WO2024103619A1 - 一种浅色水溶性植物蛋白的制备方法 - Google Patents

一种浅色水溶性植物蛋白的制备方法 Download PDF

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WO2024103619A1
WO2024103619A1 PCT/CN2023/088567 CN2023088567W WO2024103619A1 WO 2024103619 A1 WO2024103619 A1 WO 2024103619A1 CN 2023088567 W CN2023088567 W CN 2023088567W WO 2024103619 A1 WO2024103619 A1 WO 2024103619A1
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protein
water
plant
amount
soluble
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PCT/CN2023/088567
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English (en)
French (fr)
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张恺
杨家森
廖琪林
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南京纽邦生物科技有限公司
艾斯姆食品公司
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Publication of WO2024103619A1 publication Critical patent/WO2024103619A1/zh

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/14—Vegetable proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/14—Vegetable proteins
    • A23J3/16—Vegetable proteins from soybean
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/14—Vegetable proteins
    • A23J3/18—Vegetable proteins from wheat
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
    • A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
    • A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/01—Hydrolysed proteins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products

Definitions

  • the invention belongs to the technical field of deep processing of plant proteins, and particularly relates to a method for preparing light-colored water-soluble plant protein.
  • plant protein is another source of protein required by humans besides animal protein, and it plays an important physiological regulatory role in human health. Compared with animal protein, plant protein production has stronger sustainability factors and lower environmental impact. Studies have shown that people who eat plant protein are significantly less likely to suffer from cardiovascular disease than those who eat animal protein; eating more plant protein can prolong the life of patients with chronic kidney disease and also prevent diabetes. Many plant proteins have poor stability and poor absorption, which limits their application in food. Plant protein, especially cereal plant protein, has a very low solubility in aqueous media, which seriously hinders its absorption and utilization by the human body. Therefore, in order to increase the solubility of plant protein, reduce the molecular size of plant protein, promote the interaction between ionic protein and water, and weaken the interaction of protein from hydrophobic sources, it is very necessary.
  • Water-soluble plant protein is a small molecule polypeptide formed after plant protein is hydrolyzed under specific conditions. It is a type of molecular polymer with a molecular weight between 100 and 10,000 Da and a structure between amino acids and proteins. It usually has 2 to 7 amino acid residues, a low molecular weight, and is easily digested and absorbed by the human body. It also has special physiological functions and has a certain regulatory effect on human life activities.
  • Enzymatic hydrolysis has the advantages of mild action conditions, simple operation, easy control of hydrolysis conditions, less damage to the nutrients of proteins and polypeptides, and better absorption effect of the obtained polypeptides than proteins and amino acids, and generally has better biological activity.
  • plant protein contains a certain proportion of sugars, and after extracting and separating proteins from it, some sugars will still remain in it. These sugars will react with proteins to produce Maillard reactions during the enzymolysis process, producing brown or even dark brown substances, causing the color of the enzymolysis solution to deepen.
  • the main measures to inhibit the Maillard reaction include removing substances that promote browning reactions, adjusting factors that promote browning reactions, and using browning inhibitors.
  • Glucose oxidase (EC1.1.3.4, referred to as GOD) is a very important oxidoreductase, which plays many important roles in biological processes. It can convert the aldehyde group on the glucose molecule into a carboxyl group to generate gluconic acid and avoid the occurrence of browning. It is a natural food additive, non-toxic to the human body, without side effects, and is widely used in chemical, pharmaceutical, food, beverage, clinical chemistry, biotechnology and other industries. As a deglucose and deoxidant, glucose oxidase has obvious effects in inhibiting fruit juice browning and preserving freshness.
  • the present invention provides a method of adding glucose oxidase during the enzymatic hydrolysis process, which effectively reduces the browning reaction during the enzymatic hydrolysis process, reduces the pigment, and ensures that the yield of the polypeptide product is not lost.
  • the present invention provides a method for preparing light-colored water-soluble plant protein, the method comprising: adding protease and glucose oxidase to a plant protein solution for enzymatic hydrolysis to obtain the water-soluble plant protein.
  • the method further comprises: mixing the plant protein with water at a solid-liquid ratio of 1:5 to 1:15 to obtain the plant protein solution.
  • sodium sulfite is added to the above-mentioned plant protein solution.
  • the amount of sodium sulfite is 0.001% to 0.1%, preferably 0.005% to 0.04% based on the amount of the above-mentioned plant protein.
  • the plant protein includes one or more of soy protein, rapeseed protein, wheat protein, buckwheat protein, corn protein, barley protein, potato protein, rice protein, oat protein, pea protein, rice protein, pumpkin seed protein, watermelon seed protein, and sunflower seed protein.
  • the amount of glucose oxidase is 0.02% to 5%, preferably 0.1% to 0.5% of the amount of the plant protein.
  • the proteases are alkaline proteases and multi-proteases.
  • the amount of alkaline protease is 1% to 10%, preferably 1% to 3%, of the amount of plant protein, and the amount of composite protease is 0.1% to 10%, preferably 0.2% to 2.5%, of the amount of plant protein.
  • the water-soluble plant protein prepared according to the method of the present invention is used in the preparation of food, beverage, nutrient, dietary supplement, cosmetic or medicine.
