A method for preparing barley alcohol-soluble protein particles and a method for preparing an oil-in-water emulsion.

Abstract

This invention provides barley prolysin particles and their preparation method, as well as a method for preparing an oil-in-water emulsion, belonging to the field of food science and engineering technology. This invention employs proline endonuclease to enzymatically hydrolyze barley prolysin at a pH of 5.5-5.8, breaking it down into small-molecule barley prolysin polypeptides, thus reducing the size of the particles. Calcium ions in calcium chloride aqueous solution form coordination bonds with negatively charged groups such as carboxyl and hydroxyl groups on the barley prolysin polypeptide chain, regulating the charge distribution on the surface of the barley prolysin molecules and improving the dispersibility of the particles in water. Furthermore, the cycloalkenyl ether ketone active group in genipin molecules forms stable Schiff base covalent bonds with the free primary amino groups on the barley prolysin polypeptide chain, constructing a stable three-dimensional network structure and improving the thermal stability of the emulsion obtained from the barley prolysin particles.

Description

Technical Field

[0001] This invention relates to the fields of food science and engineering technology, and in particular to a barley prolysin granule and its preparation method, and a method for preparing an oil-in-water emulsion. Background Technology

[0002] In the food industry, the vast majority of foods (such as salad dressings, plant-based milk, vegetable shortening, meat products, and dairy beverages) are oil-water mixtures. Oil and water, being immiscible substances, naturally tend to separate and stratify, which severely impacts food quality, taste, and shelf life. Current technologies utilize emulsifiers to construct stable oil-water interfaces, allowing food to be uniformly and stably dispersed in an aqueous matrix as nano-sized oil droplets, fundamentally solving the problem of oil stratification.

[0003] Glycol protein is a plant-based storage protein that is insoluble in water and salt solutions, but soluble only in 70%–80% ethanol aqueous solutions. Because it originates from natural plants, is widely available, has good biocompatibility, and can be completely degraded by human digestive enzymes or microorganisms, and possesses both hydrophobic and hydrophilic groups, it can be used as a food emulsifier. However, the glycol particles prepared in existing technologies have a particle size greater than 100 nanometers, which can only stabilize oil-in-water emulsions with droplet sizes exceeding 1000 micrometers; it cannot be used to prepare food emulsions with droplet sizes all less than 100 nanometers, such as walnut milk, peanut milk, and fish oil / algae oil milk. Furthermore, heat treatment is a necessary unit operation for food emulsions; emulsions formed using fish oil or algae oil as emulsifiers in existing technologies are prone to oil-water separation, fat floating, and a rough texture after heat treatment. Summary of the Invention

[0004] The purpose of this invention is to provide barley prolysin particles and a method for preparing the same, as well as a method for preparing an oil-in-water emulsion. The barley prolysin particles prepared by the method provided by this invention have a small particle size and can be used to prepare small-sized, thermally stable oil-in-water emulsions.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: A method for preparing barley alcohol-soluble protein granules, comprising: A dispersion of barley prolyl protein raw material was mixed with proline endonuclease and subjected to enzymatic hydrolysis to obtain a barley prolyl protein polypeptide dispersion; the particle size of the barley prolyl protein raw material was 130~160nm; the pH value of the enzymatic hydrolysis reaction was 5.5~5.8; The barley alcohol-soluble protein polypeptide dispersion was mixed with calcium chloride aqueous solution and genipin ethanol solution, and then subjected to a cross-linking reaction to obtain barley alcohol-soluble protein particles.

[0006] Preferably, the mass ratio of the barley prolysin raw material to the proline endonuclease is (5~100):1.

[0007] Preferably, the temperature of the enzymatic hydrolysis reaction is 30~55℃, and the time of the enzymatic hydrolysis reaction is 0.5~2h.

[0008] Preferably, the mass ratio of barley prolysin polypeptide to calcium chloride in the calcium chloride aqueous solution is (1~5):1.

[0009] Preferably, the mass ratio of barley prolysin polypeptide in the barley prolysin polypeptide dispersion to genipin in the genipin ethanol solution is (10~40):1.

[0010] Preferably, the concentration of the genipin ethanol solution is 0.3~1g / mL.

[0011] Preferably, the crosslinking reaction is carried out at a temperature of 25-55°C for 10-100 minutes.

