboxylase activity.

[0036] Thus, the bacterium implemented within the framework of the invention exhibits glutamate decarboxylase (GAD) activity, in other words 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 widely 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 connection with 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. Bacteria carrying at least one GadA or GadB gene are, for example, selected from the group comprising Lactobacillus plantarum.

[0041] Des baccières porteuses d'au moins un gène GadA iiou 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.

[0042] 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, Limosilactobacillus fermentum and Enterococcus hermanniensis.

[0043] 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.

[0044] 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.

[0045] Advantageously, the bacterium is further cultured under conditions that promote the expression of genes in the pathway, including GadA, GadB and / or GadC.

[0046] At the end of the growth phase and for the conversion stage, the resulting culture can be used as is, or in dried or even freeze-dried form, provided that these treatments do not affect enzymatic activity. By using a bacterium equipped with the GadC-encoded transporter, whole cells can be used to perform the conversion of glutamic acid from the natural extract into GABA.

[0047] 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.

[0048] The extract, advantageously of yeast, is then incubated with the bacterial culture, possibly in the form of a bacterial lysate.

[0049] According to one embodiment, the free glutamic acid titration of the extract, particularly of yeast, 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.

[0050] According to one embodiment, the conversion is ensured by contacting or incubating the extract, advantageously of yeast, as detailed above with the bacterium, namely the bacterial must or bacterial lysate, as described above.

[0051] Incubation is advantageously carried out under conditions adapted to the bacterium used or to the enzyme in the case of a bacterial lysate.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] Other conditions suitable for this conversion stage are:

[0056] - a temperature between 30 and 40°C,

[0057] - a pH between 5 and 7;

[0058] - an incubation time of approximately 15 to 70 hours, preferably between 24 and 70 hours. The conversion step is carried out until the desired quantity of free glutamic acid is consumed. In the context of this application for the use of the final extract for masking, given the umami flavor due to glutamic acid, its conversion to GABA is advantageously greater than 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 percentage by weight being expressed relative to the dry weight of the extract. Even more advantageously, all of the glutamic acid in the extract is consumed.

[0059] 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:

[0060] - a nitrogen content of 0 to 20%, for example 7 to 20%; and / or

[0061] - a free amino acid content of 0 to 30%, for example 10 to 20%; and / or

[0062] - a total amino acid content of 20 to 50%, for example 30 to 40%.

[0063] After the conversion process has stopped, several processing options for the resulting extract can be considered:

[0064] 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.

[0065] 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.

[0066] These steps may prove unnecessary in the case of the implementation of a bacterial lysate.

[0067] Thus, at this stage, the extract may contain inactivated bacteria or be free (or essentially devoid) of bacteria.

[0068] According to another characteristic, an extract according to the invention is water-soluble.

[0069] According to a particular embodiment, the extract thus obtained is dried, advantageously by spray drying, for example using a spray drying tower. In the context of this application, it has been shown that the GABA present in the extract is resistant to these heat treatments and remains stable.

[0070] 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.

[0071] An extract according to the invention possesses masking properties of interest for any product intended for oral administration, i.e., for ingestion. One aspect of the invention therefore relates to the use of such an extract in this context.

[0072] For the purposes of the present invention, taste masking consists of reducing or completely eliminating the perception of a flavor within a product, while off-note masking consists of reducing or completely eliminating the perception of an aroma or aromatic note.

[0073] It is well known that the perception of the taste of a food or substance ("flavor") results from a multitude of mechanisms and is the outcome of taste, aroma, and trigeminal sensations. When food is placed in the mouth, chewing releases volatile and non-volatile constituents, each of which has various consequences and effects on taste perception.

[0074] Non-volatile constituents, detected by receptor cells of the gustatory system, are responsible for flavor. There are only five basic tastes: sour, bitter, sweet, salty, and umami. Volatile constituents, on the other hand, are detected by receptor cells of the olfactory system (retronasal pathway) and are responsible for aromas. Unlike flavors, there is a wide variety of aromas, which can result from several molecules. In addition, certain molecules are detected by nerve endings of the trigeminal nerve in the oral and nasal cavities, giving the trigeminal component of a food or substance, such as pungency, astringency, heat, or coldness.In particular, an extract according to the invention exhibits properties blocking bitter and sour flavors, but also properties masking undesirable aromatic notes, such as sweetener and protein notes or metallic notes, and trigeminal sensations such as astringency.

