METHOD FOR REDUCING THE BITTERNESS OF A LEGUME PROTEIN

Abstract

The invention relates to a method for reducing the bitterness of a legume protein during an extraction process, said extraction process comprising treatment with an enzyme degrading phytic acid.

Description

Title of the invention: METHOD FOR REDUCING THE BITTERNESS OF A LEGUME PROTEIN Technical field

[0001] The invention relates to the field of legume proteins, in particular legume protein isolates, even more particularly pea protein isolates. The invention relates in particular to a method for reducing the bitterness of a legume protein. Prior art

[0002] Human daily protein requirements are between 12 and 20% of the food ration. These proteins are provided by both animal products (meat, fish, eggs, dairy products) and plant foods (cereals, legumes, algae).

[0003] However, in industrialized countries, protein intake is mainly in the form of animal protein. However, numerous studies show that excessive consumption of animal protein at the expense of plant protein is one of the causes of increased cancer and cardiovascular disease.

[0004] Furthermore, animal proteins have many disadvantages, both in terms of their allergenicity, particularly proteins from milk or eggs, and in terms of the environment in relation to the harmful effects of intensive farming.

[0005] Thus, there is a growing demand from manufacturers for compounds of plant origin possessing interesting nutritional and functional properties without presenting the disadvantages of compounds of animal origin.

[0006] Since the 1970s, grain legumes, including peas in particular, have grown significantly in Europe, mainly in France, as an alternative protein resource to animal proteins for animal and human food. The term "pea" is considered here in its broadest sense and includes in particular all wild varieties of "smooth pea", and all mutant varieties of "smooth pea" and "wrinkled pea", regardless of the uses for which said varieties are generally intended (human food, animal nutrition and / or other uses). These seeds are generally non-GMO and do not require solvent deoiling.

[0007] Peas contain about 27% by weight of protein matter. Pea protein, mainly pea globulin, has been extracted and used industrially for many years. Examples of extraction processes include pea protein patent EPI400537. In this process, the seed is ground in the absence of water (known as "dry grinding") to obtain a flour. This flour will then be suspended in water to extract the protein.

[0008] Legume proteins, and in particular pea proteins, however, suffer from variable organoleptic quality because legume proteins frequently generate bitter flavor or bitterness.

[0009] Bitterness is one of the five main tastes, along with sweet, salty, sour, and umami. This flavor, generally considered harsh and unpleasant, is the characteristic flavor of products such as quinine, brucine, and caffeine. It is created by certain substances that bind to the specific bitter receptors on the taste buds on the tongue, and transmit a signal to the brain through a neurochemical mechanism.

[0010] Users of these legume proteins are aware of these taste issues and have developed formulation strategies based mainly on the use of flavorings but also on the use of salts.

[0011] For example, we can cite patent application WO 2019 / 048564 from the company RHODIA which teaches that the use of a vanilla flavoring makes it possible to reduce the bitter flavor in a finished food product such as a high-protein drink or cream.

[0012] Document WO2021 / 100729 describes the use of an agent for suppressing an abnormal flavor, and in particular bitterness, which is a cyclic compound. Legume proteins are particularly cited as ingredients whose bitterness is reduced when mixed with this agent.

[0013] Document WO2020 / 240144 in the name of the Applicant describes a particular method for manufacturing a co-atomized protein with a flavoring. The bitterness of the protein thus obtained is reduced, thus allowing its use in all known food applications.

[0014] Another solution has been described in document WO2019 / 048804, also in the name of the Applicant, for obtaining a food product based on legume proteins, for example a ready-to-drink beverage, with improved organoleptic properties. This solution is based on a particular manufacturing process for the food product using a selected compound: sodium citrate. The bitterness of the food product thus obtained is reduced.

[0015] A disadvantage of using a flavoring or masking agent is the need to label this compound on the protein ingredient. Furthermore, this requires perfect control of the flavor dosage in the protein in order to achieve the desired improvement, without "denaturing" the taste of the protein, i.e. without giving an unnatural taste, for example vanilla, to the pea protein.

[0016] The use of salt during the manufacture of a food product increases the mineral content in the finished food product, which can be prohibitive for certain uses.

[0017] All of these solutions involve adding an additional ingredient to the extracted protein to reduce the perception of the bitter taste present in the protein.

[0018] The Applicant has already succeeded in providing other proteins with reduced bitterness, by modifying the protein extraction process. Thus, pea protein compositions with reduced bitterness, using a particular manufacturing process, have been described in application WO 2019 / 053387 AL

[0019] The Applicant has also developed and described in application WO2017129921 other pea protein isolates of very particular viscosity and solubility, the manufacturing process of which includes the use of proteolytic enzymes. This application also describes food products comprising these isolates, in particular ready-to-drink beverages and ice creams, having a lower bitterness than those based on commercial pea protein isolates.

