Protein-sugar hydrolysate from sunflower meal

Abstract

The group of inventions relates to the field of processing oily raw material, and more particularly to a novel product in the form of a protein-sugar hydrolysate (PSH) obtained from sunflower meal, a method for producing same by enzymatic hydrolysis, and PSH-based food and feed products. The PSH contains 40-70% protein, 20-40% sugar, and at least 0.02% caffeic acid in free form or in bound form as part of chlorogenic acid isomer molecules. In addition, the PSH has a total branched-chain amino acid content of at least 30 g / kg of the final product (PSH), and an arginine content of at least 20 g / kg PSH. The resulting PSH 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] SUNFLOWER SEED MEAL PROTEIN-SUGAR HYDROLYSATE, ITS PRODUCTION METHOD 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—protein-sucrose hydrolysate (PSH) from sunflower meal—and a method for producing it via enzymatic hydrolysis. PSH 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.

[0009] The anti-nutritional components and characteristic properties of plant proteins contained in sunflower meal limit its direct incorporation into food and feed formulations. However, the present invention makes it possible to extract a protein-sugary hydrolysate (hereinafter referred to as "PSH") from the original sunflower meal, which can serve as the ultimate replacement for animal proteins in food products, diets for aquatic organisms (aquaculture), and farm and non-productive animals.

[0010] The term "GBS" here refers to a product obtained by enzymatic hydrolysis of proteins and sugars contained in sunflower meal.

[0011] The digestibility and assimilation of sunflower meal are negatively affected by the following factors contained in its composition:

[0012] • Chlorogenic acid;

[0013] 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 when it forms covalent bonds with them. Chlorogenic acid is also a natural antioxidant.

[0014] • Phytic acid;

[0015] 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.

[0016] • Digestive enzyme inhibitors;

[0017] Sunflower seeds contain inhibitors, particularly trypsin and Bowman-Birk chymotrypsin inhibitors, which are resistant to high temperatures and can inhibit protein digestion during digestion.

[0018] • Mild protein allergens;

[0019] 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.

[0020] • 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;

[0021] • Products of interaction between the protein part and reducing sugars (Maillard reaction);

[0022] 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.

[0023] • Oxidized forms of lipids;

[0024] 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.

[0025] • Presence of a large amount of fiber and non-starch polysaccharides;

[0026] • Potential contamination of meal with mycotoxins;

[0027] • The possibility of accumulation of heavy metals in sunflower meal, such as cadmium, lead, mercury, and arsenic.

[0028] 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.

[0029] 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%.

[0030] The disadvantages of this method include high succinic acid consumption (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 of this calls into question the economic feasibility of using this technology in industry.

[0031] Also, to increase the nutritional value and availability of sunflower meal proteins, technology for producing protein isolates can be used (Patent RU 2761654 C1).

[0032] 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 allows for the production of isolate with a protein content of 85-90% and a polysaccharide concentrate.

[0033] 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.

[0034] During the process of obtaining sunflower protein isolate, the proteins contained in sunflower meal are dissolved and then precipitated at their isoelectric point. The resulting wet protein paste is washed to remove excess salts and dried. These technological steps require significant expenditure of chemical reagents and energy. However, during digestion, the resulting protein is dispersed in digestive juices and, as it moves through the digestive tract, is broken down by enzymes into light peptides and amino acids, which are then absorbed through the intestinal mucosa and ultimately enter the bloodstream. The body expends a significant amount of energy to digest the protein component of food.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.

[0035] There are three known methods of protein hydrolysis: alkaline, acidic and enzymatic.

[0036] 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 and the destruction of the amino acids arginine, lysine and cysteine ​​often occurs; racemates of amino acids can also be formed, leading to a decrease in the digestibility of the hydrolysate.

[0037] 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.

[0038] As a result, alkaline and acid hydrolysis are not widely used in industry.

[0039] Enzymatic hydrolysis of protein substances, similar to the process of protein digestion in the digestive tract of humans and animals, seems preferable.

[0040] The closest analogue to the present invention is the method for producing protein hydrolysate from sunflower meal described in patent 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 content and a high protein content. More than 60% of the protein contained in the original meal undergoes hydrolysis.