[0075] One aspect of the invention relates to the use of an extract of the invention to reduce or even completely eliminate undesirable flavors, particularly bitter and sour ones, as well as undesirable aromatic notes, including notes of sweeteners and proteins or metallic notes, and trigeminal sensations such as astringency. It should be noted that, to date, the reduction or elimination of undesirable flavors and notes is detected by human sensory perception because other methods, such as the use of electronic language, do not allow for the precise detection of specific masking. Indeed, current detection methods are not sufficiently representative of the mechanism of human perception, which depends on a multitude of factors, some of which are not solely physiological.

[0076] As is known to those skilled in the art, the flavors to be masked, reduced, eliminated or blocked are chosen from the following group: sour, bitter, sweet, salty and umami.

[0077] Test compounds or compositions to evaluate this masking are classically chosen from the following group: bitter flavor: caffeine; quinine; L-phenylalanine; L-arginine; L-tyrosine; lysine, for example lysine HCl; caramel sauce; pea protein; soy protein; soybeans, for example in milk or yogurt; energy drink, for example rich in BCAAs; bitter brewer's yeast extract; sour flavor: jam; vegetable cheese; vegetable yogurt.

[0078] As is known to those skilled in the art, undesirable aromatic notes to be masked, reduced, eliminated or blocked are chosen from the following group: notes of sweeteners, notes of proteins, metallic notes, rancid notes, . . .

[0079] Test compounds or compositions to evaluate this masking are classically chosen from the following group: sweetener score: sucralose (splenda); aspartame; acesulfame K; thaumatin; rebaudioside A (Reb A); steviol glycosides (SG95); jam; sweetened beverage such as iced tea; energy or protein drink; protein score: pea protein; soy protein; potato protein; broad bean protein; vegan product of type "plant steak", "plant tuna", "plant cheese", "vegetarian yogurt" and "egg analogue"; metallic score: potassium chloride solution (KO); soup rich in KC1.

[0080] As is known to those skilled in the art, trigeminal sensations to be masked, reduced, suppressed or blocked include, for example, astringency, which can be tested on a ginseng extract ("Ginseng Iced Tea"), pea or soy protein, modified starch ("vegan cream cheese" application), soybeans, for example in milk or yogurt, or an energy drink, for example rich in BCAAs.

[0081] Within the scope of the invention, products intended for oral intake include, in particular, food products, pharmaceutical or nutraceutical compositions (also known as food supplements), or flavoring preparations.

[0082] In the context of the present invention, the term "product" refers to both a food, pharmaceutical, or nutraceutical product and aromatic preparations that can be used in food, pharmaceutical, or nutraceutical applications.

[0083] A food product, as defined in the present invention, can be considered any substance that provides nutrition to a living being, and particularly to a human being. A food product can therefore be in either solid or liquid form. For example, a food product could be a prepared dish, a jam, a fermented beverage such as wine or beer, an energy drink, or a protein drink for sports.

[0084] A pharmaceutical product within the meaning of the present invention means any pharmaceutical substance intended to be ingested by a human being, such as a medicine.

[0085] The amount of extract to be introduced into the product depends on the nature of the product itself and the strength of the undesirable flavors and aromatic notes to be neutralized. Typically, and particularly in the context of a food product, the extract according to the invention can represent at least 10 ppm (0.001% w / w), or even 50 ppm, or even 100 ppm. According to another embodiment, the extract represents a maximum of 3000 ppm (0.3% w / w), or even 2000 ppm, or even 1000 ppm. Any intermediate value, for example 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, or 2500 ppm, can be used. According to one particular embodiment, the extract according to the invention contains from 50 to 500 ppm, advantageously from 100 to 300 ppm, for example 200 ppm. In another particular aspect, an extract according to the invention is combined with another masking agent. This could, for example, be the ribonucleotide-rich yeast extract described in document WO2019 / 115894.Such an extract, obtained from the yeast Saccharomyces cerevisiae, is essentially defined as comprising (in percentages by weight relative to the dry weight of the extract) 25% to 55% of 5'-ribonucleotides (e.g. 50.9%) of which 5% to 20% of 5'-AMP (e.g. 10%) and 5% to 20% of 5'-GMP (e.g. 10.1%), according to a 5'-AMP / 5'GMP ratio of between 0.85 and 1.25.