[0012] The present invention also provides barley prolysin particles prepared by the preparation method described above, wherein the particle size of the barley prolysin particles is 16.3~23.1 nm.

[0013] This invention also provides a method for preparing an oil-in-water emulsion, comprising: The aqueous dispersion of barley prolysin particles described in the above technical solution is mixed with food oil and then homogenized under high pressure to obtain an oil-in-water emulsion.

[0014] Preferably, the mass ratio of the barley prolysin particles to the edible oil is (0.02~0.1):1.

[0015] This invention provides a method for preparing barley prolysin particles, comprising: mixing a dispersion of barley prolysin raw material with proline endonuclease for enzymatic hydrolysis to obtain a barley prolysin polypeptide dispersion; wherein the particle size of the barley prolysin raw material is 130-160 nm; the pH value of the enzymatic hydrolysis reaction is 5.5-5.8; and mixing the barley prolysin polypeptide dispersion with calcium chloride aqueous solution and genipin ethanol solution for cross-linking reaction to obtain barley prolysin particles. This invention employs proline endonuclease to enzymatically hydrolyze barley prolysin at a pH of 5.5-5.8, thoroughly breaking it down into small-molecule barley prolysin peptides. This reduces the size of the subsequently obtained barley prolysin particles. Furthermore, the barley prolysin peptide dispersion obtained using this invention is non-bitter, ensuring that the prepared barley prolysin particles are also non-bitter and suitable for food emulsification. Then, the barley prolysin peptide dispersion, calcium chloride aqueous solution, and genipin ethanol solution are mixed for a cross-linking reaction. The calcium ions in the calcium chloride aqueous solution react with the carboxyl groups (-COO) on the barley prolysin peptide chains. The presence of negatively charged groups such as H and hydroxyl (-OH) forms coordinate bonds, which regulate the charge distribution on the surface of barley prolysin molecules through charge neutralization, thereby improving the dispersibility of barley prolysin particles in water, preventing their aggregation, and reducing their size. Simultaneously, cross-linking with genipin allows the iridoid ketone active groups in the genipin molecule to undergo nucleophilic addition and dehydration condensation reactions with the free primary amino groups on the barley prolysin polypeptide chain, forming stable Schiff base (C=N) covalent bonds. The synergistic effect of these two components constructs a stable three-dimensional network structure, improving the thermal stability of emulsions prepared from barley prolysin particles. The results of the examples show that the particle size of barley prolysin particles prepared by the preparation method provided by the present invention can be as low as 18.5±2.2, the droplet size of the oil-in-water emulsion can be as low as 237.4±12.8, and after heat treatment, the droplet size of the oil-in-water emulsion can be as low as 248.6±21.4, and the fat floating ratio is <0.2%. Detailed Implementation

[0016] This invention provides a method for preparing barley alcohol-soluble protein particles, comprising: A dispersion of barley prolyl protein raw material was mixed with proline endonuclease and subjected to enzymatic hydrolysis to obtain a barley prolyl protein polypeptide dispersion; the particle size of the barley prolyl protein raw material was 130~160nm; the pH value of the enzymatic hydrolysis reaction was 5.5~5.8; The barley prolysin polypeptide dispersion was mixed with calcium chloride aqueous solution and genipin ethanol solution and then subjected to a cross-linking reaction to obtain barley prolysin particles.

[0017] This invention involves mixing a dispersion of barley prolyl protein particles with proline endonuclease for enzymatic hydrolysis to obtain a dispersion of barley prolyl protein polypeptides.

[0018] In this invention, the particle size of the barley prolysin raw material can be 130~160nm or 140~150nm.

[0019] In one embodiment of the present invention, the solvent for the dispersion of barley prolysin raw material can be water. In another embodiment of the present invention, the concentration of the dispersion of barley prolysin raw material can be 0.5~40 g / L. In specific embodiments of the present invention, the concentration of the dispersion of barley prolysin raw material can be 0.5 g / L, 1 g / L, 5 g / L, 10 g / L, 20 g / L, 30 g / L, or 40 g / L. The present invention ensures uniform dispersion of barley prolysin particles by limiting the concentration of the dispersion.

[0020] In one embodiment of the present invention, the aqueous dispersion of barley prolysin raw material can be prepared by a reverse-phase solvent method. In another embodiment, the aqueous dispersion of barley prolysin raw material can be prepared by mixing barley prolysin with an aqueous ethanol solution, injecting the mixture into ultrapure water, and then sequentially performing a first rotary evaporation, a second rotary evaporation, and volume adjustment to obtain the aqueous dispersion of barley prolysin raw material.

