Double-protein polypeptide soybean milk and preparation method thereof

Abstract

The invention relates to double-protein polypeptide soybean milk and a preparation method thereof, and belongs to the technical field of food processing. According to the method, the soybeans with the bud length of 4-6 cm are subjected to low-temperature stress and unfreezing temperature returning, the activity of endogenous enzymes of the soybeans is stimulated and improved, meanwhile, the soybean protein structure is loose, and enzymolysis is facilitated. Then instantaneous high-temperature heat shock is carried out, and lipoxygenase is passivated on the premise that the activity of endogenous enzymes of the soybeans is guaranteed. And grinding the soybeans subjected to high-temperature heat shock into slurry, mixing the slurry with animal milk, carrying out heat preservation treatment, and adjusting the enzymolysis temperature and pH in the heat preservation treatment process to obtain the double-protein polypeptide soybean milk disclosed by the invention. The double-protein polypeptide soybean milk disclosed by the invention has the advantages of light beany flavor, high polypeptide content, low preparation cost and good production prospect.

Description

Technical Field

[0001] The invention relates to the technical field of food processing, in particular to a double-protein polypeptide soybean milk and a preparation method thereof. Background Art

[0002] Soy protein is a plant protein with an amino acid composition similar to that of milk protein. Except for a slightly lower level of methionine, the other essential amino acids are relatively rich. The dual-protein milk beverage produced by combining soy protein with milk protein can solve the problems of poor taste of plant protein and high fat content of animal protein, thereby improving the taste of the product, making it more nutritionally balanced and having higher added value.

[0003] A mixture of oligopeptides with a molecular weight of less than 1000D, usually composed of several amino acids, obtained by hydrolysis of soy protein / animal protein, which also contains a small amount of free amino acids and sugars. Studies have shown that peptides not only have the same essential amino acids as proteins, but are also easily digested and absorbed by the human body and can quickly provide energy. Peptides have better solubility, water retention, oil absorption, foaming, emulsification and gelling properties than proteins.

[0004] Double protein polypeptide soy milk beverage is a beverage produced with soybeans and milk (milk powder) as the main raw materials. It is recommended by Chinese nutritionists as a healthy food for preventing and treating diseases such as hyperlipidemia, hypertension, and diabetes. Double protein polypeptide soy milk is rich in protein, vitamins, and minerals, and has the effects of promoting blood circulation, promoting diuresis, dispelling wind, and detoxifying. Trace elements such as zinc, copper, and magnesium in double protein polypeptide soy milk are very important for delaying human aging and reducing blood viscosity. In the preparation process of double protein polypeptide soy milk, its nutritional value mainly depends on the type and content of polypeptides in soy milk. Most commercial soy milk is prepared by directly grinding soaked soybeans into pulp. Soy protein still maintains a compact structure because there is no enzymatic hydrolysis process. Therefore, edible soy protein cannot obtain polypeptides with high nutritional value.

[0005] Soybean seeds themselves do not have a beany smell, but soy milk has a beany smell. Studies have found that the generation of beany smell is closely related to a series of chemical reactions caused by lipoxygenase (LOX) and other highly oxidative substances during the production process, mainly including LOX-induced enzymatic oxidation reactions, non-enzymatic oxidation reactions and other unknown catalytic reactions. More than half of the lipid content of soybeans is linoleic acid and linolenic acid. The beany smell formed during the pulping process is closely related to these two unsaturated fatty acids. After the soybeans soaked at room temperature are ground, the cells rupture and the lipoxygenase present in the cotyledons under the bean skin and isolated from the lipids is released. After the two come into contact, they rapidly undergo oxidation reactions under the action of oxygen and temperature in the air, producing beany smell substances.

[0006] A Chinese patent (publication number CN108634236A, published on October 12, 2018) discloses "a method for preparing an ultra-low-fat active squid-soybean mixed peptide oral liquid", which uses supercritical CO2 extraction, subcritical water extraction and ethanol to extract squid alcohol-soluble protein and soybean protein, while removing lipids and fishy substances in squid and soybeans, and obtaining functional peptides from squid and soybeans. However, the protein extraction in this invention uses supercritical CO2 extraction and subcritical water extraction methods, which have high equipment requirements and high production costs, and are not conducive to industrial production. Therefore, it is urgent to develop a method for preparing dual-protein polypeptide soymilk with low fishy smell and low production cost. Summary of the invention

[0007] Therefore, the technical problem to be solved by the present invention is to overcome the problems of the prior art in the preparation method of a double-protein polypeptide soy milk with low fishy smell and low production cost.

[0008] In order to solve the above technical problems, the present invention provides a double-protein polypeptide soymilk and a preparation method thereof. The present invention prepares a double-protein polypeptide soymilk with low beany smell and high polypeptide content by controlling the sprout length of soybean germination and the temperature, pH and other conditions during the preparation process.