[0041] The disadvantages of the known method are: 1. Lack of effect on chlorogenic and phytic acids in order to minimize their negative impact on the nutritional properties of the finished product.

[0042] 2. Disulfide covalent bonds in light protein allergens are not restored.

[0043] 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.

[0044] 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.

[0045] Taking into account the shortcomings of the known technology, the applicant set himself the following tasks, which are solved by the present invention:

[0046] 1. Obtaining easily digestible GBS, which can serve as the best possible replacement for animal proteins in food products, diets for aquatic organisms (aquaculture), agricultural and non-productive animals.

[0047] 2. Use of sugars obtained from hydrolysis of the original sunflower meal.

[0048] 3. Conversion of chlorogenic acid to quinic and caffeic acids (with subsequent removal of quinic acid).

[0049] 4. Hydrolysis of phytic acid with the release of minerals such as phosphorus, calcium, potassium, iron, etc.

[0050] 5. Restoration of disulfide bonds of light proteins with subsequent hydrolysis.

[0051] 6. Use of non-hydrolyzed residue in the technological process.

[0052] Thus, the main objective of the invention is to obtain a new product - protein-saccharide hydrolysate (PSH) and to develop a method for producing easily digestible PSH, which can serve as the maximum possible replacement for animal proteins in food products, diets for aquatic organisms, agricultural and non-productive animals, while during the processing, the maximum amount of nutrients is extracted from the original meal, the amount of waste is minimized, and the technological process for producing PSH is free from the disadvantages inherent in previously known methods from the prior art. The essence of the invention

[0053] The first object of the invention is a protein-sugary hydrolysate (PSH) containing 40-70% protein, 20-40% sugar, and at least 0.02% caffeic acid, either free or bound to chlorogenic acid isomers. Furthermore, the PSH contains at least 30 g / kg of the final product (PSH) and at least 20 g / kg of arginine. PSH is preferably obtained from sunflower meal by enzymatic hydrolysis.

[0054] Another object of the present invention is a protein food supplement that contains the aforementioned protein-sugar hydrolysate according to the present invention, which has a protein portion of 40-70%, a sugar content of 20-40%, and a caffeic acid content of at least 0.02% in free or bound form in the composition of chlorogenic acid isomer molecules, as well as a total content of branched-chain amino acids of at least 30 g / kg of the final product (BCA) and an arginine content of at least 20 g / kg BCA. Additionally, the protein food supplement may contain biologically active substances or auxiliary substances of food grade quality.

[0055] The next object of the present invention is a food product that contains the said protein-sugar hydrolysate according to the present invention, which has a protein portion of 40-70%, sugars of 20-40% and a caffeic acid content of at least 0.02% in free or bound form in the composition of chlorogenic acid isomer molecules, as well as a total content of amino acids with branched side chains of at least 30 g / kg of the final product (GBS) and an arginine content of at least 20 g / kg GBS.

[0056] Preferably, the food product is a product in solid or soft form, or a product in liquid form intended for drinking.

[0057] Another object of the present invention is a feed additive that contains the said protein-sugar hydrolysate according to the present invention, which has a protein portion of 40-70%, sugars of 20-40% and a caffeic acid content of at least 0.02% in free or bound form in the composition of chlorogenic acid isomer molecules, as well as a total content of amino acids with branched side chains of at least 30 g / kg of the final product (GBS) and an arginine content of at least 20 g / kg GBS.Another object of the present invention is a feed product that contains a protein-sugar hydrolysate according to the present invention, which has a protein portion of 40-70%, sugars of 20-40% and a caffeic acid content of at least 0.02% in free or bound form in the composition of chlorogenic acid isomer molecules, as well as a total content of amino acids with branched side chains of at least 30 g / kg of the final product (GBS) and an arginine content of at least 20 g / kg GBS.

[0058] Preferably, the feed product is intended for cattle, monogastric and non-productive animals.

[0059] Another object of the present invention is a feed product for aquaculture, which contains a protein-sugar hydrolysate according to the present invention, which has a protein portion of 40-70%, sugars of 20-40% and a caffeic acid content of at least 0.02% in free or bound form in the composition of chlorogenic acid isomer molecules, as well as a total content of amino acids with branched side chains of at least 30 g / kg of the final product (GBS) and an arginine content of at least 20 g / kg GBS.

