Hippophae rhamnoides protein peptide as well as preparation method and application thereof

Abstract

The invention provides hippophae rhamnoides protein peptide and a preparation method and application thereof, according to the preparation method, hippophae rhamnoides seed meal is used as a raw material, and the hippophae rhamnoides protein peptide is obtained through coarse crushing, pulping and shelling, centrifugal impurity removal, protein heating denaturation, enzymolysis enzyme deactivation, impurity removal clarification, concentration sterilization and spray drying in sequence. The method is green, environment-friendly, safe, sanitary and suitable for industrial production; the total nitrogen content (on a dry basis) of the sea-buckthorn protein peptide can reach 11.2% or above, and meets the requirements of GB31611 Plant Protein Peptides for Food Processing; the prepared sea-buckthorn protein peptide product can be applied to terminal products such as special medical foods, health foods, solid beverages and sports nutritious foods, and has wide application prospects.

Description

Technical Field

[0001] This invention belongs to the field of deep processing of sea buckthorn, which is both a food and a medicine, and specifically relates to a sea buckthorn protein peptide, its preparation method and application. Background Technology

[0002] Sea buckthorn ( Hippophae rhamnoides Hippophae rhamnoides (L.), also known as vinegar willow, sour thorn, or black thorn, belongs to the genus Hippophae in the family Elaeagnaceae and is widely distributed in temperate regions of Eurasia. China has the richest natural Hippophae rhamnoides germplasm resources in the world and also the largest area of ​​artificial Hippophae rhamnoides forests. As of 2018, the total area of ​​Hippophae rhamnoides in China was 2.15 × 10⁻⁶. 6 It accounts for approximately 93% of the world's total sea buckthorn area.

[0003] Sea buckthorn is a food and medicine homology substance with excellent health benefits. The Chinese Pharmacopoeia identifies its effects as promoting blood circulation, relieving cough and phlegm, and strengthening the spleen and aiding digestion. Modern medical and nutritional research has found that sea buckthorn contains various vitamins, minerals, and active ingredients, making it a hot research topic in the nutrition and health industry. Mainstream sea buckthorn products on the market include pharmaceuticals, food products, and cosmetics.

[0004] Sea buckthorn food products mainly include sea buckthorn pulp, sea buckthorn beverages, sea buckthorn oral liquid, sea buckthorn seed oil, sea buckthorn fruit oil, sea buckthorn fruit powder, sea buckthorn leaf extract, sea buckthorn proanthocyanidins, etc. Currently, the total output value of the sea buckthorn industry has exceeded 20 billion yuan. The sea buckthorn industry has good ecological, economic, and social value and a promising development prospect.

[0005] Currently, the development of sea buckthorn seeds mainly focuses on the production of sea buckthorn seed oil. The by-product, sea buckthorn seed meal, is primarily sold as animal feed with very low added value. Some of it is even discarded directly as waste, failing to achieve comprehensive utilization and polluting the environment. Therefore, it is essential to carry out intensive processing of sea buckthorn seed meal, extend the industrial chain, and turn waste into treasure.

[0006] However, the protein content of sea buckthorn seed meal is relatively low, only 20-25%, while the protein content of the most common soybean meal can reach 40-50%. This is mainly because sea buckthorn kernels have a thick, tough, brownish-brown seed coat, making separation of the kernel and shell difficult. Therefore, the development of high-quality sea buckthorn protein peptides is extremely challenging, and industrialization faces numerous difficulties. Currently, some sea buckthorn protein peptide products exist on the market, but their total nitrogen content is unsatisfactory, some only around 5%. With the implementation of the national food safety standard GB31611-2023 "Plant Protein Peptides for Food Processing," the total nitrogen content of peptide products is required to exceed 11.2%, but most sea buckthorn protein peptide products currently do not meet this requirement. Even those few products that do meet the requirements use ethanol or alkali extraction and acid precipitation processes, which are not only complex and costly but also have high explosion-proof requirements and cause significant environmental pollution, limiting their application to the laboratory stage and making industrial application unlikely.

[0007] Therefore, how to further improve the quality of sea buckthorn protein peptide products is a problem that needs to be solved. Summary of the Invention

[0008] To address the problem of low protein content in existing sea buckthorn protein peptides, the present invention aims to provide a sea buckthorn protein peptide, its preparation method, and its applications. The preparation method of the sea buckthorn protein peptide is advanced and reasonable, suitable for industrial production, and the total nitrogen content of the prepared sea buckthorn protein peptide exceeds the national standard requirements. This invention is beneficial to extending the industrial chain, enhancing the value chain, and promoting high-quality development of the sea buckthorn industry.