[0021] In one embodiment of the present invention, the mass fraction of the ethanol-water solution is 70-90%. In another embodiment, the mass ratio of the barley prolysin to the ethanol-water solution can be (0.02-0.2):1. In specific embodiments of the present invention, the mass ratio of the barley prolysin to the ethanol-water solution can be 0.02:1, 0.08:1, 0.1:1, or 0.2:1. In another embodiment, the mixing and injection are carried out under stirring; the stirring rate can be independently 50-600 rpm or 100-300 rpm. In another embodiment, the temperature of the first rotary evaporation can be 50-60°C; the time of the first rotary evaporation can be 20 min; the temperature of the second rotary evaporation can be 50-80°C; the second rotary evaporation stops when ethanol is distilled off. The present invention does not have a specific limitation on the volume adjustment; a water dispersion of the barley prolysin raw material of the desired concentration can be obtained by using methods well known in the art.

[0022] In one embodiment of the present invention, the mass ratio of the barley prolysin raw material to the proline endonuclease can be (5~100):1. In specific embodiments of the present invention, the mass ratio of the barley prolysin raw material to the proline endonuclease can be 5:1, 10:1, 20:1, 50:1, 70:1, or 100:1. The present invention ensures that the barley prolysin particles can be fully enzymatically hydrolyzed by the proline endonuclease into small-molecule, non-bitter barley prolysin polypeptides by limiting the mass ratio of the barley prolysin raw material to the proline endonuclease, thereby ensuring that the prepared barley prolysin particles are non-bitter and can be used for food emulsification.

[0023] The present invention does not have any particular limitation on the mixing of the barley prolysin particle dispersion and the proline endonuclease. The barley prolysin particle dispersion and the proline endonuclease can be mixed evenly using a mixing method well known in the art.

[0024] In this invention, during the enzymatic hydrolysis reaction, the proline residue peptides in barley prolysin are targeted and cleaved by proline endonucleases to form small-molecule barley prolysin polypeptides. In this invention, the pH value of the enzymatic hydrolysis reaction can be 5.5~5.8. In embodiments of this invention, the pH value of the enzymatic hydrolysis reaction can specifically be 5.5, 5.6, 5.7, or 5.8. This invention ensures more uniform dispersion of barley prolysin particles in water by limiting the pH value of the enzymatic hydrolysis reaction, thereby ensuring more sufficient contact with the proline endonuclease and thus a more complete enzymatic hydrolysis reaction. As one embodiment of this invention, the temperature of the enzymatic hydrolysis reaction can be 30~55℃, and the reaction time can be 0.5~2h. This invention further ensures a more complete enzymatic hydrolysis reaction by limiting the temperature and time of the enzymatic hydrolysis reaction. As one embodiment of this invention, the enzymatic hydrolysis reaction can be carried out under stirring. This invention does not have any special limitations on the stirring; any stirring method well known in the art can be used to ensure sufficient reaction of the raw materials in the enzymatic hydrolysis reaction.

[0025] In one embodiment of the present invention, after the enzymatic hydrolysis reaction is completed, the product of the enzymatic hydrolysis reaction can be ultrafiltered using an ultrafiltration membrane to obtain a permeate, namely, a barley alcohol-soluble protein polypeptide dispersion. In another embodiment of the present invention, the molecular weight cutoff of the ultrafiltration membrane can be 10 kDa.

[0026] After obtaining the aqueous solution of barley prolysin polypeptide, the present invention mixes the barley prolysin polypeptide dispersion with an aqueous solution of calcium chloride and an ethanol solution of genipin and then carries out a cross-linking reaction to obtain barley prolysin particles.