  • the prepared food, beverage, nutrient, dietary supplement, cosmetic or medicine can be in the form of capsule, tablet, powder, suspension, solution, drop, granule, liquid, syrup, functional food, beverage, toothpaste, sublingual preparation, food product, food additive, candy, sucker, lozenge, food supplement and suppository.
  • the present invention provides use of glucose oxidase for improving the color darkening of a food, dietary supplement or nutraceutical comprising a water-soluble plant protein.
  • the pigment of water-soluble plant protein is darker during the enzymatic preparation process, and more protein will be lost when decolorizing agents such as activated carbon are added later, reducing the yield.
  • the method of the present invention solves the pigment problem from the source.
  • the addition of glucose oxidase during the enzymatic hydrolysis process can effectively inhibit the browning reaction, which greatly improves the problem of dark color during the preparation of water-soluble plant protein.
  • sodium sulfite to pre-treat the plant protein solution, and then by combining chemical reagents with enzymes, the browning problem during the enzymatic hydrolysis process is better solved.
  • the water-soluble plant protein finally prepared is light in color and more acceptable in color, which is suitable for large-scale production and commercial application of water-soluble plant protein.
  • the term "comprises” or “comprising” or variations thereof refer to the following instances where the term is used in its non-limiting sense, meaning that items following the term are included, but items not specifically mentioned are not excluded. It also includes the more restrictive verbs 'consisting essentially of' and 'consisting of'.
  • Enzymatic hydrolysis of plant protein to prepare water-soluble plant protein refers to a method of using proteolytic enzymes to enzymatically hydrolyze plant protein using plant protein as a substrate to obtain water-soluble plant protein.
  • Water-soluble plant protein is obtained by hydrolysis or enzymolysis of plant protein under specific conditions.
  • the raw materials or reagents used in the present invention are commercially available. All reagents are of commercial grade and used according to the standards received.
  • the present invention provides a method for improving the color darkening during the hydrolysis preparation process of water-soluble plant protein, which comprises the following steps: heating the plant protein solution, adjusting the pH value, and then adding alkaline protease, composite protease, and glucose oxidase to the solution for enzyme catalysis. Hydrolysis reaction, thereby obtaining water-soluble plant protein.
  • Technicians can determine a suitable enzymolysis temperature or a suitable enzymolysis pH or a suitable enzymolysis time, such as enzymolyzing the plant protein with the enzyme at a pH between 4 and 9, a pH between 5 and 8.5, such as a pH between 6 and 8, such as a pH of about 8.
  • the suitable temperature for the plant protein and the enzyme may be between 20°C and 60°C, such as between 30°C and 60°C, such as about 55°C.
  • the plant protein solution can be heated to 55°C
  • the pH value can be adjusted to 8.0
  • the enzymolysis reaction can be carried out for 4 to 6 hours
  • the enzymolysis reaction speed is 150 to 300 rpm, preferably 250 rpm.
  • the amount of alkaline protease added is 1% to 10% of the amount of plant protein added, preferably 1% to 3%; the amount of composite protease added is 0.1% to 10% of the amount of plant protein added, preferably 0.2% to 2.5%; the amount of glucose oxidase added is 0.02% to 5% of the amount of plant protein added, preferably 0.1% to 0.5%.
  • the plant protein of the present invention includes one or more of soy protein, rapeseed protein, wheat protein, buckwheat protein, corn protein, barley protein, potato protein, rice protein, oat protein, pea protein, rice protein, pumpkin seed protein, watermelon seed protein, and sunflower seed protein.
  • the vegetable protein solution in the above step can be obtained by mixing vegetable protein with water at a material-liquid ratio of 1:5 to 1:15; and sodium sulfite can be added thereto, the amount of sodium sulfite added being 0.001% to 0.1% of the amount of vegetable protein added, preferably 0.005% to 0.04%.
  • the above steps also include, after the enzymatic hydrolysis reaction is completed, raising the temperature to inactivate the enzyme, the inactivation time is 10 to 30 minutes, preferably 30 minutes, and the inactivation temperature is 85 to 100° C., preferably 85° C.; filtering and collecting the filtrate, filtering with a plate-frame filter, the plate-frame filter using a 3 to 5 ⁇ m filter membrane, and adding 0.5% to 3% to the enzymatic hydrolyzate when using the plate-frame filter. perlite; then nanofiltration concentration is performed, and the nanofiltration concentrate is spray-dried.
  • the protein concentration after nanofiltration concentration is 14-16%.
  • the spray drying conditions are an inlet air temperature of 160-180°C and an outlet air temperature of 70-85°C, thereby obtaining water-soluble plant protein.
  • S2 Heat the reactor to 55°C, adjust the pH to 8.0 with 4M NaOH, add 60g alkaline protease and 30g composite protease into the reactor for enzymatic hydrolysis. During the enzymatic hydrolysis process, the temperature in the reactor is kept at 55°C and the rotation speed is 250rpm. After 5h of enzymatic hydrolysis, proceed to S3.
  • S5 spray drying the concentrated liquid to obtain water-soluble wheat protein, the spray drying conditions being an air inlet temperature of 180°C and an air outlet temperature of 75°C.