[0009] The objective of this invention is achieved through the following technical solution: The first aspect of this invention provides a method for preparing sea buckthorn protein peptides, comprising the following steps: a) Coarse grinding of sea buckthorn seed meal: Mix sea buckthorn seed meal with water, heat to 55-65℃, stir and mix evenly, and then coarsely grind using a colloid mill with a gap distance of 1-1.5mm; preferably, the gap distance of the colloid mill is controlled at 1.0-1.2mm. b) Pulping and shelling: The crushed material is pulped using a pulper with a stainless steel screen with a mesh size of 0.7-1.0 mm. During the pulping process, the sea buckthorn seed shells are intercepted and discharged, and the pulp is used after passing through the screen; preferably, the stainless steel screen of the pulper has a mesh size of 0.7-0.9 mm. c) Centrifugation to remove impurities: The slurry is separated into solid and liquid phases using a horizontal screw centrifuge. The centrifuge speed is controlled at 2800-3000 r / min. The aqueous phase is discarded, and the precipitate is kept for later use. d) Protein denaturation by heating: Place the above precipitate obtained after centrifugation into a heat denaturation tank, add water so that the total amount of material in the heat denaturation tank is 7-9 times the initial amount of sea buckthorn seed meal, and then heat to 85-100℃ and keep warm for 15-60 minutes. e) Enzymatic hydrolysis and enzyme inactivation: Cool the denatured material to 50-60℃, maintain the material temperature within this range in the enzymatic hydrolysis tank, adjust the pH value of the material to 8.0-9.5 using a strong alkali, add alkaline protease at a dosage of 0.5-1.2% of the initial sea buckthorn seed meal weight, and hydrolyze for 30-90 minutes; then add room temperature water to cool the material, controlling the material temperature at 45-55℃, add neutral protease and papain at dosages of 0.25-0.75% and 0.05-0.2% of the initial sea buckthorn seed meal weight, respectively, and continue enzymatic hydrolysis for 150-300 minutes; then raise the temperature to 85-95℃ and hold for 15-60 minutes; f) Purification and clarification: The enzymatically hydrolyzed material is cooled to 55-65℃ and centrifuged using a horizontal screw centrifuge at a speed of 2800-3000 r / min. The resulting aqueous phase is then centrifuged again using a disc centrifuge at a speed of 6500-7000 r / min. The resulting aqueous phase is then filtered using a ceramic membrane with a pore size of 50-200 nm. The turbidity of the filtered material is <1 NTU, resulting in a clarified material. g) Concentration and sterilization: Triple-effect vacuum concentration is adopted to concentrate the soluble solids concentration in the clarified material to 20-40%, and then ultra-high temperature instantaneous sterilization is adopted. The sterilization temperature is 110-135℃, the sterilization time is 30-90S, and the sterilization outlet temperature is controlled at 60-80℃. h) Spray drying: The clarified material after concentration and sterilization is spray dried to obtain sea buckthorn protein peptide powder. The inlet air temperature is 180-210℃ and the outlet air temperature is 80-110℃.

[0010] Furthermore, in step a), the sea buckthorn seed meal is a byproduct obtained after sea buckthorn seeds (sea buckthorn is a plant of the genus Hippophae in the family Elaeagnaceae) are extracted with supercritical carbon dioxide to remove sea buckthorn seed oil, and the protein content is 20-25%.

[0011] Furthermore, in step a), the ratio of sea buckthorn seed meal to water is 1:6 to 1:12.

[0012] Furthermore, in step d), the protein is heated to denature at a temperature of 90-95°C for 30-45 minutes.

[0013] Furthermore, in step e), the strong alkali is sodium hydroxide or potassium hydroxide, and the pH value of the material is adjusted to 8.0-9.0.

[0014] Further, in step e), the amount of alkaline protease used is 0.8-1.0% of the initial sea buckthorn seed meal, the enzymatic hydrolysis time is 45-75 min, the amount of neutral protease and papain used are 0.45-0.55% and 0.08-0.12% of the initial sea buckthorn seed meal, respectively, and the enzymatic hydrolysis continues for 180-240 min; then the temperature is raised to 85-90℃ and kept warm for 15-30 min.

[0015] Furthermore, in step f), the pore size of the ceramic membrane is 100-200 nm.

[0016] Furthermore, in step g), the concentration of soluble solids in the clarified material is 25-35%.

[0017] The third aspect of the present invention provides a seabuckthorn protein peptide prepared by the method described in the first aspect, wherein the total nitrogen content (on a dry basis) of the seabuckthorn protein peptide reaches 11.2% or more, which meets the total nitrogen requirements specified in GB31611 "National Food Safety Standard for Plant Protein Peptides for Food Processing".

[0018] The third aspect of this invention provides the application of the sea buckthorn protein peptide described in the second aspect in the preparation of food and health products.