[0020] In the context of its studies for improving the taste of protein, the Applicant found that it was possible to reduce the bitterness of legume proteins, and in particular, the bitterness of pea proteins. The Applicant identified that this objective of reducing bitterness could be achieved by implementing a process for extracting legume protein in which a phytic acid-degrading enzyme is used. This introduction is, according to the invention, used at particular points in this extraction process. This method is particularly useful for providing legume proteins with reduced bitterness useful for human consumption. Summary of the invention

[0021] The invention thus relates to a method for reducing the bitterness of a legume protein during a process for extracting said legume protein, said extraction process comprising: • the preparation of a legume flour suspension • the elimination of fractions rich in starch and / or fibers to form a liquid protein fraction • increasing the protein richness of the liquid protein fraction to form a protein-enriched protein fraction wherein the method comprises treating the liquid protein fraction and / or the protein-enriched protein fraction with a phytic acid-degrading enzyme.

[0022] The invention also relates to the use of an enzyme degrading phytic acid in a process for extracting legume protein to reduce the bitterness of said protein.

[0023] An advantage of the invention is that it does not require the addition of an additional compound such as a masking agent or a salt. Indeed, the Applicant has been able to observe that phytic acid-degrading enzymes, generally used to improve the digestibility of animal feed compositions, also make it possible to reduce the bitterness of a legume protein when they are used in steps of its extraction process. In bitterness reduction methods where such additional compounds are mixed with the protein to reduce its bitterness, it may then be necessary to label it, which tends to lengthen the list of constituents present in the ingredient but also that of the final food product as well. Furthermore, some of these additional compounds may be classified as additives according to certain regulations. However, consumers tend to prefer food products free of such additives.An additional advantage of this method is that it uses a specific enzyme during the legume protein extraction process. Since this enzyme is a technological aid in the extraction process, its use has no impact on the labeling of the extracted legume protein.

[0024] The invention will now be described in detail in the following paragraphs. The features described may, optionally, be implemented. They may be implemented independently of one another or in combination with one another. Detailed description of the invention

[0025] The subject of the invention is a method for reducing the bitterness of a legume protein during a process for extracting said protein.

[0026] By "reducing the bitterness of a legume protein" is meant the reduction of the perception of bitter flavor when tasting the legume protein. The determination of the reduction of this perception can be done according to the methods of the art, generally using a panel trained in tasting the legume protein as determined in the Examples section of this Application.

[0027] The term "legume protein" should be understood in the present application as a composition extracted from legumes comprising mainly polypeptide chains, or proteins, consisting of the chain of amino acid residues linked together by peptide bonds. The protein content of the legume protein is the N6.25 content, calculated by the Dumas method. Generally, the protein content is 60% or more, advantageously 80% or more, for example ranging from 80 to 95%, in particular ranging from 80 to 90%. According to the invention, protein isolate means a legume protein having a protein content of 80% or more.

[0028] Although a legume protein is primarily defined by its botanical origin and protein content, it of course generally includes other minor constituents other than protein, such as starch, lipids, fibers, sugars and / or minerals. Generally, the total starch content in the legume protein produced according to the method of the invention ranges from 0 to 20%, for example from 0 to 10%, in particular from 0.5 to 5%. This total starch content can be measured using the AO AC Method 996.11. Generally, the total fiber content can range from 0 to 20%, for example from 1 to 18%, in particular from 2 to 10%. This content can be determined by the AO AC Method 2017.16. Generally, the total lipid content ranges from 0 to 15%, for example from 1 to 10%. Total lipid content can be determined by method AO AC 996.06 acid hydrolysis. Sugar content can range from 0 to 10%, typically 0.5 to 5%.The sugar content can be determined by high-performance liquid chromatography (HPLC). The mineral content can be determined by determining the ash content. Among the minerals, legume protein includes phosphates. Legume protein generally has a free phosphate content, which can vary widely, ranging from 0.2 to 2%, for example from 0.5 to 1.9%, especially from 1.2 to 1.8%. The free phosphate content can be determined by anion chromatography. A person skilled in the art will be able to find the conditions for anion chromatography and, for illustration, a method of analysis by chromatography is given in the example section.

[0029] The legume protein obtained using the method according to the invention may comprise a phytic acid content varying from 0.1 to 1.5%, for example from 0.3 to 1.4% or from 0.5 to 1.2%. According to the invention, the phytic acid content in the legume protein is determined according to the method described in the document McKie et al., Journal of AOAC International, Vol. 99, No. 3, 2016. This determination method is an indirect method consisting of the quantification of the phosphate released during the hydrolysis of a sample by a phytase and an alkaline phosphatase. Thus, as explained in this document McKie et al., it is specified that the determination of phytic acid according to this method uses the quantification of phosphates released by the hydrolysis of phytic acid as well as phosphates released by the hydrolysis of inositol ester derivatives IPI, IP2, IP3, IP4 and IP5 present in the sample.From this quantification of the released phosphate, we deduce a quantity of phytic acid, which is a theoretical quantity of phytic acid that would be obtained. if all inositol esters consisted of phytic acid. Megazyme markets the K-PHYT kit using this method.

[0030] In the present application, the term "legume" means the family of dicotyledonous plants of the order Fabales. It is one of the largest families of flowering plants, third after Orchidaceae and Asteraceae in terms of the number of species. It has approximately 765 genera comprising more than 19,500 species. Several legumes are important cultivated plants, including soybeans, beans, especially mung beans, peas, chickpeas, field beans, peanuts, cultivated lentils, cultivated alfalfa, various clovers, broad beans, carob, licorice and lupin. Preferably, the legume is chosen from peas, field beans and mung beans, most preferably peas.