  • the water-soluble wheat protein product has a protein content of 90.2%, a polypeptide content of 85%, a peptide segment with a molecular weight less than 1000 Da accounting for 82%, and a free amino acid content of 6.1%.
  • the water-soluble wheat protein is dissolved in water at a concentration of 5%, and the color is measured to be 1245 Hazen.
  • S2 Heat the reactor to 55°C, adjust the pH to 8.0 with 4M NaOH, add 60g alkaline protease and 30g composite protease into the reactor for enzymatic hydrolysis. During the enzymatic hydrolysis process, the temperature in the reactor is kept at 55°C and the rotation speed is 250rpm. After 5h of enzymatic hydrolysis, proceed to S3.
  • S5 spray drying the concentrated liquid to obtain water-soluble wheat protein, the spray drying conditions being an air inlet temperature of 180°C and an air outlet temperature of 75°C.
  • the water-soluble wheat protein product has a protein content of 89%, a polypeptide content of 85.2%, a peptide segment with a molecular weight less than 1000 Da accounting for 84%, and a free amino acid content of 5.6%.
  • the water-soluble wheat protein is dissolved in water at a concentration of 5%, and the color is measured to be 932 Hazen.
  • S2 Heat the reactor to 55°C, adjust the pH to 8.0 with 4M NaOH, add 60g alkaline protease, 30g composite protease, and 6g glucose oxidase to the reactor.
  • Enzymolysis During the enzymolysis process, the temperature in the reactor was kept at 55°C and the rotation speed was 250 rpm. After 5 hours of enzymolysis reaction, the reactor entered S3.
  • S5 spray drying the concentrated liquid to obtain water-soluble wheat protein, the spray drying conditions being an air inlet temperature of 180°C and an air outlet temperature of 75°C.
  • the water-soluble wheat protein product has a protein content of 88.5%, a polypeptide content of 84.4%, a peptide segment with a molecular weight less than 1000 Da accounting for 82.6%, and a free amino acid content of 6.3%.
  • the water-soluble wheat protein is dissolved in water at a concentration of 5%, and the color is measured to be 748 Hazen.
  • S2 Heat the reactor to 55°C, adjust the pH to 8.0 with 4M NaOH, add 60g alkaline protease, 30g composite protease and 6g glucose oxidase into the reactor for enzymatic hydrolysis. During the enzymatic hydrolysis process, the temperature in the reactor is kept at 55°C and the rotation speed is 250rpm. After 5h of enzymatic hydrolysis, proceed to S3.
  • S5 spray drying the concentrated liquid to obtain water-soluble wheat protein, the spray drying conditions being an air inlet temperature of 180°C and an air outlet temperature of 75°C.
  • the water-soluble wheat protein product has a protein content of 89%, a polypeptide content of 83.8%, a peptide segment with a molecular weight less than 1000 Da accounting for 84%, and a free amino acid content of 5.9%.
  • the water-soluble wheat protein is dissolved in water at a concentration of 5%, and the color is measured to be 681 Hazen.
  • S1 Weigh 3 kg of rice protein with a protein content greater than 80% into a bioreactor, add 27 L of water and stir thoroughly to obtain a mixed solution, and transfer to S2.
  • S2 Heat the reactor to 55°C, adjust the pH to 8.0 with 4M NaOH, add 90g alkaline protease and 45g composite protease into the reactor for enzymatic hydrolysis. During the enzymatic hydrolysis process, the temperature in the reactor is kept at 55°C and the rotation speed is 250rpm. After 5h of enzymatic hydrolysis, proceed to S3.
  • S5 spray drying the concentrated solution to obtain water-soluble rice protein, the spray drying conditions being an air inlet temperature of 180° C. and an air outlet temperature of 75° C.
  • the water-soluble rice protein product has a protein content of 88.2%, a polypeptide content of 84.6%, a peptide segment with a molecular weight less than 1000 Da accounting for 83.5%, and a free amino acid content of 5.1%.
  • the water-soluble rice protein is dissolved in water at a concentration of 5%, and the chromaticity is measured to be 1052 Hazen.
  • S1 Weigh 3 kg of rice protein with a protein content greater than 85% into a bioreactor, add 27 L of water and stir to mix thoroughly, add 0.3 g of sodium sulfite to obtain a mixed solution, and transfer to S2.
  • S2 Heat the reactor to 55°C, adjust the pH to 8.0 with 4M NaOH, add 90g alkaline protease and 45g composite protease into the reactor for enzymatic hydrolysis. During the enzymatic hydrolysis process, the temperature in the reactor is kept at 55°C and the rotation speed is 250rpm. After 5h of enzymatic hydrolysis, proceed to S3.
  • S5 spray drying the concentrated solution to obtain water-soluble rice protein, the spray drying conditions being an air inlet temperature of 180° C. and an air outlet temperature of 75° C.
  • the water-soluble rice protein product has a protein content of 89.3%, a polypeptide content of 84.5%, a peptide segment with a molecular weight less than 1000 Da accounting for 84.2%, and a free amino acid content of 5.4%.
  • the water-soluble rice protein is dissolved in water at a concentration of 5%, and the chromaticity is measured to be 796 Hazen.