[0019] The advantages of this invention compared to the prior art are as follows: 1. In the preparation method of sea buckthorn protein peptides described in this invention, the sea buckthorn seed meal used in the coarse crushing process mainly consists of two parts: one is the sea buckthorn kernel, which is relatively loose and easy to crush into fine particles, accounting for 40-45%; the other is the sea buckthorn seed shell, which is hard and has strong toughness and is not easy to break, accounting for 55-60%. This invention uses a colloid mill for coarse crushing. By adjusting and optimizing the gap between the stator and rotor of the colloid mill, the sea buckthorn kernel can be fully crushed into fine particles, while most of the sea buckthorn seed shell breaks into coarse particles. 2. In the pulping and dehulling process of this invention, the mixture after colloid milling is pulped using a pulping machine. During high-speed rotation, the metal blades of the pulping machine strike the sea buckthorn seed shells and kernels at high speed. This process firstly effectively separates the sea buckthorn seed shells from the kernels. Simultaneously, the sea buckthorn kernels can be further crushed into fine powder, while the sea buckthorn seed shells, due to their high toughness, are not suitable for further crushing, thus facilitating the filtration and separation of the kernels and shells. A suitable screen is selected for the pulping machine. The material passes through the screen during high-speed rotation. Smaller sea buckthorn kernels and some finely ground seed shells pass through the screen, while larger seed shells are retained and separated, achieving the purpose of removing the seed shells. This operation can remove more than 65% of the total sea buckthorn seed shells from the sea buckthorn seed meal. If the sea buckthorn seed shells are not removed, alkali-soluble substances will dissolve during the enzymatic hydrolysis of the prepared polypeptides under alkaline conditions, resulting in excessive impurities in the prepared product and failure to meet national standards for total nitrogen. 3. In the centrifugal impurity removal process of this invention, the slurry after pulping is centrifuged using a horizontal screw centrifuge to obtain precipitate and an aqueous phase. The aqueous phase is discarded, as it contains a large amount of water-soluble small molecules, accounting for more than 10% of the dry matter of the material. This process achieves further impurity removal. 4. The main purpose of the protein heating denaturation process in this invention is to change the three-dimensional conformation of the sea buckthorn protein by heat treatment, making it more dispersed, so that the protease can easily contact the enzymatic sites in the later stage, which is beneficial to improving the efficiency and yield of enzymatic hydrolysis. 5. In the enzymatic hydrolysis and enzyme inactivation process of this invention, the pH value is controlled within the range of 8.0-9.5. Studies have found that when the pH value is less than 8.0, the solubility of sea buckthorn protein in water is very poor, which seriously affects the enzymatic hydrolysis effect and protein transfer rate. When the pH value is higher than 9.5, amino acids and other components in the product are prone to react and produce anti-nutritional components, which are detrimental to consumer health. The use of compound enzyme hydrolysis, with different biological enzymes having different enzymatic hydrolysis sites and synergistic effects, can make the enzymatic hydrolysis more thorough, ensuring that the content of oligopeptides with a molecular weight of less than 1000 in the product is increased to more than 50%. 6. The present invention uses spray drying to obtain sea buckthorn protein peptide products, wherein the total nitrogen content (on a dry basis) exceeds 11.2%, and the protein transfer rate from sea buckthorn seed meal raw material to sea buckthorn protein peptide products reaches more than 60%. 7. The preparation method of sea buckthorn protein peptides described in this invention is green and environmentally friendly. It does not use organic solvents such as ethanol, nor does it require highly polluting processes such as alkaline extraction and acid precipitation. Furthermore, the pH value is controlled below 9.5 during the product production process, and no anti-nutritional factors are generated during the production process. This method is not only suitable for industrial production requirements, but also produces healthy and safe polypeptide products. Attached Figure Description

[0021] The present invention will be further described below with reference to the accompanying drawings and embodiments: Figure 1 This is a schematic flowchart of the seabuckthorn protein peptide preparation method of the present invention; Figure 2 The sea buckthorn protein peptide powder prepared by the method described in this invention is shown. Detailed Implementation

[0022] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0023] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0024] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to exemplify and further explain and illustrate the content of the present invention, and are not intended to limit the present invention.

[0025] In the following embodiments, unless otherwise specified, all raw and auxiliary materials are commercially available products. The main sources of raw and auxiliary materials are shown in Table 1.

[0026] Table 1: Sources of Raw Materials The performance testing methods involved in the following embodiments are as follows: (1) Protein content detection method: Referring to GB 5009.5 "Determination of protein in food", the conversion factor of total nitrogen and protein in sea buckthorn seed meal and sea buckthorn protein peptides was calculated according to 6.25. The total nitrogen content in the examples and comparative examples is calculated on a dry basis. (2) The relative percentage of peptides with relative molecular mass in the range of 189-10000 is determined by referring to Appendix A of GB 31645-2018.