[0031] The term "pea" is to be understood in the present application as all wild varieties of "smooth pea", and all mutant varieties of "smooth pea" and "wrinkled pea".

[0032] In the invention, the method for extracting legume protein comprises: • preparing a suspension of legume flour, • the elimination of fractions rich in starch and / or fibers to form a liquid protein fraction, • increasing the protein richness of the liquid protein fraction to form a protein-enriched protein fraction, and this method comprises treating the liquid protein fraction and / or the protein-enriched protein fraction with a phytic acid-degrading enzyme.

[0033] According to the invention, the reduction in bitterness is observed for a protein obtained by the above method, comprising the treatment of the liquid protein fraction and / or the protein-enriched protein fraction with a phytic acid-degrading enzyme, in comparison with the protein obtained by a method which differs only in that it does not comprise this step of treatment with a phytic acid-degrading enzyme. This improvement could be observed significantly, as demonstrated in the Examples section.

[0034] The method of extracting the protein comprises preparing a suspension of a legume flour.

[0035] By "suspension of a legume flour" is meant an aqueous suspension of ground legumes in water.

[0036] Generally, ground legumes are ground legume seeds. These seeds typically first undergo steps well known to those skilled in the art, such as cleaning or even the removal of the outer shell of the seed (cellulosic outer envelope), by a well-known step called "dehulling".

[0037] Before grinding, additional treatments of the grains such as dry heating as described in document WO2020 / 260841 can be carried out. Also, by wet method, soaking or even wet fermentation of a seed suspension as described in document WO2015 / 071498 A1 can be applied.

[0038] The grinding can be carried out by dry grinding, that is to say by passing the previously prepared dry seeds through one or more grinders. Alternatively, the grinding can be wet grinding, that is to say by passing the previously prepared seeds in the form of a suspension of seeds in water through one or more grinders.

[0039] As a usable mill, any type of suitable technology known to those skilled in the art and adapted to the desired type of grinding can be used. These may in particular be ball mills, conical mills, helical mills, air jet mills, rotor-rotor systems or rotor-stator systems.

[0040] In the extraction process, the preparation of a legume flour suspension can thus be done by suspending in water a legume flour obtained by dry grinding of the seeds. Alternatively, the preparation of a legume flour suspension can be done by wet grinding. In this case, the legume flour suspension can be obtained directly; the water present in the suspension can be retained but can also be renewed.

[0041] Preferably, the aqueous suspension of legume flour has between 15% and 25% by weight of dry matter (DM), preferably approximately 20% by weight of DM, relative to the weight of said suspension.

[0042] In the present Application, the quantities by weight of the different constituents are expressed in dry weight.

[0043] At the end of grinding, a pH check can be carried out. Preferably, the pH of the aqueous suspension is between 5.5 and 10, for example between 7 and 10, for example between 6 and 9, generally between 7 and 9. The pH rectification can be carried out by adding acid and / or base, organic or inorganic, for example sodium hydroxide or hydrochloric acid.

[0044] The method further comprises the removal of starch-rich and / or fiber-rich fractions to form a liquid protein fraction. These starch-rich and / or fiber-rich fractions mainly comprise the insoluble parts of the suspension. Thus, these fractions are recovered conventionally by known separation methods. It can in particular be carried out by means of at least one separation step with a decanter, in particular a centrifugal decanter, or with hydrocyclones. The method can thus make it possible to recover one or more fiber-enriched fractions. and / or starch which are removed from the suspension to form the liquid protein fraction.

[0045] The method further comprises a step of increasing the protein content.

[0046] According to a first variant of the extraction method, the step of increasing the protein richness may comprise a stage of precipitation of the proteins included in the liquid protein fraction followed by a separation stage to form the protein fraction enriched in proteins. This stage may be carried out by bringing the pH of the aqueous suspension of legume flour close to the isoelectric pH of the proteins in order to precipitate them. The precipitation stage may for example be carried out at a pH ranging from 4.6 to 5.4, for example approximately 5.0. This stage may also be carried out by thermocoagulating the proteins, i.e. by heating the aqueous suspension of legume flour to a temperature ranging from 50 to 90°C. It is also possible to combine the two by carrying out this thermocoagulation after bringing the pH of the aqueous suspension of legume flour close to the isoelectric pH of the proteins, in the pH ranges previously mentioned.The protein-enriched protein fraction can be obtained after a separation step of the precipitated proteins. This separation step can be carried out by filtration or by decantation, in particular using a centrifugal decanter. This variant is particularly preferred since acid phosphatases, and in particular phytases, exhibit excellent activity at a pH close to the isoelectric pH of legume proteins; they are therefore very active without the need to modify the pH of the liquid protein fraction or the protein-enriched protein fraction.

[0047] According to a second variant of the extraction process, the step of increasing the protein richness can be carried out by known membrane filtration methods. This membrane filtration step can thus be in particular an ultrafiltration. Such processes using this method of increasing the protein richness have already been described, in particular in the document Fuhrmeister et al., Impact of processing on functional properties of protein products from wrinkled peas, Journal of Food Engineering 56 (2003) 119-129 or the patent US10143226B1.

