High-protein hydrolysate from sunflower meal

Abstract

The group of inventions relates to the field of processing oleaginous raw materials, and more particularly to a novel product in the form of a high-protein hydrolysate (HPH) obtained from sunflower meal, a method for producing same by enzymatic hydrolysis, and HPH-based food and feed products. The HPH contains 75-85% protein, 10-20% sugar, and at least 0.01% caffeic acid in free form or in bound form as part of chlorogenic acid isomer molecules. In addition, the HPH has a total branched-chain amino acid content of at least 50 g / kg of the final product (HPH), and an arginine content of at least 30 g / kg HPH. The resulting HPH does not contain antinutrients and serves as an excellent substitute for animal protein in food products and in diets for aquatic organisms, livestock and domestic pets. The claimed novel method is safe, environmentally-friendly, waste-free and allows flexibility in terms of the operations and ingredients used in the production process.

Description

[0001] HIGH-PROTEIN HYDROLYSATE FROM SUNFLOWER MEAL, METHOD OF ITS PRODUCTION AND PRODUCTS BASED ON IT

[0002] Field of technology to which the invention relates

[0003] The present invention relates to the field of oilseed processing, specifically to a new product—high-protein hydrolysate (HPH) from sunflower meal—and a method for producing it via enzymatic hydrolysis. HPH is intended for use as a protein supplement in food and feed products.

[0004] State of the art

[0005] The productivity and profitability of industries such as food processing, livestock farming, poultry farming, and aquaculture depend largely on the ingredients used. High-quality feed must meet the body's daily requirement for nutrients and energy, micro- and macronutrients, and vitamins. Products that meet this requirement contain proteins, fats, carbohydrates, minerals, and vitamins.

[0006] Protein is a vital component of any diet. Proteins are the building blocks of organ and tissue cells. Proteins are also essential for the normal functioning of the body's enzymatic, hormonal, and immune systems.

[0007] The biological value and digestibility of a protein component are primarily determined by the content of essential and conditionally essential amino acids, whose amino acid profile should be as close as possible to that of the reference protein. These amino acids cannot be synthesized by the body and must be obtained from outside sources. Examples of easily digestible proteins with high biological value include animal proteins, particularly fishmeal, dehydrated animal proteins, and animal protein hydrolysates. However, the high cost and shortage of raw materials for producing these products has necessitated the search for alternative protein sources.

[0008] For example, sunflower meal contains up to 43% protein, with digestibility comparable to that of animal proteins. Sunflower meal prices are lower than those of animal proteins, and the proximity of sunflower processing plants to food and feed industries reduces logistics costs, making sunflower meal a promising raw material for protein component production. The anti-nutritional components contained in sunflower meal and the characteristic properties of plant proteins limit its direct incorporation into food and feed formulations.However, the present invention makes it possible to isolate a high-protein hydrolysate (hereinafter referred to as “HPH”) from the original sunflower meal, where the main component is protein (75-85%) plus a small percentage of sugars, which can serve as the maximum possible replacement for animal proteins in food products, diets for aquatic organisms (aquaculture), agricultural and non-productive animals.

[0009] The digestibility and assimilation of sunflower meal are negatively affected by the following factors contained in its composition:

[0010] • Chlorogenic acid;

[0011] Sunflower meal contains 4-5% chlorogenic acid. Its drawback is that the oxidation of chlorogenic acid produces quinones, which form strong covalent bonds with amino acids in sunflower proteins (primarily lysine, cysteine, and tryptophan). Chlorogenic acid can also inactivate digestive enzymes such as alpha-amylase, glucoamylase, and pepsin enzymes if it forms covalent bonds with them. Chlorogenic acid is also a natural antioxidant.

[0012] • Phytic acid;

[0013] Sunflower seeds contain a high level of phytic acid (up to 4% of the protein fraction), which forms strong chelate complexes with micronutrients that are not absorbed by the intestines. Phytic acid also reduces the solubility and rate of protein hydrolysis in the digestive tract.

[0014] • Digestive enzyme inhibitors;

[0015] Sunflower seeds contain inhibitors, particularly trypsin and Bowman-Birk chymotrypsin inhibitors, which are resistant to high temperatures and can inhibit protein digestion during digestion.

[0016] • Mild protein allergens;

[0017] Lipid transfer proteins and light 2S albumins of sunflower, which are allergenic proteins that are resistant to the action of proteolytic enzymes during digestion due to the formation of strong disulfide bonds, can be absorbed into the blood in the small intestine and cause allergic reactions. • Low content of some essential amino acids in sunflower protein, in particular lysine, and the absence of taurine, which is found only in proteins of animal origin;

[0018] • Products of interaction between the protein part and reducing sugars (Maillard reaction);

[0019] At elevated temperatures, the Maillard reaction between carbohydrates and amino acids results in the formation of indigestible forms of amino acids (primarily lysine), which leads to a decrease in the nutritional value and digestibility of the protein.

[0020] • Oxidized forms of lipids;

[0021] At elevated temperatures in the presence of oxygen, oxidized forms of lipids are formed, which interact with methionine, cysteine, and lysine in protein molecules, leading to a decrease in the nutritional value of the protein.

[0022] • Presence of a large amount of fiber and non-starch polysaccharides;

[0023] • Potential contamination of meal with mycotoxins;

[0024] • The possibility of accumulation of heavy metals in sunflower meal, such as cadmium, lead, mercury, and arsenic.

[0025] The prior art includes methods for processing sunflower meal that can increase the biological value and digestibility of proteins contained in the meal and reduce the negative effects of anti-nutritional components and toxic factors.

[0026] A known technology for producing protein concentrate from sunflower meal (patent RU 2310355 C1) involves removing chlorogenic acid and soluble sugars from the meal by washing the meal five times with a 9% succinic acid solution, followed by three washes of the resulting protein paste with water to completely remove the succinic acid. This technology produces a protein concentrate from which soluble carbohydrates are almost completely removed, with a protein content of 50 to 80% and a chlorogenic acid concentration of no more than 0.01%.