[0027] Example 1 like Figure 1 As shown, this embodiment provides a method for preparing sea buckthorn protein peptides, including the following steps: Take an appropriate amount of sea buckthorn seed meal (total nitrogen content 3.97%, protein content 24.8%) and place it in a pre-grinding tank. Add 10 times the weight of water, stir and mix, and heat to 65℃. Grind using a colloid mill, controlling the gap between the stator and rotor of the colloid mill to 1.0mm. The ground material is then fed into a pulper for pulping. The screen used in the pulper is controlled to be 0.7mm. After pulping, the sea buckthorn seed shells are removed. The pulp is then separated into solid and liquid phases using a horizontal screw centrifuge at a speed of 3000r / min. The aqueous phase is discarded, and the precipitate is added to a heat denaturation tank. Add an appropriate amount of water to make the total amount of material equal to the initial sea buckthorn seed meal content. Nine times the initial weight of the sea buckthorn seed meal was heated to 95°C and held for 30 minutes. The denatured material was then cooled to 58-60°C using a plate heat exchanger. The material was then held at 58-60°C in an enzymatic hydrolysis tank. The pH of the material was adjusted to 9.0 using sodium hydroxide. Alkaline protease was added at 0.85% of the initial sea buckthorn seed meal weight, and the hydrolysis time was 60 minutes. Then, an appropriate amount of water was added to the hydrolysis tank to lower the material temperature to 50-52°C. Neutral protease and papain were added at 0.5% and 0.1% of the initial sea buckthorn seed meal weight, respectively, and the hydrolysis continued for 180 minutes. Finally, the temperature was raised to 90°C and held for 15 minutes. The enzymatically hydrolyzed material was cooled to 60-65℃ using a plate heat exchanger, and then centrifuged to remove impurities using a horizontal screw centrifuge at 2800 rpm. The resulting aqueous phase was then centrifuged again using a disc centrifuge at 6700 rpm. This aqueous phase was then filtered through a ceramic membrane with a pore size of 200 nm. The turbidity of the filtered material was <1 NTU, yielding a clarified material. The clarified material was then concentrated using a triple-effect vacuum system to reduce the soluble solids concentration to 30%. It was then subjected to ultra-high temperature instantaneous sterilization at 121℃ for 60 seconds, with the sterilization outlet temperature controlled at 80℃. The sterilized clarified material was then spray-dried at an inlet air temperature of 205-210℃ and an outlet air temperature of 100-110℃ to obtain sea buckthorn protein peptide powder, such as… Figure 2 As shown.

[0028] The product yield was 21.2% (product yield = seabuckthorn protein peptide product quality / initial seabuckthorn seed meal quality * 100%). The total nitrogen content of the prepared seabuckthorn protein peptide powder was 12.5% ​​(protein content 78.1%), and the protein transfer rate from seabuckthorn seed meal to seabuckthorn protein peptide powder reached 66.8%.

[0029] The relative molecular mass and molecular weight distribution range of the prepared sea buckthorn protein peptide powder were further determined, and the results are shown in Tables 2 and 3.

[0030] Table 2: Relative Molecular Mass of Sea Buckthorn Protein Peptide Powder Table 3: Molecular weight distribution of sea buckthorn protein peptide powder GB31611, the National Food Safety Standard for Plant Protein Peptides for Food Processing, requires that the relative percentage of peptides with a relative molecular mass of 189-10000 be ≥60% in terms of physicochemical indicators. As shown in Tables 2 and 3, the test results in the sea buckthorn protein peptide powder product prepared in this example show that the sum of the relative percentages of peptides with a relative molecular mass of 189-10000 reaches 86.65%, far exceeding the national standard requirements. Furthermore, it can be seen that the percentage of sea buckthorn protein peptides with a molecular weight <1000 in this product is 54.93%, exceeding 50%. Small molecule peptides have better human absorption and physiological functions, and are currently a hot research direction in bioactive peptides.

[0031] Example 2 This embodiment provides a method for preparing sea buckthorn protein peptides, including the following steps: Take an appropriate amount of sea buckthorn seed meal (total nitrogen content 3.97%, protein content 24.8%) and place it in a pre-grinding tank. Add 6 times the weight of water, stir and mix, and heat to 55℃. Grind using a colloid mill, controlling the stator and rotor gap of the colloid mill at 1.2mm. The ground material enters a pulper for pulping, using a 0.9mm screen. After pulping, remove the sea buckthorn seed shells. Separate the pulp using a horizontal screw centrifuge at 3000r / min. Discard the aqueous phase, and add the precipitate to a heat denaturation tank. Add an appropriate amount of water to make the total material amount equal to the initial sea buckthorn seed meal weight. The material was heated to 90℃ and held for 45 minutes. The denatured material was then cooled to 55-57℃ using a plate heat exchanger. The material was then kept at 55-57℃ in an enzymatic hydrolysis tank. The pH of the material was adjusted to 8.0 using sodium hydroxide. Alkaline protease was added at a dosage of 0.8% of the initial sea buckthorn seed meal weight, and the hydrolysis time was 75 minutes. Then, an appropriate amount of water was added to the hydrolysis tank to lower the material temperature to 47-50℃. Neutral protease and papain were added at dosages of 0.45% and 0.08% of the initial sea buckthorn seed meal weight, respectively, and the hydrolysis continued for 240 minutes. Finally, the temperature was raised to 85℃ and held for 30 minutes. The enzymatically hydrolyzed material was cooled to 60-65℃ using a plate heat exchanger, and then centrifuged using a horizontal screw centrifuge at 3000 rpm to remove impurities. The resulting aqueous phase was then further centrifuged using a disc centrifuge at 6700 rpm. This aqueous phase was then filtered through a ceramic membrane with a pore size of 100 nm. The turbidity of the filtered material was <1 NTU, resulting in a clarified material. The clarified material was then concentrated using a triple-effect vacuum system to reduce the soluble solids concentration to 26%. It was then subjected to ultra-high temperature (UHT) sterilization at 128℃ for 45 seconds, with the sterilization outlet temperature controlled at 70℃. Finally, the sterilized clarified material was spray-dried at an inlet air temperature of 190-200℃ and an outlet air temperature of 90-100℃ to obtain sea buckthorn protein peptide powder.