[0086] If the extract according to the invention is combined with another masking agent, its quantity can be reduced, for example, halved. Thus, and only by way of example:

[0087] - an extract according to the invention, when used alone, can be used at a level of 200 ppm in the product;

[0088] - an extract described in WO2019 / 115894, when used alone, can be used at a level of 100 ppm in the product; when the two are combined in the product, the extract according to the invention can be used at a level of 100 ppm and the extract described in WO2019 / 115894 at a level of 50 ppm.

[0089] The incorporation of the extract into a product is a step known to those skilled in the art and consists primarily of applying and / or mixing the extract with the product. It should be noted that this step can be carried out before, during, or after the product's manufacture. It can be performed using either hot or cold processes.

[0090] According to another aspect, the present invention therefore relates to a method of preparing a product having a modified quality comprising: the provision of an extract as described above; the incorporation of said extract into a product.

[0091] Thus, the present invention combines the qualities of a natural extract and GAB A for a multifaceted and enhanced masking effect on products. Furthermore, even when it is a yeast extract, it does not alter the overall taste perception of said products and, in particular, does not produce the brothy notes, aromatic yeast notes, or umami flavor characteristic of such extracts ("yeasty taste").

[0092] FIGURES Figure 1: Bitterness score (scale from 0 to 10 with 0: no bitterness and 10: strong bitterness) of Quinine (aqueous solution at 18 ppm) in the presence of:

[0093] (A) Control (no masking agent)

[0094] (BC) Extract according to the invention (EXL-I)

[0095] (AB) Anterior art masking agent (Springer® Mask 101)

[0096] (C) EXL-I + Springer® Mask 101

[0097] Figure 2: Classification with rating of the bitterness of Quinine (aqueous solution at 18 ppm) in the presence of:

[0098] (C) Control (no masking agent)

[0099] (BC) Anterior art masking agent (Springer® Mask 101)

[0100] (AB) Extract according to the invention (EXL-I)

[0101] (A) EXL-I + Springer® Mask 101

[0102] Figure 3: Evaluation of organoleptic defects in unflavored plant-based wafers under different conditions:

[0103] Control (no masking agent)

[0104] Extract according to the invention (EXL-I)

[0105] Anterior art masking agent (Springer® Mask 101)

[0106] EXL-I + Springer® Mask 101

[0107] Figure 4: Evaluation of organoleptic defects in flavored plant-based wafers under different conditions:

[0108] Control (no masking agent)

[0109] Extract according to the invention (EXL-I)

[0110] Anterior art masking agent (Springer® Mask 101)

[0111] EXL-I + Springer® Mask 101

[0112] Figure 5: Evaluation of organoleptic defects in plant-based beverages under different conditions:

[0113] Control (no masking agent)

[0114] Extract according to the invention (EXL-I)

[0115] Anterior art masking agent (Springer® Mask 101)

[0116] EXL-I + Springer® Mask 101

[0117] EXAMPLES OF ACHIEVEMENTS

[0118] 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.

[0119] 1 / Preparation of the active ingredient according to the invention 1 / Preparation of the yeast extract

[0120] A yeast extract of Saccharomyces cerevisiae was prepared and used at a concentration of between 30g / L and 300g / L.

[0121] 2 / Preparation of the bacterium carrying the glutamate decarboxylase activity

[0122] The bacterium Levilactobacillus brevis was cultured under suitable conditions on an MRS (Man Rogosa, Sharpe) type medium.

[0123] 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.

