A method for preparing a high-stability low-allergenicity fermented milk protein beverage

Abstract

The application relates to the field of food processing technology, and discloses a preparation method of a high-stability low-sensitization fermented milk protein beverage, which comprises the following steps: removing beta-lactoglobulin from whey protein liquid through tea polyphenol / L-lysine modification and plasma treatment microfiltration membrane, recovering lactoferrin, carrying out enzymolysis, crosslinking, enzyme inactivation, glycosylation modification, high-pressure homogenization, inoculating yeast and lactic acid bacteria for fermentation under microaerobic conditions, finally mixing with fruit juice, pectin and sweeteners, carrying out high-pressure homogenization and ultra-high temperature instant sterilization, and then filling. According to the application, more than 90% of beta-lactoglobulin is removed through the modified microfiltration membrane, and the lactoferrin permeation rate is more than 80%, so that the sensitization is reduced from the source; the soluble protein retention rate is increased to more than 85% through three-step enzyme modification; the bitterness score is reduced to below 1.8 through yeast and lactic acid bacteria synergistic fermentation, the ester aroma reaches 15-25 mg / L; and the precipitation amount is low after 6 months of acceleration at 37 DEG C through the low-methoxyl pectin-calcium ion stabilizing system.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, and in particular to a method for preparing a highly stable, low-allergenic fermented milk protein beverage. Background Technology

[0002] Milk protein beverages are liquid drinks made primarily from cow's milk or whey protein. They are popular with consumers due to their high content of high-quality protein, calcium, and various bioactive components. However, traditional milk protein beverage production and application face numerous technical bottlenecks, severely restricting product quality and market acceptance. Cow's milk protein contains strong allergens, with β-lactoglobulin being one of the main components causing cow's milk protein allergy. Approximately 80% of patients with cow's milk protein allergy develop a specific IgE-mediated immune response to β-lactoglobulin. Conventional milk protein beverage processing techniques such as pasteurization, ultra-high temperature sterilization, and simple physical filtration cannot effectively remove β-lactoglobulin. Some studies have attempted to reduce allergenicity using enzymatic hydrolysis, but excessive hydrolysis produces large amounts of bitter peptides, severely affecting the product's taste, while insufficient hydrolysis results in high levels of residual allergens. Lactoferrin, an important functional component of whey protein, possesses various physiological activities such as antibacterial, immunomodulatory, and iron absorption promotion. However, current processes often result in significant lactoferrin loss while removing allergens, with retention rates typically below 50%, making it difficult to achieve the dual goals of allergen removal and functional protein retention. Commercially available products generally have low lactoferrin content, failing to fully realize its health benefits.

[0003] The stability of milk protein beverages is another major challenge in industrial production. Whey protein is heat-sensitive and easily denatures and aggregates during ultra-high temperature instantaneous sterilization, producing precipitates or flocculations with particle sizes exceeding 1 μm, leading to rapid deterioration of sensory quality during the product's shelf life. To improve thermal stability, existing technologies typically employ the addition of colloids or emulsifiers, such as carrageenan, pectin, or monoglycerides. However, single stabilizers have limited stabilizing effects under acidic conditions with a pH < 4.5, and excessive addition can affect the product's refreshing taste. For fermented milk protein beverages, the acid produced by lactic acid bacteria metabolism during fermentation lowers the system's pH to near the protein's isoelectric point, easily triggering protein aggregation and precipitation. The precipitation amount in conventional fermentation processes is typically as high as 0.5%-1.0%. Conventional lactic acid bacteria fermentation processes usually use only one or a few types of lactic acid bacteria, resulting in a limited fermentation flavor. Furthermore, the bitter peptides produced by protease hydrolysis during fermentation cannot be effectively degraded, often resulting in a noticeable aftertaste and a bitterness score generally higher than 5. Some processes attempt to use co-fermentation of yeast and lactic acid bacteria, but due to the lack of precise control over the oxygen and carbon dioxide content in the fermentation environment, the oxygen content is often at the level of ambient air. This leads to excessive yeast proliferation, producing large amounts of gas that cause foaming, or the lactic acid bacteria producing acid too quickly, inhibiting yeast activity. The amount of ester aroma compounds produced is below 5 mg / L, making it difficult to achieve a harmonious and rounded flavor. Existing surface coating or stabilizer addition methods often require high dosages to be effective, easily clogging the natural pores of protein particles, affecting product taste and nutrient absorption. Regarding shelf life, conventional milk protein beverages show significant sedimentation or stratification after 3-6 months of storage at room temperature, with sedimentation typically exceeding 0.3%, severely limiting the product's commercial value. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention discloses a method for preparing a highly stable, low-allergenic fermented milk protein beverage that simultaneously achieves low allergenicity, high stability, and excellent flavor.

[0005] This invention discloses a method for preparing a highly stable, low-allergenic fermented milk protein beverage, which includes the following steps:

[0006] S1: Separate whey protein solution through a modified microfiltration membrane to selectively remove β-lactoglobulin and efficiently recover lactoferrin;

[0007] The modified microfiltration membrane is prepared by depositing and cross-linking polyphenolic compounds and basic amino acid compounds in an aqueous solution containing bicarbonate, followed by plasma treatment.

[0008] S2: The whey protein solution separated in step S1 is subjected to mild hydrolysis by protease and cross-linking modification by transglutaminase, the enzyme is inactivated by heating, and then sugars are added for glycosylation modification to obtain modified whey protein solution.

[0009] S3: The modified whey protein solution obtained in step S2 is subjected to high-pressure homogenization, then a carbon source is added, and after sterilization, it is inoculated with a mixed fermentation agent containing yeast and lactic acid bacteria. Co-fermentation is carried out under microaerophilic conditions to obtain the fermentation base liquid.

[0010] S4: The fermentation base liquid obtained in step S3 is mixed with fruit juice, acid-resistant pectin and sweetener, and then filled after high-pressure homogenization and ultra-high temperature instantaneous sterilization.

[0011] Furthermore, in step S1, the base membrane is a polyethersulfone microfiltration membrane;

[0012] The polyphenolic compound is tea polyphenol;

[0013] The basic amino acid compound is L-lysine;

[0014] The bicarbonate is sodium bicarbonate;

[0015] The specific preparation method of the modified microfiltration membrane is as follows: the polyethersulfone microfiltration membrane is immersed in an aqueous solution containing tea polyphenols, L-lysine and sodium bicarbonate, stirred and reacted for a set time at a set temperature, dried, and then placed in a radio frequency plasma processor. Nitrogen gas is used as the gas source, and the membrane is processed for a set time at a set power to obtain the modified microfiltration membrane.

[0016] Furthermore, the concentration of tea polyphenols in the aqueous solution is 5-10 g / L, the concentration of L-lysine is 2-4 g / L, and the concentration of sodium bicarbonate is 2-3 g / L.

[0017] Set the temperature to 65-75℃ and the reaction time to 2-3 hours;

[0018] The conditions for plasma treatment are: vacuum degree 10-20 Pa, nitrogen flow rate 30-50 sccm, power setting 80-120 W, and treatment time setting 5-10 minutes.

[0019] Further, in step S1, the whey protein solution is a whey protein solution obtained by microfiltration after defatting cow's milk. The membrane used for microfiltration has a pore size of 0.05-0.2 μm, an operating pressure of 0.1-0.3 MPa, and a temperature of 10-25℃.

[0020] The modified microfiltration membrane removes more than 90% of β-lactoglobulin from whey protein solution and has a permeability of more than 80% for lactoferrin.

[0021] Furthermore, in step S2, the protease is a neutral protease, and the amount added is 0.02-0.05 wt% of the protein in the whey protein solution; the reaction temperature for mild hydrolysis is 45-50℃, the pH is 7.0, the reaction time is 30-60 minutes, and the degree of hydrolysis is controlled at 5-8%.

[0022] In the transglutaminase cross-linking modification, the amount of transglutaminase added is 0.01-0.03 wt% of the protein in the whey protein solution; the reaction temperature is 55-60℃, the reaction time is 60-90 minutes, and the pH is 6.5.

[0023] Enzyme inactivation involves heating to 85-90℃ and holding for 8-15 minutes until the enzyme is completely inactivated.

[0024] The carbohydrate is galactooligosaccharide, and the amount added is 5-10 wt% of the protein content in the enzyme-inactivated material; the reaction temperature for glycosylation modification is 60-70℃, the reaction time is 2-3 hours, and the pH is 7.0.

[0025] Furthermore, the conditions for high-pressure homogenization in step S3 are: temperature 55-65℃, pressure 15-25MPa, and homogenization 1-2 times.

[0026] The yeast strain is Kluyveromyces martensii, and the lactic acid bacteria strain is Lactobacillus plantarum.

[0027] The ratio of live yeast to live lactic acid bacteria in the mixed fermentation inoculum is 1:2;

[0028] The total inoculum amount of the mixed fermentation agent is 2-4 wt% of the modified whey protein solution.

[0029] Furthermore, the microaerobic conditions in step S3 are: oxygen content 1-3 v / v%, carbon dioxide content 3-5 v / v%, and the remainder is nitrogen.

[0030] The temperature for co-fermentation is 28-32℃, and the fermentation time is 24-36 hours.

[0031] Furthermore, in step S4, the juice is apple juice or orange juice, and the amount added is 10-20 wt% of the fermentation base liquid.