[0027] The disadvantages of this method include the high consumption of succinic acid (up to 1 ton of acid per ton of meal) and the difficulty of disinfecting and disposing of wastewater. The wastewater generated during protein concentrate production is a mixture of soluble proteins, carbohydrates, and mineral salts, and its quantity can reach 35 tons per ton of finished product. All this calls into question the economic feasibility of using this technology in industry. Protein isolate production technology (Patent RU 2761654 C1) can also be used to increase the nutritional value and availability of sunflower meal proteins.

[0028] According to a well-known solution, the meal is subjected to salt or alkaline extraction. The extract is separated from the insoluble residue, and the protein is recovered from it by one of the following methods: moisture removal or precipitation at the isoelectric point. After the protein is separated from the remaining whey, a polysaccharide concentrate is obtained. This technology produces an isolate with a protein content of 85-90% and a polysaccharide concentrate.

[0029] The disadvantages of the known method include the need to repeatedly change the pH of process fluids during the process, which requires significant consumption of chemical reagents, and the accumulation of salts in wastewater and the finished product. The resulting isolate contains chlorogenic acid, and to prevent darkening of the finished isolate, additional treatment with hydrogen peroxide or the addition of sulfites to the final product is required. Hydrogen peroxide can cause oxidation of the side chains of certain amino acids, leading to disruption of the spatial structure of proteins and their aggregation. Dietary intake of sulfites cannot exceed 0.7 mg / kg of body weight per day, which significantly limits the use of this protein isolate in the food industry.

[0030] To increase the digestibility of amino acids and reduce the amount of energy expended by the body on digesting food, it is possible to hydrolyze proteins outside the body at the stage of processing the meal, followed by the isolation of a hydrolyzate containing amino acids and light peptides.

[0031] There are three known methods of protein hydrolysis: alkaline, acidic and enzymatic.

[0032] During deep acid (patent SU 1081843) and alkaline (patent RU 2601125 C2) hydrolysis of proteins to amino acids and light peptides, the formation of toxic substances lysinoalanine and lanthionine often occurs, and the destruction of the amino acids arginine, lysine and cysteine; racemates of amino acids can also be formed, leading to a decrease in the digestibility of the hydrolysate.

[0033] Acid hydrolysis of sunflower meal (SU 1081843) is known to produce a liquid hydrolysate (with a dry matter content of 10%) and a protein hydrolysis rate of 33%. However, acid hydrolysis can destroy the amino acids tryptophan, serine, and threonine. Vitamins are also destroyed in a highly acidic environment, and deamination reactions lead to the accumulation of ammonia nitrogen in the finished hydrolysate. Neutralization of the acid saturates the hydrolysate with salts, requiring additional processing steps to remove them.

[0034] As a result, alkaline and acid hydrolysis are not widely used in industry.

[0035] Enzymatic hydrolysis of protein substances, similar to the process of protein digestion in the digestive tract of humans and animals, seems preferable.

[0036] The closest analogue to the present invention is a method for producing protein hydrolysate from sunflower meal, described in application CN 106834402 A. According to this known method, enzymatic hydrolysis is carried out in two stages. In the first stage, hydrolysis is carried out using an alkaline exoprotease. After hydrolysis is complete and the enzyme is inactivated, a complex of neutral endoproteases is added to the hydrolysate. After hydrolysis is complete, the hydrolysate is separated from the undissolved residue and dried in a spray dryer. The result is a powdered hydrolysate with a low ash (mineral) content and a high protein content. More than 60% of the protein contained in the original meal is hydrolyzed.

[0037] The disadvantages of the known method are:

[0038] 1. No impact on chlorogenic and phytic acids in order to minimize their negative impact on the nutritional properties of the finished product.

[0039] 2. Disulfide covalent bonds in light protein allergens are not restored.

[0040] 3. Adjusting the pH of the solution from 3.5 to 8.0 inevitably leads to the accumulation of salts in quantities that require the mandatory use of effective methods for desalting the finished product and the subsequent process of reagent disposal.

[0041] 4. After hydrolysis, a protein-depleted, unhydrolyzed residue with a high moisture content remains. The processes for recycling the unhydrolyzed residue and the economic feasibility of implementing this technology on an industrial scale are not disclosed.

[0042] Taking into account the shortcomings of the prior art, the applicant has set himself the following tasks, which are solved by the present invention: 1. Obtaining an easily digestible high-protein hydrolysate, which can serve as the maximum possible replacement for animal proteins in food products, diets for aquatic organisms (aquaculture), agricultural and non-productive animals.

[0043] 2. Conversion of chlorogenic acid to quinic and caffeic acids (with subsequent removal of quinic acid).

[0044] 3. Hydrolysis of phytic acid with the release of minerals such as phosphorus, calcium, potassium, iron, etc.

[0045] 4. Restoration of disulfide bonds of light proteins with subsequent hydrolysis.

[0046] 5. Use of the unhydrolyzed residue (cake) in the technological process. Thus, the primary objective of the invention is to develop a method for producing an easily digestible high-protein hydrolysate that can serve as the best possible replacement for animal proteins in food products, diets for aquatic organisms, and farm and non-productive animals. This method also maximizes the extraction of nutrients from the original meal during processing, minimizes waste, and eliminates the disadvantages of previously known methods.

[0047] The essence of the invention

[0048] The first object of the invention is a high-protein hydrolysate (HPH) containing 75-85% protein, 10-20% sugar, and at least 0.01% caffeic acid, either free or bound to chlorogenic acid isomers. Furthermore, the HPH contains at least 50 g / kg of branched-chain amino acids and at least 30 g / kg of arginine. HPH is preferably obtained from sunflower meal by enzymatic hydrolysis.

[0049] Another object of the present invention is a protein food supplement that contains the aforementioned high-protein hydrolysate according to the present invention, which has a protein content of 75-85%, sugars of 10-20%, and a content of caffeic acid in free or bound form in the composition of chlorogenic acid isomer molecules of at least 0.01%, as well as a total content of branched-chain amino acids of at least 50 g / kg of the final product (BCA) and an arginine content of at least 30 g / kg BCA. Additionally, the protein food supplement may contain biologically active substances or auxiliary substances of food grade quality.