[0060] The next main object of the invention is a method for producing protein-sugary hydrolysate (PSH) from sunflower meal, according to which sunflower meal is crushed; the crushed fraction is mixed with water until a homogeneous suspension is formed under constant stirring; enzymatic hydrolysis is carried out in a neutral medium in two stages simultaneously or sequentially, where the first stage includes introducing into the suspension an enzyme for hydrolyzing chlorogenic acid into caffeic and quinic acids, adding carbohydrase enzymes for hydrolyzing carbohydrates and lipase and phytase enzymes for hydrolyzing fats and phytic acid, respectively; in the second stage, enzymatic hydrolysis of proteins is carried out by adding proteolytic enzymes; after the end of the enzymatic hydrolysis, the resulting suspension is diluted with water and separated into protein-sugary hydrolysate (PSH) and unhydrolyzed residue / cake;Finally, the GBS solution is concentrated and dried to yield GBS, which has a protein content of 40-70%, sugar content of 20-40%, and a content of caffeic acid in free or bound form in the composition of chlorogenic acid isomer molecules of at least 0.02%. In a preferred embodiment of the method, sunflower meal is ground to a fraction size of 50-400 μm.

[0061] Enzymatic hydrolysis is carried out in a neutral aqueous medium. Preferably, enzymatic hydrolysis is carried out in water purified by reverse osmosis.

[0062] It is preferable when the water for hydrolysis additionally contains a mixture of light thiols - a source of glutathione.

[0063] 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.

[0064] In a preferred embodiment of the method, anhydrous magnesium citrate is additionally added to the water.

[0065] In a preferred embodiment of the method, mixing of the crushed fraction and water is carried out at a water ratio of 1:5 - 1:10.

[0066] In a preferred embodiment of the method, enzymatic hydrolysis is carried out for 60-160 minutes in the first stage, and 60-160 minutes in the second stage. If enzymatic hydrolysis is carried out simultaneously (i.e., both stages at once), it is carried out for 90-180 minutes.

[0067] In a preferred embodiment of the method, after completion of the enzymatic hydrolysis, the suspension is diluted with water to a water ratio of 1:11 - 1:15.

[0068] In a preferred embodiment of the method, chlorogenate hydrolase or tannase is added as the enzyme for hydrolyzing chlorogenic acid. This enzyme is added in an amount of 0.002 - 0.2% by weight of the sunflower meal.

[0069] 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 proportions. Lipase and phytase enzymes are also added in equal proportions.

[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] Preferably, neutral protease is added in an amount of 0.005-0.5 wt.%, neutral endoprotease is added in an amount of 0.005-0.25 wt.%, and neutral exoprotease in an amount of 0.005-0.25 wt.% of the sunflower meal weight. In a preferred embodiment of the method, the enzymes are added in the form of multienzyme complexes and / or as individual enzymes.

[0072] In a preferred embodiment of the method, before the concentration stage, the GBS is clarified and purified from substances such as quinic acid, heavy metals, nitrates, chlorides, sulfates, silicates, and hydroxides.

[0073] In a preferred embodiment of the method, the concentration of GBS is carried out to a dry matter content of 60% or less, preferably to 20-40%.

[0074] In a preferred embodiment of the method, the unhydrolyzed residue / cake is dewatered, dried, and granulated. Waste-free production is ensured when the dry granules of the unhydrolyzed residue are burned 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 the present method for producing protein-saccharide hydrolysate.

[0075] The present method results in yet another object of the invention: a protein-sugar hydrolysate obtained by the aforementioned method from sunflower meal via enzymatic hydrolysis in a neutral aqueous medium. The hydrolysate has a protein content of 40-70%, a sugar content of 20-40%, and a caffeic acid content of at least 0.02%, either free or bound to chlorogenic acid isomer molecules. Furthermore, the resulting final product (FPP) has a total branched-chain amino acid content of at least 30 g / kg of the final product (FPP) and an arginine content of at least 20 g / kg FPP.

[0076] Further in the description, additional advantages and details of the invention will be disclosed.