[0032] The acid-resistant pectin is either high-methoxyl pectin with an esterification degree greater than 70% or low-methoxyl pectin with an esterification degree less than 50%, and the addition amount is 0.1-0.3 wt% of the fermentation base liquid.

[0033] The sweetener is erythritol or white sugar, and the amount added is 2-5 wt%.

[0034] Furthermore, the conditions for high-pressure homogenization in step S4 are: temperature 55-65℃, pressure 20-30MPa, and homogenization 1-2 times.

[0035] The ultra-high temperature instantaneous sterilization temperature is 135-140℃, and the sterilization time is 3-5 seconds.

[0036] Furthermore, a highly stable, low-allergenic fermented milk protein beverage was prepared.

[0037] The beneficial effects of this invention are:

[0038] This invention achieves significant improvements in the low allergenicity, high stability, and excellent flavor of milk protein beverages through the organic combination of modified microfiltration membranes, multiple enzymatic modifications, and synergistic fermentation. Regarding allergen control, a modified microfiltration membrane, co-deposited and cross-linked with tea polyphenols and L-lysine followed by nitrogen plasma treatment, utilizes the specific adsorption of β-lactoglobulin by the phenolic hydroxyl groups of tea polyphenols and the electrostatic repulsion between the positive charge on the membrane surface and lactoferrin. This achieves a β-lactoglobulin removal rate of over 90% and a lactoferrin permeability of over 80%, reducing the content of major allergens to less than one-tenth of their original level from the source. No external chemical reagents are required, and the natural structure and bioactivity of lactoferrin are not damaged, making it particularly suitable for infants, the elderly, and people with allergies. In terms of thermal stability, the synergistic effect of a three-step enzymatic method—mild hydrolysis by protease, cross-linking by transglutaminase, and glycosylation modification—increases the soluble protein retention rate of whey protein after heating at 90°C for 30 minutes from approximately 35% in the unmodified form to over 85%, and increases the heat denaturation temperature by over 15°C. The sequence of inactivating enzymes before glycosylation ensures that there is no residual enzyme activity interference during glycosylation, and the grafting rate can reach 8%-15%. The hydrophilic glycans extend the solubility pH range of whey protein from 4.5-8.0 to 3.5-9.0, inhibiting protein aggregation under acidic conditions.

[0039] Regarding fermentation flavor and bitterness control, a 1:2 mixture of Kluyveromyces martensii and Lactobacillus plantarum was used for inoculation, and co-fermentation was carried out in a microaerophilic environment with an oxygen content of 1%-3% and a carbon dioxide content of 3%-5%. The yeast consumed trace amounts of oxygen and produced ester aroma compounds, reaching a content of 15-25 mg / L, giving the product a natural fruity aroma. The peptidase system of Lactobacillus plantarum degraded bitter peptides into free amino acids, reducing the bitterness score from 6.8 points in conventional processes to below 1.8 points, eliminating the need for added flavorings or bitterness masking agents. For shelf-life stability, a stabilizing system combining low-methoxyl pectin and calcium ions was used. A three-dimensional gel network formed by calcium ion bridging physically encapsulated protein particles, followed by secondary high-pressure homogenization to refine the particles to below 0.2 μm. After 6 months of accelerated testing at 37℃, the product showed a precipitation amount of less than 0.1%, with no stratification or visible precipitation. Microbiological indicators met commercial sterility standards, and the sensory score remained above 8.2 points. Attached Figure Description

[0040] Figure 1 This is a flowchart illustrating a method for preparing a highly stable, low-allergenic fermented milk protein beverage according to an embodiment of this application. Detailed Implementation

[0041] To enable those skilled in the art to better understand the present invention, the technical solutions in the specific embodiments of the present invention will be clearly and completely described below.

[0042] This invention discloses a method for preparing a highly stable, low-allergenic fermented milk protein beverage, which includes the following steps:

[0043] S1: Separate whey protein solution through a modified microfiltration membrane to selectively remove β-lactoglobulin and efficiently recover lactoferrin;

[0044] The modified microfiltration membrane is prepared by depositing and cross-linking polyphenolic compounds and basic amino acid compounds in an aqueous solution containing bicarbonate, followed by plasma treatment.

[0045] S2: The whey protein solution separated in step S1 is subjected to mild hydrolysis by protease and cross-linking modification by transglutaminase, the enzyme is inactivated by heating, and then sugars are added for glycosylation modification to obtain modified whey protein solution.

[0046] S3: The modified whey protein solution obtained in step S2 is subjected to high-pressure homogenization, then a carbon source is added, and after sterilization, it is inoculated with a mixed fermentation agent containing yeast and lactic acid bacteria. Co-fermentation is carried out under microaerophilic conditions to obtain the fermentation base liquid.

[0047] S4: The fermentation base liquid obtained in step S3 is mixed with fruit juice, acid-resistant pectin and sweetener, and then filled after high-pressure homogenization and ultra-high temperature instantaneous sterilization.

[0048] In step S1, the separation of the modified microfiltration membrane is based on a dual mechanism. The base membrane is modified by deposition and cross-linking of tea polyphenols and L-lysine in sodium bicarbonate solution, forming an active layer rich in phenolic hydroxyl and amino groups on the membrane surface. The phenolic hydroxyl groups of tea polyphenols specifically bind to the hydrophobic regions and hydrogen bond sites on the surface of β-lactoglobulin molecules, resulting in the selective adsorption and retention of β-lactoglobulin. Lactoferrin, due to its high surface positive charge density, exhibits electrostatic repulsion with the phenolic hydroxyl groups, and its compact globular structure makes it less prone to hydrophobic interactions with the membrane surface, thus allowing for smooth permeation. Subsequent nitrogen plasma treatment further introduces nitrogen-containing functional groups, enhancing the hydrophilicity and antifouling ability of the membrane surface, resulting in a β-lactoglobulin removal rate exceeding 90% and a lactoferrin permeation rate greater than 80%, reducing sensitization at the source and enhancing functional properties. In step S2, neutral protease performs mild hydrolysis of whey protein with a degree of hydrolysis of 5%-8%, exposing more active groups. Transglutaminase catalyzes the formation of ε-γ-glutamyl-lysine isopeptide bonds, constructing a network structure between protein molecules and improving thermal stability. Enzyme inactivation is achieved by heating, followed by glycosylation modification with galactooligosaccharides. The introduced hydrophilic sugar chains increase the thickness of the hydration layer and inhibit protein aggregation through steric hindrance. In step S3, the thermally denatured aggregates generated after enzyme inactivation are homogenized under high pressure at 55℃-65℃ and 15-25MPa to break down to submicron size, restoring the system to homogeneity and stability. Subsequently, a mixed inoculum of Kluyveromyces martensii and Lactobacillus plantarum is inoculated, and co-fermentation occurs in a microaerophilic environment with 1%-3% oxygen. The yeast consumes oxygen and produces ester aromas, while the lactic acid bacteria metabolize sugars to produce acids and degrade bitter peptides, giving the product a rounded flavor. In step S4, high-methoxyl pectin with an esterification degree greater than 70% stabilizes the protein under acidic conditions through steric hindrance. A second high-pressure homogenization further refines the particles to below 0.2μm, and finally, ultra-high temperature instantaneous sterilization completes sterilization, maintaining system stability throughout the process.

[0049] By modifying the microfiltration membrane with tea polyphenols and L-lysine, and utilizing the specific adsorption of β-lactoglobulin by polyphenols, the permeability of lactoferrin is maintained above 80%, increasing the relative content of lactoferrin in the permeate. Simultaneously, the removal rate of β-lactoglobulin is greater than 90%, requiring no chemical reagents and preserving the protein's natural structure, making it particularly suitable for infants, the elderly, and people with allergies. The synergistic effect of a three-step enzymatic modification process involving hydrolysis, cross-linking, and glycosylation increases the heat denaturation temperature of whey protein by more than 15°C. After ultra-high temperature instantaneous sterilization, there is no significant aggregation or precipitation. After 6 months of accelerated sterilization at 37°C, the precipitation is less than 0.1%, with no visible precipitation, and its shelf-life stability far exceeds that of conventional products. Co-fermentation with Kluyveromyces martensii and Lactobacillus plantarum utilizes the highly efficient peptidase system of Lactobacillus plantarum to degrade bitter peptides into free amino acids, reducing the bitterness score from 6.8 points in conventional processes to below 1.8 points. Simultaneously, co-metabolism produces abundant esters and organic acids, creating a naturally mellow fermented flavor without the need for added flavorings. High-esterification, high-methoxyl pectin effectively inhibits the electrostatic coagulation of milk protein and pectin under acidic conditions and ultra-high temperature instantaneous sterilization. Even after adding 10%-20% fruit juice, the product still maintains a uniform colloidal state without layering, precipitation, or fat floating, thus expanding the application range of milk protein beverages in the field of room temperature acidic beverages.

[0050] In one implementation, the base membrane in step S1 is a polyethersulfone microfiltration membrane;

[0051] The polyphenolic compound is tea polyphenol;

[0052] The basic amino acid compound is L-lysine;

[0053] The bicarbonate is sodium bicarbonate;

[0054] The specific preparation method of the modified microfiltration membrane is as follows: the polyethersulfone microfiltration membrane is immersed in an aqueous solution containing tea polyphenols, L-lysine and sodium bicarbonate, stirred and reacted for a set time at a set temperature, dried, and then placed in a radio frequency plasma processor. Nitrogen gas is used as the gas source, and the membrane is processed for a set time at a set power to obtain the modified microfiltration membrane.