  • S1 Weigh 3 kg of rice protein with a protein content greater than 80% into a bioreactor, add 27 L of water and stir thoroughly to obtain a mixed solution, and transfer to S2.
  • S2 Heat the reactor to 55°C, adjust the pH to pH 8.0 with 4M NaOH, add 90g alkaline protease, 45g composite protease and 3g glucose oxidase into the reactor for enzymatic hydrolysis. During the enzymatic hydrolysis process, the temperature in the reactor is kept at 55°C and the rotation speed is 250rpm. After 5h of enzymatic hydrolysis, proceed to S3.
  • S5 spray drying the concentrated solution to obtain water-soluble rice protein, the spray drying conditions being an air inlet temperature of 180° C. and an air outlet temperature of 75° C.
  • the water-soluble rice protein product has a protein content of 90.2%, a polypeptide content of 83.6%, a peptide segment with a molecular weight less than 1000 Da accounting for 86.2%, and a free amino acid content of 6.6%.
  • the water-soluble rice protein is dissolved in water at a concentration of 5%, and the chromaticity is measured to be 670 Hazen.
  • S1 Weigh 3 kg of rice protein with a protein content greater than 85% into a bioreactor, add 27 L of water and stir to mix thoroughly, add 0.3 g of sodium sulfite to obtain a mixed solution, and transfer to S2.
  • S2 Heat the reactor to 55°C, adjust the pH to pH 8.0 with 4M NaOH, add 90g alkaline protease, 45g composite protease and 3g glucose oxidase into the reactor for enzymatic hydrolysis. During the enzymatic hydrolysis process, the temperature in the reactor is kept at 55°C and the rotation speed is 250rpm. After 5h of enzymatic hydrolysis, proceed to S3.
  • S5 spray drying the concentrated solution to obtain water-soluble rice protein, the spray drying conditions being an air inlet temperature of 180° C. and an air outlet temperature of 75° C.
  • the water-soluble rice protein product has a protein content of 89.5%, a polypeptide content of 83.8%, a peptide segment with a molecular weight less than 1000 Da accounting for 85.6%, and a free amino acid content of 4.3%.
  • the water-soluble rice protein is dissolved in water at a concentration of 5%, and the chromaticity is measured to be 625 Hazen.