[0077] Brief description of the figures

[0078] Figure 1 shows aquaculture feed pellets with a diameter of 2 mm, in which 70% of the fish meal is replaced by GBS60.

[0079] Disclosure of invention

[0080] To maximize the utilization of the nutrients contained in sunflower meal, in addition to the protein and mineral components, the carbohydrates it contains are hydrolyzed by carbohydrase enzymes, which convert the carbohydrates into simple and higher sugars, which are then incorporated into the finished hydrolysate. For this purpose, mixtures of individual enzymes with the required activity are formulated, or ready-made multienzyme complexes, available commercially, are used.

[0081] 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 is passed through an ion exchange column equipped with an anion exchange resin after enzymatic hydrolysis.

[0082] 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.

[0083] 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.

[0084] To increase the availability of enzymes to the substrate and increase the yield of hydrolyzed product, the enzyme lipase is added.

[0085] In more detail, the method includes the following operations and processes:

[0086] 1. Enzymatic hydrolysis is carried out in a neutral aqueous medium.

[0087] In the best embodiment of the method, 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 used. A mixture of light thiols is then added to it: 0.01 to 0.2% by weight of deactivated yeast with a high glutathione content. The yeast is added to the solution to restore disulfide bonds in lipid transfer proteins (LTPs) and 2β-albumins, which are allergenic proteins.

[0088] Next, anhydrous magnesium citrate is added from 0.2 to 1.5 mmol / l, which then acts as a cofactor in the hydrolysis reaction, and heated to 45-60°C.

[0089] 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.

[0090] 3. Enzymes are added to the resulting suspension. First, enzymes that hydrolyze chlorogenic acid into caffeic and quinic acids, chlorogenate hydrolase or tannase in an amount of 0.002 - 0.2 wt.%, then 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, finally, in an equal proportion to each other lipase (hydrolyzing fats) in the amount of 0.002 - 0.2% and phytase (hydrolyzing phytic acid) in the amount of 0.002 - 0.2% of the mass of sunflower meal. First-stage enzymatic hydrolysis is carried out: hydrolysis of chlorogenic acid, carbohydrate hydrolysis, hydrolysis of fat and phytic acid for 60 - 160 minutes at a temperature of 45-60°C.When carrying out enzymatic hydrolysis, it is permissible to use both individual enzymes with specific activity and ready-made multi-enzyme complexes with a wide spectrum of action, for example, a multi-enzyme carbohydrase complex, a multi-enzyme proteolytic complex, a multi-enzyme complex with lipase and phytase activities, or a combination of individual enzymes with each other and / or with any of the ready-made multi-enzyme complexes that are available on the market.

[0091] 4. After carbohydrate hydrolysis is complete, proteolytic enzymes are added to the suspension, 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. Protein enzymatic hydrolysis (the 2nd stage of enzymatic hydrolysis) is carried out for 60-160 minutes at a temperature of 45-60°C.

[0092] Enzymatic hydrolysis is conventionally divided into two stages - carbohydrate and protein (based on the yield of the main components of the HBS). Alternatively, these two stages of carbohydrate and protein hydrolysis are carried out simultaneously. In this case, immediately after the addition of carbohydrase enzymes, lipase, phytase, proteolytic enzymes are added and simultaneous (both stages at once) enzymatic hydrolysis is carried out for 90 - 180 minutes at a temperature of 45-60 ° C. 5. 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 an unhydrolyzed residue and a protein-sugary hydrolysate (PSH).

[0093] 6. Next, the resulting protein-sugary 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 HBS is further purified to remove small particles of unhydrolyzed solid residue. In the ion-exchange columns, undesirable substances are removed. The cation exchanger is used to remove heavy metals, such as lead, mercury, arsenic, and cadmium; the anion exchanger removes 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 HBS is then spray-dried, preferably to a final product moisture content of 4-6%; the finished powder is packaged in a sealed container.

[0094] 7. 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 HBS production process. The calorific value of the resulting cake granules, tested using a colorimeter, was found to be 4320 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.

[0095] As a result of the proposed method, GBS is obtained in the form of a powder, completely soluble in water, with a protein portion of 40-70%, sugars (such as glucose, fructose, maltose, maltotriose, oligosaccharides) - 20-40% and with a caffeic acid content of at least 0.02% in free or bound form in the composition of chlorogenic acid isomer molecules.