[0124] 3 / Conversion of glutamic acid to GABA

[0125] The conversion is carried out at a temperature between 30 and 40°C and a pH between 5 and 7. Inoculation of the extract with the bacterial culture 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.

[0126] The resulting GABA-rich extract is then subjected to thermal inactivation and then centrifuged.

[0127] The composition of the final extract obtained, in dry form, is given in the table below:

[0128] Table 1

[0129] II / Masking properties of the asset according to the invention

[0130] In the following examples, an extract according to the invention, as obtained in (I), is referred to as "EXL-I". Furthermore, an extract as described in WO2019 / 115894 is commercially available under the name "Springer® Mask 101".

[0131] 1 / Effects of an extract according to the invention on various organoleptic defects. The effectiveness of an extract according to the invention at 200 ppm was tested on a wide range of molecules or extracts in aqueous solution, or on food products, during an expert tasting. The tested products were submitted to the panel of five experts for a comparative organoleptic analysis. Each expert tasted the control product in a blind comparison with the same products containing an extract according to the invention.

[0132] The results are summarized and presented in the table below, which lists the molecules, extracts or foods for which the active ingredient according to the invention has proven effective, with mention of the corrected organoleptic defect.

[0133] Table 2

[0134] These results demonstrate that the extract according to the invention has a very broad spectrum of action, making it possible to correct both flavor defects (bitterness, sweet persistence), aromatic notes (cardboard, earthy, vegetal, metallic, licorice, fish), and trigeminal sensations (astringency).

[0135] 2 / Sensory analysis of the effect of an extract according to the invention on bitterness In order to refine the previous study and to make a statistical analysis of it, a new study was carried out using a panel of 21 experts, using a bitter solution of Quinine (aqueous solution at 18 ppm), known to be a reference bitter molecule in the food field and tested in the previous study.

[0136] Protocol:

[0137] Two anonymized samples, at room temperature, were submitted to a trained panel of 21 people:

[0138] One contained quinine (aqueous solution at 18 ppm) and the other contained quinine (aqueous solution at 18 ppm) + an extract according to the invention (EXL-I, 200 ppm).

[0139] The following question was posed to the panel: "Which sample is the most bitter?"

[0140] Sensory analysis was carried out using the 2AFC (“two-alternative forced choice”) method followed by a statistical analysis of the results.

[0141] Results:

[0142] The collected results are presented in the table below.

[0143] p-value = 0.0069

[0144] These bitterness ranking data demonstrate that the sample containing an extract according to the invention is significantly less bitter than the control (without masking agent), with a very high 99% confidence level.

[0145] 3 / Association of an extract according to the invention with another masking agent

[0146] Further studies under the same conditions as above were conducted to investigate the combined effect of an extract according to the invention (EXL-I) with another yeast extract already marketed as a masking agent under the name Springer® Mask 101.

[0147] Four conditions were tested in parallel: yeast extract according to the invention (“EXL-I”) at 200 ppm; commercial yeast extract “Springer® Mask 101” at 100 ppm; a combination of the two extracts: EXL-I at 100 ppm + Springer® Mask 101 at 50 ppm; and a control without masking agent. The tests were conducted with 22 trained judges, using an aqueous quinine solution (18 ppm) as a bitterness control molecule.

[0148] The bitterness score of the different products tested was determined using a scale rating from 0 to 10 (0: no bitterness; 10: strong bitterness).

[0149] The results obtained are reported in the table below and illustrated in diagram form in Figure 1.

[0150] Table 4

[0151] Based on the scores obtained (Figure 1), a significant decrease in bitterness was observed, with the best result for the combination of the two products, the extract according to the invention ranking second.

[0152] A multiple pairwise comparison ranking was then carried out using the Nemenyi test (two-sided test).

[0153] The results obtained are reported in the table below and illustrated in diagram form in Figure 2.

[0154] Table 5

[0155] The ranking test confirms that the products are different. On the ranking test with bitterness rating (Figure 2), the same ranking emerges as with the bitterness profile, with the best performance for the combination Springer® Mask 101 + EXL-I, the extract according to the invention being positioned as the 2nd best solution, ahead of Springer® Mask 101, itself less bitter than the control.