  • the plant protein in Example 3-4 or 7-8 is replaced with one or more of soy protein, rapeseed protein, wheat protein, buckwheat protein, corn protein, barley protein, potato protein, rice protein, oat protein, pea protein, rice protein, pumpkin seed protein, watermelon seed protein, and sunflower seed protein, and the appropriate reaction conditions are adjusted to prepare the corresponding water-soluble plant protein.
  • the color of the obtained water-soluble plant protein is 560 Hazen-750 Hazen.

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Abstract

本发明提供了一种浅色水溶性植物蛋白的制备方法，该方法包括：将植物蛋白溶液中加入蛋白酶，及葡萄糖氧化酶进行酶解反应，得到水溶性植物蛋白。该方法有效减轻酶解过程中的褐变反应，既减轻了色素，又可以保证多肽产物的收率无损失。

Description

一种浅色水溶性植物蛋白的制备方法 技术领域
本发明属于植物蛋白深加工技术领域，具体涉及一种浅色水溶性植物蛋白的制备方法。
背景技术
植物蛋白作为一种可再生资源，是除动物蛋白外，另一种人类所需的蛋白质来源，对人体健康具有重要的生理调节作用。与动物蛋白相比植物蛋白生产具有较强的可持续性因素和较低的环境影响。有研究表明，相比食用动物蛋白，食用植物蛋白的人患心血管疾病的几率要显著低于食用动物蛋白的人；多食用植物蛋白，可使慢性肾病患者的生命延长，同时也可起到预防糖尿病的作用。许多植物蛋白的稳定性差，吸收效果差，限制了其在食品中的应用。植物蛋白，尤其是谷物类植物蛋白，在水性介质中的溶解度很低，严重阻碍了人体对其的吸收与利用，因此为了提高植物蛋白的溶解度，降低植物蛋白质的分子大小，促进离子型蛋白质与水的相互作用，减弱疏水性来源的蛋白质的相互作用就显得十分必要。
水溶性植物蛋白是植物蛋白在特定条件下水解后形成的一种小分子多肽，是一类分子量介于100～10000Da，结构介于氨基酸和蛋白质之间的分子聚合物，通常带有2～7个氨基酸残基，分子量低，易于被人体消化和吸收，同时具有特殊生理功能，对人体的生命活动具有一定的调节作用。酶法水解植物蛋白制备水溶性植物蛋白是指使 用蛋白质水解酶，以植物蛋白为底物对其进行酶解，借以制得水溶性植物蛋白的方法。酶法水解具有作用条件温和，操作简便，水解条件易控制，对蛋白质和多肽营养成分破坏较小，且获得的多肽比蛋白质和氨基酸的吸收效果好，一般具有较好的生物活性等优点。但植物蛋白中含有一定比例的糖类，且从中提取分离蛋白后，仍会有部分糖留在其中，这些糖在酶解过程中会与蛋白质作用发生美拉德反应，产生褐色甚至黑褐色的物质，造成酶解液颜色加深。目前，抑制美拉德反应的主要措施有去除促进褐变反应的物质，调节促进褐变反应的影响因子，使用褐变抑制剂等方法。
水溶性植物蛋白在制备过程中色素较深的问题，会大大影响其商业价值，若通过后续加入活性炭等脱色剂脱色时会损失较多蛋白，大大降低收率。因此，本领域中存在寻找改善水溶性植物蛋白酶解液颜色加深的合适的方法的需要。
发明内容
葡萄糖氧化酶(GlucoseOxidase，E.C.1.1.3.4，简称GOD)是一种非常重要的氧化还原酶，在生物过程中具有许多重要作用，能将葡萄糖分子上的醛基转变为羧基，生成葡萄糖酸，避免褐变的发生。它是一种天然的食品添加剂，对人体无毒，无副作用，在化工、医药、食品、饮料、临床化学、生物技术等行业得到广泛应用。作为去葡萄糖和脱氧剂，葡萄糖氧化酶在抑制果汁褐变，保鲜等方面具有明显效果。
为了解决上述的问题，本发明提供了一种在酶解过程中加入葡萄糖氧化酶的方法，有效减轻酶解过程中的褐变反应，既减轻了色素，又保证了多肽产物的收率无损失。
在第一方面，本发明提供了一种浅色水溶性植物蛋白的制备方法，该方法包括：将植物蛋白溶液中加入蛋白酶，及葡萄糖氧化酶进行酶解反应，得到水溶性植物蛋白。
在一些实施方案中，该方法还包括：按料液比1:5～1:15的比例将植物蛋白与水混合以获得上述植物蛋白溶液。
在一些实施方案中，向上述植物蛋白溶液中加入亚硫酸钠。
在一些实施方案中，亚硫酸钠的量为上述植物蛋白的量的0.001％～0.1％，优选0.005％～0.04％。
在一些实施方案中，植物蛋白包括大豆蛋白、油菜籽蛋白、小麦蛋白、荞麦蛋白、玉米蛋白、大麦蛋白、马铃薯蛋白、稻米蛋白、燕麦蛋白、豌豆蛋白、大米蛋白、南瓜籽蛋白、西瓜籽蛋白、葵花籽蛋白中的一种或多种。