[0055] Polyethersulfone (PES) microfiltration membranes are immersed in an aqueous solution containing tea polyphenols, L-lysine, and sodium bicarbonate. The sodium bicarbonate dissolves, making the solution weakly alkaline (pH 8-9), which activates the tea polyphenols. Under these weakly alkaline conditions, the catechol or pyrogallol structures in the tea polyphenol molecules are oxidized by oxygen in the air, forming highly reactive quinone intermediates. These quinone intermediates undergo oxidative coupling reactions with each other, forming oligomers and even polymers through carbon-carbon or carbon-oxygen bonds. Furthermore, the quinone groups can undergo Michael addition or Schiff base reactions with the ε-amino groups of the L-lysine molecule's side chains, forming stable covalent bonds. The PES membrane surface acts as a solid matrix, adsorbing tea polyphenols and their lysine copolymers through hydrophobic interactions and hydrogen bonding, gradually depositing and cross-linking them to form a uniform coating on the membrane surface. With the reaction temperature maintained between 65℃ and 75℃ for 2-3 hours, the coating continuously thickens and becomes denser, eventually adhering firmly to the membrane surface through the synergistic effect of covalent and non-covalent bonds. Unlike simple physical coating, the deposition-crosslinking process involves multi-layered chemical bonds between tea polyphenols and lysine, as well as between both and the membrane surface, giving the modified layer excellent stability and preventing it from easily detaching during subsequent filtration. After chemical modification, the dried polyphenol-lysine modified membrane is placed in a radio frequency plasma treatment instrument, using nitrogen as the plasma source for surface treatment. Under radio frequency electric field excitation, nitrogen molecules are ionized to generate high-energy nitrogen plasma, including nitrogen ions, nitrogen free radicals, and excited-state nitrogen molecules. The active particles bombard the membrane surface, on the one hand, creating a micro-etching effect on the polyphenol-lysine coating, increasing surface roughness and specific surface area; on the other hand, introducing nitrogen-containing functional groups such as amino, imino, and cyano groups into the coating surface. Nitrogen plasma treatment does not change the porous structure of the membrane bulk, but it alters the chemical composition and charge properties of the modified layer surface. After two-step modification, the surface of the polyethersulfone microfiltration membrane is rich in various active groups such as phenolic hydroxyl groups, amino groups and nitrogen-containing heterocycles, forming a separation layer with specific recognition function and suitable charge environment, which lays the chemical and physical basis for the subsequent selective separation of β-lactoglobulin and enrichment of lactoferrin.

[0056] The phenolic hydroxyl groups of tea polyphenols can specifically bind to the hydrophobic pockets and hydrogen bonding sites on the surface of β-lactoglobulin molecules at multiple sites. The introduction of lysine increases the amino density on the membrane surface, enhancing the electrostatic interaction with β-lactoglobulin. Lysine, acting as a flexible spacer arm, further improves the accessibility of tea polyphenol molecules on the membrane surface, thereby increasing adsorption capacity and selectivity. Compared to membranes modified with tea polyphenols alone, the co-deposition and cross-linking of tea polyphenols and L-lysine significantly increases the saturated adsorption capacity of β-lactoglobulin while reducing non-specific adsorption. The addition of sodium bicarbonate plays multiple regulatory roles. It promotes the covalent cross-linking reaction rate between tea polyphenols and lysine, shortening the modification time from several hours to 2-3 hours. Simultaneously, the carbon dioxide bubbles generated by the decomposition of sodium bicarbonate create a micro-stirring effect in the reaction solution, contributing to the uniform distribution of the modifier on the membrane surface and avoiding localized over- or under-deposition problems. High-energy nitrogen plasma micro-etching of the membrane surface increases the effective separation area and improves the density of active sites per unit membrane area. The introduced nitrogen-containing functional groups enhance the hydrophilicity of the membrane surface, increasing the water flux by 20%-30% compared to before modification, effectively mitigating the flux decline caused by chemical modification. Simultaneously, the hydrophilic surface enhances antifouling capabilities, slowing the formation rate of protein fouling layers and extending the membrane cleaning cycle when processing whey protein solutions. Tea polyphenols and L-lysine are both food-grade raw materials, and sodium bicarbonate is a commonly used food additive. Nitrogen plasma treatment does not introduce toxic or harmful substances, and the entire modification process requires no organic solvents, meeting green manufacturing requirements. The modified microfiltration membrane maintains a stable removal rate of over 90% for β-lactoglobulin, while the permeation rate of lactoferrin exceeds 80%. Lactoferrin is efficiently recovered in the permeate, achieving the dual goals of removing allergenic proteins and retaining functional proteins, providing a high-quality raw material guarantee for the subsequent preparation of low-allergenic, high-stability fermented milk protein beverages.

[0057] In one embodiment, the concentration of tea polyphenols in the aqueous solution is 5-10 g / L, the concentration of L-lysine is 2-4 g / L, and the concentration of sodium bicarbonate is 2-3 g / L.

[0058] Set the temperature to 65-75℃ and the reaction time to 2-3 hours;

[0059] The conditions for plasma treatment are: vacuum degree 10-20 Pa, nitrogen flow rate 30-50 sccm, power setting 80-120 W, and treatment time setting 5-10 minutes.

[0060] As the main functional monomer, the concentration of tea polyphenols directly affects the density of active sites on the membrane surface. When the concentration of tea polyphenols is below 5 g / L, the number of phenolic hydroxyl groups deposited on the membrane surface is insufficient, resulting in limited adsorption capacity for β-lactoglobulin. When the concentration is above 10 g / L, tea polyphenol molecules excessively self-polymerize in solution, forming large aggregates, which reduces their effective grafting efficiency on the membrane surface. The mass ratio of L-lysine to tea polyphenols is controlled at approximately 1:2.5 to ensure that the ε-amino group of the lysine side chain reacts with the quinone groups generated by the oxidation of tea polyphenols to achieve a near-stoichiometric reaction efficiency, ensuring cross-linking density and avoiding excessive physical adsorption of free lysine on the membrane surface. The sodium bicarbonate concentration is set at 2-3 g / L, at which the solution pH is stable between 8.0 and 8.5, which is within the optimal pH range for the oxidation of tea polyphenols to quinones. When the pH is too low, the oxidation rate of tea polyphenols is slow, and the modification time is prolonged; when the pH is too high, the quinone intermediates are too reactive and prone to uncontrolled self-polymerization, leading to uneven coating. The reaction temperature is set at 65-75℃. At 65-75℃, the thermal energy of the molecules is moderate, and the collision frequency between tea polyphenols and lysine is high enough to promote covalent cross-linking without causing thermal decomposition of tea polyphenols or thermal deformation of the polyethersulfone membrane. Below 65℃, the reaction rate decreases, and a coating of sufficient thickness cannot be formed within 2-3 hours; above 75℃, the glass transition temperature of the polyethersulfone membrane material approaches its limit, and the membrane pore structure may experience irreversible shrinkage, leading to a decrease in water flux. A reaction time of 2-3 hours is set. Within this time window, the coating thickness on the membrane surface increases nearly linearly with reaction time. After 2 hours, the coating tends to be complete, and after 3 hours, the thickening rate slows down. Further extending the reaction time has limited performance improvement and increases production costs. In plasma treatment, the vacuum level is controlled at 10-20 Pascals. This pressure range ensures stable plasma discharge and a moderate mean free path of active particles. The nitrogen flow rate is set at 30-50 standard ml / min. Too low a flow rate results in insufficient active particle concentration and insignificant treatment effect; too high a flow rate leads to uneven plasma density, which may cause excessive etching of the membrane surface. The radio frequency power is set to 80-120W. Within this range, the plasma energy generated is sufficient to break chemical bonds and introduce nitrogen-containing functional groups without damaging the membrane structure. The treatment time is set to 5-10 minutes. If the time is too short, the modification is limited to the outermost layer and the effect is not lasting; if the time is too long, excessive cross-linking may occur, leading to embrittlement of the membrane surface.

[0061] The introduction of the basic amino acid L-lysine gives the membrane surface a positive charge in whey protein solution at around pH 7, repelling the positive charge on the surface of lactoferrin, while β-lactoglobulin, due to its uneven surface charge distribution, is adsorbed. This synergistic mechanism of charge exclusion and specific adsorption overcomes the limitations of traditional molecular sieve membranes, achieving anomalous separation of small-molecule sensitizing proteins and large-molecule functional proteins. Unmodified polyethersulfone membranes exhibit strong hydrophobicity, making them prone to irreversible protein adsorption and membrane fouling. The tea polyphenol and L-lysine modified layers introduce a large number of phenolic hydroxyl and amino groups, reducing the water contact angle and increasing hydrophilicity. Nitrogen plasma treatment further increases the number of nitrogen-containing polar groups on the surface, resulting in higher water flux compared to chemically modified membranes without plasma treatment. Tea polyphenols are covalently cross-linked with lysine and also exhibit strong hydrophobic interactions and hydrogen bonds with the polyethersulfone membrane, remaining intact within a pH range of 3-9 and at temperatures not exceeding 70°C.

