Preparation method and application of high-protein cow milk

Abstract

The invention relates to the field of food processing, and discloses a preparation method and application of high-protein cow milk, and the preparation method comprises the following steps: mixing pea protein isolate and feruloylated beet pectin in water; laccase is added into the mixed solution for an enzymatic grafting reaction in a controlled oxidation-reduction microenvironment, dispersion liquid containing the functional protein-pectin grafted copolymer is obtained, and construction of the microenvironment comprises the steps that oxygen reduction treatment is conducted before the reaction, and a preset low-concentration dissolved oxygen window is maintained in the reaction; and then mixing the dispersion liquid with a cow milk base material, and performing homogenization and ultrahigh-temperature sterilization treatment to obtain the high-protein cow milk. The hydrophilic polysaccharide chain is covalently grafted on the surface of the protein to form steric hindrance, and the reaction path is accurately controlled to inhibit side reaction, so that the prepared high-protein cow milk can still keep high physical stability, does not stratify or precipitate and is uniform in system even after being subjected to ultrahigh-temperature sterilization and long-term storage.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of food processing, in particular to a preparation method of high-protein cow milk and application thereof. BACKGROUND

[0002] With the increasing demand for health and nutrition from consumers, high-protein dairy products are favored in the market. In order to increase the protein content of cow milk products, a common technical approach is to add an additional protein source to the cow milk base. Plant proteins such as pea protein have become an important supplement or replacement source of cow milk protein due to their sustainability, cost-effectiveness and non-allergenic properties.

[0003] However, when introducing exogenous plant proteins directly into the cow milk system and producing commercially, a key technical challenge is faced. Cow milk itself is a complex colloidal system, and plant proteins differ significantly from cow milk proteins in isoelectric point, surface charge and three-dimensional structure. During necessary heat processing such as ultra-high temperature instant sterilization, high temperature induces denaturation of plant proteins, exposing their internal hydrophobic groups, resulting in irreversible aggregation of protein molecules. This aggregation phenomenon causes physical instability problems such as rough texture, precipitation and even phase separation in the final product, seriously affecting the shelf life, shelf performance and consumer acceptance of the product.

[0004] To solve this instability problem, the existing technology usually adopts the way of adding food colloids and other stabilizers in the system. These stabilizers physically delay or hinder the sedimentation of protein particles by increasing the viscosity of the system or forming a gel network. However, this method does not fundamentally improve the thermal stability of plant proteins, and the stabilizing effect provided is limited, and excessive additives may have a negative impact on the taste and flavor of the product. Therefore, the existing technology still lacks a technical solution that can fundamentally improve the thermal stability of plant proteins in the cow milk matrix to ensure that high-protein cow milk products can maintain their uniform and stable state after severe heat treatment. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application provides a preparation method of high-protein cow milk and application thereof, which solves the problem that exogenous plant proteins are prone to aggregation and precipitation after heat processing due to insufficient thermal stability in the cow milk system, resulting in poor physical stability of the final product.

[0006] To solve the above technical problems, the present application provides a preparation method of high-protein cow milk and application thereof.

[0007] The first aspect of the present application provides a preparation method of high-protein cow milk, which comprises the following steps: S1: preparing a mixed solution: mixing pea protein isolate and feruloylated sugar beet pectin in water to obtain a mixed solution; S2: Enzymatic grafting reaction: under a controlled redox microenvironment, laccase is added to the mixture to perform an enzymatic grafting reaction, to obtain a dispersion containing functional protein-pectin graft copolymer; S3: Enzyme inactivation: the dispersion obtained in S2 is subjected to a heating treatment to inactivate the laccase, to obtain a functional protein-pectin graft copolymer dispersion; S4: Product preparation: the functional protein-pectin graft copolymer dispersion is mixed with a milk base, and then subjected to homogenization and ultra-high temperature sterilization treatment, to obtain a high-protein milk.