[0050] The next object of the present invention is a food product that contains the said high-protein hydrolysate according to the present invention, which has a protein portion of 75-85%, sugars of 10-20% and a content of caffeic acid in free or bound form in the composition of chlorogenic acid isomer molecules of at least 0.01%, as well as a total content of amino acids with branched side chains of at least 50 g / kg of the final product (VBG) and an arginine content of at least 30 g / kg VBG.

[0051] Preferably, the food product is a product in solid or soft form, or a product in liquid form intended for drinking.

[0052] Another object of the present invention is a feed additive that contains the said high-protein hydrolysate according to the present invention, which has a protein portion of 75-85%, sugars of 10-20% and a content of caffeic acid in free or bound form in the composition of chlorogenic acid isomer molecules of at least 0.01%, as well as a total content of amino acids with branched side chains of at least 50 g / kg of the final product (VBG) and an arginine content of at least 30 g / kg VBG.

[0053] Another object of the present invention is a feed product that contains the said high-protein hydrolysate according to the present invention, which has a protein portion of 75-85%, sugars of 10-20% and a content of caffeic acid in free or bound form in the composition of chlorogenic acid isomer molecules of at least 0.01%, as well as a total content of amino acids with branched side chains of at least 50 g / kg of the final product (VBG) and an arginine content of at least 30 g / kg VBG.

[0054] Preferably, the feed product is intended for cattle, monogastric and unproductive animals at weaning as a starter feed.

[0055] Another object of the present invention is a feed product as a starter feed for aquaculture, which has a protein portion of 75-85%, sugar - 10-20% and a content of caffeic acid in free or bound form in the composition of the molecules of chlorogenic acid isomers of at least 0.01%, as well as a total content of amino acids with branched side chains of at least 50 g / kg of the final product (VBG) and an arginine content of at least 30 g / kg VBG. The next main object of the invention is a method for obtaining a high-protein hydrolysate (VBG) from sunflower meal, according to which the sunflower meal is crushed; the crushed fraction is mixed with water, continuously stirred until a homogeneous suspension is formed, then enzymatic hydrolysis is carried out in a neutral medium in two stages,

[0056] where the first stage involves the introduction of carbohydrase enzymes into the suspension to hydrolyze carbohydrates and lipase and phytase enzymes to hydrolyze fats and phytic acid, respectively; hydrolysis of the suspension is carried out, after the enzymatic hydrolysis is completed, the resulting primary suspension is diluted with water and the high-protein non-hydrolyzed solid residue is separated from the low-protein hydrolysate,

[0057] and in the second stage, the high-protein non-hydrolyzed solid residue is diluted with water to form a secondary suspension, in which enzymatic hydrolysis of proteins is carried out by adding proteolytic enzymes and an enzyme for hydrolyzing chlorogenic acid into caffeic and quinic acids; after the end of the enzymatic hydrolysis, the resulting secondary suspension is separated into a high-protein hydrolysate and a non-hydrolyzed residue / cake;

[0058] Finally, the high-protein hydrolyzate is concentrated and dried, yielding a protein fraction of 75-85%, sugar of 10-20%, and a content of caffeic acid in free or bound form in the molecules of chlorogenic acid isomers of at least 0.01%.

[0059] In a preferred embodiment of the method, sunflower meal is ground to a fraction size of 50 - 400 microns.

[0060] Enzymatic hydrolysis is carried out in a neutral aqueous medium, preferably in water purified by reverse osmosis.

[0061] In a preferred embodiment of the method, a co-factor, anhydrous magnesium citrate, is additionally added when mixing the crushed fraction with water, and, if necessary, this addition is repeated in the second stage for the secondary suspension.

[0062] In a preferred embodiment of the method, carbohydrase enzymes such as xylanase, beta-glucanase, cellulase, mannanase, alpha-amylase, glucoamylase, and pectinase are added in approximately equal ratios. Lipase and phytase enzymes are also added in equal ratios. In a preferred embodiment of the method, mixing of the crushed fraction and water is carried out at a water ratio of 1:5 to 1:10.

[0063] In a preferred embodiment of the method, enzymatic hydrolysis in the first stage is carried out for 60 - 160 minutes.

[0064] In a preferred embodiment of the method, after completion of the enzymatic hydrolysis at the first stage, the primary suspension is diluted with water at a water ratio of 1:11 - 1:15.

[0065] In a preferred embodiment of the method, in the second stage, the high-protein non-hydrolyzed solid residue is diluted with water at a water ratio of 1:5 - 1:10.

[0066] In a preferred embodiment of the method, deactivated yeast with a high glutathione content is used as a mixture of light thiols - a source of glutathione.

[0067] In a preferred embodiment of the method, deactivated yeast is introduced in an amount of 0.01 to 0.3 wt.%.

[0068] In a preferred embodiment of the method, chlorogenate hydrolase or tannase is introduced as an enzyme for hydrolyzing chlorogenic acid.

[0069] In a preferred embodiment of the method, the enzyme for hydrolyzing chlorogenic acid is introduced in an amount of 0.002 - 0.15 wt.% of the weight of the high-protein non-hydrolyzed residue.

[0070] In a preferred embodiment of the method, neutral protease and / or a mixture of neutral endoprotease and neutral exoprotease are used as proteolytic enzymes.

[0071] In a preferred embodiment of the method, neutral protease is added in an amount of 0.005 - 0.6 wt.%, neutral endoprotease is added in an amount of 0.005 - 0.3 wt.%, and neutral exoprotease in an amount of 0.005 - 0.3 wt.% of the weight of the high-protein non-hydrolyzed residue.

[0072] In a particular embodiment of the method, enzymes are introduced in the form of multienzyme complexes and / or in the form of individual enzymes.

[0073] In a preferred embodiment of the method, the enzymatic hydrolysis in the second stage is carried out for 90 - 180 minutes. In a preferred embodiment of the method, after completion of the hydrolysis in the second stage, the secondary suspension is additionally diluted with water at a hydromodulus of 1:11 - 1:15 before separation.

[0074] In a particular embodiment of the method, before the concentration stage, the high-protein hydrolysate is clarified and purified from such undesirable substances as quinic acid, heavy metals, nitrates, nitrites, chlorides, sulfates, silicates, and hydroxides.