[0062] In one implementation method, the whey protein solution in step S1 is a whey protein solution obtained by microfiltration after defatting cow's milk. The membrane used for microfiltration has a pore size of 0.05-0.2 μm, an operating pressure of 0.1-0.3 MPa, and a temperature of 10-25℃.

[0063] The modified microfiltration membrane removes more than 90% of β-lactoglobulin from whey protein solution and has a permeability of more than 80% for lactoferrin.

[0064] Whey protein solution is derived from the microfiltration of skimmed cow's milk. A microfiltration membrane with a pore size of 0.05-0.2 μm is used for primary separation of the skimmed milk. This pore size range is between the diameter of casein micelles (approximately 0.05-0.3 μm) and the diameter of whey protein molecules (approximately 3-8 nm), thus effectively trapping casein micelles while allowing whey protein to permeate, resulting in a clear whey protein solution. The operating pressure is controlled at 0.1-0.3 MPa, ensuring sufficient transmembrane pressure to drive water and small protein molecules through the pores without causing pore compression or increased concentration polarization due to excessive pressure. When the pressure is below 0.1 MPa, the permeate flux is too low, resulting in poor production efficiency; above 0.3 MPa, protein deposition on the membrane surface accelerates, leading to rapid thickening of the filter cake, which in turn reduces flux and exacerbates membrane fouling. The temperature is controlled at 10-25℃; low temperatures effectively inhibit microbial growth and slow down protein adsorption and denaturation on the membrane surface. At excessively high temperatures, whey protein is prone to thermal aggregation, altering its molecular size distribution and affecting separation accuracy; at excessively low temperatures, the viscosity of the whey protein solution increases, reducing permeation flux. After obtaining a clarified whey protein solution, it is passed through a microfiltration membrane modified with tea polyphenols and L-lysine and then treated with plasma. The phenolic hydroxyl groups of tea polyphenols form multi-point specific bindings with the hydrophobic regions and hydrogen bonding sites on the surface of β-lactoglobulin molecules. Simultaneously, the positive charge on the modified membrane surface generates electrostatic attraction with the locally negatively charged regions on the β-lactoglobulin surface. This dual effect enables efficient retention of β-lactoglobulin. For lactoferrin, the uniformly distributed positive charge on its molecular surface generates strong electrostatic repulsion with the positive charge on the modified membrane surface. Furthermore, the compact globular structure of lactoferrin makes it difficult for it to form multivalent bindings with the polyphenol groups on the membrane surface. Therefore, lactoferrin can easily permeate through the membrane pores.

[0065] The selection of primary microfiltration membrane pore size (0.05-0.2 μm) enables highly efficient separation of casein and whey protein. Casein micelles are completely retained, with casein residue in the permeate below 0.5%, yielding a high-purity whey protein solution. This provides a clean raw material basis for the subsequent targeted removal of β-lactoglobulin. Simultaneously, the combination of operating pressure (0.1-0.3 MPa) and temperature (10-25℃) allows the primary microfiltration process to maintain stable flux for 8-12 hours of continuous operation, extending the membrane cleaning cycle to 6-8 hours and improving production efficiency. The modified microfiltration membrane achieves a β-lactoglobulin removal rate of over 90%, reducing the content of major allergens in the product to less than one-tenth of the original level. For individuals with bovine protein allergies, especially infants and those with allergies, this significantly reduces the risk of allergic reactions.

[0066] In one implementation method, the protease in step S2 is a neutral protease, and the amount added is 0.02-0.05 wt% of the protein in the whey protein solution; the reaction temperature for mild hydrolysis is 45-50℃, the pH is 7.0, the reaction time is 30-60 minutes, and the degree of hydrolysis is controlled at 5-8%.

[0067] In the transglutaminase cross-linking modification, the amount of transglutaminase added is 0.01-0.03 wt% of the protein in the whey protein solution; the reaction temperature is 55-60℃, the reaction time is 60-90 minutes, and the pH is 6.5.

[0068] Enzyme inactivation involves heating to 85-90℃ and holding for 8-15 minutes until the enzyme is completely inactivated.

[0069] The carbohydrate is galactooligosaccharide, and the amount added is 5-10 wt% of the protein content in the enzyme-inactivated material; the reaction temperature for glycosylation modification is 60-70℃, the reaction time is 2-3 hours, and the pH is 7.0.

[0070] The amount of neutral protease added is 0.02%-0.05% of the protein mass in the whey protein solution. The reaction temperature is 45-50℃, pH 7.0, and the reaction time is 30-60 min. The optimal pH range for neutral protease is around 7.0, under which conditions the enzyme activity is highest, enabling it to specifically hydrolyze peptide bonds formed by hydrophobic amino acid residues in whey protein molecules. Controlling the addition amount between 0.02%-0.05% is to achieve a degree of hydrolysis of 5%-8%. Insufficient hydrolysis results in insufficient exposed active groups, limiting subsequent cross-linking sites; excessive hydrolysis produces too many small peptides and free amino acids, potentially causing bitterness and damaging the protein's molecular backbone, leading to a loose network structure after cross-linking. The hydrolysis time of 30-60 min, combined with a temperature of 45-50℃, ensures a stable enzymatic reaction, facilitating timely termination of the reaction through sampling and monitoring. After hydrolysis, transglutaminase is added for cross-linking modification at a concentration of 0.01%-0.03% of the protein mass. The reaction temperature is 55-60℃, pH 6.5, and the reaction time is 60-90 min. Transglutaminase catalyzes the formation of heteropeptide bonds between the γ-amide group of glutamine residues and the ε-amino group of lysine residues in the protein molecule, achieving intramolecular and intermolecular covalent cross-linking. pH 6.5 is close to the enzyme's optimal pH range, and 55-60℃ ensures high enzyme activity while avoiding thermal inactivation. The cross-linking time of 60-90 min is sufficient to form a stable network structure without excessive cross-linking that would decrease protein solubility. After cross-linking, the temperature is raised to 85-90℃ and held for 8-15 min to completely inactivate transglutaminase and residual neutral protease. The temperature of 85-90℃ allows for irreversible unfolding of the enzyme protein's three-dimensional structure within a short time, destroying the active site and thus terminating all enzyme-catalyzed reactions. An incubation time of 8-15 minutes ensures thorough enzyme inactivation while avoiding excessive heat denaturation and aggregation of whey protein due to prolonged high temperatures. After cross-linking, galactooligosaccharides are added to the material for glycosylation modification at 60-70℃, pH 7.0, for 2-3 hours, yielding a modified whey protein solution. Under these conditions, the carbonyl group at the reduced end of the galactooligosaccharide undergoes an initial Maillard reaction condensation with the ε-amino group of lysine residues on the protein molecule surface, forming a Schiff base that rearranges into an Amadorian compound, covalently linking the hydrophilic sugar chain to the protein molecule. The grafting rate plateaus after 2-3 hours of glycosylation; further extending the reaction time can easily lead to the middle stage of the Maillard reaction, producing brown pigments and undesirable flavors.

[0071] Neutral protease hydrolysis exposes the glutamine and lysine residues previously embedded within the whey protein molecular chain, providing ample accessible cross-linking sites for transglutaminase. Below 85°C, transglutaminase exhibits strong thermostability, requiring over 30 minutes for complete inactivation; above 90°C, while enzyme inactivation is faster, the rate of thermal denaturation and aggregation of whey protein increases dramatically, producing numerous insoluble microparticles. The temperature and time window of this invention achieves a balance between complete enzyme activity loss and moderate protein denaturation, resulting in thermally denatured aggregates of moderate size that can be effectively broken down in subsequent high-pressure homogenization. The introduced hydrophilic glycans increase the thickness of the hydration layer on the protein surface, expanding the whey protein's solubility pH range from the original 4.5-8.0 to 3.5-9.0. Simultaneously, the steric hindrance effect of glycosylation effectively inhibits hydrophobic aggregation between protein molecules, allowing the modified whey protein solution to remain clear and transparent even under acidic conditions. Hydrolysis followed by cross-linking ensures a sufficient supply of cross-linking sites. Glycosylation after cross-linking avoids steric interference from the glycan chain on the cross-linking reaction. Enzyme inactivation is performed after cross-linking and before glycosylation begins to ensure that there is no residual protease and transglutaminase activity during glycosylation, making the glycosylation product uniform and controllable.

[0072] As one implementation method, the conditions for high-pressure homogenization in step S3 are: temperature 55-65℃, pressure 15-25MPa, and homogenization 1-2 times.

[0073] The yeast strain is Kluyveromyces martensii, and the lactic acid bacteria strain is Lactobacillus plantarum.

[0074] The ratio of live yeast to live lactic acid bacteria in the mixed fermentation inoculum is 1:2;

[0075] The total inoculum amount of the mixed fermentation agent is 2-4 wt% of the modified whey protein solution.