[0032] The product yield was 22.8% (product yield = seabuckthorn protein peptide product quality / initial seabuckthorn seed meal quality * 100%). The total nitrogen content of the prepared seabuckthorn protein peptide powder was 11.8% (protein content 73.8%), and the protein transfer rate from seabuckthorn seed meal to seabuckthorn protein peptide powder reached 67.8%.

[0033] Comparative Example 1 This comparative example provides a method for preparing sea buckthorn protein peptides. Compared with Example 1, the b) pulping and dehulling process is omitted in this comparative example, and the method specifically includes the following steps: Take an appropriate amount of sea buckthorn seed meal (total nitrogen content 3.97%, protein content 24.8%) and place it in a pre-grinding tank. Add 10 times its weight of water, stir and mix, and heat to 65℃. Grind using a colloid mill, controlling the gap between the stator and rotor of the colloid mill to 1.0 mm. Separate the pulverized material using a horizontal screw centrifuge at a speed of 3000 r / min. Discard the aqueous phase, and add the precipitated material to a heat denaturation tank. Add an appropriate amount of water to make the total amount of material 9 times the initial weight of sea buckthorn seed meal. Heat to 95℃ and maintain the temperature for 30 minutes. The material is cooled to 58-60℃ using a plate heat exchanger and then kept at 58-60℃ in an enzymatic hydrolysis tank. The pH is adjusted to 9.0 using sodium hydroxide, and alkaline protease is added at 0.85% of the initial sea buckthorn seed meal mass. The hydrolysis time is 60 minutes. Then, an appropriate amount of water is added to the hydrolysis tank to lower the material temperature to 50-52℃. Neutral protease and papain are added at 0.5% and 0.1% of the initial sea buckthorn seed meal mass, respectively, and hydrolysis continues for 180 minutes. The temperature is then raised to 90℃ and held for 15 minutes. The hydrolyzed material is cooled to 60-65℃ using a plate heat exchanger and centrifuged at 2800 rpm using a horizontal screw centrifuge to remove impurities. The resulting aqueous phase is then centrifuged again using a disc centrifuge at 6700 rpm. Finally, the aqueous phase is filtered through a ceramic membrane with a pore size of 200 nm. The turbidity of the filtered material is <1 NTU, resulting in a clarified material. The clarified material is concentrated using triple-effect vacuum to reduce the soluble solids concentration to 30%, followed by ultra-high temperature instantaneous sterilization at 121℃ for 60 seconds, with the sterilization outlet temperature controlled at 80℃. The sterilized clarified material is then spray-dried at an inlet air temperature of 205-210℃ and an outlet air temperature of 100-110℃ to obtain sea buckthorn protein peptide powder.

[0034] The product yield was 34.5% (product yield = seabuckthorn protein peptide product quality / initial seabuckthorn seed meal quality * 100%). The total nitrogen content of the prepared seabuckthorn protein peptide powder was 6.6% (protein content 41.3%), and the protein transfer rate from seabuckthorn seed meal to seabuckthorn protein peptide powder was 57.4%.

[0035] Comparative Example 2 This comparative example provides a method for preparing sea buckthorn protein peptides. Compared with Example 1, this comparative example omits the centrifugation purification process (c) and specifically includes the following steps: Take an appropriate amount of sea buckthorn seed meal (total nitrogen content 3.97%, protein content 24.8%) and place it in a pre-grinding tank. Add 10 times the weight of water, stir and mix, and heat to 65℃. Grind it using a colloid mill, controlling the gap between the stator and rotor of the colloid mill to 1.0mm. The ground material enters a pulper for pulping, using a 0.7mm screen. After pulping, remove the sea buckthorn seed shells. Heat the pulp to 95℃ and keep it at that temperature for 30 minutes. Cool the material to 58-60℃ using a plate heat exchanger and keep it at 58-60℃ in an enzymatic hydrolysis tank. Adjust the pH of the material to 9.0 using sodium hydroxide. Add alkaline protease at a dosage of 0.85% of the initial sea buckthorn seed meal weight and hydrolyze for 60 minutes. Then, add an appropriate amount of water to the enzymatic hydrolysis tank to lower the material temperature to 50-52℃. Add neutral protease and papain at dosages of 0.5% and 0.1% of the initial sea buckthorn seed meal weight, respectively, and continue enzymatic hydrolysis for 180 minutes. The temperature was then raised to 90℃ and held for 15 minutes. The enzymatically hydrolyzed material was cooled to 60-65℃ using a plate heat exchanger and centrifuged at 2800 rpm to remove impurities. The resulting aqueous phase was then centrifuged again using a disc centrifuge at 6700 rpm. This aqueous phase was then filtered through a ceramic membrane with a pore size of 200 nm. The turbidity of the filtered material was <1 NTU, yielding a clarified material. The clarified material was concentrated using triple-effect vacuum to reduce the soluble solids concentration to 30%. It was then subjected to ultra-high temperature instantaneous sterilization at 121℃ for 60 seconds, with the sterilization outlet temperature controlled at 80℃. The sterilized clarified material was then spray-dried at an inlet air temperature of 205-210℃ and an outlet air temperature of 100-110℃ to obtain sea buckthorn protein peptide powder.