[0048] At the end of this step of increasing the protein richness, the protein fraction enriched in proteins can have the contents indicated in paragraphs [0029-0030] above for the legume protein, in proteins, fibers, starch, lipids, sugars and minerals.

[0049] According to the invention, the extraction process comprises treating the liquid protein fraction and / or the protein-enriched protein fraction with a phytic acid-degrading enzyme. As both variants are possible and the reaction conditions are similar in both cases, the terms "treatment of the protein fraction” group together the two processing variants of the liquid protein fraction and / or the protein-enriched protein fraction.

[0050] By "phytic acid degrading enzyme" is meant according to the invention an enzyme capable of degrading phytic acid or phytates when it is used in an effective amount under its optimal conditions of use.

[0051] This enzyme may be a phosphatase, for example an acid phosphatase and more particularly a phytase. These enzymes are known to have the capacity to reduce the amount of phytic acid which is an antinutritional factor preventing the intestinal absorption of micronutrients such as iron, magnesium, zinc and copper. Phytic acid is mainly present in protein compositions of plant origin, in particular cereals and legumes. Certain animals such as pigs and poultry are particularly sensitive to the presence of phytic acid or phytate in their diet. Thus, to this day, these enzymes have mainly been used for animal nutrition, by integrating them as such into nutritional compositions for animals, in particular those intended for pigs and poultry.Alternatively, phytases have also been used by making them act on proteins, in order to provide protein feeds that are more easily assimilated by animals. To the Applicant's knowledge, although new phytases can now be used to provide ingredients for human consumption, there is not yet on the market a legume protein manufactured by a process that uses such enzymes during its extraction process.

[0052] According to the invention, it is also possible to use several phytases (such as for example the mixture described in document WO2007 / 006952 A1), several acid phosphatases or a mixture of phytase(s) and acid phosphatase(s).

[0053] Preferably, the phytic acid degrading enzyme is a phytase or a mixture of phytases. Phytases are hexakisphosphohydrolases that hydrolyze the phosphoester bonds present in phytic acid or phytates. Thus, they catalyze the hydrolysis of myo-inositol (1,2,3,4,5,6) hexakisphosphate into inorganic monophosphates and myo-inositol phosphates of lower degree of phosphorylation (IP5 to IPI) and into free myo-inositol in certain cases.

[0054] There are three subclasses of phytases, differentiated by the position of the first hydrolyzed phosphate. 3-phytases (EC 3.1.3.8) hydrolyze the first phosphate at position 3, 6-phytases (EC 3.1.3.26) hydrolyze the first phosphate at position 6, 5-phytases (EC 3.1.3.72) hydrolyze the first phosphate at position 5.

[0055] Phytases can be obtained by a wide variety of organisms: plants, animals, and especially microorganisms. These phytases can have different biochemical characteristics, in particular their pH-dependent activity and temperature stability may differ.

[0056] Among the microorganisms producing phytases, we will notably mention fungi, bacteria and yeasts.

[0057] The mushrooms can be of the Aspergillus, Penicillium, Mucor and Rhizopus genus. Aspergillus type fungi include Aspergillus niger, Aspergillus oryzae, Aspergillus ficuum, Aspergillus awamori, Aspergillus nidulans and Aspergillus terreus and Trichoderma reesei.

[0058] The bacteria can be Pseudomonas sp., Klebsiella sp., Escherichia coli, Enterobacter sp., Bacillus sp. including Bacillus subtilis.

[0059] Yeasts are for example Saccharomyces cerevisiae, Candida tropicalis, Torulopsis candida, Debaryomyces castellii, Debaryomyces occidentalis, Kluyveromyces fragilis and Peniophora lycii.

[0060] Phytases can also be extracted from plants, including endogenous phytases from cereals such as wheat, rye and barley.

[0061] According to the invention, the expression "an obtainable phytase" encompasses a phytase produced naturally by the particular strain and recovered from that strain and either encoded by a DNA sequence isolated from that strain and produced in a host organism transformed with said DNA sequence. The phytase may be obtained by fermentation of a phytase-producing microorganism in a suitable nutrient medium, followed by isolation of the phytase by methods known in the art. The medium used for cultivation may be any medium suitable for cultivating the microorganism in question. The medium preferably contains carbon and nitrogen sources and other inorganic salts.

[0062] The preferred phytases useful in the invention are of type EC 3.1.3.8, for example SUMIZYME PHY, Shin Nihon and EC 3.1.3.26, for example NATUPHOS E 10000, BASF.

[0063] The phytic acid degrading enzymes may further comprise other enzymatic activities, such as, for example, amylase, protease, cellulase, hemicellulase and / or pectinase secondary activities.

[0064] The method may also use other enzymatic steps, for example steps of enzymatic proteolysis of the protein-enriched protein fraction. The enzymatic proteolysis is generally carried out in a known manner, conventionally using proteases. According to one embodiment, the extraction method comprises proteolysis of the protein-enriched protein fraction. According to one embodiment, the extraction method does not comprise proteolysis of the protein-enriched protein fraction. This proteolysis step may make it possible to modify the degree of hydrolysis (DH) of the legume protein. The legume protein may have a degree of hydrolysis of less than 15%, advantageously less than 10%, preferably less than 6%, for example between 3 and 5%. A person skilled in the art will know how to adapt the conditions of enzymatic proteolysis, or will even not carry out such a step in order to obtain the desired DH.