[0009] Soybean seeds are living organisms with inherent enzyme systems such as proteases, amylases and cellulases. A series of physiological and biochemical changes will occur during the germination of soybeans. Endogenous enzymes are activated and gradually hydrolyze the soybean's own storage substances such as protein, starch and fat, and convert them into forms that are easily absorbed and utilized by plants, providing nutrition for the growth of embryos. Among them, soybean endogenous enzymes are divided into endopeptidases and exopeptidases. During the germination of soybeans, part of the activated protease is obtained by the activation of the enzyme source, and the other part is converted from proteins synthesized by biochemical reactions. In the early stage of germination, enzymes such as serine endopeptidase are highly active, and then enzymes such as cysteine ​​endopeptidase are activated. Exopeptidases such as tripeptidyl peptidase and aminopeptidase, as well as enzymes such as serine carboxypeptidase, glutamate decarboxylase, and aspartate aminopeptidase also play a role in the hydrolysis of soybean protein. Since soybean endogenous proteases are a series of enzymes, the pH when hydrolyzing proteins ranges from 3.5 to 9.0. Some enzymes are most active under acidic conditions, and other enzymes work under alkaline conditions. Based on the above principle, in the present invention, when the soybean endogenous enzyme acts on the double protein homogenate, the pH is successively set to the acidic and alkaline ranges to give full play to the effect of the entire soybean endogenous enzyme system.

[0010] At the same time, the present invention, by regulating the enzymolysis conditions, improves the activity of soybean endogenous protease while passivating the activity of lipoxygenase, so that the obtained double-protein soy milk has a high polypeptide content and a light beany smell. Specifically, by subjecting the germinated soybeans to low-temperature stress and thawing and warming, the activity of endogenous proteases is stimulated and improved, and the structure of soybean protein is loosened to facilitate enzymolysis. Then, an instantaneous high-temperature heat shock is performed to passivate the lipoxygenase, while the soybean endogenous enzyme is still active. After adding animal protein, the enzymolysis time and temperature of the double-protein soy milk are continuously adjusted so that the soybean endogenous enzyme simultaneously catalyzes the hydrolysis of soybean protein and milk protein into polypeptides. Therefore, the double-protein polypeptide soy milk of the present invention has a light beany smell and is rich in polypeptides, and has a high nutritional value.

[0011] The first object of the present invention is to provide a method for preparing double-protein polypeptide soymilk, comprising the following steps:

[0012] S1, germinating soybeans in the dark to obtain germinated soybeans;

[0013] S2, subjecting the germinated soybeans to low temperature stress;

[0014] S3, thawing and warming the germinated soybeans after low temperature stress;

[0015] S4, subjecting the thawed and warmed sprouted soybeans to high temperature heat shock;

[0016] S5, pulping the germinated soybeans after high temperature heat shock, and mixing with animal milk to obtain a double protein homogenate;

[0017] S6, heat-insulating the double protein homogenate, and adding a stabilizer to obtain the double protein polypeptide soy milk;

[0018] Wherein, the sprout length of the sprouted soybean in step S1 is 4-6 cm.

[0019] Furthermore, the temperature for germination in the dark in step S1 is 25-30°C.

[0020] Furthermore, the temperature of the low temperature stress in step S2 is -12 to -4°C, and the time of the low temperature stress is 7-10 hours.

[0021] Furthermore, in step S3, the temperature of thawing and warming the sprouted soybeans is 25-30°C, and the warming time is 5-6 hours. The sprouted soybeans are first subjected to low temperature stress and then thawed and warmed, which can stimulate and increase the activity of soybean endogenous enzymes and make the soybean protein structure loose for enzymatic hydrolysis.

[0022] Furthermore, the high temperature heat shock in step S4 is to raise the temperature of the thawed and warmed sprouted soybeans to 85-90° C. and maintain it for 3-6 minutes to inactivate the lipoxygenase.

[0023] Furthermore, the pulping in step S5 is to add water to the germinated soybeans after high-temperature heat shock for grinding, and the amount of water added is 7-10 times the volume of the germinated soybeans to obtain soy milk.

[0024] Furthermore, in step S5, the volume ratio of the soy milk to the animal milk is (3-5):1.

[0025] Furthermore, the animal milk in step S5 includes cow's milk.

[0026] Furthermore, the temperature of the heat preservation treatment in step S6 is 35-50°C.

[0027] Furthermore, the heat preservation treatment in step S6 is divided into two steps:

[0028] (1) adjusting the pH of the double protein homogenate to 3.5-5.0 and performing a heat preservation treatment, wherein the heat preservation treatment time is 60-90 minutes;

[0029] (2) The pH of the double protein homogenate is adjusted to 8-9.5 and a second heat preservation treatment is performed. The second heat preservation treatment time is 70-100 minutes.

[0030] Furthermore, the heat preservation treatment in step S6 is supplemented by intermittent ultrasonic treatment, wherein the ultrasonic power of the intermittent ultrasonic treatment is 750-850W, the frequency is 40-50kHz, and the intermittent ratio is (2-3): 1, that is, ultrasonic treatment for 4 minutes, stop for 2 minutes, and automatically cycle back and forth. During the enzymolysis process, the use of ultrasonic treatment can fully expose the active center of the enzyme, which is conducive to full contact between the enzyme and the substrate, and can accelerate the enzymatic reaction rate.

[0031] The second object of the present invention is to provide a double-protein polypeptide soymilk prepared by the above preparation method.

[0032] Beneficial effects of the present invention:

[0033] (1) The present invention utilizes the plant endogenous protease system activated and formed by soybean germination to hydrolyze protein under two pH conditions, first acid and then alkaline, to give full play to the role of the plant endogenous protease system and obtain more peptide substances.