[0036] The product yield was 26.8% (product yield = seabuckthorn protein peptide product quality / initial seabuckthorn seed meal quality * 100%). The total nitrogen content of the prepared seabuckthorn protein peptide powder was 9.9% (protein content 61.9%), and the protein transfer rate from seabuckthorn seed meal to seabuckthorn protein peptide powder reached 66.9%.

[0037] Comparative Example 3 This comparative example provides a method for preparing sea buckthorn protein peptides. Compared with Example 1, this comparative example omits d) protein heating denaturation treatment and specifically includes the following steps: Take an appropriate amount of sea buckthorn seed meal (total nitrogen content 3.97%, protein content 24.8%) and place it in a pre-grinding tank. Add 10 times the weight of water, stir and mix, and heat to 65℃. Grind using a colloid mill, controlling the gap between the stator and rotor of the colloid mill to 1.0 mm. The ground material enters a pulper for pulping. The screen used in the pulper is controlled to be 0.7 mm. After pulping, the sea buckthorn seed shells are removed. The pulp is separated into solid and liquid phases using a horizontal screw centrifuge at a speed of 3000 r / min. The aqueous phase is discarded, and the precipitate is added to... Add an appropriate amount of water to the enzymatic hydrolysis tank to make the total amount of material 9 times the initial weight of sea buckthorn seed meal. Maintain the temperature of the material in the enzymatic hydrolysis tank at 58-60℃. Adjust the pH of the material to 9.0 using sodium hydroxide. Add alkaline protease at 0.85% of the initial weight of sea buckthorn seed meal. Hydrolyze for 60 minutes. Then, add an appropriate amount of water to the enzymatic hydrolysis tank to lower the material temperature to 50-52℃. Add neutral protease and papain at 0.5% and 0.1% of the initial weight of sea buckthorn seed meal, respectively. Continue enzymatic hydrolysis for 180 minutes. Then, raise the temperature to 90℃ and hold for 15 minutes. Cool the hydrolyzed material to 60-65℃ using a plate heat exchanger. Centrifuge using a horizontal screw centrifuge at 2800 rpm to remove impurities. The resulting aqueous phase is then centrifuged using a disc centrifuge at 6700 rpm. The resulting aqueous phase is then filtered through a ceramic membrane with a pore size of 200 nm. The turbidity of the filtered material is <1 NTU, resulting in a clarified material. The clarified material is concentrated using triple-effect vacuum to reduce the soluble solids concentration to 30%, followed by ultra-high temperature instantaneous sterilization at 121℃ for 60 seconds, with the sterilization outlet temperature controlled at 80℃. The sterilized clarified material is then spray-dried at an inlet air temperature of 205-210℃ and an outlet air temperature of 100-110℃ to obtain sea buckthorn protein peptide powder.

[0038] The product yield was 18.9% (product yield = seabuckthorn protein peptide product quality / initial seabuckthorn seed meal quality * 100%). The total nitrogen content of the prepared seabuckthorn protein peptide powder was 12.4% (protein content 77.5%), and the protein transfer rate from seabuckthorn seed meal to seabuckthorn protein peptide powder reached 59.1%.

[0039] Comparative Example 4 This comparative example provides a method for preparing sea buckthorn protein peptides. Compared with Example 1, the pH is adjusted to 7.5 before enzymatic hydrolysis in this comparative example, specifically including the following steps: Take an appropriate amount of sea buckthorn seed meal (total nitrogen content 3.97%, protein content 24.8%) and place it in a pre-grinding tank. Add 10 times the weight of water, stir and mix, and heat to 65℃. Grind using a colloid mill, controlling the gap between the stator and rotor of the colloid mill to 1.0 mm. The ground material enters a pulper for pulping. The screen used in the pulper is controlled to be 0.7 mm. After pulping, the sea buckthorn seed shells are removed. The pulp is separated into solid and liquid phases using a horizontal screw centrifuge at a speed of 3000 r / min. The aqueous phase is discarded, and the precipitate is added to a heat denaturation tank. Add an appropriate amount of water to make the total amount of material equal to the initial sea buckthorn seed meal. Nine times the weight of the sea buckthorn seed meal was heated to 95℃ and held for 30 minutes. The material was then cooled to 58-60℃ using a plate heat exchanger. The material was then held at 58-60℃ in an enzymatic hydrolysis tank. The pH of the material was adjusted to 7.5 using sodium hydroxide. Alkaline protease was added at 0.85% of the initial sea buckthorn seed meal weight, and the hydrolysis time was 60 minutes. Then, an appropriate amount of water was added to the hydrolysis tank to lower the material temperature to 50-52℃. Neutral protease and papain were added at 0.5% and 0.1% of the initial sea buckthorn seed meal weight, respectively, and the hydrolysis continued for 180 minutes. Finally, the temperature was raised to 90℃ and held for 15 minutes. The enzymatically hydrolyzed material was cooled to 60-65℃ using a plate heat exchanger, and then centrifuged to remove impurities using a horizontal screw centrifuge at 2800 rpm. The resulting aqueous phase was then centrifuged again using a disc centrifuge at 6700 rpm. This aqueous phase was then filtered through a ceramic membrane with a pore size of 200 nm. The turbidity of the filtered material was <1 NTU, yielding a clarified material. The clarified material was then concentrated using a triple-effect vacuum system to reduce the soluble solids concentration to 30%. It was then subjected to ultra-high temperature (UHT) sterilization at 121℃ for 60 seconds, with the sterilization outlet temperature controlled at 80℃. Finally, the sterilized clarified material was spray-dried at an inlet air temperature of 205-210℃ and an outlet air temperature of 100-110℃ to obtain sea buckthorn protein peptide powder.