[0096] Optimally, the protein yield is approximately 60%, sugars approximately 28%, and caffeic acid approximately 1.9%. The upper limit for caffeic acid may be around 2.5% according to calculations, but it still depends on the initial amount in the raw material, so limiting it to an upper limit is not practical. Furthermore, the resulting final product (HBS) has a total branched-chain amino acid content of at least 30 g / kg of the final product (HBS) and an arginine content of at least 20 g / kg HBS.

[0097] 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.

[0098] Arginine promotes the release of growth hormones and accelerates muscle mass gain. It is also essential for the effective functioning of the body's immune system under stress, plays a key role in normalizing lipid metabolism and energy balance, and has a hepatoprotective effect.

[0099] The amino acid profile of the hydrolyzed protein product obtained using this invention is similar to that of fishmeal, with the exception of lysine. The hydrolysate is completely water-soluble and contains 40-70% crude protein. When using the hydrolyzed protein product in feed / food additives or food / feed products, the lysine content is adjusted, if necessary, by adding synthetic lysine. The amino acid taurine is absent from the hydrolyzed protein product. This can also be adjusted by using the hydrolyzed protein product.

[0100] GBS 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. GBS 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.

[0101] The GBS 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 0.02% caffeic acid, either free or bound to chlorogenic acid isomer molecules (as part of the polyphenol complex). The advantage of GBS with caffeic acid is that it is absorbed at 95% + / - 4%, compared to chlorogenic acid's 33% + / - 17%. This prevents binding to amino acids during gastrointestinal tract digestion, enhancing the nutritional value of the product, and also possesses antioxidant and antimicrobial properties.

[0102] 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)].

[0103] GBS 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 GBS has a wide range of applications in the food and feed industries. GBS is an excellent alternative to animal proteins in compound feed for aquatic organisms (aquaculture), non-productive animals, farm animals, and poultry. For example, GBS is suitable for pets (cats and dogs); ornamental birds; farm animals (pigs, cows, and sheep); and poultry (chicken, turkey, quail, ducks, geese, and ostriches). This list of animals and birds is provided for illustrative purposes only and is not exhaustive.

[0105] GBS 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 or the elderly.This list of products is provided for example purposes only and is not limiting.

[0106] The technical results / advantages of the present invention are:

[0107] - The method is safe and environmentally friendly due to the use of neutral media, 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, with reduced 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;

[0108] - flexibility of the technological process, the possibility of carrying out both step-by-step and simultaneous enzymatic hydrolysis, the possibility of using ready-made multi-enzyme complexes that are available on the market;

[0109] - waste-free method: closed production cycle with minimal wastewater generation and energy generation by burning non-hydrolyzed residue / cake.

[0110] - maximum use of useful ingredients contained in the original raw materials during the isolation of hydrolyzate;

[0111] - the nutritional and feed value of GBS as an alternative plant protein. The method ensures the production of an easily digestible and highly nutritious GBS composition, which is a worthy replacement for animal proteins in feed and food products;

[0112] - the food and feed purity of the resulting GBS is an additional advantage of the present invention. GBS is minimally or virtually free of undesirable and toxic substances. It does not contain chlorogenic acid, anti-nutritional substances such as phytic acid, digestive enzyme inhibitors, mild protein allergens, Maillard reaction products, or oxidized lipid forms. GBS is free of mycotoxins and heavy metals such as cadmium, lead, mercury, and arsenic. The carbohydrate portion of GBS 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 are 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.

[0113] Examples of the invention

[0114] The examples below illustrate the practical implementation of the GBS product and the method for its production, but in no way limit all possible options for the practical implementation of the method and GBS-based products.

[0115] Example 1

[0116] 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. Then, 0.2 g of inactivated yeast with a high glutathione content and 0.6 g of anhydrous magnesium citrate were added. The resulting suspension was heated to 50°C with constant stirring at 20 rpm.