[0034] (2) The present invention subjects the germinated soybeans to low temperature stress and thaws to warming, thereby stimulating and further improving the activity of endogenous enzymes and making the soybean protein structure loose, thereby facilitating enzymatic hydrolysis.

[0035] (3) After adding animal milk to soy milk, the present invention adjusts the enzymatic hydrolysis time and temperature of the double-protein soy milk so that the endogenous enzyme of soybean catalyzes the soy protein and the animal protein at the same time, and hydrolyzes the soy protein into a double-protein polypeptide with a small molecular weight.

[0036] (4) In the process of enzymatic hydrolysis of double-protein soymilk of the present invention, enzymatic hydrolysis is assisted by intermittent ultrasonic treatment, so that the active center of the protease is fully exposed, which is conducive to full contact with the protein action site and improves the enzymatic reaction speed.

[0037] (5) The present invention reduces the activity of lipoxygenase while increasing the activity of endogenous soybean enzymes by controlling high-temperature heat shock and enzymatic hydrolysis conditions. The obtained dual-protein polypeptide soymilk has a light bean smell, a high polypeptide content, and has antioxidant and ACE inhibitory properties. The preparation process is simple and low-cost, and has good production prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0039] Figure 1 The present invention is a flow chart of the method for preparing double-protein polypeptide soymilk. DETAILED DESCRIPTION

[0040] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0041] Example 1

[0042] (1) Soybean germination: Select soybean seeds with high germination rate harvested in the current year as raw materials for germination, and remove impurities and insect eggs by air separation; wash the soybeans with running water, place the soybeans in a container and soak them in water, keep the water temperature between 25-30°C, soak for 4-6 hours, and change the water once in the middle; place the soaked soybeans in a germination barrel, cover the barrel mouth with a layer of wet cloth, and germinate at 25-30°C in the dark for 24-36 hours, watering every 4-6 hours until the soybean sprouts are 4-6 cm long.

[0043] (2) Low temperature stress: The germinated soybeans were frozen at -10°C for 8 hours.

[0044] (3) Thawing and warming: The germinated soybeans subjected to low temperature stress were placed in the air to thaw and warm to 27°C for 5 hours.

[0045] (4) High temperature heat shock: The thawed and warmed sprouted soybeans were instantly heated to 87°C for 5 minutes to inactivate the activity of lipoxygenase.

[0046] (5) Pulping and mixing: Add water to the germinated soybeans after high-temperature heat shock and grind them into pulp. The amount of water added is 9 times the volume of the germinated soybeans to obtain soy milk. Add milk to the soy milk. The volume ratio of soy milk to milk is 4:1. Mix them in a blender to obtain a double protein homogenate.

[0047] (6) Insulation treatment: After the double protein homogenate was heated to 45°C, the pH was adjusted to 4.0 with citric acid-sodium citrate buffer, and the protein in the homogenate was hydrolyzed by endogenous soybean protease for 70 min. Then, the pH of the reaction solution was adjusted to 8.5 with sodium bicarbonate-sodium carbonate buffer, and the hydrolysis was continued for 90 min. During the insulation treatment, intermittent ultrasonic treatment was used with an ultrasonic power of 800 W, a frequency of 45 kHz, and an intermittent ratio of 2:1, that is, ultrasonic treatment for 4 min, stop for 2 min, and automatic reciprocating.

[0048] A stabilizer is added to the double-protein slurry after enzymatic hydrolysis, and after homogenization and sterilization, the double-protein polypeptide soymilk of this embodiment is obtained.

[0049] Example 2

[0050] (1) Soybean germination: Select soybean seeds with high germination rate harvested in the current year as raw materials for germination, and remove impurities and insect eggs by air separation; wash the soybeans with running water, place the soybeans in a container and soak them in water, keep the water temperature between 25-30°C, soak for 4-6 hours, and change the water once in the middle; place the soaked soybeans in a germination barrel, cover the barrel mouth with a layer of wet cloth, and germinate at 25-30°C in the dark for 24-36 hours, watering every 4-6 hours until the soybean sprouts are 4-6 cm long.

[0051] (2) Low temperature stress: The germinated soybeans were frozen at -12°C for 8 hours.

[0052] (3) Thawing and warming: The germinated soybeans subjected to low temperature stress were placed in the air to thaw and warm to 25°C for 5 h.

[0053] (4) High temperature heat shock: The thawed and warmed sprouted soybeans were instantly heated to 85°C for 3 minutes to inactivate the activity of lipoxygenase.

[0054] (5) Pulping and mixing: Add water to the germinated soybeans after high-temperature heat shock and grind them into pulp. The amount of water added is 9 times the volume of the germinated soybeans to obtain soy milk. Add milk to the soy milk. The volume ratio of soy milk to milk is 3:1. Mix them in a blender to obtain a double protein slurry.

[0055] (6) Insulation treatment: After the double protein homogenate was heated to 35°C, the pH was adjusted to 3.5 with citric acid-sodium citrate buffer, and the protein in the double protein homogenate was hydrolyzed by endogenous soybean protease for 60 min. Then, the pH of the reaction solution was adjusted to 8 with sodium bicarbonate-sodium carbonate buffer, and the hydrolysis was continued for 70 min. During the insulation treatment, intermittent ultrasonic treatment was used with an ultrasonic power of 800 W, a frequency of 45 kHz, and an intermittent ratio of 2:1, that is, ultrasonic treatment for 4 min, stop for 2 min, and automatic reciprocating.