[0075] In a preferred embodiment of the method, the high-protein hydrolysate is concentrated to a dry matter content of 60% or less, preferably to 20-40%.

[0076] In a specific embodiment of the method, the unhydrolyzed residue (cake) is dewatered, dried, and granulated. Waste-free production is achieved by burning the dry granules of the unhydrolyzed residue as biofuel in the present method, and the energy obtained from combustion is used to generate steam and electricity for use in the process of producing high-protein hydrolysate (HPH).

[0077] The result of the present method is another object of the invention - a high-protein hydrolysate obtained by the said method from sunflower meal by enzymatic hydrolysis, which has a protein content of 75-85%, sugar - 10-20% and a content of caffeic acid in free or bound form in the composition of chlorogenic acid isomer molecules of at least 0.01%. In addition, the resulting final product (FBP) has a total content of amino acids with branched side chains of at least 50 g / kg of the final product (FBP) and an arginine content of at least 30 g / kg FBP.

[0078] Unlike classical protein isolates, previously mentioned in patent RU 2761654 C1, during the production of a high-protein hydrolysate according to the present invention, disulfide bonds are reduced in the original protein, which allows for complete hydrolysis to amino acids and light peptides to obtain a completely and easily digestible product free of antinutrients (antinutrients). In the known method, the protein is not fully digestible due to the fact that antinutrients (antinutrients) are retained in the native isolate, and disulfide bonds cannot be hydrolyzed in the digestive tract, where proteases can only hydrolyze covalent-peptide bonds, and covalent bonds - disulfide bonds.

[0079] The bonds do not break even at high temperatures. Therefore, the claimed method and VBG have all the advantages.

[0080] Further in the description, additional advantages and details of the invention will be disclosed.

[0081] Brief description of the figures

[0082] Figure 1 shows aquaculture feed pellets with a diameter of 2 mm in which 70% of the fish meal is replaced by WBG obtained according to Example 1.

[0083] Disclosure of invention

[0084] To maximize the extraction of nutrients from sunflower meal, mixtures of individual enzymes with the required activity are created, or ready-made multi-enzyme complexes available on the market are used.

[0085] To prevent darkening of the hydrolysate and reduce the chlorogenic acid content in the finished product, sunflower meal is treated with the enzyme chlorogenate hydrolase or tannase, which hydrolyzes chlorogenic acid into caffeic and quinic acids. Caffeic acid is a powerful antioxidant and increases the biological value of the finished hydrolysate. On the other hand, quinic acid can form strong covalent bonds with the amino groups of amino acids, particularly lysine. Therefore, to remove quinic acid, the hydrolysate after enzymatic hydrolysis is passed through an ion exchange column equipped with an anion exchange resin.

[0086] To hydrolyze chelate complexes of phytic acid and increase the bioavailability of minerals phosphorus, calcium, potassium and iron during hydrolysis, the enzyme phytase is used.

[0087] To restore disulfide bonds in sunflower allergen proteins that are resistant to proteolytic enzymes, enzymatic hydrolysis is performed in water containing reduced glutathione. Commercially available light thiol mixtures containing deactivated yeast (high in glutathione) are used as the glutathione source.

[0088] To increase the availability of enzymes to the substrate and increase the yield of hydrolyzed product, the enzyme lipase is added.

[0089] The method, in more detail, involves the following steps and processes: 1. Enzymatic hydrolysis is carried out in two stages in a neutral aqueous medium. In the best embodiment, water is purified from salts and organic compounds using reverse osmosis, i.e., osmotic water is used. For enzymatic hydrolysis, water with a pH of 6.5-7.5 and a temperature of 25-30°C is preferably used. Then, if necessary, a mixture of light thiols is added to it - from 0.01 to 0.3 wt.% of deactivated yeast with a high glutathione content. Yeast is added to the solution to restore disulfide bonds in lipid transfer proteins (LTPs) and 2S-albumins, which are allergenic proteins.

[0090] Next, if necessary, add anhydrous magnesium citrate from 0.3 to 1.7 mmol / l, which acts as a cofactor in the hydrolysis reaction, and heat to 45-60°C.

[0091] 2. Sunflower meal is ground to obtain a homogeneous, fine powder with the desired particle size distribution (usually 50-400 µm). The ground meal is placed in a hydrolyzer, where it is mixed with water under continuous stirring until a homogeneous suspension is formed. It is desirable to maintain a certain water-to-meal ratio, for example, with a water-to-solid ratio of 1:5 to 1:10.

[0092] 3. Enzymes are added to the resulting suspension, namely carbohydrase enzymes that hydrolyze carbohydrates, in approximately equal proportions: xylanase in an amount of 0.005 - 0.2%, beta-glucanase in an amount of 0.005 - 0.2%, cellulase in an amount of 0.005 - 0.2%, mannanase in an amount of 0.005 - 0.2%, alpha-amylase in an amount of 0.005 - 0.2%, glucoamylase in an amount of 0.005 - 0.2%, pectinase in an amount of 0.005 - 0.2%, and in equal proportions lipase (hydrolyzing fats) in an amount of 0.002 - 0.2% and phytase (hydrolyzing phytic acid) in an amount of 0.002 - 0.2% of the sunflower meal weight. First-stage enzymatic hydrolysis is performed: carbohydrate hydrolysis, fat hydrolysis, and phytic acid hydrolysis for 60-160 minutes at 45-60°C.When performing enzymatic hydrolysis, it is permissible to use either individual enzymes with specific activity or ready-made broad-spectrum multienzyme complexes, such as a multienzyme carbohydrase complex, a multienzyme complex with lipase and phytase activity, or a combination of individual enzymes and / or any of the ready-made multienzyme complexes available on the market. 4. After carbohydrate hydrolysis is complete, the primary suspension is diluted with water, and the high-protein, unhydrolyzed solid residue, which is further processed, is separated in a centrifuge from the low-protein hydrolysate, which is removed from the process.