[0027] In one embodiment of the present invention, the mass concentration of the barley alcohol-soluble protein polypeptide dispersion can be 0.1% to 5%. In specific embodiments of the present invention, the mass concentration of the barley alcohol-soluble protein polypeptide dispersion can be 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5%. The present invention ensures uniform distribution of cross-linking sites and controllable reaction rate by limiting the concentration of the barley alcohol-soluble protein polypeptide dispersion, ultimately forming a structurally uniform cross-linked network. In another embodiment of the present invention, the concentration of calcium chloride in the calcium chloride aqueous solution in the mixture of the barley alcohol-soluble protein polypeptide dispersion and the calcium chloride aqueous solution can be 10 to 30 mmol / L. This invention limits the concentration of calcium chloride in the calcium chloride aqueous solution in the mixture of barley alcohol-soluble protein peptide dispersion and calcium chloride aqueous solution to ensure sufficient coordination between calcium ions in the calcium chloride aqueous solution and the negatively charged groups in the barley alcohol-soluble protein peptide, thereby improving the dispersibility of barley alcohol-soluble protein particles in water; and promotes the expansion of barley alcohol-soluble protein peptide molecules, exposing more cross-linking active sites to facilitate cross-linking with genipin.

[0028] In one embodiment of the present invention, the mass ratio of barley prolysin polypeptide to calcium chloride in the calcium chloride aqueous solution in the barley prolysin polypeptide dispersion can be (1~5):1. In the embodiments of the present invention, the mass ratio of barley prolysin polypeptide to calcium chloride in the calcium chloride aqueous solution in the barley prolysin polypeptide dispersion can specifically be 1:1, 3:1 or 5:1. This invention limits the mass ratio of barley prolysin peptides to calcium chloride in a calcium chloride aqueous solution to ensure that calcium ions in calcium chloride fully form coordination bonds with negatively charged groups such as carboxyl groups (-COOH) and hydroxyl groups (-OH) on the barley prolysin peptide chains. This neutralization effect regulates the charge distribution on the surface of barley prolysin molecules, improves the dispersibility of barley prolysin particles in water, prevents aggregation of barley prolysin particles, and thus reduces the size of barley prolysin particles. It also promotes the expansion of barley prolysin peptide molecules, exposing more cross-linking active sites to facilitate cross-linking with genipin.

[0029] In one embodiment of the present invention, the concentration of the genipin ethanol solution can be 0.3~1 g / mL. In specific embodiments of the present invention, the concentration of the genipin ethanol solution can be 0.3 g / mL, 0.5 g / mL, 0.8 g / mL, or 1 g / mL. The present invention precisely controls the covalent cross-linking density by limiting the mass ratio of genipin to ethanol in the genipin ethanol solution, thereby synergistically constructing a stable three-dimensional network structure with coordination cross-linking, and improving the thermal stability of the emulsion prepared from barley prolysin particles.

[0030] In one embodiment of the present invention, the mass ratio of barley prolysin polypeptide in the barley prolysin polypeptide dispersion to genipin in the genipin ethanol solution can be (10~40):1. In embodiments of the present invention, the mass ratio of barley prolysin polypeptide in the barley prolysin polypeptide dispersion to genipin in the genipin ethanol solution can specifically be 10:1, 20:1, 30:1, or 40:1. The present invention ensures that the iridoid ketone active groups in the genipin molecule fully undergo nucleophilic addition and dehydration condensation reactions with the free primary amino groups on the barley prolysin polypeptide molecular chain to form stable Schiff base (C=N) covalent bonds, which, together with coordination crosslinking, construct a stable three-dimensional network structure, thereby improving the thermal stability of the emulsion prepared from barley prolysin particles.

[0031] In one embodiment of the present invention, the mixing temperature of the barley prolysin polypeptide dispersion with the calcium chloride aqueous solution and genipin ethanol solution can be 30-40°C. The present invention does not impose any particular limitation on the mixing of the barley prolysin polypeptide dispersion with the calcium chloride aqueous solution and genipin ethanol solution; any mixing method well known in the art can be used to mix the barley prolysin polypeptide dispersion with the calcium chloride aqueous solution and genipin ethanol solution.

[0032] In this invention, during the cross-linking reaction, calcium ions in the calcium chloride aqueous solution coordinate with negatively charged groups such as carboxyl groups (-COOH) and hydroxyl groups (-OH) on the barley prolysin polypeptide chain, forming coordinate bonds. Simultaneously, the iridoid ketone active groups in the genipin ethanol solution undergo nucleophilic addition and dehydration condensation reactions with the free primary amino groups on the barley prolysin polypeptide molecular chain, forming stable Schiff base (C=N) covalent bonds. These two reactions work synergistically to construct a stable three-dimensional network structure. As one embodiment of this invention, the temperature of the cross-linking reaction can be 25~55℃, and the reaction time can be 10~100 min or 30~70 min. This invention, by limiting the temperature and time of the cross-linking reaction, ensures that covalent cross-linking and coordination cross-linking proceed simultaneously and uniformly, forming a structurally regular three-dimensional network.