[0065] According to the invention, the treatment on a protein fraction comprises the introduction into said treated fraction of at least one phytic acid-degrading enzyme and the action of the enzyme in order to reduce the quantities of phytic acid. The introduced enzyme is generally in the form of an enzymatic solution. According to one embodiment, the quantity of phytic acid-degrading enzyme, expressed as dry mass relative to the dry mass of the protein fraction, ranges from 0.1 to 3%, for example from 0.2 to 1%. It is possible to dilute or concentrate the treated protein fraction using known concentration and dilution methods. One of the advantages of the method of the invention is that it is also possible not to modify the dry matter of the protein fraction and to introduce the enzyme directly into it.Typically, during the action of the enzyme, the dry matter mass of the protein fraction during the treatment step with the phytic acid-degrading enzyme ranges from 3 to 30%, for example from 4 to 20%, for example between 5 and 15%. Typically, during the action of the enzyme, the pH of the protein fraction ranges from 4 to 7, for example from 4.5 to 6, especially from 4.6 to 5.4, for example about 5.0. The duration of the treatment can also vary, it can range from a few minutes to a few hours, for example from 10 to 240 minutes, especially from 15 to 100 minutes, generally from 20 to 80 minutes.

[0066] The conditions of pH, temperature, dry matter, quantity of enzyme and time will be easily adapted by a person skilled in the art so that the enzyme is effective: according to the invention, the treatment step is carried out so as to reduce the quantities of phytic acid in the protein fraction. Preferably, the treatment step with the phytic acid-degrading enzyme is carried out until the phytic acid content in the treated fraction is less than 1% by mass relative to the dry mass of this fraction.

[0067] By way of illustration, according to the first variant of the extraction process where the step of increasing the protein richness comprises a protein precipitation stage followed by a separation stage, the step of treating the liquid protein fraction with an enzyme degrading phytic acid can be carried out before, during or after having brought the pH of the aqueous suspension of legume flour close to the isoelectric pH. Preferably, the treatment step is carried out simultaneously or after, most preferably simultaneously. This embodiment is illustrated in Example 4 below.

[0068] Alternatively, still according to this same first variant, the step of treating the protein fraction enriched in proteins with an enzyme degrading phytic acid can be carried out on the protein fraction enriched in proteins recovered during the separation step, optionally after dilution thereof. This embodiment is notably illustrated in Example 2 below.

[0069] It is possible to resolubilize the recovered protein-enriched protein fraction and then centrifuge it. This step makes it possible to wash the protein and remove compounds other than proteins, such as salts. However, this step is not necessary and according to a preferred embodiment of the method of the invention, the extraction process does not include steps of resolubilization and centrifugation of the protein-enriched protein fraction.

[0070] The method may further comprise, after the step of treatment with the phytic acid-degrading enzyme, a step of heat treatment of the protein-enriched protein fraction. This step is carried out to ensure satisfactory bacteriological quality and / or to modify the functionalities of the legume protein. This step may be carried out directly after recovery of the protein-enriched protein fraction. This heat treatment may be carried out at a temperature ranging from 75 to 160°C and for 0.1 seconds to 30 minutes. Advantageously, this heat treatment may also serve to functionalize the protein composition. It is therefore preferably carried out according to a conventional scale of 100°C to 160°C for 0.01s to 15s, preferably between 1 and 2 seconds followed by immediate cooling.

[0071] The method may also further comprise a step of drying the protein-enriched protein fraction. Generally, this drying step is carried out so as to achieve a dry matter content greater than 80%, preferably greater than 90% by weight of dry matter relative to the weight of said protein-enriched protein fraction. Any technique well known to those skilled in the art is used for this purpose, such as, for example, freeze-drying, flash drying or drum drying, or even atomization. The method may also comprise a grinding or micronization step. Atomization is the preferred technology, in particular multiple-effect atomization. The legume protein may be in the form of a powder having a particle size d50, which may vary widely, for example, from 10 to 200 μm.