在一些实施方案中，葡萄糖氧化酶的量为所述植物蛋白的量的0.02％～5％，优选0.1％～0.5％。
在一些实施方案中，蛋白酶为碱性蛋白酶和复合蛋白酶。
在一些实施方案中，碱性蛋白酶的量为植物蛋白的量的1％～10％，优选1％～3％，复合蛋白酶的量为植物蛋白的量的0.1％～10％，优选0.2％～2.5％。
在第二方面，根据本发明的方法制得的水溶性植物蛋白在制备食品、饮料、营养品、膳食补充剂、化妆品或药品中的应用。所制备的食品、饮料、营养品、膳食补充剂、化妆品或药品可以是胶囊、片剂、粉末、悬浮液、溶液剂、滴剂、颗粒剂、液体、糖浆、功能化食品、饮料、牙膏、舌下制品、食品制品、食品添加剂、糖果、吸盘、锭剂、食品补充剂和栓剂的形式。
在第三方面，本发明提供了葡萄糖氧化酶在用于改善包含水溶性植物蛋白的食物、膳食补充剂或营养品的颜色变深中的用途。
水溶性植物蛋白在酶解制备过程中色素较深，后去加入活性炭等脱色剂时会损失较多蛋白，降低收率。本发明的方法从源头上解决了色素问题，酶解过程中添加了葡萄糖氧化酶可有效抑制褐变反应，使水溶性植物蛋白制备过程中颜色深的问题得到极大的改善。另外，通过使用亚硫酸钠前处理植物蛋白溶液，进而通过将化学试剂与酶相结合的方法，更好的解决了酶解过程中的褐变问题，最终制备出的水溶性植物蛋白颜色浅，色泽上更易被接受，适宜水溶性植物蛋白规模化生产及商业化应用。
具体实施方式
在上面的发明内容和本部分发明详述，以及下面的权利要求书中，提到了本发明的特定特征，应当理解，本发明的说明书中公开了包括这些特定特征的所有可能的组合。例如，当在本发明的特定方面或实施例或特定权利要求中公开了特定特征时，则该特征也可尽可能的和/或与本发明的其他特定方面和实施例结合使用。
如本文所用，术语“或”旨在包括“和”和“或”。换句话说，术语“或”也可以被替换为“和/或”。
如本文所用，除非上下文另有明确指示，否则单数形式“一(a/an)”和“所述(the)”旨在也包括复数形式。
如本文所用，术语“包含”或“包括”或其变化形式指以下情况，其中该术语以其非限制性含义使用，指包括该词语后的项目，但并不排除未特别提及的项目。其还包括更限制性的动词‘基本上由……组成’和‘由……组成’。
酶法水解植物蛋白制备水溶性植物蛋白是指使用蛋白质水解酶，以植物蛋白为底物对其进行酶解，借以制得水溶性植物蛋白的方法。
水溶性植物蛋白通过植物蛋白在特定条件下水解或酶解后制得。
实施例
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用实验室操作步骤均为相应领域内广泛使用的常规步骤。
本发明中所用的原料或试剂除特别说明之外，均市售可得。所有的试剂都是商品级的，并按照收到的标准使用。
本发明提供一种改善水溶性植物蛋白酶解制备过程中颜色变深的方法，其包括如下步骤：将植物蛋白溶液加热，调节pH值，然后向该溶液中加入碱性蛋白酶，复合蛋白酶，及葡萄糖氧化酶进行酶 解反应，进而得到水溶性植物蛋白。技术人员能够确定合适的酶解温度或合适的酶解pH或合适的酶解时间，例如将植物蛋白与酶一起在介于4与9之间的pH，介于5与8.5之间的pH，例如介于6与8之间的pH，例如约8的pH下酶解。植物蛋白与酶一起的合适温度可介于20℃与60℃之间，例如介于30℃与60℃之间，例如约55℃。具体而言，例如可将植物蛋白溶液加热至55℃，调节pH值为8.0，酶解反应进行4～6h，酶解反应转速为150～300rpm，优选为250rpm。其中，碱性蛋白酶加入量为植物蛋白加入量的1％～10％，优选1％～3％；复合蛋白酶加入量为植物蛋白加入量的0.1％～10％，优选0.2％～2.5％；葡萄糖氧化酶加入量为植物蛋白加入量的0.02％～5％，优选0.1％～0.5％。
本发明的植物蛋白包括大豆蛋白、油菜籽蛋白、小麦蛋白、荞麦蛋白、玉米蛋白、大麦蛋白、马铃薯蛋白、稻米蛋白、燕麦蛋白、豌豆蛋白、大米蛋白、南瓜籽蛋白、西瓜籽蛋白、葵花籽蛋白中的一种或多种。
上述步骤的植物蛋白溶液可以通过按料液比1:5～1:15的比例将植物蛋白与水混合获得；并且可以向其中添加亚硫酸钠，亚硫酸钠的加入量为植物蛋白加入量的0.001％～0.1％，优选0.005％～0.04％。
另外，上述步骤还包括酶解反应完成后，升高温度进行灭酶，灭酶时间为10～30min，优选为30min，灭酶温度为85～100℃，优选为85℃；过滤并收集滤液，采用板框过滤器进行过滤，板框过滤器采用3～5μm的滤膜，采用板框过滤时需向酶解液中添加0.5％～3％ 的珍珠岩；然后进行纳滤浓缩，将纳滤浓缩液喷雾干燥，纳滤浓缩之后的蛋白量浓度为14～16％，喷雾干燥条件为进风温度160～180℃，出风温度70～85℃，进而得到水溶性植物蛋白。
实施例1
S1：称取3kg蛋白含量大于80％的小麦蛋白至生物反应器中，加入27L水，充分搅拌混匀得到混合溶液，转入S2。
S2：将反应器加热至55℃，用4M NaOH调pH至8.0，向反应器中加入60g碱性蛋白酶，30g复合蛋白酶进行酶解，恒定酶解过程中反应器内温度为55℃，转速为250rpm，酶解反应5h后转入S3。
S3：将酶解液加热至85℃，维持温度不变30min，使酶灭活，转入S4。