[0033] In one embodiment of the present invention, after the crosslinking reaction is completed, the product of the crosslinking reaction can be sequentially diluted with water, ultrafiltered through an ultrafiltration membrane to obtain a retentate, and then freeze-dried to obtain barley prolysin particles. In one embodiment of the present invention, the amount of water added can be 3 to 50 times, 10 to 40 times, or 20 to 30 times the mass of the product after the crosslinking reaction. In one embodiment of the present invention, the molecular weight cutoff of the ultrafiltration membrane can be 10 kDa. In one embodiment of the present invention, ultrafiltration is stopped when the volume of the retentate is 1 / 6 of the permeate. The present invention does not have any particular limitation on the freeze-drying process; any freeze-drying method well known in the art can be used.

[0034] This invention employs proline endonuclease to enzymatically hydrolyze barley prolysin at a pH of 5.5-5.8, thoroughly breaking it down into small-molecule barley prolysin peptides. This reduces the size of the subsequently obtained barley prolysin particles. Furthermore, the barley prolysin peptide dispersion obtained using this invention is non-bitter, ensuring that the prepared barley prolysin particles are also non-bitter and suitable for food emulsification. Then, the barley prolysin peptide dispersion, calcium chloride aqueous solution, and genipin ethanol solution are mixed for a cross-linking reaction. The calcium ions in the calcium chloride aqueous solution react with the carboxyl groups (-COO) on the barley prolysin peptide chains. The presence of negatively charged groups such as H and hydroxyl (-OH) forms coordinate bonds, which regulate the charge distribution on the surface of barley prolysin molecules through charge neutralization, thereby improving the dispersibility of barley prolysin particles in water, preventing their aggregation, and reducing their size. Simultaneously, cross-linking with genipin allows the iridoid ketone active groups in the genipin molecule to undergo nucleophilic addition and dehydration condensation reactions with the free primary amino groups on the barley prolysin polypeptide chain, forming stable Schiff base (C=N) covalent bonds. The synergistic effect of these two components constructs a stable three-dimensional network structure, improving the thermal stability of emulsions prepared from barley prolysin particles.

[0035] The present invention also provides barley alcohol-soluble protein particles prepared by the preparation method described in the above technical solution.

[0036] In this invention, the particle size of the barley prolysin particles is 16.3~23.1 nm. As one embodiment of this invention, the particle size of the barley prolysin particles can be 16.3~20.7 nm, 19.1~21.3 nm, or 20.5~23.1 nm.

[0037] The barley prolysin particles provided by this invention have small particle size and can be used to prepare food emulsions.

[0038] This invention also provides a method for preparing an oil-in-water emulsion, comprising: The aqueous dispersion of barley prolysin particles described in the above technical solution is mixed with food oil and then homogenized under high pressure to obtain an oil-in-water emulsion.

[0039] As one embodiment of the present invention, the method for preparing the aqueous dispersion of barley alcohol-soluble protein particles can be: mixing barley alcohol-soluble protein particles with water to obtain an aqueous dispersion of barley alcohol-soluble protein particles.

[0040] In one embodiment of the present invention, the mass ratio of barley prolysin particles to water in the aqueous dispersion of the barley prolysin particles can be (0.1~0.3):1. The present invention precisely controls the dispersion stability of the emulsion by limiting the mass ratio of barley prolysin particles to water in the aqueous dispersion of the barley prolysin particles, thus avoiding demulsification and stratification.

[0041] In one embodiment of the present invention, the edible oil can be soybean oil, corn oil, or peanut oil. In another embodiment, the mass ratio of barley prolysin particles to edible oil can be (0.02~0.1):1 or (0.05~0.08):1. The present invention, by limiting the mass ratio of barley prolysin particles to edible oil, ensures the saturated adsorption of oil droplets by the barley prolysin, forming an interface film of moderate thickness and good elasticity, effectively preventing oil droplet aggregation and ensuring that the emulsion does not separate during long-term storage.

[0042] This invention does not impose any particular limitation on the mixing of the aqueous dispersion of barley prolysin particles with the edible oil; any mixing method well-known in the art can be used to mix the aqueous dispersion of barley prolysin particles with the edible oil until homogeneous. This invention also does not impose any particular limitation on the parameters of the high-pressure homogenization; any high-pressure homogenization parameters well-known in the art can be used to form an oil-in-water emulsion.