[0056] A stabilizer is added to the double-protein slurry after enzymatic hydrolysis, and after homogenization and sterilization, the double-protein polypeptide soymilk of this embodiment is obtained.

[0057] Example 3

[0058] (1) Soybean germination: Select soybean seeds with high germination rate harvested in the current year as raw materials for germination, and remove impurities and insect eggs by air separation; wash the soybeans with running water, place the soybeans in a container and soak them in water, keep the water temperature between 25-30°C, soak for 4-6 hours, and change the water once in the middle; place the soaked soybeans in a germination barrel, cover the barrel mouth with a layer of wet cloth, and germinate at 25-30°C in the dark for 24-36 hours, watering every 4-6 hours until the soybean sprouts are 4-6 cm long.

[0059] (2) Low temperature stress: The germinated soybeans were frozen at -4°C for 10 hours.

[0060] (3) Thawing and warming: The germinated soybeans subjected to low temperature stress were placed in the air to thaw and warm to 30°C for 5 h.

[0061] (4) High temperature heat shock: The thawed and warmed sprouted soybeans were instantly heated to 90°C for 6 minutes to inactivate the activity of lipoxygenase.

[0062] (5) Pulping and mixing: Add water to the germinated soybeans after high-temperature heat shock and grind them into pulp. The amount of water added is 9 times the volume of the germinated soybeans to obtain soy milk. Add milk to the soy milk. The volume ratio of soy milk to milk is 5:1. Mix them in a blender to obtain a double protein slurry.

[0063] (6) Insulation treatment: After the double protein homogenate was heated to 50°C, the pH was adjusted to 5 with citric acid-sodium citrate buffer, and the protein in the homogenate was hydrolyzed by endogenous soybean protease for 90 min. Then, the pH of the reaction solution was adjusted to 9.5 with sodium bicarbonate-sodium carbonate buffer, and the hydrolysis was continued for 100 min. During the insulation treatment, intermittent ultrasonic treatment was used with an ultrasonic power of 800 W, a frequency of 45 kHz, and an intermittent ratio of 2:1, that is, ultrasonic treatment for 4 min, stop for 2 min, and automatic reciprocating.

[0064] A stabilizer is added to the double-protein slurry after enzymatic hydrolysis, and after homogenization and sterilization, the double-protein polypeptide soymilk of this embodiment is obtained.

[0065] Comparative Example 1

[0066] The heat preservation treatment in this comparative example is to use soybean endogenous protease to enzymatically hydrolyze the protein in the homogenate under acidic conditions, and the remaining steps are the same as those in Example 1. The specific operation is as follows:

[0067] (1) Soybean germination: Select soybean seeds with high germination rate harvested in the current year as raw materials for germination, and remove impurities and insect eggs by air separation; wash the soybeans with running water, place the soybeans in a container and soak them in water, keep the water temperature between 25-30°C, soak for 4-6 hours, and change the water once in the middle; place the soaked soybeans in a germination barrel, cover the barrel mouth with a layer of wet cloth, and germinate at 25-30°C in the dark for 24-36 hours, watering every 4-6 hours until the soybean sprouts are 4-6 cm long.

[0068] (2) Low temperature stress: The germinated soybeans were frozen at -10°C for 8 hours.

[0069] (3) Thawing and warming: The germinated soybeans subjected to low temperature stress were placed in the air to thaw and warm to 27°C for 5 hours.

[0070] (4) High temperature heat shock: The thawed and warmed-up sprouted soybeans were instantly heated to 90°C for 5 minutes to inactivate the activity of lipoxygenase.

[0071] (5) Pulping and mixing: Add water to the germinated soybeans after high-temperature heat shock to grind the soybeans, and the amount of water added is 9 times the volume of the germinated soybeans to obtain soy milk; add milk to the soy milk, and the volume ratio of soy milk to milk is 4.5:1. Stir and mix in a blender to obtain a double protein homogenate.

[0072] (6) Insulation treatment: After the double protein homogenate is heated to 45° C., the pH is adjusted to 4.0 with citric acid-sodium citrate buffer, and the protein in the homogenate is hydrolyzed by endogenous soybean protease for 160 min. During the insulation treatment, intermittent ultrasonic treatment is performed with an ultrasonic power of 800 W and a frequency of 45 kHz. The intermittent ratio and intermittent cycle time are the same as those in Example 1.

[0073] A stabilizer was added to the double protein slurry after enzymatic hydrolysis, and the double protein polypeptide soymilk of this comparative example was prepared after homogenization and sterilization.

[0074] Comparative Example 2

[0075] The heat preservation treatment in this embodiment is to use soybean endogenous protease to enzymatically hydrolyze the protein in the homogenate under alkaline conditions. The remaining steps are the same as those in Example 1. The specific operations are as follows:

[0076] (1) Soybean germination: Select soybean seeds with high germination rate harvested in the current year as raw materials for germination, and remove impurities and insect eggs by air separation; wash the soybeans with running water, place the soybeans in a container and soak them in water, keep the water temperature between 25-30°C, soak for 4-6 hours, and change the water once in the middle; place the soaked soybeans in a germination barrel, cover the barrel mouth with a layer of wet cloth, and germinate at 25-30°C in the dark for 24-36 hours, watering every 4-6 hours until the soybean sprouts are 4-6 cm long.