[0072] Generally, the legume protein thus obtained may be used in food and beverage products which may include it in an amount of up to 100% by weight relative to the total dry weight of the food or beverage product, for example in an amount of from about 1% by weight to about 80% by weight relative to the total dry weight of the food or beverage product. All intermediate amounts (i.e. 2%, 3%, 4%... 77%, 78%, 79% by weight) are not suitable. weight relative to the total weight of the food or beverage product) may be used, as may any intermediate ranges based on these amounts. Food or beverage products that may be affected include bakery products; sweet bakery products (including, but not limited to, rolls, cakes, pies, pastries, and biscuits); pre-made sweet baking mixes for the preparation of sweet bakery products; pie fillings and other sweet fillings (including, but not limited to, fruit pie fillings and nut pie fillings such as pecan pie fillings, and fillings for cookies, cakes, pastries, confectionery products, and the like, such as fat-based cream fillings); desserts, gelatins, and puddings;frozen desserts (including, but not limited to, frozen dairy desserts such as ice cream – including regular ice cream, soft-serve ice cream, and all other types of ice cream – and frozen non-dairy desserts such as non-dairy ice cream, sorbet, and the like); carbonated beverages (including, but not limited to, carbonated soft drinks); non-carbonated beverages (including, but not limited to, non-carbonated soft drinks such as flavored waters, fruit juices, and sweet tea or coffee-based beverages); beverage concentrates (including, but not limited to, liquid concentrates and syrups as well as non-liquid “concentrates,” such as freeze-dried and / or powdered preparations); yogurts (including, but not limited to, full-fat, reduced-fat, and fat-free dairy yogurts, as well as non-dairy and lactose-free yogurts);snack bars (including, but not limited to, cereal, nut, and / or fruit bars); bread products (including, but not limited to, leavened and unleavened breads, yeast breads and undyed breads such as soda breads, breads comprising any type of wheat flour, breads comprising any type of flour other than wheat (such as potato, rice, and rye flours), gluten-free breads); bread mixes for the preparation of bread products; sauces, syrups, and dressings; sweet spreads (including, but not limited to, jellies, jams, butters, spreads, and other preserves, preserves, and other spreadable preserves); confectionery products (including, but not limited to, jelly beans, soft candies, hard candies, chocolates and gums);sweetened and unsweetened breakfast cereals (including, but not limited to, extruded breakfast cereals, flaked breakfast cereals, and puffed breakfast cereals); and cereal coating compositions for preparing sweetened breakfast cereals. Other types of foods and beverages not mentioned herein but which conventionally comprise one or more proteins may also be contemplated within the scope of the present invention. ; In particular, animal feed (such as pet food) is explicitly considered. It can also be used, possibly after extrusion texturization, in meat-like products such as emulsified sausages or plant-based burgers. It can also be used in egg replacement formulations.

[0073] The food or beverage product may be used in specialized nutrition, for specific populations, for example for babies or infants, the elderly, athletes, or in clinical nutrition (for example tube feeding or enteral nutrition).

[0074] Legume protein can be used as the sole source of protein, but can also be used in combination with other plant or animal proteins. The term "plant protein" refers to all proteins derived from cereals, oilseed plants, legumes and tuberous plants, as well as all proteins derived from algae and microalgae or fungi, used alone or in mixtures, chosen from the same family or from different families. In the present application, the term "cereals" refers to cultivated plants of the grass family producing edible grains, for example wheat, rye, barley, maize, sorghum or rice. Cereals are often ground into flour, but are also supplied as grains and sometimes as whole plants (fodder).The tubers can be carrot, cassava, konjac, potato, Jerusalem artichoke, sweet potato. The animal protein can be for example egg or milk proteins, such as whey proteins, casein proteins or caseinate. The pea protein composition can thus be used in combination with one or more of these proteins or amino acids in order to improve the nutritional properties of the final product, for example to improve the PDCAAS of the protein or to provide other functionalities or modify.

[0075] The legume protein obtained has improved organoleptic properties and in particular a reduced bitterness compared to the same legume protein not having been prepared by the method according to the invention comprising the treatment of the liquid protein fraction and / or the enriched protein fraction with an enzyme degrading phytic acid. Another observed advantage is that the solubility in water at acidic pH, for example between 3 and 5 and in particular at pH4 is increased, as demonstrated in the examples section. This solubility at pH4 can range from 26 to 50%, for example from 30 to 45%. The method for determining the solubility is described in the Examples section below. The legume protein of the invention can thus be used in drinks at acidic pH, which will have an improved texture.

[0076] The invention will now be illustrated using the following examples, which are intended to be non-limiting. Examples

[0077] Methods

[0078] Evaluation of bitterness

[0079] The organoleptic study of the different pea protein powders obtained is carried out using a panel and following the protocol below.

[0080] The panel is composed of 30 people trained for 2 to 4 years. Their performances are frequently checked in terms of sensitivity, consensus and repeatability.

[0081] The tasting matrices consist of suspensions of each of the powders at 4% by weight in Evian® water, homogenized using an immersion mixer.

[0082] The tasting conditions are as follows: individual box, white walls, calm atmosphere, red light, late morning, products coded with 3 digits, presented in random order, and use of apple and / or water to wash the oral cavity.

[0083] The methodology used is called “BLOCK PROFILING”. This method is called Quantitative Descriptive Analysis (QDA): the panelists rate each product on an intensity scale (from 0 to 10) using different indicators that correspond, for example, to flavors, flavours, or particular notes.

[0084] The witness is always presented first and is presented blindly 1 session out of 2.

[0085] The panelists conduct the tasting exercise in blocks: they evaluate each product individually (starting with the control) in a first block (indicators: salty, bitter, astringent, sandy - with a nose clip), then they analyze all the products in a second block (indicators: peas, broth, walnuts, almonds). Finally, they repeat the exercise in a third block (indicators: potato, cereal). The products are evaluated during multiple sessions, to reach 10 evaluations. The arithmetic mean of these 10 evaluations is then carried out for each indicator. The reduction in bitterness is calculated for the tests according to the invention (2, 4 and 6) according to the following formula: (Control score - Invention sample score) / Control score x 100.

[0086] Protein content:

[0087] Percentage by dry mass of protein N6.25 using the Dumas method (ISO 16634).

[0088] Determination of phytic acid content:

[0089] The phytic acid content is determined by the Megazyme K-PHYT kit according to the method described in McKie et al., Journal of AOAC International, Vol. 99, No. 3, 2016.