[0093] 5. In the second stage of enzymatic hydrolysis, the high-protein non-hydrolyzed solid residue is diluted with water to form a secondary suspension. If necessary, a mixture of light thiols is added - a source of glutathione and the cofactor magnesium citrate anhydrous. Enzymatic hydrolysis of proteins is carried out by adding proteolytic enzymes to the secondary suspension. Proteolytic enzymes are added, such as neutral protease in an amount of 0.005 - 0.5 wt.% and / or a mixture of neutral endoprotease in an amount of 0.005 - 0.25 wt.% with neutral exoprotease in an amount of 0.005 - 0.25 wt.% of the sunflower meal weight. Proteolytic enzymes are added separately, but it is possible to introduce proteolytic enzymes in the form of a complex enzyme preparation. Simultaneously, hydrolysis of chlorogenic acid is carried out. That is, enzymes that hydrolyze chlorogenic acid into caffeic and quinic acid, chlorogenate hydrolase or tannase are added in an amount of 0.002 - 0.2 wt%.Thus, the second stage of enzymatic hydrolysis involves protein hydrolysis and chlorogenic acid hydrolysis. Enzymatic hydrolysis is carried out for 60-160 minutes at a temperature of 45-60°C.

[0094] 6. After completion of the enzymatic hydrolysis of proteins, the suspension is diluted with water, preferably osmotic water, mainly with a temperature of 25-30°C and pH = 6.5 - 7.5, the hydromodulus is brought to a ratio of 1:11 - 1:15 and the suspension is separated in a decanter into non-hydrolyzed residue / cake and high-protein hydrolysate (HPH).

[0095] 7. Further, if necessary, the obtained high-protein hydrolysate is clarified in a separator and passed sequentially through two ion-exchange columns equipped with a cation exchanger and anion exchanger. In the separator, the high-protein hydrolysate is further purified from small particles of unhydrolyzed solid residue. In the ion-exchange columns, undesirable substances are removed. The cation exchanger is used to purify heavy metals, such as lead, mercury, arsenic, and cadmium; the anion exchanger is used to purify nitrates, nitrites, chlorides, sulfates, silicates, and hydroxides. The hydrolysate is then concentrated in an evaporation unit to a dry matter content of 60% or less, preferably 20-40% based on absolutely dry matter. The high-protein hydrolysate is then spray-dried, preferably to a final product moisture content of 4-6%; the finished powder is packaged in a sealed container. The by-product of enzymatic hydrolysis, the unhydrolyzed residue (cake), is dehydrated, dried and granulated.The cake granules are burned in the process, and the energy generated during combustion is used to generate steam and electricity for use in the VBG production process. The optimal calorific value of the resulting cake granules was tested by combustion in a colorimeter and found to be approximately 4,320 kcal / kg. This ensures waste-free, environmentally friendly production. Alternatively, the unhydrolyzed residue can be used as an additional source of fiber and protein in feed, or as a base for mushroom cultivation.

[0096] As a result of the proposed method, VBG is obtained in the form of a powder, completely soluble in water, with a protein portion of 75-85%, a small amount of sugars (such as glucose, fructose, maltose, maltotriose, oligosaccharides) - 10-20% and with a caffeic acid content of at least 0.01% in free or bound form in the composition of chlorogenic acid isomer molecules.

[0097] Optimally, the protein yield is around 80%, sugars around 10%, and caffeic acid around 1.9%. The upper limit for caffeic acid can be around 2.5%, but it still depends on the initial amount in the raw material, so limiting it to an upper limit is not practical.

[0098] In addition, the resulting final product (FBP) has a total content of branched-chain amino acids of at least 50 g / kg of the final product (FBP) and an arginine content of at least 30 g / kg of FBP.

[0099] The total content of branched-chain amino acids (BCAAs) is made up of three amino acids: valine, leucine, and isoleucine. These BCAAs are metabolized not in the liver, but directly in skeletal muscle, stimulating protein synthesis and positively affecting the ability to transport amino acids, ultimately leading to accelerated muscle mass gain. BCAAs also have a positive effect on intestinal function.

[0100] Arginine promotes the release of growth hormones and accelerates muscle mass gain. It is also essential for the effective functioning of the immune system under stress, plays a key role in normalizing lipid metabolism and energy balance, and has a hepatoprotective effect. The amino acid profile of the hydrolyzed fish meal protein obtained using this invention is close to that of fishmeal protein, with the exception of lysine. The hydrolysate is completely water-soluble and contains 75-85% crude protein. When using the hydrolyzed fish meal protein in feed / food additives or food / feed products, the lysine content can be adjusted, if necessary, by adding synthetic lysine. The hydrolyzed fish meal protein does not contain the amino acid taurine. This can also be adjusted when using the hydrolyzed fish meal protein.

[0101] VBG contains hydrolyzed sugars, which serve as an energy source for protein digestion. It also contains minerals such as calcium and phosphorus, iron, selenium, zinc, copper, and iodine. VBG contains caffeic acid, an antioxidant with immunomodulatory and anti-inflammatory properties. It is maximally absorbed by the body and does not form orthoquinone bonds with light proteins in the digestive tract.

[0102] The VBG obtained using this method compares favorably with protein products obtained using known methods (where chlorogenic acid is not hydrolyzed) in that the final product contains at least 0.01% caffeic acid, either free or bound to chlorogenic acid isomer molecules (as part of the polyphenol complex). The advantage of VBG with caffeic acid is that it is absorbed at 95% + / - 4%, compared to 33% + / - 17% for chlorogenic acid. This prevents binding to amino acids during gastrointestinal tract digestion, enhancing the nutritional value of the product, and also possesses antioxidant and antimicrobial properties.

[0103] The content of caffeic acid was determined by two methods: in free form using ultra-performance liquid chromatography [Analysis of chlorogenic acid isomers and caffeic acid in 89 herbal infusions (tea); Dillenburg et al.; Journal of Food Composition and Analysis, Vol. 73, October 2018, pp. 76-82] and in bound form as part of chlorogenic acid isomer molecules using a spectrophotometric method [Development and validation of a method for the spectrophotometric determination of caffeic and chlorogenic acids, Novas D.S.; Actual problems of modern medicine and pharmacy - 2023. Belarusian State Medical University, Minsk (19.04-20.04)]. VBG is stored in a sealed container, protected from moisture and direct sunlight, at a temperature of 25 °C for 12 months from the date of manufacture.