[0040] The product yield was 14.7% (product yield = seabuckthorn protein peptide product quality / initial seabuckthorn seed meal quality * 100%). The total nitrogen content of the prepared seabuckthorn protein peptide powder was 11.0 (protein content 68.8%), and the protein transfer rate from seabuckthorn seed meal to seabuckthorn protein peptide powder reached 40.8%.

[0041] The data from the above embodiments and comparative examples are compared in Table 4 below.

[0042] Table 4: Comparison of Data from Examples and Comparative Examples As shown in Table 4, in the preparation of sea buckthorn seed protein peptide powder in Examples 1 and 2, coarse crushing and pulping were used to remove most of the sea buckthorn seed shells, and the slurry was centrifuged by horizontal screw centrifugation to remove water-soluble components. The material before enzymatic hydrolysis was prepared by a combination of heat denaturation treatment and other methods. The total nitrogen content can reach more than 11.2%. In Example 1, the amount of water used before coarse crushing was greater, and the amount of impurities removed during horizontal screw centrifugation was more thorough. Therefore, the total nitrogen content in the product is higher, reaching 12.5%.

[0043] In Comparative Example 1, no crushing, pulping, or dehulling was used. Although the product yield increased significantly, the total nitrogen content was only 6.6%. This was mainly because the sea buckthorn shells released more impurities under alkaline conditions, resulting in a significant decrease in protein content. In Comparative Example 2, direct enzymatic hydrolysis after pulping, without descaling, reduced the total nitrogen content of the resulting sea buckthorn protein peptide powder to 9.9%. In Comparative Example 3, no heat denaturation treatment was performed before enzymatic hydrolysis. Although the total nitrogen content was not significantly affected, the product yield was only 18.9%. In Comparative Example 4, the pH was adjusted to 7.5 before enzymatic hydrolysis. Under these conditions, the product yield was only 14.7%, and the protein transfer rate dropped to 40.7%. This was mainly because the protein in the sea buckthorn seed meal had poor solubility in water at this pH, resulting in tight protein encapsulation and unsatisfactory enzymatic hydrolysis, severely impacting both product yield and protein transfer rate.

[0044] For effective enzymatic hydrolysis of sea buckthorn protein peptides, the pH must be controlled above 8.0 to achieve good hydrolysis results and a high protein transfer rate. However, under these conditions, components in the sea buckthorn seed shell will dissolve, leading to a decrease in product yield. Currently, GB31611, "Plant Protein Peptides for Food Processing," requires the total nitrogen content of plant protein peptides to be >11.2% (equivalent to a protein content higher than 70% based on a protein coefficient of 6.25). Therefore, dehulling is required in the industrial production of sea buckthorn protein peptides. The current conventional approach is to extract sea buckthorn protein from the seed meal, using alkaline extraction at a pH of 10-12. After protein extraction, acid precipitation is used to adjust the pH to 4.5-5.0. After protein precipitation and separation, the protein is washed with water until neutral to obtain sea buckthorn protein, which is then used to prepare sea buckthorn protein peptides. This process is complex, using large amounts of strong acids and alkalis, and the amount of water used for protein washing is also very large, which is very detrimental to environmental protection. The approach of this invention is exactly the opposite of the conventional approach. It considers removing the seabuckthorn seed shells and other impurities from the seabuckthorn seed meal, and then performing a post-processing process on the remaining seabuckthorn protein.

[0045] This invention employs a combination of colloid milling and pulping to remove most of the sea buckthorn seed shells. The process is simple, easy to operate and industrialize, environmentally friendly, and maintains a pH below 9.5 throughout the production process, preventing the formation of anti-nutritional factors and ensuring the safety and reliability of the produced sea buckthorn protein peptides. Furthermore, this invention combines water washing for impurity removal with heat denaturation to disrupt the protein's spatial structure, promoting higher product yield and protein content. Ultimately, the product meets the total nitrogen content requirements of GB31611 "Plant Protein Peptides for Food Processing".