[0090] Determination of free phosphate content:

[0091] The determination of the free phosphate content is carried out according to the method of the art known anion chromatography. The legume protein sample is analyzed, following the instructions in the user manual, on a Thermo ICS-2100 chromatograph equipped with an AG 11 HC DIONEX pre-column and an AS 11 HC DIONEX column, all coupled to a conductometric detector, using as eluting with a potassium hydroxide solution.

[0092] Measurement of solubility in water at different pHs

[0093] This measurement is based on diluting the sample in distilled water, centrifuging it and analyzing the supernatant.

[0094] Operating mode:

[0095] In a 400 ml beaker, introduce 150 g of distilled water at a temperature of 20°C + / - 2°C, mix with a magnetic bar and add precisely 5 g of the sample to be tested.

[0096] Adjust or not the pH to the desired value with NaOH or 0.1 N HCl (pH 4 or pH 7).

[0097] Complete the water content to 200 g.

[0098] Mix for 30 minutes at 1000 rpm and centrifuge for 15 minutes at 3000 g-

[0099] Collect 25 g of the supernatant.

[0100] Introduce into a previously dried and tared crystallizer.

[0101] Place in an oven at 103°C + / - 2°C for 1 hour.

[0102] Then place in a desiccator (with desiccant) to cool to room temperature and weigh.

[0103] The soluble solids content, expressed in % by weight, is given by the

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110] [YES]

[0112] following formula: (ml - m2) x (200 + P) x 100 .....................................= % solubility PI xP Or: o P = weight, in g, of the sample = 5 g o ml = weight, in g, of the crystallizer after drying o m2 = weight, in g, of the empty crystallizer o PI = weight, in g, of the collected sample = 25 g Example 1 (witness)

[0113] The following protocol is applied:

[0114] Mixing of 2 kg (expressed in commercial mass) of smooth yellow pea flour (86% dry matter and 27% protein N6.25 in dry weight) with demineralized water (20°C) => Suspension at 17% dry matter with a RAYNERI type benchtop agitator with dispersion turbine for 30 minutes

[0115] Centrifugation 1000 g - 5 minutes / BECKMAN laboratory centrifuge

[0116] Recovery of 7 kg of supernatant at approximately 7% dry matter in a stainless steel container and elimination of the fiber / starch residue

[0117] Acidification => pH5 with HCl IN

[0118] Thermofloculation: Heat and hold for 10 minutes at 60°C, (hot plate) with stirring

[0119] Centrifugation 5000 g - 5 minutes / BECKMAN laboratory centrifuge

[0120] Recovery of the protein sediment (or protein fraction enriched in protein) and elimination of the supernatant

[0121] Dissolving the sediment with demineralized water (20°C) => 3 kg of suspension at 12% dry matter with a RAYNERI type benchtop agitator with dispersion turbine for 30 minutes

[0122] Neutralization (with stirring) => pH7 with NaOH IN

[0123] Heat treatment type pasteurization: heating => 80°C (in stirred crystallizer) + holding for 3 minutes

[0124] Spray drying with a BUCHI laboratory atomizer

[0125] Example 2

[0126] The protocol of this test, which differs mainly from the control protocol by the use in the extraction process of a phytase before the neutralization step, is applied. Thus, this test is identical to that of Example 1 except that between the sediment dilution and neutralization step, the following additional steps are carried out:

[0127] Heating => 55°C on a stirred hotplate

[0128] Introduction of 0.5% dry weight of phytase relative to the dry weight of suspension (SUMIZYME PHY, Shi Nihon), i.e. an enzymatic activity of 3,600 FTU according to the supplier's data, in 3 kg of suspension at 12% dry matter and hydrolysis with stirring for 20 minutes.

[0129] Example 3 (comparative example)

[0130] The objective of this test is to evaluate the impact of the use of the enzyme SUMIZYME PHY and the heat treatment used during hydrolysis on the properties of the protein. The protocol of Example 3 thus differs from that of Example 2 only in that no phytase is introduced after heating the diluted sediment to 55°C. Thus, the dried protein in Example 3 exhibits exactly the same thermal history as the dried protein in Example 2.

[0131] Example 4

[0132] The protocol of this test differs primarily from the protocol of Example 2 in that the phytase is introduced into the extraction process after the fiber and starch removal step rather than after the protein sediment dispersion step. Thus, this test is identical to that of Example 1 except that between the acidification and thermoflocculation steps, the following additional steps are performed:

[0133] Heating of the acidified supernatant => 55°C on a stirred hot plate

[0134] Introduction of 0.5% dry weight of phytase relative to the dry weight of supernatant and hydrolysis with stirring for 20 minutes.

[0135] The invention was also evaluated on a larger scale (pilot scale) in the following examples 5 and 6.