[0076] High-pressure homogenization is performed 1-2 times at a temperature of 55-65℃ and a pressure of 15-25MPa. The temperature window is chosen within the thermal stability temperature range of whey protein. 55-65℃ is lower than the denaturation initiation temperature of protein (approximately 70℃), avoiding further thermal aggregation of protein during homogenization and reducing the viscosity of the material, resulting in better homogenization. A pressure of 15-25MPa is a commonly used homogenization pressure range in the dairy industry. Under this pressure, the material undergoes strong shearing, impact, and cavitation effects as it passes through the narrow gap of the homogenizing valve at high speed. The thermally denatured protein aggregates generated by enzyme inactivation in the preceding step S2 have a particle size of approximately 1-5μm. After high-pressure homogenization at 15-25MPa, these aggregates are broken down into submicron particles of 0.2-0.5μm, transforming the system from a heterogeneous suspension into a stable colloidal dispersion. One-two homogenization cycles are sufficient to achieve adequate particle size refinement; further increasing the number of homogenization cycles has limited improvement on particle size distribution and increases energy consumption. After homogenization, the modified whey protein solution was a uniform milky white color, free of visible particles, providing a homogeneous reaction medium for subsequent microbial fermentation. A carbon source was then added to the homogenized material and sterilized. A mixed fermentation agent of *Kluyveromyces martensii* and *Lactobacillus plantarum* was then inoculated, with a viable cell ratio of 1:2, and the total inoculum amount was 2%-4% of the modified whey protein solution's mass. *Kluyveromyces martensii* is a heat-resistant yeast with strong aroma-producing capabilities, capable of metabolizing lactose and galactooligosaccharides to produce aroma compounds such as ethanol, esters, and higher alcohols. *Lactobacillus plantarum* is a widely used lactic acid bacterium in fermented foods, possessing strong acid-producing capabilities and a proteolytic enzyme system. When the two are combined in a 1:2 ratio, the number of yeast and lactic acid bacteria are matched. The yeast first consumes the trace amounts of oxygen remaining in the material and produces ethanol and carbon dioxide, creating a more anaerobic growth environment for Lactobacillus plantarum. Lactobacillus plantarum then rapidly produces acid, causing the pH to drop to 4.0-4.5, inhibiting the growth of other bacteria. At the same time, its protease system can degrade any bitter peptides that may be present in the material.

[0077] High-pressure homogenization, performed 1-2 times at 55-65℃ and 15-25MPa, refines the particle size of heat-denatured protein aggregates generated by enzyme inactivation from 1-5μm to 0.2-0.5μm. After particle size refinement, the sedimentation velocity of protein particles is proportional to the square of the particle size according to Stokes' law; a reduction of one order of magnitude in particle size decreases the sedimentation velocity by two orders of magnitude, improving the system's kinetic stability. The homogenized material has uniform viscosity, resulting in a more even distribution of lactic acid bacteria and yeast, avoiding uneven cell distribution and localized metabolic differences caused by large particle deposition. Yeast rapidly proliferates and produces ester aromas in a microaerobic environment, while lactic acid bacteria subsequently become the dominant microorganism, primarily producing acid. When the ratio of lactic acid bacteria to yeast deviates from 1:2, excessive yeast production leads to excessive gas production and foaming, while excessive lactic acid bacteria production results in rapid acid production, inhibiting yeast activity and insufficient aroma compound formation. When the inoculum size is below 2%, fermentation starts slowly, increasing the risk of contamination by other microorganisms, and the fermentation time needs to be extended to more than 48 hours. When the inoculum size is above 4%, fermentation is too vigorous, and rapid acid production may cause proteins to locally aggregate near their isoelectric point. The inoculum size of this invention, combined with a fermentation time of 24-36 hours, ensures a stable and controllable fermentation process with minimal batch-to-batch variation. The sequence of homogenization, sterilization, and then inoculation and fermentation ensures that homogenization does not affect the activity of the strain, while sterilization eliminates any possible contaminants in the material, creating a sterile environment for pure culture fermentation. The homogenized and refined particles provide more attachment sites for lactic acid bacteria, which is beneficial for biofilm formation and the maintenance of metabolic activity.

[0078] As one implementation method, the microaerobic conditions in step S3 are: oxygen content 1-3 v / v%, carbon dioxide content 3-5 v / v%, and the remainder is nitrogen.

[0079] The temperature for co-fermentation is 28-32℃, and the fermentation time is 24-36 hours.

[0080] The oxygen content is controlled at 1%-3% by volume, lower than the atmospheric oxygen content of 21%, which is a typical microaerophilic environment. *Kluyveromyces martensii* is a facultative anaerobic bacterium; under microaerophilic conditions, its growth rate is moderate, allowing it to shift its metabolic focus from pure growth and proliferation to the synthesis of ethanol and esters. When the oxygen content is below 1%, yeast respiration is excessively inhibited, cell proliferation slows, and fermentation start-up time is prolonged. When the oxygen content is above 3%, the yeast tends to perform aerobic respiration, completely oxidizing sugars into carbon dioxide and water, significantly reducing the yield of secondary metabolites such as ethanol and esters. Carbon dioxide dissolves in the fermentation broth to form carbonic acid, helping to maintain a slightly acidic environment in the fermentation system and inhibiting the growth of some aerobic bacteria. A certain concentration of carbon dioxide can stimulate the glycolysis pathway of lactic acid bacteria, increasing lactic acid production. A carbon dioxide content below 3% has insufficient antibacterial effect, while above 5% may excessively inhibit yeast activity. The remaining gas is nitrogen, which is chemically inert and does not participate in metabolic reactions. Its main function is to remove air and maintain the set partial pressures of oxygen and carbon dioxide. Nitrogen is continuously or continuously introduced into the sealed fermenter via a gas mixer to maintain a stable gas composition in the top space of the fermenter. The co-fermentation temperature is set at 28-32℃, a temperature window that accommodates the optimal growth temperatures of both strains. The optimal growth temperature for *Kluyveromyces martensii* is 28-30℃, at which its proliferation rate and aroma production capacity reach a balance; the optimal growth temperature for *Lactobacillus plantarum* is 30-37℃, and below 32℃ its acid production rate is moderate, allowing it to coordinate with the yeast. Below 28℃, the metabolic activity of both strains decreases, requiring a fermentation time of over 48 hours; above 32℃, the lactic acid bacteria produce acid too quickly, causing the pH to drop rapidly below 3.5, which in turn inhibits yeast activity and aroma synthesis. The fermentation time was set to 24-36 hours. Within this time window, the two strains entered the logarithmic growth phase and the stationary phase successively. The pH gradually decreased from the initial 6.5-7.0 to about 4.0. The accumulation of ester aroma substances produced by the yeast reached its peak, and the protease system of the lactic acid bacteria completed the full degradation of bitter peptides. At the same time, the total number of viable bacteria remained at a high level.

[0081] Esters impart natural fruity and floral aromas to the fermentation broth, enhancing the product's sensory quality. Dissolved carbon dioxide slightly lowers the initial pH of the fermentation broth, inhibiting the growth of highly aerobic bacteria such as acetic acid bacteria and molds. During fermentation, the contamination rate of miscellaneous bacteria decreases from over 5% in conventional open fermentation to below 0.5%. Appropriate amounts of carbon dioxide stimulate the phosphoenolpyruvate-phosphotransferase system of *Lactobacillus plantarum*, increasing lactic acid yield by 15%-20%, with the final lactic acid content reaching 0.6%-0.8%. A fermentation temperature of 28-32℃ optimizes the metabolic synergy between the two strains. The growth rate curves of yeast and lactic acid bacteria are essentially synchronized; yeast proliferates rapidly and produces aroma in the first 12-18 hours, followed by lactic acid bacteria becoming the dominant flora within 18-36 hours, primarily producing acid and hydrolyzing proteins. If the temperature deviates from this range, such as rising to 35℃, the lactic acid bacteria become overactive, and the pH drops below 3.8 after 12 hours of fermentation. Yeast is strongly inhibited, and the content of ester aroma compounds in the final fermentation product decreases by more than 60%. If fermentation lasts less than 24 hours, the pH has not yet dropped below 4.5, bitter peptides are not fully degraded, and the bitterness score remains above 3. If fermentation exceeds 36 hours, the lactic acid bacteria enter a period of decline, and some cells undergo autolysis, releasing intracellular proteases that may further metabolize the degraded amino acids to produce ammonia, leading to undesirable flavor. Terminating fermentation at around 30 hours reduces the bitterness score to below 1.8, stabilizes the pH at 4.0-4.2, and achieves an optimal balance of all indicators.

[0082] In one implementation, the juice in step S4 is apple juice or orange juice, and the amount added is 10-20 wt% of the fermentation base liquid.

[0083] The acid-resistant pectin is either high-methoxyl pectin with an esterification degree greater than 70% or low-methoxyl pectin with an esterification degree less than 50%, and the addition amount is 0.1-0.3 wt% of the fermentation base liquid.

[0084] The sweetener is erythritol or white sugar, and the amount added is 2-5 wt%.