[0077] (2) Low temperature stress: The germinated soybeans were frozen at -10°C for 8 hours.

[0078] (3) Thawing and warming: The germinated soybeans subjected to low temperature stress were placed in the air to thaw and warm to 27°C for 5 hours.

[0079] (4) High temperature heat shock: The thawed and warmed-up sprouted soybeans were instantly heated to 90°C for 5 minutes to inactivate the activity of lipoxygenase.

[0080] (5) Pulping and mixing: Add water to the germinated soybeans after high-temperature heat shock to grind the soybeans, and the amount of water added is 9 times the volume of the germinated soybeans to obtain soy milk; add milk to the soy milk, and the volume ratio of soy milk to milk is 4.5:1. Stir and mix in a blender to obtain a double protein homogenate.

[0081] (6) Insulation treatment: After the double protein homogenate was heated to 45° C., the pH of the reaction solution was adjusted to 8.5 with sodium bicarbonate-sodium carbonate buffer, and the protein in the homogenate was hydrolyzed by endogenous soybean protease for 160 min. During the insulation treatment, intermittent ultrasonic treatment was performed with an ultrasonic power of 800 W and a frequency of 45 kHz. The intermittent ratio and intermittent cycle time were the same as those in Example 1.

[0082] A stabilizer was added to the double protein slurry after enzymatic hydrolysis, and the double protein polypeptide soymilk of this comparative example was prepared after homogenization and sterilization.

[0083] Comparative Example 3

[0084] In this comparative example, only germinated soybean pulp is used, and no milk is added. The remaining steps are the same as those in Example 1. The specific operations are as follows:

[0085] (1) Soybean germination: Select soybean seeds with high germination rate harvested in the current year as raw materials for germination, and remove impurities and insect eggs by air separation; wash the soybeans with running water, place the soybeans in a container and soak them in water, keep the water temperature between 25-30°C, soak for 4-6 hours, and change the water once in the middle; place the soaked soybeans in a germination barrel, cover the barrel mouth with a layer of wet cloth, and germinate at 25-30°C in the dark for 24-36 hours, watering every 4-6 hours until the soybean sprouts are 4-6 cm long.

[0086] (2) Low temperature stress: The germinated soybeans were frozen at -10°C for 8 hours.

[0087] (3) Thawing and warming: The germinated soybeans subjected to low temperature stress were placed in the air to thaw and warm to 27°C for 5 hours.

[0088] (4) High temperature heat shock: The thawed and warmed-up sprouted soybeans were instantly heated to 90°C for 5 minutes to inactivate the activity of lipoxygenase.

[0089] (5) Pulping: Add water to the germinated soybeans after high temperature heat shock and grind them into pulp. The amount of water added is 9 times the volume of the germinated soybeans to produce soy milk.

[0090] (6) Heat preservation treatment: After the soy milk is heated to 45° C., the pH value is adjusted to 5 with citric acid-sodium citrate buffer, and the protein in the soy milk is hydrolyzed by endogenous soybean protease for 90 min. Then, the pH value of the reaction solution is adjusted to 9.5 with sodium bicarbonate-sodium carbonate buffer, and the hydrolysis is continued for 100 min. During the heat preservation treatment, intermittent ultrasonic treatment is performed, with an ultrasonic power of 800 W and a frequency of 45 kHz. The intermittent ratio and intermittent cycle time are the same as those in Example 1.

[0091] A stabilizer is added to the enzymatically hydrolyzed soybean milk slurry, and the soybean milk of this comparative example is prepared after homogenization and sterilization.

[0092] Comparative Example 4

[0093] Compared with Example 1, the only difference is that the high temperature heat shock step is omitted in this comparative example, and the remaining operations are the same as those in Example 1. The specific operations are as follows:

[0094] (1) Soybean germination: Select soybean seeds with high germination rate harvested in the current year as raw materials for germination, and remove impurities and insect eggs by air separation; wash the soybeans with running water, place the soybeans in a container and soak them in water, keep the water temperature between 25-30°C, soak for 4-6 hours, and change the water once in the middle; place the soaked soybeans in a germination barrel, cover the barrel mouth with a layer of wet cloth, and germinate at 25-30°C in the dark for 24-36 hours, watering every 4-6 hours until the soybean sprouts are 4-6 cm long.

[0095] (2) Low temperature stress: The germinated soybeans were frozen at -10°C for 8 hours.

[0096] (3) Thawing and warming: The germinated soybeans subjected to low temperature stress were placed in the air to thaw and warm to 27°C for 5 hours.

[0097] (4) Pulping and mixing: Add water to the germinated soybeans after high-temperature heat shock to grind the soybeans, and the amount of water added is 9 times the volume of the germinated soybeans to obtain soy milk; add milk to the soy milk, and the volume ratio of soy milk to milk is 4.5:1. Stir and mix in a blender to obtain a double protein homogenate.