[0043] The oil-in-water emulsion prepared by the method provided by this invention has small particle size and good thermal stability.

[0044] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0045] Example 1 A method for preparing barley alcohol-soluble protein granules, comprising: Preparation of barley prolysin: Barley lees were dried to a fixed weight at 70°C in an electric drying oven (DHG-9070B, Jinan, China), pulverized, and passed through a 40-mesh sieve. Then, 100g of the mixture was mixed with 1000mL of 75% ethanol aqueous solution and stirred with a magnetic stirrer for 5min. The mixture was then heated in a 40°C water bath (HH-S6, Jinan, China) for 120min and centrifuged for 15min. The supernatant was poured into a 20L stainless steel pot, 8L of water was added for precipitation, and the mixture was centrifuged for 15min to obtain barley prolysin.

[0046] 3g of barley prolysin was mixed with 50mL of 70% ethanol aqueous solution at room temperature and stirred at 500rpm until completely dissolved. The solution was then injected into 150mL of ultrapure water stirred at 500rpm using a syringe. After injection, stirring was continued for 1min. The solution was then rotary evaporated at 40℃ for 10min, and then the temperature was increased to 50℃ under negative pressure for rotary evaporation. After the ethanol was removed, the sample was collected, and pure water was added to make up to 200mL. The mixture was stirred evenly to obtain a dispersion of barley prolysin raw material. The mass concentration of the dispersion of barley prolysin raw material was 15g / L. The barley prolysin particle dispersion was mixed with proline endonuclease and enzymatically hydrolyzed at 300 rpm and 40°C for 2 hours. The mixture was then passed through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to obtain the permeate, which was the barley prolysin polypeptide dispersion. The mass ratio of barley prolysin raw material to proline endonuclease was 50:1; the mass concentration of the barley prolysin polypeptide dispersion was 5%; the pH of the enzymatic hydrolysis reaction was 5.8; and the pH of the enzymatic hydrolysis reaction was adjusted using 1 mol / L sodium hydroxide. At 35°C, an aqueous solution of calcium chloride was added to a dispersion of barley prolysin peptides to achieve a calcium chloride concentration of 30 mmol / L. Then, 0.5 mL of an ethanol solution containing 0.1 g genipin was added. After reacting at 40°C for 12 h, the mixture was diluted with water and passed through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa. Filtration was stopped when the retentate volume was approximately 1 / 6 of the permeate volume, yielding the retentate. This retentate was then freeze-dried at -20°C to obtain barley prolysin particles. The mass ratio of genipin to ethanol in the genipin ethanol solution was 0.1 g:0.5 mL. The mass ratio of prolysin peptides in the aqueous solution to calcium chloride in the aqueous solution was 3.5:1. The mass ratio of barley prolysin peptides in the dispersion to genipin in the genipin ethanol solution was 30:1. The amount of water added during dilution was 10 times the mass of the product after the cross-linking reaction.

[0047] Example 2 The difference between this implementation and Example 1 is that the pH value of the enzymatic hydrolysis reaction is replaced with 5.5; the rest remains the same.

[0048] Example 3 The difference between this implementation and Example 1 is that: a calcium chloride aqueous solution is added to the highland barley alcohol-soluble protein polypeptide dispersion to make the concentration of calcium chloride in the calcium chloride aqueous solution 10 mmol / L, and the mass ratio of alcohol-soluble protein polypeptide in the alcohol-soluble protein polypeptide aqueous solution to calcium chloride in the calcium chloride aqueous solution is replaced with 2.5:1; the rest remain unchanged.

[0049] Comparative Example 1 The difference between this comparative example and Example 1 is that the pH value of the enzymatic hydrolysis reaction is replaced with 5; the rest remains the same.

[0050] Comparative Example 2 The difference between this comparative example and Example 1 is that the pH value of the enzymatic hydrolysis reaction is replaced with 4.5; the rest remains the same.

[0051] Comparative Example 3 The difference between this comparative example and Example 1 is that the calcium chloride aqueous solution is replaced with a magnesium chloride aqueous solution; the rest remains the same.

[0052] Comparative Example 4 The difference between this comparative example and Example 1 is that the calcium chloride aqueous solution is replaced with ultrapure water; the rest remains the same.