[0104] The resulting VBG has a wide range of applications in the food and feed industries. VBG is an excellent alternative to animal proteins in compound feed for non-productive animals, farm animals, and poultry. For example, VBG is suitable for pets (cats and dogs); ornamental birds; farm animals (pigs, cows, and sheep); and poultry (chicken, turkey, quail, duck, geese, and ostriches). This list of animals and birds is provided for illustrative purposes only and is not exhaustive.

[0105] VBG can successfully replace animal proteins in the production of starter and grower feeds for aquaculture. When used in hydrolysis processes with food-grade enzymes, this hydrolysate can be used as a protein supplement in the food industry to enrich meat, dairy, and bakery products with amino acids, as well as other food products where plant protein is preferred. It can also be used in various beverages and shakes, including those for the elderly, children, and athletes, as well as vegetarian and vegan products. It can also be incorporated into dietary, therapeutic, sports, and restorative nutrition formulas. For example, a protein supplement can additionally contain amino acids such as taurine and lysine, and biologically active substances such as vitamins and minerals, and be available in powder or tablet form. This powder can also be used to prepare a shake for children, those losing weight, fasting, or the elderly.This list of products is provided for example purposes only and is not limiting.

[0106] The technical results / advantages of this group of inventions are:

[0107] - The method is safe and environmentally friendly due to the use of neutral environments, enzymes, and non-aggressive, safe reagents. All processes are environmentally friendly, conducted in a neutral environment, without the formation of salts during neutralization of the reaction medium, which requires lower reagent consumption and a gentle technology that prevents amino acid racemization. Operating in a neutral and non-aggressive environment means the equipment meets minimal requirements for corrosion and chemical resistance. - The technological process is flexible, allowing the use of ready-made multi-enzyme complexes instead of commercially available individual enzymes.

[0108] - waste-free method: closed production cycle with minimal wastewater generation and energy generation by burning non-hydrolyzed residue / cake.

[0109] - maximum use of useful ingredients contained in the original raw materials during the isolation of hydrolyzate;

[0110] - the nutritional and feed value of VBG as an alternative plant protein. The method ensures the production of an easily digestible and highly nutritious VBG composition, which is a worthy replacement for animal proteins in feed and food products;

[0111] - the food and feed purity of the resulting WBG is an additional advantage of the present invention. WBG is minimally or virtually free of undesirable and toxic substances. It is free of anti-nutritional substances such as phytic acid, digestive enzyme inhibitors, mild protein allergens, Maillard reaction products, and oxidized lipid forms. WBG is free of mycotoxins and heavy metals such as cadmium, lead, mercury, and arsenic. The carbohydrate portion of WBG is hydrolyzed to water-soluble monosaccharides and oligosaccharides. Known analogs tend to remove as much chlorogenic acid as possible to avoid the formation of bright green hydroquinones, which is an indication of the presence of chlorogenic acid in the product.In the present invention, when hydrolysis is carried out in a neutral medium (which prevents the formation of colored hydroquinones), chlorogenic acid is hydrolyzed by chlorogenate hydrolase or tannase into caffeic and quinic acids.

[0112] Examples of the invention

[0113] The examples below illustrate the practical implementation of the VBG product and the method for its production, but in no way limit all possible options for the practical implementation of the method and products based on VBG.

[0114] Example 1

[0115] The original sunflower meal was ground in a rotary vortex mill to a particle size of 100-150 µm. One kilogram of the ground meal was placed in a hydrolyzer with an anchor stirrer and a heating jacket. 6 kilograms of osmotic water with a pH of 6.7 were added, and 0.6 g of anhydrous magnesium citrate was added. The resulting suspension was heated to 50°C with constant stirring at 20 rpm.

[0116] During the first stage of enzymatic hydrolysis, the following enzymes were added to the suspension: 2 g of a ready-to-use concentrated multienzyme complex containing carbohydrase enzymes (xylanase, beta-glucanase, cellulase, mannanase, pectinase, alpha-amylase, and glucoamylase in equal proportions), lipase, and phytase in equal proportions. The first stage of enzymatic hydrolysis was carried out for 120 minutes at 50°C with constant stirring at 20 rpm.

[0117] Upon completion of the hydrolysis process, 6 kg of osmotic water with a pH of 6.7 was added to the hydrolyzer, and the suspension was stirred for 7 minutes. The high-protein, unhydrolyzed solid residue was then separated from the low-protein hydrolysate in a decanter.

[0118] Next, the high-protein solid residue was placed in a hydrolyzer, 5 kg of osmotic water with a pH of 6.7 was added, anhydrous magnesium citrate was added in an amount of 0.6 g and the suspension was stirred for 7 minutes.

[0119] Added to the suspension:

[0120] 1. Neutral protease - 2 g.

[0121] 2. Ready-made mixture of neutral endoproteases and exoproteases - 2 g.

[0122] 3. Chlorogenate hydrolase - 0.2 g.

[0123] 4. Inactivated yeast with high glutathione content in the amount of -1.5 g.

[0124] Then, the second stage enzymatic hydrolysis was carried out for 120 min at a temperature of 50°C with constant stirring at a speed of 20 rpm.

[0125] Upon completion of the hydrolysis process, 6 kg of osmotic water with a pH of 6.7 were additionally added to the hydrolyzer and the suspension was stirred for 7 min. After this, the high-protein hydrolysate and the unhydrolyzed residue (cake) were separated in a decanter. The hydrolysate was clarified in a separator, after which it was passed sequentially through two ion-exchange columns: the first, equipped with a sodium cation exchanger and the second, with an anion exchanger. After ion exchange, the hydrolysate was evaporated in an evaporation unit to a dry matter content of 30% and dried in a spray dryer with an inlet air temperature of 180 °C and an outlet air temperature of 80 °C. The yield of the high-protein hydrolysate was 305 g. The protein portion of the WBG was 84%, the carbohydrate portion / sugars - 7.1%, the mineral portion - 5.3%, caffeic acid - 0.5%, moisture - 4.3%. The remaining insignificant mass (up to 100%) in VBG is accounted for by neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid, and other hydrolysis products.