[0098] (5) Insulation treatment: After the double protein homogenate was heated to 45° C., the pH value was adjusted to 5 with citric acid-sodium citrate buffer, and the protein in the homogenate was hydrolyzed by endogenous soybean protease for 90 min. Then, the pH value of the reaction solution was adjusted to 9.5 with sodium bicarbonate-sodium carbonate buffer, and the hydrolysis was continued for 100 min. During the insulation treatment, intermittent ultrasonic treatment was performed, with an ultrasonic power of 800 W and a frequency of 45 kHz. The intermittent ratio and intermittent cycle time were the same as those in Example 1.

[0099] A stabilizer was added to the double-protein slurry after enzymatic hydrolysis, and the double-protein polypeptide soymilk of this comparative example was prepared after homogenization and sterilization.

[0100] Comparative Example 5

[0101] Compared with Example 1, the only difference is that the heat preservation treatment step is omitted in this comparative example, and the remaining operations are the same as those in Example 1. The specific operations are as follows:

[0102] (1) Soybean germination: Select soybean seeds with high germination rate harvested in the current year as raw materials for germination, and remove impurities and insect eggs by air separation; wash the soybeans with running water, place the soybeans in a container and soak them in water, keep the water temperature between 25-30°C, soak for 4-6 hours, and change the water once in the middle; place the soaked soybeans in a germination barrel, cover the barrel mouth with a layer of wet cloth, and germinate at 25-30°C in the dark for 24-36 hours, watering every 4-6 hours until the soybean sprouts are 4-6 cm long.

[0103] (2) Low temperature stress: The germinated soybeans were frozen at -10°C for 8 hours.

[0104] (3) Thawing and warming: The germinated soybeans subjected to low temperature stress were placed in the air to thaw and warm to 27°C for 5 hours.

[0105] (4) High temperature heat shock: The thawed and warmed-up sprouted soybeans were instantly heated to 90°C for 5 minutes to inactivate the activity of lipoxygenase.

[0106] (5) Pulping and mixing: Add water to the germinated soybeans after high-temperature heat shock to grind the soybeans, and the amount of water added is 9 times the volume of the germinated soybeans to obtain soy milk; add milk to the soy milk, and the volume ratio of soy milk to milk is 4.5:1. Stir and mix in a blender to obtain a double protein homogenate.

[0107] A stabilizer was added to the double protein homogenate, and after homogenization and sterilization, the double protein polypeptide soymilk of this comparative example was obtained.

[0108] Comparative Example 6

[0109] Compared with Example 1, the only difference is that the sprout length of the sprouted soybean in this comparative example is 1-2 cm, and the rest of the operations are the same as those in Example 1. The specific operations are as follows:

[0110] (1) Soybean germination: Select soybean seeds with high germination rate harvested in the current year as raw materials for germination, and remove impurities and insect eggs by air separation; wash the soybeans with running water, place the soybeans in a container and soak them in water, keep the water temperature between 25-30°C, soak for 4-6 hours, and change the water once in the middle; place the soaked soybeans in a germination barrel, cover the barrel mouth with a layer of wet cloth, and germinate at 25-30°C in the dark for 6-10 hours, and water them every 4-6 hours until the soybean sprouts are 1-2 cm long.

[0111] (2) Low temperature stress: The germinated soybeans were frozen at -10°C for 8 hours.

[0112] (3) Thawing and warming: The germinated soybeans subjected to low temperature stress were placed in the air to thaw and warm to 27°C for 5 hours.

[0113] (4) High temperature heat shock: The thawed and warmed-up sprouted soybeans were instantly heated to 90°C for 5 minutes to inactivate the activity of lipoxygenase.

[0114] (5) Pulping and mixing: Add water to the germinated soybeans after high-temperature heat shock to grind the soybeans, and the amount of water added is 9 times the volume of the germinated soybeans to obtain soy milk; add milk to the soy milk, and the volume ratio of soy milk to milk is 4.5:1. Stir and mix in a blender to obtain a double protein homogenate.

[0115] (6) Heat preservation treatment: After the double protein homogenate was heated to 45°C, the pH value was adjusted to 5 with citric acid-sodium citrate buffer, and the protein in the double protein homogenate was hydrolyzed by endogenous soybean protease for 90 min. Then, the pH value of the reaction solution was adjusted to 9.5 with sodium bicarbonate-sodium carbonate buffer, and the hydrolysis was continued for 100 min. During the heat preservation treatment, intermittent ultrasonic treatment was performed, with an ultrasonic power of 800 W and a frequency of 45 kHz. The intermittent ratio and intermittent cycle time were the same as those in Example 1.

[0116] A stabilizer was added to the double-protein homogenate after enzymatic hydrolysis, and the double-protein polypeptide soymilk of this comparative example was obtained after homogenization and sterilization.

[0117] Comparative Example 7

[0118] Compared with Example 1, the only difference is that the sprout length of the sprouted soybean in this comparative example is 8-12 cm, and the rest of the operations are the same as those in Example 1. The specific operations are as follows:

[0119] (1) Soybean germination: Select soybean seeds with high germination rate harvested in the current year as raw materials for germination, and remove impurities and insect eggs by air separation; wash the soybeans with running water, place the soybeans in a container and soak them in water, keep the water temperature between 25-30°C, soak for 4-6 hours, and change the water once in the middle; place the soaked soybeans in a germination barrel, cover the barrel mouth with a layer of wet cloth, and germinate at 25-30°C in the dark for 48-60 hours, watering every 4-6 hours until the soybean sprouts are 8-12 cm long.