[0008] The technical solution provided by the present application is based on the following technical concept: through the catalytic action of laccase, a free radical coupling reaction is initiated between the tyrosine residues on the surface of pea protein isolate molecules and the ferulic acid residues on the molecular chain of feruloylated sugar beet pectin, to form stable carbon-carbon or carbon-oxygen covalent bonds. This reaction enables the hydrophilic sugar beet pectin molecular chain to be covalently grafted to the surface of the pea protein isolate, forming a new functional protein-pectin graft copolymer. The sugar beet pectin molecular chain grafted on the surface of the protein physically hinders the mutual approach and aggregation of protein molecules during heat treatment through steric hindrance effect, thereby endowing the pea protein isolate with heat stability in a milk system.

[0009] In step S2, the construction and maintenance of the controlled redox microenvironment aims to guide the enzymatic reaction towards the desired protein-polysaccharide heterocoupling and inhibit non-target homocoupling side reactions. Specifically, the process includes: before adding the laccase, the mixture is subjected to oxygen reduction treatment; during the enzymatic grafting reaction, the dissolved oxygen concentration of the reaction system is maintained at a pre-set low concentration window by micro-inhaling oxygen-containing gas. The former oxygen reduction treatment step aims to reduce the initial dissolved oxygen content in the system to inhibit the self-crosslinking of proteins caused by excessive oxidation at the initial stage of the reaction; the subsequent step of maintaining a low concentration dissolved oxygen window provides the necessary electron acceptor for the catalytic cycle of laccase, while avoiding side reactions caused by excessive oxygen.

[0010] In a specific embodiment, the oxygen reduction treatment in step S2 reduces the dissolved oxygen concentration of the mixture to less than 1.0 mg / L; and the pre-set low concentration window is 1.0-2.5 mg / L.

[0011] In a specific embodiment, to further control the reaction process, ascorbic acid can also be added to the mixture after the oxygen reduction treatment in step S2 and before the addition of the laccase. As an oxidation-reduction mediator, ascorbic acid can regulate the progress of free radical reactions in the system. For example, the amount of ascorbic acid added can be 0.05 to 0.2 parts by mass of the dry basis of pea protein isolate.

[0012] In a specific embodiment, the dry basis mass ratio of the pea protein isolate to the feruloylated sugar beet pectin in step S1 can be set to 10:1 to 20:1.

[0013] In a specific embodiment, the feruloylated sugar beet pectin used in step S1 can have a ferulic acid content of 0.5 parts to 1.5 parts to provide sufficient reaction sites.

[0014] In a specific embodiment, the process parameters of the enzymatic grafting reaction in step S2 can be set to a reaction temperature of 45-55℃ and a reaction time of 1.5-3.0 hours.

[0015] In a specific embodiment, the homogenization treatment in step S4 can be performed under conditions of a first stage pressure of 15-25 MPa and a second stage pressure of 3-5 MPa.

[0016] In a specific embodiment, the ultra-high temperature sterilization treatment in step S4 can be performed at a temperature of 137-142℃ for 4-6 seconds.

[0017] The second aspect of the present application provides the use of the functional protein-pectin graft copolymer dispersion prepared in S3 in improving the heat stability of cow milk. The functional protein-pectin graft copolymer, due to the hydrophilic sugar beet pectin molecular chains grafted on its surface, can effectively prevent protein aggregation in the cow milk system, especially during heat treatment, thereby maintaining the uniformity and stability of the cow milk product.

[0018] The present application provides a high-protein cow milk preparation method and its application. It has the following beneficial effects: 1. The technical solution provided by the present application can significantly improve the physical stability of cow milk products containing exogenous plant proteins after heat treatment such as ultra-high temperature sterilization. By performing an enzymatic grafting reaction under controlled conditions, feruloylated sugar beet pectin molecular chains are covalently linked to the surface of pea protein isolate molecules, forming a functional protein-pectin graft copolymer with a specific structure. The polysaccharide chains on the surface of the copolymer physically hinder the close proximity and aggregation of protein molecules during heat treatment through steric hindrance effect, thereby effectively inhibiting the occurrence of precipitation or delamination, ensuring the long-term uniformity of the final product system.

[0019] 2、The present application realizes the accurate guidance of the reaction path by constructing and maintaining a controlled redox microenvironment in the enzymatic grafting reaction step. The controlled environment, especially the dynamic regulation of the dissolved oxygen concentration, can selectively promote the protein-polymer hetero-coupling reaction catalyzed by laccase, while effectively inhibiting the non-target homo-crosslinking side reaction between proteins that may be caused by excessive oxidation. This control of the reaction path ensures that the functional protein-pectin graft copolymer generated has uniform structural properties, thereby ensuring the quality consistency between batches of the final product.