S4：酶解液自然降温至55℃，向酶解液中加入300g珍珠岩，搅拌混匀，通过板框过滤器进行过滤，收集滤液25L。将滤液进行纳滤浓缩，浓缩液的蛋白量为14.4％，转入S5。
S5：将浓缩液进行喷雾干燥制得水溶性小麦蛋白，喷雾干燥条件为进风口温度180℃，出风口温度75℃。
水溶性小麦蛋白产品蛋白质含量90.2％，多肽含量85％，小于1000Da分子量肽段占82％，游离氨基酸含量6.1％。水溶性小麦蛋白按照5％浓度溶于水，测得色度为1245Hazen。
实施例2
S1：称取3kg蛋白含量大于80％的小麦蛋白至生物反应器中，加入27L水充分搅拌混匀，添加0.6g亚硫酸钠得到混合溶液，转入S2。
S2：将反应器加热至55℃，用4M NaOH调pH至8.0，向反应器中加入60g碱性蛋白酶，30g复合蛋白酶进行酶解，恒定酶解过程中反应器内温度为55℃，转速为250rpm，酶解反应5h后转入S3。
S3：将酶解液加热至85℃，维持温度不变30min，使酶灭活，转入S4。
S4：酶解液自然降温至55℃，向酶解液中加300g珍珠岩进行搅拌混匀，通过板框过滤器进行过滤，收集滤液25L。将滤液进行纳滤浓缩，浓缩液的蛋白量为14.8％，转入S5。
S5：将浓缩液进行喷雾干燥制得水溶性小麦蛋白，喷雾干燥条件为进风口温度180℃，出风口温度75℃。
水溶性小麦蛋白产品蛋白质含量89％，多肽含量85.2％，小于1000Da分子量肽段占84％，游离氨基酸含量5.6％。水溶性小麦蛋白按照5％浓度溶于水，测得色度为932Hazen。
实施例3
S1：称取3kg蛋白含量大于80％的小麦蛋白至生物反应器中，加入27L水，充分搅拌混匀得到混合溶液，转入S2。
S2：将反应器加热至55℃，用4M NaOH调pH至8.0，向反应器中加入60g碱性蛋白酶，30g复合蛋白酶，6g葡萄糖氧化酶进行 酶解，恒定酶解过程中反应器内温度为55℃，转速为250rpm，酶解反应5h后转入S3。
S3：将酶解液加热至85℃，维持30min，使酶灭活，转入S4。
S4：酶解液自然降温至55℃，向酶解液中加300g珍珠岩进行搅拌混匀，通过板框过滤器进行过滤，收集滤液25L。将滤液进行纳滤浓缩，浓缩液的蛋白量为14.5％，转入S5。
S5：将浓缩液进行喷雾干燥制得水溶性小麦蛋白，喷雾干燥条件为进风口温度180℃，出风口温度75℃。
水溶性小麦蛋白产品蛋白质含量88.5％，多肽含量84.4％，小于1000Da分子量肽段占82.6％，游离氨基酸含量6.3％。水溶性小麦蛋白按照5％浓度溶于水，测得色度为748Hazen。
实施例4
S1：称取3kg蛋白含量大于80％的小麦蛋白至生物反应器中，加入27L水充分搅拌混匀，添加0.6g亚硫酸钠得到混合溶液，转入S2。
S2：将反应器加热至55℃，用4M NaOH调pH至8.0，向反应器中加入60g碱性蛋白酶，30g复合蛋白酶，6g葡萄糖氧化酶进行酶解，恒定酶解过程中反应器内温度为55℃，转速为250rpm，酶解反应5h后转入S3。
S3：将酶解液加热至85℃，维持30min，使酶灭活，转入S4。
S4：酶解液自然降温至55℃，向酶解液中加300g珍珠岩进行搅拌混匀，通过板框过滤器进行过滤，收集滤液25L。将滤液进行纳滤浓缩，浓缩液的蛋白量为15.2％，转入S5。
S5：将浓缩液进行喷雾干燥制得水溶性小麦蛋白，喷雾干燥条件为进风口温度180℃，出风口温度75℃。
水溶性小麦蛋白产品蛋白质含量89％，多肽含量83.8％，小于1000Da分子量肽段占84％，游离氨基酸含量5.9％。水溶性小麦蛋白按照5％浓度溶于水，测得色度为681Hazen。
实施例5
S1：称取3kg蛋白含量大于80％的大米蛋白至生物反应器中，加入27L水充分搅拌混匀得到混合溶液，转入S2。
S2：将反应器加热至55℃，用4M NaOH调pH至8.0，向反应器中加入90g碱性蛋白酶，45g复合蛋白酶进行酶解，恒定酶解过程中反应器内温度为55℃，转速为250rpm，酶解反应5h后转入S3。
S3：将酶解液加热至85℃，维持30min，使酶灭活，转入S4。
S4：酶解液自然降温至55℃，向酶解液中加600g珍珠岩进行搅拌混匀，通过板框过滤器进行过滤，收集滤液24L。将滤液进行纳滤浓缩，浓缩液的蛋白量为14.2％，转入S5。
S5：将浓缩液进行喷雾干燥制得水溶性大米蛋白，喷雾干燥条件为进风口温度180℃，出风口温度75℃。
水溶性大米蛋白产品蛋白质含量88.2％，多肽含量84.6％，小于1000Da分子量肽段占83.5％，游离氨基酸含量5.1％。水溶性大米蛋白按照5％浓度溶于水，测得色度为1052Hazen。
实施例6
S1：称取3kg蛋白含量大于85％的大米蛋白至生物反应器中，加入27L水充分搅拌混匀，添加0.3g亚硫酸钠得到混合溶液，转入S2。
S2：将反应器加热至55℃，用4M NaOH调pH至8.0，向反应器中加入90g碱性蛋白酶，45g复合蛋白酶进行酶解，恒定酶解过程中反应器内温度为55℃，转速为250rpm，酶解反应5h后转入S3。
S3：将酶解液加热至85℃，维持30min，使酶灭活，转入S4。