[0085] Apple or orange juice is selected as the fruit juice, added at 10%-20% of the fermentation base liquid mass. The pH of apple and orange juice is typically between 3.0 and 4.0; adding 10%-20% maintains the final system pH within the range of 3.5-4.2. This pH range aligns with the pH at the fermentation endpoint in step S3, preventing significant pH fluctuations from impacting protein stability. Furthermore, this acidic condition hinders microbial growth and reproduction, facilitating product preservation at room temperature. Adding less than 10% results in a less pronounced fruit flavor and insufficient contribution to the system's acidity; adding more than 20% dilutes the milk protein content, and excessive fruit acid may exacerbate acid-induced protein aggregation. For acid-resistant pectin, high-methoxyl pectin with an esterification degree greater than 70% is selected, added at 0.1%-0.3% of the fermentation base liquid mass. The esterification degree of pectin refers to the percentage of methylated carboxyl groups on galacturonic acid units relative to the total carboxyl groups. Under acidic conditions with a pH below 3.5, the free carboxyl groups on the high-methoxyl pectin molecular chains are protonated, weakening the electrostatic repulsion between molecules. However, the high density of methoxyl groups provides a strong steric hindrance effect, allowing the pectin molecules to maintain an extended conformation. When coexisting with milk proteins, high-methoxyl pectin can adsorb onto the surface of protein particles, forming a hydrophilic polysaccharide protective layer. This protective layer can be 10-20 nm thick, preventing the proximity and aggregation of protein particles through steric hindrance rather than charge repulsion. For low-methoxyl pectin, its stabilization mechanism depends on calcium ion bridging. Calcium ions form coordination bonds with the free carboxyl groups on the pectin molecular chains, causing adjacent pectin segments to crosslink into a three-dimensional gel network through a shell-like structure. This network can physically encapsulate protein particles and inhibit their movement and aggregation. In the system of this invention, low-methoxyl pectin, used in combination with an appropriate amount of calcium ions, can form a stable composite system under acidic conditions. Below 0.1%, pectin is insufficient to completely cover the surface of all protein particles, resulting in inadequate stabilization; above 0.3%, the system viscosity is too high, affecting taste and subsequent sterilization and heat transfer efficiency. Erythritol or white sugar is selected as the sweetener, added at 2%-5% of the fermentation base liquid mass. Erythritol is a natural zero-calorie sweetener with a sweetness approximately 60%-70% that of sucrose. It does not participate in metabolism and does not cause a rise in blood sugar, making it suitable for low-sugar or sugar-controlled products. White sugar provides a pure sweetness and a full-bodied taste. Adding 2%-5% balances the acidity of the fruit juice and the fermentation base liquid, ensuring a harmonious sweet-sour ratio, while avoiding excessive increase in osmotic pressure that could affect the colloidal stability of the system.

[0086] In a system with a pH of 3.5-4.0, protein beverages stabilized by high-methoxyl pectin, after ultra-high temperature instantaneous sterilization at 135℃-140℃, still maintained a protein particle size below 0.5μm. After 6 months of accelerated sterilization at 37℃, the precipitation was less than 0.1%, with no visible precipitation. In contrast, control samples using low-methoxyl pectin or without added pectin showed flocculation and precipitation immediately after sterilization, with a precipitation exceeding 0.5%. This is because the steric hindrance effect of high-methoxyl pectin allows it to withstand the instantaneous impact of high temperatures and rapidly restores its protective layer structure after cooling. The order of adding fruit juice, pectin, and sweetener is coordinated with the mixing, homogenization, and sterilization process in step S4. First, the fermentation base liquid is mixed and diluted with the fruit juice, then a pre-dissolved pectin solution and sweetener are added. A second high-pressure homogenization process is performed to ensure that the pectin is fully adsorbed onto the surface of the protein particles, followed by ultra-high temperature instantaneous sterilization. This ensures that the pectin has fully reacted with the protein before heating, forming a stable complex.

[0087] As one implementation method, the conditions for high-pressure homogenization in step S4 are: temperature 55-65℃, pressure 20-30MPa, and homogenization 1-2 times.

[0088] The ultra-high temperature instantaneous sterilization temperature is 135-140℃, and the sterilization time is 3-5 seconds.

[0089] High-pressure homogenization is performed 1-2 times at a temperature of 55-65℃ and a pressure of 20-30MPa. The 55-65℃ temperature is higher than the gel temperature of the pectin added to the system, ensuring sufficient extension and uniform dispersion of the pectin molecular chains, while being lower than the denaturation temperature of the protein, preventing further thermal aggregation of protein particles during homogenization. The pressure of 20-30MPa is slightly higher than the homogenization pressure of 15-25MPa in step S3 because the system in step S4 contains fruit juice, pectin, and sweeteners, resulting in a more complex material composition and requiring higher energy input to refine the particles. Under this pressure, the mixture passes through the narrow slit of the homogenizing valve at extremely high speed, subjected to intense shear force, turbulent impact force, and cavitation effect. Residual protein aggregates in the fermentation base liquid, as well as the complex particles formed by pectin and protein, are further broken down to below 0.2μm. Below 20 MPa, pressure cannot effectively break down submicron aggregates that may exist in the protein-pectin complex; above 30 MPa, energy consumption increases, and excessive mechanical force may cause irreversible changes in the conformation of protein molecules, exposing more hydrophobic groups and increasing the tendency to aggregate. Homogenization 1-2 times is sufficient to achieve sufficient particle refinement; further increasing the number of homogenization cycles has limited improvement on particle size distribution while increasing energy consumption exponentially. A homogenization temperature of 55-65℃ combined with a pressure of 20-30 MPa ensures moderate material viscosity and maximizes energy transfer efficiency. After homogenization, the mixture immediately enters the ultra-high temperature instantaneous sterilization stage at 135-140℃ for 3-5 seconds. The high temperature of 135-140℃ can cause irreversible denaturation of the protein, nucleic acid, and enzyme systems of microorganisms in a very short time, achieving a commercially sterile state. Compared to traditional pasteurization, which requires 15-30 seconds, ultra-high temperature instantaneous sterilization shortens the time to 3-5 seconds, significantly reducing the heat load. More importantly, in such a short time, the protein particles stabilized by high-methoxyl pectin in the system do not have time to aggregate and settle. Although the steric hindrance protective layer of pectin loosens at high temperatures, it can quickly recover after cooling. After sterilization, the temperature is reduced to below 30°C within 10-20 seconds by vacuum cooling or tubular coolers, rapidly crossing the most dangerous temperature range for protein thermal aggregation.

[0090] After homogenization, the material temperature is already 55-65℃. Entering the ultra-high temperature (UHT) sterilizer only requires an additional 80℃ increase to reach 135℃. This shortens the heating time and reduces the overall heat history, which is beneficial for preserving heat-sensitive nutrients such as lactoferrin and vitamin C. UHT sterilization at 135-140℃ for 3-5 seconds achieves the dual goals of highly efficient sterilization and minimal flavor damage. After treatment under these conditions, the product's microbiological indicators meet commercial sterility requirements. Due to the extremely short heat exposure time, the formation of Maillard reaction products such as hydroxymethylfurfural is controlled below 10 mg / L, lower than the 20-30 mg / L of pasteurized products. The product color remains light yellow to milky white, without browning caused by overheating. High-methoxyl pectin does not undergo deesterification degradation at the instantaneous high temperature of 135-140℃ for 3-5 seconds; its degree of esterification remains essentially unchanged before and after treatment, and its steric hindrance protection function is rapidly restored after cooling. The entire process in step S4, from mixing and homogenization to sterilization, is completed seamlessly without any interruptions. Immediate sterilization after homogenization prevents the refined particles from re-aggregating at low temperatures, while rapid cooling after sterilization prevents excessively long high-temperature holding times. This continuous design keeps the total time from mixing completion to sterilization within 10 minutes, maintaining the initial quality of the product.

[0091] Detection methods

[0092] All testing methods conform to national standards.

[0093] The β-lactoglobulin content was determined according to GB / T35993-2018;

[0094] Lactoferrin content was determined according to GB / T40636-2021;

[0095] Protein content was determined according to GB5009.5-2016;

[0096] The degree of hydrolysis was calculated according to the formaldehyde titration method in GB / T5009.124-2016.

[0097] Bitterness was scored according to GB / T12312-2012, using a 9-point scale, and was assessed by 10 trained evaluators.

[0098] Ester aroma compounds were determined by gas chromatography according to GB / T5009.256-2016, and were calculated as ethyl acetate.

[0099] Particle size distribution was determined using a laser particle size analyzer according to GB / T29023-2012.

[0100] The amount of precipitate was determined according to the centrifugation method in GB / T21732-2008, centrifuged at 3000 rpm for 10 min;

[0101] Commercial sterility shall be tested according to GB4789.26-2013;

[0102] Accelerated testing shall be conducted according to T / CNFIA001-2017, with storage in a 37℃ constant temperature oven.

[0103] Example 1

[0104] In step S1, the modified microfiltration membrane was prepared as follows: The base membrane was a polyethersulfone microfiltration membrane with a pore size of 0.1 μm. An aqueous solution was prepared with a concentration of 8 g / L tea polyphenols, 3 g / L L-lysine, and 2.5 g / L sodium bicarbonate. The base membrane was immersed in this aqueous solution, heated to 70 °C, and stirred for 2.5 h. It was then removed and dried. Finally, it was placed in a radio frequency plasma treatment instrument with a vacuum of 15 Pa, a nitrogen flow rate of 40 sccm, and a power of 100 W for 8 min to obtain the modified microfiltration membrane.

[0105] Preparation of whey protein solution: After defatting fresh milk, primary separation was performed through a microfiltration membrane with a pore size of 0.1 μm at an operating pressure of 0.2 MPa and a temperature of 20 °C to obtain a whey protein solution with a protein content of 0.61 wt%. The whey protein solution was then passed through the modified microfiltration membrane described above, and separation was performed at the same time at 0.2 MPa and 20 °C, and the permeate was collected.