[0156] Furthermore, a synergy between the 2 masking products is noted, since the combination of the 2 at half the concentration is more effective than each one taken alone at its concentration considered effective.

[0157] 4 / Sensory analysis of the effect of an extract according to the invention in an unsweetened soy yogurt

[0158] Further studies were conducted to investigate the effect of an extract according to the invention (EXL-I) in comparison with another yeast extract already marketed as a masking agent called "EXL-0" corresponding to a yeast extract as obtained in point I but without the conversion step 3 / , or commercial pure synthetic GABA (Ref. A2129, Sigma-Aldrich).

[0159] The three samples, containing EXL-I, EXL-0, and GABA respectively, were evaluated by a panel of four sensory evaluators. The products were presented in a random order, with the panelists comparing each sample to a control sample of unsweetened soy yogurt.

[0160] A descriptive test was used, allowing panelists to provide detailed feedback on the sensory attributes of each sample.

[0161] The recipes used are presented below:

[0162] The results obtained are reported in the table below.

[0163] The average ranking of the samples by the tasters is as follows:

[0164] 1) EXL-I (test 1) - the most appreciated and best performing overall,

[0165] 2) GABA (test 3) - Second best, despite increased acidity,

[0166] 3) EXL-0 (test 2) - the least liked and least efficient of the three.

[0167] Conclusions:

[0168] The best performing and most appreciated sample overall was the extract according to the invention, "EXL-I (test 1)". This sample showed the best masking performance, with fewer buttery, bitter, and cardboard notes compared to the control, while retaining some astringency.

[0169] The GABA sample (test 3) received the second-highest score, even though it was perceived as more acidic. The reduction in bitterness, bean flavor, and cardboard notes still represents an improvement over the control.

[0170] 5 / Sensory analysis of the effect of an extract according to the invention on Stevia

[0171] Further studies, under the same conditions as above, were carried out to study the effect of an extract according to the invention (EXL-I) in comparison with another yeast extract already marketed as a masking agent called "EXL-0" corresponding to a yeast extract as obtained in point I but without the conversion step 3 / or commercial pure synthetic GABA (Ref. A2129, Sigma-Aldrich).

[0172] The 4 samples were tested in parallel: yeast extract according to the invention “EXL-I” at 200 ppm; commercial yeast extract “EXL-0” at 200 ppm;

[0173] Pure GABA at 16 ppm, control at 100 ppm of Stevia SG95 without masking agent. Evaluation by a panel of 4 sensory evaluators. The products were presented in random order.

[0174] A descriptive test was used, allowing panelists to provide detailed feedback on the sensory attributes of each sample.

[0175] The notes of sweetness, persistence, licorice and bitterness of the different products tested were determined using a scale rating from 0 to 10 (0: none; 10: strong).

[0176] The results obtained are reported in the table below:

[0177] Conclusions:

[0178] The extract according to the invention is more effective at masking licorice notes and reducing sweetness than EXL-0 and GABA.

[0179] 6 / Sensory analysis of the effect of an extract according to the invention on herbal cakes and drinks

[0180] Further studies were conducted to investigate the effect of an extract according to the invention (EXL-I) in comparison with another yeast extract already marketed as a masking agent under the name "Springer® Mask 101" for the reduction of bitterness and the masking of secondary notes in two standard applications containing bean proteins.

[0181] Sensory evaluation:

[0182] Test type: Descriptive analysis

[0183] Participants: 4 internal panelists (members of the sensory center and culinary center teams)

[0184] Method: Samples were presented to panelists in a blind test, along with a control sample containing no added product. Panelists were then asked to rate selected attributes on a scale of 1 to 10. Sensory attributes were selected by the Global CC team following initial product evaluations.

[0185] Selected applications: Plant-based cakes (unflavored)

[0186] Herbal-based cakes (flavored) Herbal drink

[0187] 6-1 / Plant-Based Patties (Unflavored) Water and textured fava bean protein were mixed on one side, and the dry ingredients on the other. Crushed water / ice was placed in a blender, along with sunflower oil, then the hydrated protein and the dry ingredients. The mixture was blended until smooth. The patties were formed and baked at 175°C for 7 minutes. The results obtained are illustrated in diagram form in Figure 3.