[0020] 3、The present application fundamentally improves the intrinsic thermal stability of pea protein isolate, rather than relying on the physical blocking effect of external additives. By preparing functional protein-pectin graft copolymer, the plant protein component obtained has higher resistance to heat processing and milk environment. This makes it possible to introduce higher content of plant protein into the milk system without sacrificing the physical stability of the product, thereby providing a feasible technical basis for developing milk products with higher protein content. DETAILED DESCRIPTION

[0021] The technical solutions in the embodiments of the present application will be described below in a clear and complete manner. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor are within the scope of protection of the present application.

[0022] Embodiment: Embodiment 1

[0023] The present embodiment provides a preparation method of high-protein milk, and the specific steps are as follows: S1: Prepare a mixed solution by taking 15.0 kg of pea protein isolate (protein content 85 parts) and 1.0 kg of feruloylated sugar beet pectin (ferulic acid content 1.0 part), and adding 134 kg of purified water with a temperature of 45℃, and stirring for 1 hour at low speed. Adjust the pH value of the mixed solution to 6.8 using 2M sodium hydroxide solution.

[0024] S2: Enzymatic grafting reaction The mixture obtained in S1 was pumped into a closed reactor. High purity nitrogen was bubbled into the mixture until the dissolved oxygen concentration was reduced to 0.8 mg / L as indicated by the online dissolved oxygen sensor. After stopping the nitrogen bubbling, 15 g of ascorbic acid (0.1 parts by mass of the dry pea protein isolate) was added and stirred for 5 minutes to dissolve. Then, laccase (enzyme activity 1000 U / g) was added at a dosage of 150 g (10 U / g of pea protein isolate). The jacket temperature of the reactor was set and maintained at 50°C. After starting the reaction, sterile air was bubbled into the reaction system at a controlled rate to dynamically maintain the dissolved oxygen concentration at 1.8 mg / L. The reaction was continued under stirring for 2.5 hours.

[0025] S3: Enzyme inactivation reaction After the reaction was completed, the mixture was pumped into a plate heat exchanger and rapidly heated to 90°C, and maintained at this temperature for 45 seconds, and then cooled to below 25°C to obtain a functional protein-pectin graft copolymer dispersion.

[0026] S4: Product preparation The functional protein-pectin graft copolymer dispersion obtained in S3 was mixed with skimmed milk base to make the total protein content in the final product 6.0 g / 100 mL. The mixed mixture was preheated to 70°C, and then homogenized under the conditions of 20 MPa in the first stage and 4 MPa in the second stage. The homogenized mixture was subjected to heat sterilization treatment at 140°C for 5 seconds by UHT equipment. The sterilized product was filled under sterile conditions to obtain a high-protein milk sample.

[0027] Example 2

[0028] The present embodiment provides a method for preparing a high-protein milk, and the specific steps are as follows: S1: Preparation of the mixture 10.0 kg of pea protein isolate and 1.0 kg of feruloylated sugar beet pectin (ferulic acid content 0.5 parts) were added to 109 kg of purified water at a temperature of 40°C, and stirred for 1 hour. The pH of the mixture was adjusted to 6.5 using a citric acid solution.

[0029] S2: Enzymatic grafting reaction The mixture obtained in S1 was pumped into a closed reactor. High purity nitrogen was bubbled into the mixture until the dissolved oxygen concentration was reduced to 0.9 mg / L. After stopping the nitrogen bubbling, 5 g of ascorbic acid (0.05 parts by mass of the dry pea protein isolate) was added and stirred for 5 minutes to dissolve. Then, laccase was added at a dosage of 50 g (5 U / g of pea protein isolate). The jacket temperature of the reactor was set and maintained at 45°C. After starting the reaction, sterile air was bubbled into the reaction system to dynamically maintain the dissolved oxygen concentration at 1.0 mg / L. The reaction was continued under stirring for 1.5 hours.