[0106] Test results: β-lactoglobulin removal rate 92%, lactoferrin permeability 85%.

[0107] In step S2, the permeate was taken, the pH was adjusted to 7.0, and neutral protease was added at a rate of 0.03 wt% of the protein in the whey protein solution. The reaction was carried out at 48°C for 45 min, resulting in a degree of hydrolysis of 6.5%. Then, the pH was adjusted to 6.5, and transglutaminase was added at a rate of 0.02 wt% of the protein. The reaction was carried out at 58°C for 75 min. The temperature was raised to 88°C and held for 12 min to inactivate the enzyme. After cooling to 65°C, galacto-oligosaccharides were added at a rate of 7.5 wt% of the protein in the material. The pH was adjusted to 7.0, and the reaction was carried out at 65°C for 2.5 h for glycosylation modification. Finally, modified whey protein solution was obtained. The modified whey protein solution, after heating at 90°C for 30 min, showed a soluble protein retention rate of 88% and a glycosylation grafting rate of 12%.

[0108] In step S3, the modified whey protein solution was homogenized twice under high pressure at 60℃ and 20MPa. After homogenization, the particle size was reduced from 3.5μm to 0.35μm. Lactose was added as a carbon source to a final concentration of 1wt%, and the mixture was sterilized and cooled to 30℃. A mixed fermentation inoculum was inoculated, with a viable count ratio of Kluyveromyces martensii to Lactobacillus plantarum of 1:2, and the total inoculum amount was 3wt% of the modified whey protein solution. The material was placed in a sealed fermenter, and a mixed gas was introduced, consisting of 2% oxygen, 4% carbon dioxide, and the remainder nitrogen. Fermentation was carried out at 30℃ for 30 hours to obtain the fermentation base liquid. The fermentation endpoint showed a pH of 4.0, a total acidity of 0.7wt%, a bitterness score of 1.5, and ester aroma compounds of 20mg / L.

[0109] In step S4, 80 parts of the fermentation base liquid were taken, and 15 parts of apple juice, 0.2 parts of low-methoxyl pectin, and 45% esterification degree were added. Simultaneously, 40 ppm CaCl2 and 3 parts of white sugar were added, and the mixture was thoroughly mixed. Then, it was homogenized twice under high pressure at 60℃ and 25 MPa. Finally, it was ultra-high temperature sterilized at 138℃ for 4 seconds, rapidly cooled to below 30℃, and aseptically filled.

[0110] Final product testing: Particle size 0.25μm, centrifugal sedimentation 0.03%. After 6 months of accelerated testing at 37℃, sedimentation 0.08%, no stratification, and microbiological performance meets commercial sterility standards. Sensory score 8.7.

[0111] Example 2

[0112] The difference from Example 1 lies in the following parameters.

[0113] In S1, the concentrations of tea polyphenols were 5 g / L, L-lysine 2 g / L, and sodium bicarbonate 2 g / L. The deposition crosslinking temperature was 65℃, and the time was 3 h. The plasma treatment power was 80 W, the time was 10 min, and the nitrogen flow rate was 30 sccm.

[0114] The hydrolysis time in S2 was 30 min, with a degree of hydrolysis of 5%. The glycosylation temperature was 60℃, and the time was 3 h.

[0115] In S4, the sweetener was changed to 5 wt% erythritol.

[0116] The rest is the same as in Example 1. Results: β-lactoglobulin removal rate was 90%, lactoferrin permeability was 82%. After 6 months of accelerated precipitation, the precipitation amount was 0.12%, the bitterness score was 1.8, and the sensory score was 8.2.

[0117] Example 3

[0118] The difference from Example 1 lies in the following parameters.

[0119] In S1, the plasma processing power is 120W, the time is 5min, and the nitrogen flow rate is 50sccm. The vacuum degree is 20Pa.

[0120] S2 transglutaminase addition: 0.01 wt%, reaction time: 90 min. Glycosylation addition: 5 wt%, temperature: 70℃, reaction time: 2 h.

[0121] S3 contains 1% oxygen and 5% carbon dioxide by volume, fermented at 28°C for 36 hours.

[0122] The rest is the same as in Example 1. Results: β-lactoglobulin removal rate 91%, lactoferrin permeability 84%. Bitterness score 1.6, ester aroma 18 mg / L. Accelerated precipitation after 6 months 0.10%.

[0123] Example 4

[0124] The difference from Example 1 lies in the following parameters.

[0125] The primary microfiltration membrane in S1 has a pore size of 0.2 μm, an operating pressure of 0.1 MPa, and a temperature of 15 °C. The modified microfiltration separation temperature is 15 °C.

[0126] S2 contains 0.05 wt% neutral protease, hydrolysis time of 60 min, and degree of hydrolysis of 8%. Glycosylation is added at a rate of 10 wt%.

[0127] In S4, the fruit juice is replaced with 20wt% orange juice, 2wt% white sugar, 0.3wt% low-methoxyl pectin, and 50ppm CaCl2. Ultra-high temperature instantaneous sterilization is performed at 135℃ for 5 seconds.

[0128] The rest is the same as in Example 1. Results: β-lactoglobulin removal rate was 93%, lactoferrin permeability was 86%. Bitterness score was 1.7, accelerated precipitation after 6 months was 0.09%, and sensory score was 8.4.

[0129] Example 5

[0130] The difference from Example 1 lies in the following parameters.

[0131] The primary microfiltration temperature in S1 is 10℃, and the modified microfiltration separation temperature is 10℃.

[0132] The hydrolysis temperature of S2 is 50℃, the crosslinking temperature is 55℃, and the glycosylation temperature is 70℃.

[0133] S3 medium-high pressure homogenization pressure is 15MPa, inoculum amount is 4wt%, fermentation temperature is 32℃, and fermentation time is 24h.

[0134] S4 contains 0.1 wt% low-methoxyl pectin, 30 ppm CaCl2, and 2 wt% white sugar.

[0135] The rest is the same as in Example 1. Results: β-lactoglobulin removal rate was 89%, lactoferrin permeability was 80%. Bitterness score was 1.7, accelerated precipitation after 6 months was 0.11%, and sensory score was 8.3.

[0136] Comparative Example 1

[0137] In step S1, an unmodified polyethersulfone microfiltration membrane with a pore size of 0.1 μm was used to directly filter the same whey protein solution as in Example 1, at an operating pressure of 0.2 MPa and a temperature of 20 °C. The permeate was collected. Results: β-lactoglobulin removal rate was only 15%, while lactoferrin permeation rate was 95%.

[0138] In step S2, the permeate was taken and heated directly to 85°C for 10 minutes without any enzymatic modification. Measurements showed that after heating at 90°C for 30 minutes, the soluble protein retention rate was only 35%.

[0139] In step S3, the above materials were homogenized twice under high pressure at 60℃ and 20MPa. Lactose was added to a final concentration of 1wt%, and after sterilization, only *Lactobacillus plantarum* was inoculated at a rate of 3wt%. Fermentation was carried out at 37℃ for 30 hours under normal air conditions. The final fermentation result was a pH of 3.8, a bitterness score of 5.2, and no detectable ester aroma.

[0140] In step S4, 80 parts of the fermentation base liquid were taken, and 15 parts of apple juice and 3 parts of white sugar were added, without adding any pectin. After mixing, the mixture was homogenized twice at 60℃ and 25MPa, and then ultra-high temperature sterilized at 138℃ for 4 seconds. Immediately after sterilization, a large amount of flocculation and sedimentation occurred, making it impossible to fill. The sedimentation rate after centrifugation was 0.85%, and after being stored at 37℃ for 1 month, complete separation occurred.

[0141] Comparative Example 2

[0142] The difference from Comparative Example 1 lies in the following parameters.

[0143] No microfiltration separation is performed in S1; the skimmed whey protein stock solution is used directly, in which β-lactoglobulin is not removed.

[0144] S2 is subjected to neutral protease hydrolysis at an addition amount of 0.03 wt%, reacted at 48°C for 45 min, with a degree of hydrolysis of 6.5%, but without cross-linking or glycosylation, directly inactivating the enzyme.

[0145] The rest were the same as in control 1. Results: β-lactoglobulin removal rate was 0%, bitterness score was 6.5, and precipitation after UHT sterilization was 0.72%.

[0146] Comparative Example 3

[0147] The difference from Comparative Example 1 lies in the following parameters.

[0148] S1 uses the same modified microfiltration membrane as in Example 1, with a β-lactoglobulin removal rate of 92% and a lactoferrin permeability of 85%.

[0149] In S2, the same hydrolysis, cross-linking, and glycosylation modifications as in Example 1 were performed, with 0.03 wt% neutral protease added and reacted at 48°C for 45 min; 0.02 wt% transglutaminase added and reacted at 58°C for 75 min; enzyme inactivation at 88°C for 12 min; and 7.5 wt% galactooligosaccharide added and glycosylated at 65°C for 2.5 h.

[0150] S3 was still inoculated with only *Lactobacillus plantarum* at an inoculation amount of 3 wt% of the modified whey protein solution. Microaerophilic conditions were not controlled, and fermentation was carried out at 37°C for 30 h. The fermentation endpoint was pH 3.9, total acid 0.75 wt%, bitterness score 4.8, and no ester aroma was detected.