[0126] The protein composition (see Table 1) includes the amino acid Arginine - 103.27 g / kg. The total content of branched-chain amino acids is made up of three amino acids - valine, leucine, and isoleucine - 140.26 g / kg: Valine - 55.77 g / kg, Leucine - 51.46 g / kg. Isoleucine - 33.03 g / kg. The amino acid composition of WBG is presented in more detail below in Table 1.

[0127] Table 1. Amino acid composition of VBG

[0128]

[0129] Example 2

[0130] The VBG obtained in Example 1 was used in the production of compound feed. A feeding trial was also conducted on juvenile sterlet using compound feed developed by JSC Aston at a training university. In this feed, 100% of the fishmeal in the starter feed was replaced with VBG.

[0131] Table 2. Composition of the starting experimental feed.

[0132]

[0133] Feed pellets containing WBG were obtained (see Fig. 1). They were used in an aquaculture feeding experiment and compared with a commercially available starter feed with a known formula (Control). The starter control feed consisted of fishmeal, wheat gluten, wheat, krill meal, lecithin, yeast extract, betaine, probiotic, and vitamins and minerals. The protein component of the control feed consisted primarily of animal-based ingredients (fishmeal, krill meal). A comparison table of the amino acid compositions of the feeds tested is presented below (see Table 3).

[0134] Table 3. Comparison of amino acid composition of starter feeds

[0135]

[0136]

[0137] Table 4. Assessment of the nutritional value of feed

[0138]

[0139] During the experiment, 20 juvenile sterlets were collected and placed under identical conditions, controlled by a common life-support system for the experimental tanks. Feeding was based on body weight and amounted to 2% of the total body weight per day. Feeding was administered four times daily in equal portions at the same time using automatic feeders.

[0140] During the experiment, which lasted 35 days, the following results were obtained (see Table 4).

[0141] Table 4. Weight of test groups, grams, on the date of weighing

[0142]

[0143]

[0144] From 08 / 05 / 2024 to 09 / 09 / 2024, the growth of sterlet weight increased and amounted to the following in each group:

[0145] Starter feed Control - 111% or 352 g.

[0146] Starter experimental feed - 151% or 482 g.

[0147] The clinical condition of the fish examined was assessed as good. The survival rate was 100%. No deviations from the species' physiological norm were recorded.

[0148] Example 3

[0149] The high-protein sunflower meal hydrolysate (HPH) obtained in Example 1, with a protein content of 84%, was used to prepare a nutritious amino acid (protein) mixture. The nutritious mixture formula is presented in Table 5.

[0150] Table 5. Nutrient mixture, composition

[0151]

[0152] To add taste and aroma, a strawberry flavoring agent based on natural ingredients was additionally used.

[0153] Example 4

[0154] The high-protein hydrolyzed sunflower meal (HPH) obtained in Example 1, with a protein content of 84%, was used to prepare chicken feed. The grain ingredients for the feed were first crushed in a grain crusher, then all components were mixed in a "drunken barrel" mixer and passed through a granulator.

[0155] The formula for the compound feed is presented in Table 6.

[0156] Table 6

[0157]

[0158] Example 5

[0159] The VBG obtained in Example 1 was used to prepare compound feed for broilers based on plant materials.

[0160] The formula for the compound feed is presented in Table 7.

[0161] Table 7

[0162]

[0163] Example 6

[0164] The VBG obtained in example 1 was used to prepare cooked sausages from poultry meat using traditional technology.

[0165] The sausage recipe is presented in Table 8.

[0166] Table 8

[0167]

[0168]

[0169] Example 7

[0170] The VBG obtained in Example 1 was used to prepare a non-milk-type drink based on plant materials.

[0171] The recipe for the “non-milk” drink is presented in Table 9.

[0172] Table 9

[0173]

[0174] In other food or feed products according to the present invention, VBG can be successfully added according to traditional technologies instead of any other plant or animal protein used in the product.

[0175] In the present invention, the authors do not claim specific quantities of enzymes or specific parameters of the technological process, since they are selected empirically by a specialist in this field who has experience in experimentation, depending on the quality, composition and / or dispersion of the raw materials, specific equipment, its volume, the volume of the substrate for fermentation, pH, temperature, hydrolysis time, enzyme activity, etc.

Claims

CLAUSES OF THE INVENTION E High-protein hydrolysate (HPH) obtained from sunflower meal by enzymatic hydrolysis, which has a protein content of 75-85%, sugars of 10-20% and a content of caffeic acid in free or bound form in the composition of chlorogenic acid isomer molecules of at least 0.01%.

2. The VBG according to item 1, in which the total content of amino acids with branched side chains is not less than 50 g / kg of the final product (VBG) and the arginine content is not less than 30 g / kg VBG.

3. A protein food supplement, characterized in that it contains VBG according to paragraph 1 or paragraph 2, which has a protein portion of 75-85%, sugars of 10-20% and a content of caffeic acid in free or bound form in the composition of chlorogenic acid isomer molecules of at least 0.01%.

4. A protein food supplement according to paragraph 3, characterized in that it additionally contains biologically active or auxiliary substances of food quality category.

5. A food product characterized in that it contains VBG according to I.1 or I.2, which has a protein portion of 75-85%, sugars of 10-20% and a content of caffeic acid in free or bound form in the composition of chlorogenic acid isomer molecules of at least 0.01%.

6. A food product according to item 5, characterized in that it is a product in hard or soft form, intended for chewing.

7. A food product according to item 5, characterized in that it is a product in liquid form intended for drinking.

8. A feed additive characterized in that it contains VBG according to paragraph 1 or paragraph 2, which has a protein portion of 75-85%, sugars of 10-20% and a content of caffeic acid in free or bound form in the composition of chlorogenic acid isomer molecules of at least 0.01%.

9. A feed product characterized in that it contains a VBG according to paragraph 1 or paragraph 2, which has a protein portion of 75-85%, sugars of 10-20% and a content of caffeic acid in free or bound form in the composition of chlorogenic acid isomer molecules of at least 0.01%.

10. A feed product according to item 9, which is intended for cattle, monogastric and non-productive animals at weaning as a starter feed.