[0188] Conclusions

[0189] The witness exhibited strong flavors of toast, bitterness, and earth with a lingering aftertaste.

[0190] Test 1 (EXL-I) reduced bitter and earthy tastes, with a lesser impact on bitterness.

[0191] Tests 2 (Springer® Mask 101) and 3 (Springer® Mask 101 + EXL-I) both significantly reduced bitterness and improved the overall flavor profile by decreasing buttery, earthy and toasty notes.

[0192] Trial 3 proved to be the most neutral, with results similar to those of trial 2.

[0193] 6-2 / Herbal (flavored) patties

[0194] The flavoured cakes were prepared in the same way as in the previous point 6-1.

[0195] The results obtained are illustrated in diagram form in Figure 4.

[0196] Conclusions

[0197] The control sample had a bitter, buttery, and earthy taste with strong astringency, a slight umami, and a lasting mushroom and toast flavor.

[0198] Test 1 (EXL-I) effectively reduced bitterness and astringency, slightly enhanced umami, improved the perception of beef / meat flavors and was the preferred sample.

[0199] Test 2 (Springer® Mask 101) reduced bitterness, astringency and hay notes while enhancing beef and meat flavors.

[0200] Test 3 (Springer® Mask 101+ EXL-I) gave similar results to test 2, with a reduction in bitterness and astringency, a slight increase in umami and an improvement in the meaty sensation.

[0201] 6-3 / Herbal drink

[0202] The dry ingredients were mixed together. Water and oil were added to the Thermomix bowl, and the mixture was blended until completely smooth. The mixture was pasteurized by heating it to 85°C for 10 minutes, then transferred to a heatproof container where it was left to cool. Once cooled, the mixture was stored in the refrigerator.

[0203] The results obtained are illustrated in diagram form in Figure 5.

[0204] Conclusions

[0205] The witness had a slightly sweet taste with strong buttery, starchy and cardboard flavors, accompanied by bitterness, astringency and earthy notes.

[0206] Test 1 with the extract according to the invention (EXL-I) reduced the buttery, cardboard and hay notes, as well as the astringency.

[0207] Test 2 (Springer® Mask 101) was perceived as slightly less sweet than the control and other samples, with a reduction in bitter and earthy notes.

[0208] Test 3 (Springer® Mask 101+ EXL-I) reduced the buttery and cardboard notes and was noted by some tasters as having a sweetness intensity closer to the control, with a more impactful flavor than the other two test samples.

[0209] 6-4 / General Conclusions

[0210] The effectiveness of the extract according to the invention depends on the specific application and the desired aroma modifications.

[0211] For unflavored vegetable patties and plant-based drinks, a synergy between the two masking products is noted, since the combination of the extract according to the invention with another yeast extract already commercially available (test 3) at half the concentration is more effective than each one alone at its concentration considered effective in reducing bitterness and improving the overall flavor profile.

[0212] For beef-flavored vegetable patties, the extract according to the invention (Test 1) is the preferred choice, as it effectively reduces bitterness and astringency while improving the perception of beef or meat flavors.

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 selected from the group consisting of: Levilactobacilus brevis, Lentilactobacilus buchneri and Lactococcus lactis.

10. Extract according to claim 9, characterized in that the bacterium is Levilactobacillus brevis. Tl 11. Extract according to any one of claims 8 to 10, 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.

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

13. Product comprising an extract according to any one of claims 1 to 12, advantageously a food product, wherein the extract is present in an amount between 10 ppm and 3000 ppm, preferably between 50 ppm and 500 ppm, for example 200 ppm.

14. Product according to claim 13, characterized in that it further contains another masking agent.

15. Use of an extract according to any one of claims 1 to 12 or of a product according to claim 13 or 14, for masking flavours, in particular bitter and sour, undesirable notes, in particular sweeteners, proteins and metallic notes, or trigeminal sensations such as astringency in a product, advantageously a food, pharmaceutical, nutraceutical, or flavouring preparation.

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