[0053] Comparative Example 5 The difference between this comparative example and Example 1 is that the addition of genipin is omitted; everything else remains the same.

[0054] Comparative Example 6 The difference between this comparative example and Example 1 is that the pH value of the enzymatic hydrolysis reaction is replaced with 6; the rest remains the same.

[0055] Comparative Example 7 The difference between this comparative example and Example 1 is that the mass ratio of genipin to ethanol in the genipin ethanol solution is replaced with 0.02 g: 0.5 mL; the mass ratio of barley prolysin polypeptide to genipin in the genipin ethanol solution is replaced with 20: 1; the rest remain unchanged.

[0056] Comparative Example 8 The difference between this comparative example and Example 1 is that the barley gliadin is replaced with zein. Preparation of zein: Corn yellow powder was dried to a fixed weight at 70°C in an electric drying oven (DHG-9070B, Jinan, China), pulverized, and passed through a 40-mesh sieve. Then, 100g of the powder was mixed with 1000mL of 75% ethanol aqueous solution, stirred with a magnetic stirrer for 5min, heated in a 40°C water bath (HH-S6, Jinan, China) for 120min, centrifuged for 15min, and the supernatant was poured into a 20L stainless steel pot. 8L of water was added for precipitation, and the mixture was centrifuged for 15min to obtain zein. The rest remained unchanged.

[0057] Comparative Example 9 The difference between this comparative example and Example 1 is that the barley gliadin is replaced with oat gliadin, and the pH value of the enzymatic hydrolysis reaction is replaced with 5. Oat gliadin preparation: 1 kg of defatted oat flour is weighed and added to a 75% ethanol solution to make the mass ratio of oat flour to 75% ethanol aqueous solution 1:8. The mixture is then magnetically stirred at 4°C for 3 h, and then centrifuged at 10000 r / min for 15 min to separate the supernatant (gliadin solution) and the precipitate. The supernatant is freeze-dried to obtain oat gliadin; the rest remains unchanged.

[0058] Application Example 1 A method for preparing an oil-in-water emulsion is as follows: The aqueous dispersion of barley prolysin particles prepared in Example 1 was mixed with soybean oil and then homogenized under high pressure at 30 MPa to obtain an oil-in-water emulsion. The mass ratio of barley prolysin particles to water in the aqueous dispersion of barley prolysin particles was 0.02:1, and the mass ratio of barley prolysin particles to edible oil was 0.1:1.

[0059] Application Example 2 The difference between this application example and application example 1 is that the barley alcohol-soluble protein particles prepared in example 1 are replaced with the barley alcohol-soluble protein particles prepared in example 2.

[0060] Application Example 3 The difference between this application example and application example 1 is that the barley alcohol-soluble protein particles prepared in example 1 are replaced with the barley alcohol-soluble protein particles prepared in example 3.

[0061] Comparative Application Example 1 The difference between this comparative application example and application example 1 is that the barley alcohol-soluble protein particles prepared in example 1 are replaced with the barley alcohol-soluble protein particles prepared in comparative example 1.

[0062] Comparative Application Example 2 The difference between this comparative application example and application example 1 is that the barley alcohol-soluble protein particles prepared in example 1 are replaced with the barley alcohol-soluble protein particles prepared in comparative example 2.

[0063] Comparative Application Example 3 The difference between this comparative application example and application example 1 is that the barley alcohol-soluble protein particles prepared in example 1 are replaced with the barley alcohol-soluble protein particles prepared in comparative example 3.

[0064] Comparative Application Example 4 The difference between this comparative application example and application example 1 is that the barley alcohol-soluble protein particles prepared in example 1 are replaced with the barley alcohol-soluble protein particles prepared in comparative example 4.

[0065] Comparative Application Example 5 The difference between this comparative application example and application example 1 is that the barley alcohol-soluble protein particles prepared in example 1 are replaced with the barley alcohol-soluble protein particles prepared in comparative example 5.

[0066] Comparative Application Example 6 The difference between this comparative application example and application example 1 is that the barley alcohol-soluble protein particles prepared in example 1 are replaced with the barley alcohol-soluble protein particles prepared in comparative example 6.

[0067] Comparative Application Example 7 The difference between this comparative application example and application example 1 is that the barley alcohol-soluble protein particles prepared in example 1 are replaced with the barley alcohol-soluble protein particles prepared in comparative example 7.