11. A feed product as a starter feed for aquaculture, characterized in that it contains VBG according to item 1 or item 2, which has a protein content of 75-85%, sugars of 10-20% and a content of caffeic acid in free or bound form in the composition of chlorogenic acid isomer molecules of at least 0.01%.

12. A method for producing high-protein hydrolysate (HPH) from sunflower meal by enzymatic hydrolysis, according to which the sunflower meal is ground; the ground fraction is mixed with water, continuously stirred until a homogeneous suspension is formed, then enzymatic hydrolysis is carried out in a neutral medium in two stages, where the first stage involves the introduction of carbohydrase enzymes into the suspension to hydrolyze carbohydrates and lipase and phytase enzymes to hydrolyze fats and phytic acid, respectively; hydrolysis of the suspension is carried out, after the enzymatic hydrolysis is completed, the resulting primary suspension is diluted with water and the high-protein non-hydrolyzed solid residue is separated from the low-protein hydrolysate, and in the second stage, the high-protein non-hydrolyzed solid residue is diluted with water to form a secondary suspension, in which enzymatic hydrolysis of proteins is carried out by adding proteolytic enzymes and an enzyme for hydrolyzing chlorogenic acid into caffeic and quinic acids; after the end of the enzymatic hydrolysis, the resulting secondary suspension is separated into a high-protein hydrolysate and a non-hydrolyzed residue / cake; Finally, the high-protein hydrolyzate is concentrated and dried, yielding a protein fraction of 75-85%, sugar of 10-20%, and a content of caffeic acid in free or bound form in the molecules of chlorogenic acid isomers of at least 0.01%.

13. The method according to item 12, in which the sunflower meal is ground to a fraction size of 50–400 µm.

14. The method according to item 12 or item 13, in which the enzymatic hydrolysis is carried out in water purified by reverse osmosis.

15. The method according to any one of paragraphs 12 to 14, in which the water, when mixed with the crushed fraction, additionally contains a mixture of light thiols - a source of glutathione.

16. The method according to any one of paragraphs 12 to 15, in which the water additionally contains a mixture of light thiols - a source of glutathione in the second stage of enzymatic hydrolysis.

17. The method according to i.15 or i.16, in which deactivated yeast with a high glutathione content is used as a mixture of light thiols - a source of glutathione.

18. The method according to item 17, in which deactivated yeast is introduced in an amount of 0.01 to 0.3 wt.%.

19. The method according to any of paragraphs 12 to 18, in which, in addition to mixing the crushed fraction with water, a co-factor is added - anhydrous magnesium citrate, and, if necessary, this is repeated in a second stage for a secondary suspension.

20. The method according to any one of claims 12 to 19, in which carbohydrase enzymes such as xylanase, beta-glucanase, cellulase, mannanase, alpha-amylase, glucoamylase, pectinase are introduced in equal proportions, as well as lipase and phytase in equal proportions.

21. The method according to any of paragraphs 12 - 20, in which the mixing of the crushed fraction and water is carried out at a water ratio of 1:5 - 1:

10.

22. The method according to any one of paragraphs 12 to 21, wherein the enzymatic hydrolysis in the first stage is carried out for 60 to 160 minutes.

23. The method according to any of paragraphs 12-22, in which, after completion of the enzymatic hydrolysis in the first stage, the primary suspension is diluted with water at a water ratio of 1:11 - 1:

15.

24. The method according to any one of paragraphs 12 to 23, wherein in the second stage the high-protein non-hydrolyzed solid residue is diluted with water at a water ratio of 1:5 to 1:

10.

25. The method according to any one of paragraphs 12 to 24, wherein chlorogenate hydrolase or tannase is introduced as an enzyme for hydrolyzing chlorogenic acid.

26. The method according to item 25, in which the enzyme for hydrolyzing chlorogenic acid is introduced in an amount of 0.002 - 0.15 wt.% of the weight of the high-protein non-hydrolyzed solid residue.

27. The method according to any one of paragraphs 12 to 26, wherein a neutral protease and / or a mixture of a neutral endoprotease and a neutral exoprotease are used as proteolytic enzymes.

28. The method according to item 27, in which neutral protease is added in an amount of 0.005-0.6 wt.%, neutral endoprotease is added in an amount of 0.005-0.3 wt.%, and neutral exoprotease in an amount of 0.005-0.3 wt.% of the weight of the high-protein non-hydrolyzed solid residue.

29. The method according to any one of paragraphs 12 to 28, wherein the enzymes are introduced in the form of multienzyme complexes and / or in the form of individual enzymes.

30. The method according to any one of paragraphs 12 to 29, wherein the enzymatic hydrolysis in the second stage is carried out for 90 to 180 minutes.

31. The method according to any one of paragraphs 12 to 30, in which, after completion of hydrolysis in the second stage, the secondary suspension is further diluted with water at a water ratio of 1:11-1:15 before separation.

32. The method according to any one of paragraphs 12 to 31, in which, before the stage of concentration, the high-protein hydrolysate is clarified and purified from substances such as quinic acid, heavy metals, nitrates, nitrites, chlorides, sulfates, silicates, and hydroxides.

33. The method according to any one of paragraphs 12 to 32, wherein the concentration of the high-protein hydrolysate is carried out to a dry matter content of 60% or less, preferably to 20-40%.

34. The method according to item 12, wherein the non-hydrolyzed residue / cake is dewatered, dried and granulated.

35. The method according to art. 34, in which dry granules of unhydrolyzed residue / cake are burned as biofuel in the present method, and the energy obtained from burning is directed to the production of steam and electricity for use in the technological process of the present method for producing high-protein hydrolysate.

36. High-protein hydrolysate (HPH), obtained by the method according to any of paragraphs 12 to 35 from sunflower meal by enzymatic hydrolysis, which has a protein content of 75-85%, sugars of 10-20% and a content of caffeic acid in free or bound form in the composition of chlorogenic acid isomer molecules of at least 0.01%.

37. VBG according to clause 36, in which the total content of amino acids with branched side chains is not less than 50 g / kg of the final product (VBG) and the arginine content is not less than 30 g / kg VBG.

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