[0117] The following enzymes were added to the suspension:

[0118] 1. Chlorogenate hydrolase - 0.2 g and

[0119] 2. A ready-made multi-enzyme complex containing carbohydrases: xylanase, beta-glucanase, cellulase, mannanase, pectinase, alpha-amylase and glucoamylase in equal proportions, as well as lipase with phytase in equal proportions - 1.5 g.

[0120] Enzymatic hydrolysis was carried out for 120 minutes. After the first stage of enzymatic hydrolysis, proteolytic enzymes were added to the suspension:

[0121] 1. Neutral protease - 2 g and

[0122] 2. Ready-made mixture of neutral endoproteases and exoproteases - 2 g.

[0123] Enzymatic hydrolysis (second stage) was carried out for 120 min.

[0124] Upon completion of the process, 6 kg of osmotic water were added to the hydrolyzer and the suspension was stirred for 7 min. After this, the hydrolysate was separated from the non-hydrolyzed residue 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 (inlet air temperature 180°C, outlet 80°C). The yield of protein-sugar hydrolysate was 500 g. The protein portion of HBS was 60% (hereinafter - HBS60). Sugars - 25% (including 12% - glucose, 14% - fructose, 74% - oligosaccharides). Crude ash - 8%. Caffeic acid - 1.9%. Moisture - 4%. Minor amounts of GBS60 mass are accounted for by acids such as neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid, and their hydrolysis products.

[0125] Protein composition: Arginine - 67.82 g / kg. The total content of branched-chain amino acids is made up of the sum of three amino acids - valine, leucine and isoleucine - and is equal to 89 g / kg: Valine - 33.37 g / kg, Leucine - 35.11 g / kg. Isoleucine - 20.52 g / kg. The amino acid composition of GBS60 is presented in more detail below in Table 1.

[0126] Table 1. Amino acid composition of GBS60

[0127] Example 2: The original sunflower meal was ground in a rotary vortex mill to a particle size of 200-250 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 6.8 were added. Then, 0.2 g of inactivated yeast with a high glutathione content and 0.6 g of anhydrous magnesium citrate were added. The resulting suspension was heated to 50°C with constant stirring at 20 rpm.

[0128] The following individual enzymes were added to the suspension:

[0129] 1. Tannase - 0.2 g;

[0130] 2. Neutral protease - 1.5 g;

[0131] 3. Individual carbohydrases: xylanase, beta-glucanase, cellulase, mannanase, alpha-amylase, glucoamylase and pectinase in the same ratio to each other (1: 1: 1: 1: 1: 1: 1) in a total amount of 2 g;

[0132] 4. Lipase and phytase were added to this mixture also in the same ratio (1:1) in a total amount of 0.2 g.

[0133] Enzymatic hydrolysis was carried out simultaneously (both stages at once) for 150 min.

[0134] At the end of the process, 5 kg of osmotic water was added to the hydrolyzer and the suspension was stirred for 10 minutes. The hydrolysate was then separated from the unhydrolyzed residue in a decanter.

[0135] To remove any remaining anti-nutrients, the hydrolysate was clarified in a separator and then 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 evaporator to a dry matter content of 40% and dried in a spray dryer (inlet air temperature 180°C, outlet air temperature 80°C).

[0136] The yield of protein-sugar hydrolysate was 391 g. The protein content of GBS was 45%. Sugars - 36% (including 12% glucose, 14% fructose, 74% oligosaccharides). Crude ash - 10.9%. Caffeic acid - 2.6%. Moisture - 4.3%. Minor amounts of GBS mass (approximately 1.2% are accounted for by acids such as neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid and their hydrolysis products).

[0137] Protein composition: Arginine - 47.51 g / kg. The total content of branched-chain amino acids is made up of the sum of three amino acids - valine, leucine, and isoleucine - and is equal to 62.3 g / kg: Valine - 23.37 g / kg, Leucine - 24.59 g / kg. Isoleucine - 14.37 g / kg. The amino acid composition of the obtained product is presented in more detail below in Table 2.

[0138] Table 2. Amino acid composition of the obtained product

[0139] Example 3

[0140] The GBS60 hydrolysate obtained in Example 1 was used in the production of compound feed.

[0141] A test feeding of juvenile sharptooth catfish with compound feed was also conducted at the training institute, in which 50% of the fish meal was replaced with GBS60.