[0030] S3: After the enzyme inactivation reaction, the slurry was rapidly heated to 85°C and maintained at this temperature for 60 seconds, and then cooled to below 25°C to obtain a functional protein-pectin graft copolymer dispersion.

[0031] S4: The dispersion obtained in S3 was mixed with a milk base so that the total protein content in the final product was 5.0 g / 100 mL. The mixed slurry was preheated to 65°C, and then homogenized under the conditions of a first stage pressure of 15 MPa and a second stage pressure of 3 MPa. The homogenized slurry was subjected to a holding sterilization treatment at a temperature of 137°C for 6 seconds. The sterilized product was filled under aseptic conditions to obtain a high-protein milk sample 2.

[0032] Example 3

[0033] This example provides a method for preparing a high-protein milk, and the specific steps are as follows: S1: 20.0 kg of pea protein isolate and 1.0 kg of feruloylated sugar beet pectin (ferulic acid content 1.5 parts) were added to 189 kg of purified water at a temperature of 50°C, and the mixture was hydrated by low-speed stirring for 1 hour. A 2M sodium hydroxide solution was used to adjust the pH of the mixture to 7.0.

[0034] S2: The mixture obtained in S1 was pumped into a sealed reaction kettle. High-purity nitrogen was introduced into the slurry until the dissolved oxygen concentration was reduced to 0.7 mg / L. After stopping the nitrogen flow, 40 g of ascorbic acid (0.2 parts based on the dry mass of the pea protein isolate) was added and stirred for 5 minutes to dissolve it. Then, laccase was added in an amount of 400 g (i.e., 20 U / g of pea protein isolate). The jacket temperature of the reaction kettle was set and maintained at 55°C. After the reaction was started, the dissolved oxygen concentration of the reaction system was dynamically maintained at 2.5 mg / L by introducing a small amount of sterile air. The reaction was continuously stirred for 3.0 hours under these conditions.

[0035] S3: After the reaction was completed, the slurry was rapidly heated to 95°C and maintained at this temperature for 30 seconds, and then cooled to below 25°C to obtain a functional protein-pectin graft copolymer dispersion.

[0036] S4: The dispersion obtained in S3 was mixed with a milk base so that the total protein content in the final product was 8.0 g / 100 mL. The mixed slurry was preheated to 75°C, and then homogenized under the conditions of a first stage pressure of 25 MPa and a second stage pressure of 5 MPa. The homogenized slurry was subjected to a holding sterilization treatment at a temperature of 142°C for 4 seconds. The sterilized product was filled under aseptic conditions to obtain a high-protein milk sample 3 Comparative Example 1 The difference compared with Example 1 is that no laccase is added into the mixed solution in the enzymatic grafting reaction of S2. All the other steps, raw material dosages and process parameters are exactly the same as in Example 1.

[0037] Comparative Example 2 The difference compared with Example 1 is that the enzymatic grafting reaction of S2 is carried out in an open reaction kettle under normal pressure, without performing the oxygen reduction treatment and the trace oxygen control treatment by passing sterile air. All the other steps, raw material dosages and process parameters are exactly the same as in Example 1.

[0038] Comparative Example 3 The difference compared with Example 1 is that no ascorbic acid is added into the mixed solution in the enzymatic grafting reaction of S2. All the other steps, raw material dosages and process parameters are exactly the same as in Example 1.

[0039] Comparative Example 4 The difference compared with Example 1 is that in this comparative example, the equal amount of pea protein isolate and feruloylated sugar beet pectin in S1 of Example 1 are directly mixed with the milk base in S4 by the physical mixing method, without performing the whole pretreatment process defined in S1 to S3 of Example 1. All the other process parameters for the finished product preparation are exactly the same as in Example 1. Test Example 1: Test of initial physical properties of products 1. Experimental steps This test example aims to test the initial particle size distribution of each sample after preparation is completed, without storage.

[0040] Take 10 mL of high-protein milk samples prepared in Examples 1-3 and Comparative Examples 1-4. Use a laser diffraction particle size analyzer for measurement. Use purified water as the dispersant. Slowly drop the sample into the instrument circulating pool until the obscuration rate displayed on the screen reaches the range of 10-15 parts. After turning on the ultrasonic dispersion function (power 40 W) for 60 seconds, start collecting data. Repeat the measurement three times for each sample and take the average value. Record the volume average particle size and D90 value (i.e. the particle size corresponding to the cumulative volume fraction of 90 parts) of each sample.