[0120] (2) Low temperature stress: The germinated soybeans were frozen at -10°C for 8 hours.

[0121] (3) Thawing and warming: The germinated soybeans subjected to low temperature stress were placed in the air to thaw and warm to 27°C for 5 hours.

[0122] (4) High temperature heat shock: The thawed and warmed-up sprouted soybeans were instantly heated to 90°C for 5 minutes to inactivate the activity of lipoxygenase.

[0123] (5) Pulping and mixing: Add water to the germinated soybeans after high-temperature heat shock to grind the soybeans, and the amount of water added is 9 times the volume of the germinated soybeans to obtain soy milk; add milk to the soy milk, and the volume ratio of soy milk to milk is 4.5:1. Stir and mix in a blender to obtain a double protein homogenate.

[0124] (6) Heat preservation treatment: After the double protein homogenate was heated to 45°C, the pH value was adjusted to 5 with citric acid-sodium citrate buffer, and the protein in the double protein homogenate was hydrolyzed by endogenous soybean protease for 90 min. Then, the pH value of the reaction solution was adjusted to 9.5 with sodium bicarbonate-sodium carbonate buffer, and the hydrolysis was continued for 100 min. During the heat preservation treatment, intermittent ultrasonic treatment was performed, with an ultrasonic power of 800 W and a frequency of 45 kHz. The intermittent ratio and intermittent cycle time were the same as those in Example 1.

[0125] A stabilizer was added to the double-protein homogenate after enzymatic hydrolysis, and the double-protein polypeptide soymilk of this comparative example was obtained after homogenization and sterilization.

[0126] Test Case

[0127] (I) Sensory evaluation

[0128] The dual protein polypeptide soymilk obtained in each example and comparative example was evaluated based on sensory indicators (overall evaluation score is 10 points). The results are shown in Table 1. The dual protein polypeptide soymilk obtained in Examples 1-3 has uniform color and obvious bean flavor, slight bean smell and no precipitation, so it can be expanded for production.

[0129] Table 1 Sensory quality evaluation results of dual protein polypeptide soymilk

[0130]

[0131] (II) Determination of polypeptide content and performance

[0132] 1. Peptide content determination

[0133] The Folin-phenol method was used for determination, and bovine serum albumin was used as the standard protein for the standard curve.

[0134] Peptide extraction method: sample addition concentration is 0.5 mol.L -1 , pH 5.0 phosphate-citrate buffer, the ratio of sample to extract was 1:50 (W:V), and the extraction was carried out at 25°C and 100 r / min for 2 h. Phosphotungstic acid solution was added to make the concentration of phosphotungstic acid reach 1.5% to precipitate large molecular proteins. The mixture was centrifuged at 5000 r / min for 15 min, and the supernatant was filtered with medium-speed quantitative filter paper. The filtrate was used to determine the peptide content.

[0135] 2. Antioxidant Determination

[0136] The antioxidant activity was determined by the DPPH method, and the specific operation was as follows:

[0137] The extract was prepared into sample solutions with different concentration mass gradients (0.05±0.5 mg / mL), 0.4 mL of sample solution and 4.5 mL of 0.1 mmol / L DPPH methanol solution (Abs=1.00±0.02) were aspirated, and the mixture was shaken thoroughly and reacted in the dark for 30 min. The absorbance was measured at a wavelength of 517 nm, and the absorbance of vitamin C was used as a control.

[0138] The scavenging rate of DPPH free radicals was calculated according to the following formula:

[0139]

[0140] Where: A0 represents the absorbance value of the control group;

[0141] A1 represents the absorbance value of the sample group.

[0142] 3. In vitro ACE inhibitory activity assay

[0143] The ACE inhibitory activity of the peptide was measured by enzyme labeling. 1mmol / L FAPGG solution, 0.1U / mL ACE solution and 0.5mmol / L peptide solution (P1, P2, P3, P4) were prepared in 80mmol / L HEPES buffer (containing 300mmol / L NaCl, H8.2). Then 40μL 1mmol / L FAPGG solution, 50μL 0.5mmol / L peptide solution, 10μL 0.1U / mL ACE solution and 100μL HEPES buffer were added to the detection wells, and HEPES buffer was used instead of the peptide solution as a blank control. After incubation at 37°C for 5min, the absorbance of the sample well (A1) and the blank well (B1) were measured at a wavelength of 340nm. After the test, the absorbance of the sample well (A2) and the blank well (B2) were measured again after incubation at 37°C for 30min, and the ACE inhibition rate was calculated according to the following formula:

[0144] ACE inhibition rate (%) = (△B-△A) / △B-100

[0145] Among them, △A=A1-A2, △B=B1-B2.

[0146] The results are shown in Table 2. After measurement, the double protein polypeptide soy milk of Examples 1-3 has a high polypeptide content, and has antioxidant and ACE inhibitory properties, and has good health care effects. The polypeptide content of Comparative Examples 1-7 is relatively low. Comparative Examples 1, 2, and 5 all changed the steps of heat preservation treatment, resulting in less polypeptide content. It is speculated that it may be caused by insufficient enzymatic hydrolysis of soybean protein and animal protein during the heat preservation treatment. The polypeptide content of Comparative Examples 6 and 7 is also less, which shows that there is a large difference in the content of soybean endogenous enzymes when the soybean sprouts to different bud lengths during the germination process, and there are also significant differences in the enzymatic hydrolysis effects of soybean protein and animal protein.