[0142] The following compound feed recipes were tested (see Table 3).

[0143] Table 3

[0144]

[0145] Feed pellets containing GBS60 were obtained (see Fig. 1). They were used in an aquaculture feeding experiment, and the following results were obtained (see Table 4).

[0146] Table 4

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

[0148] Example 4

[0149] The GBS60 obtained in Example 1 was used to prepare compound feed for weaned piglets aged 43 to 60 days. The grain ingredients of the compound feed were first crushed in a grain crusher, then all components were mixed in a "drunken barrel" mixer and passed through a pellet mill.

[0150] The formula for the compound feed is presented in Table 5.

[0151] Table 5

[0152] Example 5: The protein-sugary hydrolysate obtained in Example 1 with a protein content of 60% (PSH60) was used to prepare chicken feed. The grain ingredients of the feed were first crushed in a grain crusher, then all components were mixed in a "drunken barrel" mixer and passed through a granulator.

[0153] The formula for the compound feed is presented in Table 6.

[0154] Table 6

[0155] Example 6

[0156] The GBS60 obtained in Example 1 was used to prepare compound feed for broilers based on plant materials.

[0157] The formula for the compound feed is presented in Table 7.

[0158] Table 7

[0159] Example 7

[0160] The GBS60 obtained in example 1 was used to prepare cooked sausages from poultry meat using traditional technology.

[0161] The sausage recipe is presented in Table 8.

[0162] Table 8

[0163] Example 8

[0164] The GBS60 obtained in Example 1 was used to prepare a non-milk-type drink based on plant-based raw materials.

[0165] The recipe for the “non-milk” drink is presented in Table 9. Table 9

[0166] According to the present invention, GBS can be successfully added to other food or feed products using traditional technologies in place of any other plant or animal protein used in the product. The present invention makes no claims regarding specific enzyme amounts or process parameters, as these are determined empirically by a specialist in the field with experience in experimentation, depending on the quality, composition, and / or dispersion of the feedstock, the specific equipment, its volume, the volume of the fermentation substrate, pH, temperature, hydrolysis time, enzyme activity, and other factors.

Claims

CLAUSES OF THE INVENTION 1. Protein-sugary hydrolysate (PSH) obtained from sunflower meal by enzymatic hydrolysis, which has a protein content of 40-70%, sugars of 20-40% and a content of caffeic acid in free or bound form in the composition of chlorogenic acid isomer molecules of at least 0.02%.

2. GBS according to item 1, in which the total content of amino acids with branched side chains is not less than 30 g / kg of the final product (GBS) and the arginine content is not less than 20 g / kg GBS.

3. A protein food supplement, characterized in that it contains GBS according to paragraph 1 or paragraph 2, which has a protein portion of 40-70%, sugars of 20-40%, and a content of caffeic acid in free or bound form in the composition of chlorogenic acid isomer molecules of at least 0.02%.

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 HBS according to paragraph 1 or paragraph 2, which has a protein portion of 40-70%, sugars of 20-40% and a content of caffeic acid in free or bound form in the composition of chlorogenic acid isomer molecules of at least 0.02%.

6. A food product according to item 5, characterized in that it is a product in solid or soft form.

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 GBS according to paragraph 1 or paragraph 2, which has a protein portion of 40-70%, sugars of 20-40%, and a content of caffeic acid in free or bound form in the composition of chlorogenic acid isomer molecules of at least 0.02%.

9. A feed product characterized in that it contains GBS according to paragraph 1 or paragraph 2, which has a protein portion of 40-70%, sugars of 20-40% and a content of caffeic acid in free or bound form in the composition of chlorogenic acid isomer molecules of at least 0.02%.

10. The feed product according to paragraph 9, which is intended for cattle, monogastric and non-productive animals.

11. A feed product for aquaculture, characterized in that it contains GBS according to paragraph 1 or paragraph 2, which has a protein portion of 40-70%, sugars of 20-40% and a content of caffeic acid in free or bound form in the composition of chlorogenic acid isomer molecules of at least 0.02%.