[0046] Finally, it should be noted that the above description is only used to illustrate the technical solutions of the present invention and is not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention.

Claims

1. A method for preparing sea buckthorn protein peptides, characterized in that, The preparation method includes the following steps: a) Coarse grinding of sea buckthorn seed meal: Mix sea buckthorn seed meal with water, heat to 55-65℃, stir and mix evenly, and then coarsely grind using a colloid mill with a gap distance of 1-1.5mm. b) Pulping and shelling: The crushed material is pulped using a pulping machine with a stainless steel screen with a mesh size of 0.7-1.0mm. During the pulping process, the sea buckthorn seed shells are intercepted and discharged, and the pulp is used after passing through the screen. c) Centrifugation to remove impurities: The slurry is separated into solid and liquid phases using a horizontal screw centrifuge. The centrifuge speed is controlled at 2800-3000 r / min. The aqueous phase is discarded, and the precipitate is kept for later use. d) Protein denaturation by heating: Place the above precipitate obtained after centrifugation into a heat denaturation tank, add water so that the total amount of material in the heat denaturation tank is 7-9 times the initial amount of sea buckthorn seed meal, and then heat to 85-100℃ and keep warm for 15-60 minutes. e) Enzymatic hydrolysis and enzyme inactivation: Cool the denatured material to 50-60℃, maintain the material temperature in the enzymatic hydrolysis tank within this range, adjust the pH value of the material to 8.0-9.5 using a strong alkali, add alkaline protease at a dosage of 0.5-1.2% of the initial sea buckthorn seed meal weight, and hydrolyze for 30-90 minutes; then add room temperature water to cool the material, controlling the material temperature to 45-55℃, add neutral protease and papain at dosages of 0.25-0.75% and 0.05-0.2% of the initial sea buckthorn seed meal weight, respectively, and continue enzymatic hydrolysis for 150-300 minutes; then raise the temperature to 85-95℃ and hold for 15-60 minutes; f) Purification and clarification: The enzymatically hydrolyzed material is cooled to 55-65℃ and centrifuged using a horizontal screw centrifuge at a speed of 2800-3000 r / min. The resulting aqueous phase is then centrifuged again using a disc centrifuge at a speed of 6500-7000 r / min. The resulting aqueous phase is then filtered using a ceramic membrane with a pore size of 50-200 nm. The turbidity of the filtered material is <1 NTU, resulting in a clarified material. g) Concentration and sterilization: Triple-effect vacuum concentration is adopted to concentrate the soluble solids concentration in the clarified material to 20-40%, and then ultra-high temperature instantaneous sterilization is adopted. The sterilization temperature is 110-135℃, the sterilization time is 30-90S, and the sterilization outlet temperature is controlled at 60-80℃. h) Spray drying: The clarified material after concentration and sterilization is spray dried to obtain sea buckthorn protein peptide powder. The inlet air temperature is 180-210℃ and the outlet air temperature is 80-110℃.

2. The method for preparing sea buckthorn protein peptides according to claim 1, characterized in that, In step a), the seabuckthorn seed meal is a byproduct obtained after seabuckthorn seeds have undergone supercritical carbon dioxide extraction to remove seabuckthorn seed oil, and the protein content is 20-25%.

3. The method for preparing sea buckthorn protein peptides according to claim 1, characterized in that, In step a), the ratio of sea buckthorn seed meal to water is 1:6 to 1:

12.

4. The method for preparing sea buckthorn protein peptides according to claim 1, characterized in that, In step d), the protein is heated to denature at a temperature of 90-95℃ for 30-45 minutes.

5. The method for preparing sea buckthorn protein peptides according to claim 1, characterized in that, In step e), the strong alkali is sodium hydroxide or potassium hydroxide, and the pH value of the material is adjusted to 8.0-9.

0.

6. The method for preparing sea buckthorn protein peptides according to claim 1, characterized in that, In step e), the amount of alkaline protease used is 0.8-1.0% of the initial sea buckthorn seed meal, the enzymatic hydrolysis time is 45-75 min, the amount of neutral protease and papain used is 0.45-0.55% and 0.08-0.12% of the initial sea buckthorn seed meal, respectively, and the enzymatic hydrolysis continues for 180-240 min; then the temperature is raised to 85-90℃ and kept warm for 15-30 min.

7. The method for preparing sea buckthorn protein peptides according to claim 1, characterized in that, In step f), the pore size of the ceramic membrane is 100-200 nm.

8. The method for preparing sea buckthorn protein peptides according to claim 1, characterized in that, In step g), the concentration of soluble solids in the clarified material is 25-35%.

9. The sea buckthorn protein peptide prepared by the method according to any one of claims 1 to 8, characterized in that, The total nitrogen content of the sea buckthorn protein peptides is above 11.2% on a dry basis.

10. The application of the sea buckthorn protein peptide as described in claim 9 in the preparation of food and health products.

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