[0041] 2. Experimental data Table 1: Test results of initial particle size of each sample

[0042] Results and explanations The data in Table 1 show that the samples prepared by the methods described in Example 1, Example 2 and Example 3 have significantly smaller volume average particle size and D90 values than the samples prepared by the methods of Comparative Examples 1-4. The samples prepared in Comparative Examples 1, 2, 3 and 4 all exhibit larger volume average particle size and D90 values. This indicates that the final products prepared in Examples 1-3 have smaller size and more concentrated distribution of the internal particles.

[0043] Comparing the results of Example 1 with Comparative Examples 1 and 4, both of which were prepared under conditions lacking either the laccase catalysis step or the entire grafting reaction process, respectively, both of the final products formed large size particles. The technical mechanism of this phenomenon is that the method of the present application generates a functional protein-pectin graft copolymer by catalyzing the formation of covalent bonds between pea protein isolate and feruloylated sugar beet pectin by laccase. The hydrophilic pectin molecular chains grafted on the surface of the protein form a steric hindrance layer, which effectively inhibits the mutual aggregation of protein molecules during the subsequent ultra-high temperature sterilization heat treatment, thereby limiting the formation of large size aggregates. Without this covalent grafting structure, the protein will aggregate during heat treatment.

[0044] Comparing the results of Example 1 with Comparative Examples 2 and 3, both of which were prepared under conditions without applying a controlled redox microenvironment or lacking ascorbic acid, respectively, the particle size of the final products is also significantly larger than Example 1. This indicates that the controlled redox microenvironment plays a decisive role in controlling the final particle size. Under uncontrolled oxidation conditions (Comparative Example 2), the laccase catalyzed radical reaction pathway is uncontrolled, which tends to initiate homogenous crosslinking between protein molecules, forming large size aggregates. The lack of ascorbic acid (Comparative Example 3) also leads to a weakening of the control effect. Therefore, in the enzymatic grafting reaction step, it is necessary to construct a controlled redox microenvironment containing oxygen reduction and oxygen control in order to prepare a small particle size, uniformly dispersed system.

[0045] Test Example 2: Product accelerated storage stability test 1. Experimental procedure This test example aims to evaluate the physical stability of each sample under simulated long-term storage conditions.

[0046] The high-protein milk samples prepared in Examples 1-3 and Comparative Examples 1-4 were each aseptically packed in a sealed container. All samples were stored in a constant temperature incubator at 37°C in the dark for 14 days. After the storage period, the samples were removed and allowed to return to room temperature (20-25°C). The samples were first visually observed for appearance, and whether there was visible stratification, flocculation or precipitation. Then, 50 g of each sample was accurately weighed into a centrifuge tube and centrifuged at 5000g for 20 minutes. The supernatant was carefully discarded, and the centrifuge tube was inverted on filter paper for 2 minutes to remove residual liquid. The mass of the precipitate at the bottom of the centrifuge tube was weighed. The centrifugal precipitation rate (parts) was calculated according to the following formula: Centrifugal precipitation rate (parts) = (mass of precipitate / 50 g) x 100 parts.

[0047] 2. Experimental data Table 2: Results of accelerated storage stability test of each sample

[0048] Results and explanations The test results in Table 2 show that the samples prepared by the methods described in Examples 1, 2 and 3 all maintained a uniform appearance without visible precipitate after undergoing accelerated storage, and their centrifugal precipitation rates were all maintained at a very low level. In contrast, the samples prepared by the methods of Comparative Examples 1-4 all showed different degrees of precipitation under the same storage conditions, and their centrifugal precipitation rates were significantly higher. This result shows that the products prepared by the methods provided by the present application have physical stability under heat storage conditions.