[0068] Comparative Application Example 8 The difference between this comparative application example and application example 1 is that the barley prolysin particles prepared in example 1 are replaced with the zein particles prepared in comparative example 8.

[0069] Comparative Application Example 9 The difference between this comparative application example and application example 1 is that the barley prolysin particles prepared in example 1 are replaced with oat prolysin particles prepared in comparative example 9.

[0070] Take 0.1 mL of the dispersion of alcohol-soluble protein particles prepared in Examples 1-3 and Comparative Examples 1-9 respectively, mix with 10 mL of ultrapure water, and test with a Malvern nanoparticle size analyzer. The dynamic light scattering angle is set to 90° and the test temperature is 25℃. The results are shown in Table 1.

[0071] Take 10 mL of the oil-in-water emulsions prepared in Application Examples 1-3 and Comparative Application Examples 1-9 respectively into glass tubes, place them in an oil bath at 95°C, and heat at a constant temperature for 10 min after the center temperature of the oil-in-water emulsions rises to 95°C. Then, after the heat-treated samples are left to stand at room temperature for 24 hours, record the volume of the fat floating layer in the emulsion to test its stability. The results are shown in Table 1.

[0072] Take 10 μL of each of the above-mentioned oil-in-water emulsions before and after heating into centrifuge tubes, add pure water to dilute to 10 mL, and measure their particle size using a nanoscale Malvern particle size analyzer. The results are shown in Table 1.

[0073] Table 1. Particle size of barley prolysin in Examples 1-3 and Comparative Examples 1-9, stability data of oil-in-water emulsions in Application Examples 1-3 and Comparative Application Examples 1-9, and particle size data before and after heat treatment.

[0074] As shown in Table 1, compared with the prolysin particles prepared in Comparative Examples 1-9, the barley prolysin particles prepared in Examples 1-3 of the present invention have smaller particle sizes; compared with the oil-in-water emulsions prepared in Comparative Application Examples 1-9, the oil-in-water emulsions prepared in Application Examples 1-3 of the present invention have smaller particle sizes and better thermal stability.

[0075] In summary, the barley prolysin particles prepared by the method provided by this invention have small particle size and can be used to prepare water-in-oil emulsions with small size and good thermal stability.

[0076] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing barley alcohol-soluble protein granules, comprising: A dispersion of barley prolyl protein raw material was mixed with proline endonuclease and subjected to enzymatic hydrolysis to obtain a barley prolyl protein polypeptide dispersion; the particle size of the barley prolyl protein raw material was 130~160nm; the pH value of the enzymatic hydrolysis reaction was 5.5~5.8; The barley prolysin polypeptide dispersion was mixed with calcium chloride aqueous solution and genipin ethanol solution and then subjected to a cross-linking reaction to obtain barley prolysin particles.

2. The preparation method according to claim 1, characterized in that, The mass ratio of the barley prolysin raw material to the proline endonuclease is (5~100):

1.

3. The preparation method according to claim 1, characterized in that, The enzymatic hydrolysis reaction is carried out at a temperature of 30~55℃ for 0.5~2h.

4. The preparation method according to claim 1, characterized in that, The mass ratio of barley prolysin polypeptide to calcium chloride in the calcium chloride aqueous solution is (1~5):

1.

5. The preparation method according to claim 1 or 4, characterized in that, The mass ratio of barley prolysin polypeptide in the barley prolysin polypeptide dispersion to genipin in the genipin ethanol solution is (10~40):

1.

6. The preparation method according to claim 1, characterized in that, The concentration of the genipin ethanol solution is 0.3~1g / mL.

7. The preparation method according to claim 1, characterized in that, The cross-linking reaction is carried out at a temperature of 25-55°C for 10-100 minutes.

8. The barley alcohol-soluble protein granules prepared by the preparation method according to any one of claims 1 to 7, characterized in that, The particle size of the barley prolysin particles is 16.3~23.1 nm.

9. A method for preparing an oil-in-water emulsion, comprising: The aqueous dispersion of the barley prolysin particles described in claim 8 was mixed with edible oil and then homogenized under high pressure to obtain an oil-in-water emulsion.

10. The preparation method according to claim 9, characterized in that, The mass ratio of the barley prolysin granules to the edible oil is (0.02~0.1):1.

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