12. A method for producing protein-sugary hydrolysate (PSH) from sunflower meal by enzymatic hydrolysis, comprising grinding sunflower meal; mixing the ground fraction with water until a homogeneous suspension is formed with constant stirring; carrying out enzymatic hydrolysis in a neutral medium in two stages simultaneously or sequentially, where the first stage involves introducing into the suspension an enzyme for hydrolyzing chlorogenic acid into caffeic and quinic acids, adding carbohydrase enzymes for hydrolyzing carbohydrates and lipase and phytase enzymes for hydrolyzing fats and phytic acid, respectively; in the second stage, enzymatic hydrolysis of proteins is carried out by adding proteolytic enzymes; after completion of enzymatic hydrolysis, the resulting suspension is diluted with water and separated into protein-sugary hydrolysate (PSH) and unhydrolyzed residue / cake;Finally, the GBS solution is concentrated and dried, yielding GBS that has a protein portion of 40-70%, sugar of 20-40%, and a content of caffeic acid in free or bound form in the composition of chlorogenic acid isomer molecules of at least 0.02%.

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 of paragraphs 12-14, wherein the water additionally contains a mixture of light thiols - a source of glutathione.

16. The method according to i.15, in which deactivated yeast with a high glutathione content is used as a source of glutathione.

17. The method according to item 16, in which deactivated yeast is introduced in an amount of 0.01 to 0.2 wt.%.

18. The method according to any of paragraphs 12-17, in which anhydrous magnesium citrate is additionally added to the water.

19. The method according to any of paragraphs 12 - 18, in which the mixing of the crushed fraction and water is carried out at a water ratio of 1:5 - 1:

10.

20. The method according to any one of paragraphs 12 to 19, wherein the enzymatic hydrolysis in the first stage is carried out for 60 to 160 minutes, and in the second stage for 60 to 160 minutes.

21. The method according to any one of paragraphs 12 to 20, in which the enzymatic hydrolysis is carried out simultaneously in two stages for 90 to 180 minutes.

22. The method according to any of paragraphs 12 - 21, in which, after completion of the enzymatic hydrolysis, the suspension is diluted with water to a water ratio of 1:11 - 1:

15.

23. The method according to any one of paragraphs 12 to 22, wherein chlorogenate hydrolase or tannase is introduced as an enzyme for hydrolyzing chlorogenic acid.

24. The method according to item 23, in which the enzyme for hydrolyzing chlorogenic acid is introduced in an amount of 0.002 - 0.2 wt.% of the mass of sunflower meal.

25. The method according to any one of claims 12 to 24, 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.

26. The method according to any one of paragraphs 12 to 25, wherein a neutral protease and / or a mixture of a neutral endoprotease and a neutral exoprotease are used as proteolytic enzymes.

27. The method according to item 26, in which neutral protease is added in an amount of 0.005 - 0.5 wt.%, neutral endoprotease is added in an amount of 0.005 - 0.25 wt.%, and neutral exoprotease in an amount of 0.005 - 0.25 wt.% of the mass of sunflower meal.

28. The method according to any one of paragraphs 12 to 27, wherein the enzymes are introduced in the form of multienzyme complexes and / or in the form of individual enzymes.

29. The method according to any one of paragraphs 12 to 28, in which, before the stage of concentration of the solution, the GBS is clarified and purified from substances such as quinic acid, heavy metals, nitrates, nitrites, chlorides, sulfates, silicates, and hydroxides.

30. The method according to any one of paragraphs 12 to 29, in which the concentration of the GBS solution is carried out to a dry matter content of 60% or less, preferably to 20-40%.

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

32. The method according to art. 31, in which dry granules of unhydrolyzed residue / cake are burned as biofuel in the present method, and the resulting combustion The energy is directed to the production of steam and electrical energy for use in the technological process of the present method for producing protein-sucrose hydrolysate.

33. A protein-sugar hydrolysate obtained by the method according to any of paragraphs 12 to 32 from sunflower meal by enzymatic hydrolysis, which has a protein content of 40-70%, sugars of 20-40% and a content of caffeic acid in free or bound form in the composition of chlorogenic acid isomer molecules of at least 0.02%.

34. GBS according to item 33, in which the total content of amino acids with branched side chains is not less than 30 g / kg of the final product (GBS) and the arginine content is not less than 20 g / kg GBS.

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