S4：酶解液自然降温至55℃，向酶解液中加600g珍珠岩进行搅拌混匀，通过板框过滤器进行过滤，收集滤液24L。将滤液进行纳滤浓缩，浓缩液的蛋白量为15.5％，转入S5。
S5：将浓缩液进行喷雾干燥制得水溶性大米蛋白，喷雾干燥条件为进风口温度180℃，出风口温度75℃。
水溶性大米蛋白产品蛋白质含量89.3％，多肽含量84.5％，小于1000Da分子量肽段占84.2％，游离氨基酸含量5.4％。水溶性大米蛋白按照5％浓度溶于水，测得色度为796Hazen。
实施例7
S1：称取3kg蛋白含量大于80％的大米蛋白至生物反应器中，加入27L水充分搅拌混匀得到混合溶液，转入S2。
S2：将反应器加热至55℃，用4M NaOH调pH至pH8.0，向反应器中加入90g碱性蛋白酶，45g复合蛋白酶，3g葡萄糖氧化酶进行酶解，恒定酶解过程中反应器内温度为55℃，转速为250rpm，酶解反应5h后转入S3。
S3：将酶解液加热至85℃，维持30min，使酶灭活，转入S4。
S4：酶解液自然降温至55℃，向酶解液中加600g珍珠岩进行搅拌混匀，通过板框过滤器进行过滤，收集滤到24L。将滤液进行纳滤浓缩，浓缩液的蛋白量为14.1％，转入S5。
S5：将浓缩液进行喷雾干燥制得水溶性大米蛋白，喷雾干燥条件为进风口温度180℃，出风口温度75℃。
水溶性大米蛋白产品蛋白质含量90.2％，多肽含量83.6％，小于1000Da分子量肽段占86.2％，游离氨基酸含量6.6％。水溶性大米蛋白按照5％浓度溶于水，测得色度为670Hazen。
实施例8
S1：称取3kg蛋白含量大于85％的大米蛋白至生物反应器中，加入27L水充分搅拌混匀，添加0.3g亚硫酸钠得到混合溶液，转入S2。
S2：将反应器加热至55℃，用4M NaOH调pH至pH8.0，向反应器中加入90g碱性蛋白酶，45g复合蛋白酶，3g葡萄糖氧化酶进行酶解，恒定酶解过程中反应器内温度为55℃，转速为250rpm，酶解反应5h后转入S3。
S3：将酶解液加热至85℃，维持30min，使酶灭活，转入S4。
S4：酶解液自然降温至55℃，向酶解液中加600g珍珠岩进行搅拌混匀，通过板框过滤器进行过滤，收集滤到24L。将滤液进行纳滤浓缩，浓缩液的蛋白量为14.6％，转入S5。
S5：将浓缩液进行喷雾干燥制得水溶性大米蛋白，喷雾干燥条件为进风口温度180℃，出风口温度75℃。
水溶性大米蛋白产品蛋白质含量89.5％，多肽含量83.8％，小于1000Da分子量肽段占85.6％，游离氨基酸含量4.3％。水溶性大米蛋白按照5％浓度溶于水，测得色度为625Hazen。
实施例9
将实施例3-4或7-8的植物蛋白替换成包括大豆蛋白、油菜籽蛋白、小麦蛋白、荞麦蛋白、玉米蛋白、大麦蛋白、马铃薯蛋白、稻米蛋白、燕麦蛋白、豌豆蛋白、大米蛋白、南瓜籽蛋白、西瓜籽蛋白、葵花籽蛋白中的一种或多种，并调整合适的反应条件制备得到相应的水溶性植物蛋白，所得到的水溶性植物蛋白的色度为560Hazen-750Hazen。
尽管本文已经说明了本发明的特定实施方案和实施例，但本领域技术人员将理解，可以在不脱离本发明的原则的情况下进行任何修改和变动。上面的实施例和说明并不限制本发明的范围。本发明的实施方案的任何组合，以及其任何明显的扩展或类似物，均在本发明的范围内。此外，本发明涵盖旨在实现相同目的而存在的任何布置，以及落入所附权利要求书的范围内的所有这些变动和修改。

Claims (10)

  1. 一种浅色水溶性植物蛋白的制备方法，所述方法包括：将植物蛋白溶液中加入蛋白酶，及葡萄糖氧化酶进行酶解反应，得到水溶性植物蛋白。
  2. 根据权利要求1所述的方法，其特征在于，所述方法还包括：按料液比1:5～1:15的比例将植物蛋白与水混合以获得所述植物蛋白溶液。
  3. 根据权利要求2所述的方法，其特征在于，向所述植物蛋白溶液中加入亚硫酸钠。
  4. 根据权利要求3所述的方法，其特征在于，所述亚硫酸钠的量为所述植物蛋白的量的0.001％～0.1％。
  5. 根据权利要求1或2所述的方法，其特征在于，所述植物蛋白是大豆蛋白、油菜籽蛋白、小麦蛋白、荞麦蛋白、玉米蛋白、大麦蛋白、马铃薯蛋白、稻米蛋白、燕麦蛋白、豌豆蛋白、大米蛋白、南瓜籽蛋白、西瓜籽蛋白、葵花籽蛋白中的一种或多种。
  6. 根据权利要求1或2所述的方法，其特征在于，所述葡萄糖氧化酶的量为所述植物蛋白的量的0.02％～5％。
  7. 根据权利要求1所述的方法，其特征在于，所述蛋白酶为碱性蛋白酶和复合蛋白酶。
  8. 根据权利要求7所述的方法，其特征在于，所述碱性蛋白酶的量为所述植物蛋白的量的1％～10％，所述复合蛋白酶的量为所述植物蛋白的量的0.1％～10％。
  9. 根据权利要求1-3任一项所述的方法制得的水溶性植物蛋白在制备食品、饮料、营养品、膳食补充剂、化妆品或药品中的应用。
  10. 葡萄糖氧化酶在用于改善包含水溶性植物蛋白的食物、膳食补充剂或营养品的颜色变深中的用途。
PCT/CN2023/088567 2022-11-15 2023-04-17 一种浅色水溶性植物蛋白的制备方法 WO2024103619A1 (zh)

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