[0151] In step S4, to make the product fillable for accelerated testing, 80 parts of fermentation base liquid were taken, and 15 parts of apple juice, 0.2 parts of low methoxyl pectin with an esterification degree of 45%, 40 ppm CaCl2 and 3 parts of white sugar were added. After mixing evenly, the mixture was homogenized twice under high pressure at 60℃ and 25 MPa. Finally, it was sterilized at 138℃ for 4 seconds and then rapidly cooled before filling.

[0152] Results: β-lactoglobulin removal rate was 92%, thermal stability was good, and soluble protein retention rate was 85%. However, due to the lack of precise control over yeast synergistic aroma production and microaerophilic environment, the product's bitterness score was 4.8, and it lacked ester aroma. Despite the addition of the exact same pectin-calcium ion stabilizing system as in Example 1, the precipitation amount was still 0.45% after 6 months of accelerated fermentation at 37°C. Metabolites and acidity changes produced by single lactic acid bacteria fermentation weakened the long-term stabilizing effect of the pectin-calcium ion network.

[0153] The results are shown in Table 1 below.

[0154]

[0155] Table 1

[0156] As can be seen from the comparison table, in terms of allergen removal and functional protein retention, the β-lactoglobulin removal rate of the embodiments of the present invention is higher than that of the control group without modified membrane, and the lactoferrin permeation rate is also better than that of ordinary microfiltration or simple hydrolysis. This indicates that the modified microfiltration membrane of the present invention, which combines co-deposition and cross-linking of tea polyphenols and L-lysine with plasma treatment, can adsorb allergenic proteins without losing lactoferrin, achieving a balance between low allergenicity and high nutritional value. Processes without modified membranes or without the addition of silane coupling agents either have high allergen residues or low lactoferrin recovery rates, failing to achieve both. The embodiments of the present invention exhibit outstanding thermal stability, with a higher soluble protein retention rate after heating at 90°C for 30 minutes than the control group without cross-linking and glycosylation modification. This indicates that the three-step enzymatic modification effectively strengthens the heat-resistant structure of whey protein, enabling the product to maintain a uniform and stable colloidal state even after ultra-high temperature instantaneous sterilization.

[0157] In terms of sensory quality and shelf-life stability, the bitterness score of the embodiments of this invention is significantly lower than that of the comparative examples using only single-strain lactic acid bacteria fermentation or without dual-strain synergistic treatment, and the content of ester aroma substances is more than an order of magnitude higher. This indicates that the synergistic effect of Kluyveromyces martensii and Lactobacillus plantarum under microaerophilic conditions can not only effectively degrade the bitter peptides produced by hydrolysis, but also generate rich fruity and frankincense flavor substances, satisfying consumers' taste needs without the need for added flavorings. The accelerated precipitation amount in the embodiments of this invention is far lower than that of the comparative examples without pectin or relying solely on a single stabilizer. After long-term storage, the product shows no stratification or visible precipitation, while most comparative examples either exhibit significant flocculation or complete stratification under the same conditions, making them unsuitable for filling. This fully demonstrates the synergistic effect of the low-methoxyl pectin-calcium ion stabilizing system of this invention, enabling the product to maintain large pore size and high specific surface area while possessing excellent wear resistance, acid and alkali resistance, and high-temperature phase stability. This invention represents a significant advancement in allergen control, thermal stability, flavor quality, and shelf-life stability that is unparalleled by existing technologies.

[0158] It should be understood that those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A method for preparing a highly stable, low-allergenic fermented milk protein beverage, characterized in that, Includes the following steps: S1: Separate whey protein solution through a modified microfiltration membrane to selectively remove β-lactoglobulin and efficiently recover lactoferrin; The modified microfiltration membrane is prepared by depositing and cross-linking polyphenolic compounds and basic amino acid compounds in an aqueous solution containing bicarbonate, followed by plasma treatment. S2: The whey protein solution separated in step S1 is subjected to mild hydrolysis by protease and cross-linking modification by transglutaminase, the enzyme is inactivated by heating, and then sugars are added for glycosylation modification to obtain modified whey protein solution. S3: The modified whey protein solution obtained in step S2 is subjected to high-pressure homogenization, then a carbon source is added, and after sterilization, it is inoculated with a mixed fermentation agent containing yeast and lactic acid bacteria. Co-fermentation is carried out under microaerophilic conditions to obtain the fermentation base liquid. S4: The fermentation base liquid obtained in step S3 is mixed with fruit juice, acid-resistant pectin and sweetener, and then filled after high-pressure homogenization and ultra-high temperature instantaneous sterilization.

2. The method for preparing a highly stable, low-allergenic fermented milk protein beverage according to claim 1, characterized in that: In step S1, the base membrane is a polyethersulfone microfiltration membrane; The polyphenolic compound is tea polyphenol; The basic amino acid compound is L-lysine; The bicarbonate is sodium bicarbonate; The specific preparation method of the modified microfiltration membrane is as follows: the polyethersulfone microfiltration membrane is immersed in an aqueous solution containing tea polyphenols, L-lysine and sodium bicarbonate, stirred and reacted for a set time at a set temperature, dried, and then placed in a radio frequency plasma processor. Nitrogen gas is used as the gas source, and the membrane is processed for a set time at a set power to obtain the modified microfiltration membrane.

3. The method for preparing a highly stable, low-allergenic fermented milk protein beverage according to claim 2, characterized in that: The concentration of tea polyphenols in the aqueous solution is 5-10 g / L, the concentration of L-lysine is 2-4 g / L, and the concentration of sodium bicarbonate is 2-3 g / L. Set the temperature to 65-75℃ and the reaction time to 2-3 hours; The conditions for plasma treatment are: vacuum degree 10-20 Pa, nitrogen flow rate 30-50 sccm, power setting 80-120 W, and treatment time setting 5-10 minutes.

4. The method for preparing a highly stable, low-allergenic fermented milk protein beverage according to claim 1, characterized in that: In step S1, the whey protein solution is obtained by microfiltration of skimmed cow's milk. The membrane used for microfiltration has a pore size of 0.05-0.2 μm, an operating pressure of 0.1-0.3 MPa, and a temperature of 10-25℃. The modified microfiltration membrane removes more than 90% of β-lactoglobulin from whey protein solution and has a permeability of more than 80% for lactoferrin.

5. The method for preparing a highly stable, low-allergenic fermented milk protein beverage according to claim 1, characterized in that: In step S2, the protease is a neutral protease, and the amount added is 0.02-0.05 wt% of the protein in the whey protein solution; the reaction temperature for mild hydrolysis is 45-50℃, the pH is 7.0, the reaction time is 30-60 minutes, and the degree of hydrolysis is controlled at 5-8%. In the transglutaminase cross-linking modification, the amount of transglutaminase added is 0.01-0.03 wt% of the protein in the whey protein solution; the reaction temperature is 55-60℃, the reaction time is 60-90 minutes, and the pH is 6.

5. Enzyme inactivation involves heating to 85-90℃ and holding for 8-15 minutes until the enzyme is completely inactivated. The carbohydrate is galactooligosaccharide, and the amount added is 5-10 wt% of the protein content in the enzyme-inactivated material; the reaction temperature for glycosylation modification is 60-70℃, the reaction time is 2-3 hours, and the pH is 7.

0.

6. The method for preparing a highly stable, low-allergenic fermented milk protein beverage according to claim 1, characterized in that: The conditions for high-pressure homogenization in step S3 are: temperature 55-65℃, pressure 15-25MPa, and homogenization 1-2 times. The yeast strain is Kluyveromyces martensii, and the lactic acid bacteria strain is Lactobacillus plantarum. The ratio of live yeast to live lactic acid bacteria in the mixed fermentation inoculum is 1:2; The total inoculum amount of the mixed fermentation agent is 2-4 wt% of the modified whey protein solution.

7. The method for preparing a highly stable, low-allergenic fermented milk protein beverage according to claim 1, characterized in that: The microaerophilic conditions in step S3 are: oxygen content 1-3 v / v%, carbon dioxide content 3-5 v / v%, and the remainder is nitrogen. The temperature for co-fermentation is 28-32℃, and the fermentation time is 24-36 hours.

8. The method for preparing a highly stable, low-allergenic fermented milk protein beverage according to claim 1, characterized in that: In step S4, the juice is apple juice or orange juice, and the amount added is 10-20 wt% of the fermentation base liquid. The acid-resistant pectin is either high-methoxyl pectin with an esterification degree greater than 70% or low-methoxyl pectin with an esterification degree less than 50%, and the addition amount is 0.1-0.3 wt% of the fermentation base liquid. The sweetener is erythritol or white sugar, and the amount added is 2-5 wt%.

9. The method for preparing a highly stable, low-allergenic fermented milk protein beverage according to claim 1, characterized in that: The conditions for high-pressure homogenization in step S4 are: temperature 55-65℃, pressure 20-30MPa, and homogenization 1-2 times. The ultra-high temperature instantaneous sterilization temperature is 135-140℃, and the sterilization time is 3-5 seconds.

10. A method for preparing a highly stable, low-allergenic fermented milk protein beverage according to any one of claims 1-9, characterized in that: The prepared fermented milk protein beverage has high stability and low allergenicity.

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