[0136] Example 5 - pilot scale witness

[0137] The pilot protocol below was carried out:

[0138] Mixing of 80kg dry weight of smooth yellow pea flour with demineralized water (20°C) => Suspension at 15% dry matter in a tank equipped with axial and peripheral stirring blades for 30 minutes

[0139] Decantation in a Flottweg decanter type Z2 - 4000 g

[0140] Transfer of the supernatant (approximately 7% dry matter) into a stirred tank equipped with a double jacket and elimination of the fiber / starch pellet

[0141] Acidification => pH5 with HCl

[0142] Thermoflocculation: heating and holding for 10 minutes at 60°C in a double-jacketed tank heated by steam

[0143] Decantation in a Flottweg decanter type Z2 - 4000 g

[0144] Recovery of protein sediment and removal of supernatant

[0145] Dissolving sediment with demineralized water (20°C) in a stirred tank => Suspension at 12% dry matter with a propeller stirrer for 30 minutes

[0146] Neutralization (with stirring) => pH7 with NaOH IN

[0147] Heat treatment by direct steam injection for 10s at 135°C then flash at 70°C

[0148] Spray drying in a NUBILOSA pilot sprayer: drying temperature 190-195°C; product outlet temperature 90 - 95°C

[0149] Example 6 - pilot scale test

[0150] The protocol below, which differs mainly from the protocol of Example 5 by the use of a phytase in the extraction process before the neutralization step. Thus, this test is identical to that of Example 5 except in that that between the protein sediment dilution and neutralization step, the following additional steps are carried out:

[0151] Heating => 55°C on a stirred hotplate in a stirred tank with a double jacket

[0152] Introduction of 0.5% dry weight of phytase relative to the dry weight of suspension, or 30,000 FTU, in 25 kg of suspension at 12% dry matter and hydrolysis with stirring for 20 minutes

[0153] Properties of the proteins obtained in examples 1 to 6:

[0154] The properties of the products obtained are reported in the Table below: Protein richness N6.25 Phytic acid content Free phosphate content Solubility pH4 (%) Solubility pH7 (%) Reduction Bitterness (%) % MS LABO Example 1 2.1 0.07 26 54 Example 2 0.8 1.6 40 40 38% Example 3 NM NM 18 51 Example 4 0.4 0.6 39 47 24% pilot Example 5 87.6 2.0 0.07 15 70 Example 6 87.9 0.4 1.1 27 30 26% The tests demonstrate that the use of phytase in the extraction process allows for a reduction in the bitterness of the protein formed, compared to a process that does not include such treatment.

[0155] In the variant where the treatment step with a phytase is carried out on the protein fraction, the phosphate content is lower.

[0156] The protein further exhibits improved acidic pH solubility in assays including treatment with phytase.

[0157] Further tests were carried out with a phytase NATUPHOS E 10000, BASF, instead of SUMIZYME PHY and similar results were obtained.

Claims

Claims

1. A method for reducing the bitterness of a legume protein having a degree of hydrolysis of less than 15%, said legume being selected from pea, field bean and mung bean, during a process for extracting said legume protein, said extraction process comprising: - preparing a legume flour suspension - removing starch-rich and / or fiber-rich fractions to form a liquid protein fraction - increasing the protein richness of the liquid protein fraction to form a protein-enriched protein fraction, characterized in that the process comprises treating the liquid protein fraction and / or the protein-enriched protein fraction with a phytic acid-degrading enzyme.

2. Method according to claim 1 characterized in that the step of increasing the protein richness comprises a stage of precipitation of the proteins included in the liquid protein fraction followed by a separation stage to form the protein fraction enriched in proteins.

3. Method according to claim 2 characterized in that the precipitation stage is carried out at a pH ranging from 4.6 to 5.4, for example approximately 5.

0.

4. Method according to claim 1 characterized in that the step of increasing the protein richness comprises membrane filtration.

5. Method according to one of the preceding claims, characterized in that the extraction process comprises a step of treating the liquid protein fraction with an enzyme degrading phytic acid.

6. Method according to one of the preceding claims, characterized in that the extraction process comprises a step of treating the protein fraction enriched in proteins with an enzyme degrading phytic acid.

7. Method according to one of the preceding claims, characterized in that the enzyme degrading phytic acid is a phytase.

8. Method according to one of the preceding claims, characterized in that the method further comprises, after the treatment step with the phytic acid degrading enzyme, a heat treatment step of the protein-enriched protein fraction.

9. Method according to one of the preceding claims, characterized in that the mass percentage of dry matter of the protein fraction during the treatment step with the phytic acid-degrading enzyme is 4 to 20%, in particular 5 to 15%.

10. Method according to one of the preceding claims, characterized in that the step of treatment with the enzyme degrading phytic acid is carried out until the phytic acid content in the treated fraction is less than 1% by mass relative to the dry mass of this fraction.

11. Method according to one of the preceding claims, characterized in that the legume protein has a degree of hydrolysis of less than 10%, preferably less than 6%, for example between 3 and 5%.

12. Method according to one of the preceding claims, characterized in that the quantity of phytic acid-degrading enzyme, expressed in dry mass relative to the dry mass of the liquid protein fraction or of the protein-enriched protein fraction, ranges from 0.1 to 3%, for example from 0.2 to 1%.

13. Method according to one of the preceding claims, characterized in that the method further comprises a step of drying the protein fraction enriched with proteins.

14. Use of a phytic acid degrading enzyme in a process for extracting legume protein, said legume being selected from pea, field bean and mung bean, to reduce the bitterness thereof, the legume protein having a degree of hydrolysis of less than 15%.