[0147] Table 2 Results of determination of polypeptide content and performance in double protein polypeptide soymilk

[0148] Group Peptide content (mg / mL) DPPH clearance rate (%) ACE inhibition rate (%) Example 1 451 73 82 Example 2 398 69 78 Example 3 409 65 81 Comparative Example 1 56 35 34 Comparative Example 2 84 28 49 Comparative Example 3 92 23 25 Comparative Example 4 289 57 65 Comparative Example 5 95 12 53 Comparative Example 6 75 34 42 Comparative Example 7 109 25 39

[0149] (III) Determination of the content of the main substances producing beany smell

[0150] Determination of volatile substances: 10 mL of the test solution was taken and placed in a 20 mL headspace bottle, 10 μL of internal standard benzyl alcohol (0.1 g dissolved in 10 mL methanol, final mass concentration 10 mg / L) was added, the bottle mouth was sealed, the extraction head was inserted, and the extraction was carried out in a 40 ° C water bath for 40 min, and the extraction head was about 1 cm away from the liquid surface. Subsequently, thermal desorption was performed at the GC injection port for 5 min (250 ° C) for GC-MS analysis. Chromatographic conditions: Chromatographic column: (DB-WAX, 30m×0.25mm, 0.25 μm), injection port temperature: 230 ° C, program temperature rise: 40 ° C, hold for 3 min, 6 ° C / min to 100 ° C, then 10 ° C / min to 230 ° C, hold for 7 min, injection mode: splitless injection (1.0 mL / min), carrier gas: helium (99.999%). Mass spectrometry conditions: interface temperature: 250°C, ion source temperature: 200°C, ionization mode: EI, electron energy: 70 eV, scanning mass range m / z: 29-500.

[0151] After the samples were analyzed by GC-MS, the mass spectra obtained were automatically retrieved from the random Xcalibμr workstation NIST 2002 standard spectral library to retrieve the mass spectrum data of each component. At the same time, the literature was consulted to conduct a qualitative analysis of the components of each volatile aroma substance. The volatile components were quantitatively analyzed (mg / L) by the internal standard method. The results are shown in Table 3. The contents of the main substances producing beany smell in Comparative Examples 4, 6, and 7 were all relatively high, and the beany smell of Comparative Examples 4, 6, and 7 was heavier, indicating that the bud length and high temperature heat shock would affect the activity of lipoxygenase.

[0152] Table 3 Bean smell test results of double protein polypeptide soy milk

[0153]

[0154] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A method for preparing double-protein polypeptide soymilk, characterized in that: The following steps are involved: S1, germinating soybeans in the dark to obtain germinated soybeans; S2, subjecting the germinated soybeans to low temperature stress; S3, thawing and warming the germinated soybeans after low temperature stress; S4, subjecting the thawed and warmed sprouted soybeans to high temperature heat shock; S5, pulping the germinated soybeans after high temperature heat shock, and mixing with animal milk to obtain a double protein homogenate; S6, heat-insulating the double protein homogenate, and adding a stabilizer to obtain the double protein polypeptide soy milk; Wherein, the sprout length of the sprouted soybean in step S1 is 4-6 cm.

2. The preparation method according to claim 1, characterized in that: The temperature for germination in the dark in step S1 is 25-30°C.

3. The preparation method according to claim 1, characterized in that: The temperature of the low temperature stress in step S2 is -12 to -4°C, and the time of the low temperature stress is 7 to 10 hours.

4. The preparation method according to claim 1, characterized in that: The thawing and warming-up temperature in step S3 is 25-30° C., and the warming-up time is 5-6 hours.

5. The preparation method according to claim 1, characterized in that: The high temperature heat shock in step S4 is to raise the temperature of the thawed and warmed sprouted soybeans to 85-90° C. and maintain it for 3-6 minutes.

6. The preparation method according to claim 1, characterized in that: The pulping in step S5 is to add water to the germinated soybeans after high-temperature heat shock for grinding, and the amount of water added is 7-10 times the volume of the germinated soybeans to obtain soy milk.

7. The preparation method according to claim 6, characterized in that: The volume ratio of the soy milk to the animal milk is (3-5):1; the animal milk includes cow's milk.

8. The preparation method according to claim 1, characterized in that: The heat preservation treatment in step S6 is divided into two steps: (1) adjusting the pH of the double protein homogenate to 3.5-5.0 with citric acid-sodium citrate buffer at 35-50° C. and performing a heat treatment once, wherein the heat treatment time is 60-90 min; (2) adjusting the pH of the double protein homogenate to 8-9.5 with sodium bicarbonate-sodium carbonate buffer at 35-50° C. and performing a secondary heat preservation treatment for 70-100 min.

9. The preparation method according to claim 1, characterized in that: During the heat preservation treatment in step S6, intermittent ultrasonic treatment is performed, wherein the ultrasonic power of the intermittent ultrasonic treatment is 750-850W, the frequency is 40-50kHz, and the intermittent ratio is (2-3):

1.

10. Double-protein polypeptide soymilk prepared by the preparation method according to any one of claims 1 to 9.

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