[0049] Comparing the results of Comparative Example 1 with Comparative Examples 1 and 4, both of which lack the laccase catalysis step or the entire enzymatic grafting process, the products of both examples produced a large amount of precipitate after storage. The technical mechanism is that the method of the present application forms a covalent grafting structure between pea protein isolate and feruloylated sugar beet pectin through laccase catalysis. This structure stably connects the hydrophilic pectin molecular chain to the protein surface, forming an effective steric hindrance layer. This steric hindrance layer remains intact even under long-term heat action, continuously preventing the mutual aggregation and sedimentation of protein molecules, thereby maintaining the long-term physical stability of the system. In the absence of this covalent grafting structure, the protein and polysaccharide are only physically mixed, and cannot prevent the aggregation and sedimentation of the protein in a thermodynamically unstable state.

[0050] Comparative Example 1 and Comparative Examples 2 and 3, the latter two of which were conducted under uncontrolled oxidation environment or in the absence of ascorbic acid, respectively, the long-term stability of the products was also significantly reduced. This indicates that it is necessary to construct a controlled redox microenvironment in the enzymatic grafting reaction step. Under uncontrolled oxidation conditions (Comparative Example 2), the laccase-catalyzed radical reaction pathway lacks specificity, and is prone to initiate homocoupling between proteins and proteins, forming insoluble macromolecular aggregates that gradually settle during storage. In the absence of ascorbic acid (Comparative Example 3), the mildness and selectivity of the reaction are reduced, which also affects the structural integrity of the final grafted product, resulting in a decrease in its stabilization ability. Therefore, by controlling the reaction microenvironment to ensure the efficient progress of the protein-polysaccharide heterocoupling reaction, it is a technical prerequisite to obtain a long-term stable product.

[0051] While embodiments of the present application have been shown and described, it is to be understood that various modifications, substitutions, combinations, and alterations can be made to the embodiments without departing from the principles and spirit of the application, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a high protein cow milk, characterized by, The method comprises the following steps: S1: preparing a mixed solution: mixing pea protein isolate and feruloylated sugar beet pectin in water to obtain a mixed solution; S2: enzymatic grafting reaction: adding laccase to the mixed solution under a controlled redox microenvironment to perform an enzymatic grafting reaction, to obtain a dispersion liquid containing a functional protein-pectin graft copolymer; The construction and maintenance of the controlled redox microenvironment comprises: before adding the laccase, performing a deoxygenation treatment on the mixed solution; During the enzymatic grafting reaction, the dissolved oxygen concentration of the reaction system is maintained at a preset low concentration window by micro-inhaling oxygen-containing gas; S3: enzyme inactivation: performing a heating treatment on the dispersion liquid obtained in S2 to inactivate the laccase, to obtain a functional protein-pectin graft copolymer dispersion liquid; S4: preparing a finished product: mixing the functional protein-pectin graft copolymer dispersion liquid with a milk base, and then performing homogenization and ultra-high temperature sterilization treatment, to obtain high-protein milk.

2. The method of claim 1, wherein, In S2, the deoxygenation treatment reduces the dissolved oxygen concentration of the mixed solution to less than 1.0 mg / L; and the preset low concentration window is 1.0-2.5 mg / L.

3. The method of claim 1, wherein, In S1, the dry basis mass ratio of the pea protein isolate to the feruloylated sugar beet pectin is 10:1 to 20:

1.

4. The method of claim 1, wherein, In S2, after the deoxygenation treatment and before the addition of the laccase, a step of adding ascorbic acid to the mixed solution is further included.

5. The method of claim 4, wherein, The addition amount of the ascorbic acid is 0.05-0.2 parts by mass of the pea protein isolate on a dry basis.

6. The method of claim 1, wherein, In S2, the temperature of the enzymatic grafting reaction is 45-55℃, and the reaction time is 1.5-3.0 hours.

7. The method of claim 1, wherein, In S4, the homogenization treatment is performed at a first stage pressure of 15-25 MPa and a second stage pressure of 3-5 MPa.

8. The method of claim 1, wherein, The feruloylated sugar beet pectin used in S1 has a ferulic acid content of 0.5-1.5 parts.

9. The method of claim 1, wherein, The ultra-high temperature sterilization treatment is performed at a temperature of 137-142℃ for 4-6 seconds.

10. Use of the high-protein milk according to claim 1 in the preparation of a high-stability milk product.

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