Method for preparing whey protein

Abstract

The present application relates to a method for preparing alpha-lactalbumin-enriched whey protein. The method comprises the following steps: a step of defatting a milk source to obtain skim milk; a step of heat aggregation reaction, in which the skim milk is heated to 80-99 DEG C for main sterilization treatment to obtain a heat aggregation reaction product, and the holding time of the sterilization treatment is 10-60 s; and a step of whey separation, in which the alpha-lactalbumin-enriched whey protein solution is obtained through microfiltration treatment. The preparation method of the present application is simple, greatly realizes the enrichment of alpha-lactalbumin, does not introduce any exogenous components during the whole preparation process, is pure in material, has small one-time equipment investment, low consumable maintenance cost, and is convenient for large-scale industrial production.

Description

Technical Field

[0001] This invention belongs to the field of deep processing of dairy products, and specifically relates to a method for preparing whey protein enriched with α-lactalbumin. Background Technology

[0002] Whey protein is rich in a variety of bioactive components and has enormous potential for nutritional and functional applications. It is mainly composed of β-lactoglobulin, α-lactalbumin, bovine serum albumin, immunoglobulins, and lactoferrin. These proteins not only provide high-quality amino acids but have also attracted widespread attention due to their unique structure and bioactivity.

[0003] Alpha-lactalbumin is a protein primarily found in mammalian milk, especially breast milk and cow's milk. It is an important globulin, belonging to the whey protein family. Alpha-lactalbumin has various physiological functions, including participation in lactose synthesis, aiding in lactose production by binding to β-1,4-glucosyltransferase (lactose synthase). Besides its role in lactation, alpha-lactalbumin has other potential biological functions, such as anti-tumor, antibacterial, and immunomodulatory effects. It is rich in essential amino acids, particularly tryptophan, and is therefore considered to have high nutritional value, often appearing as a nutrient in foods and supplements. Furthermore, alpha-lactalbumin is a hot research topic, especially regarding its potential health benefits such as antioxidant and anti-inflammatory effects. In some cases, it is also used as a drug carrier or in medical research.

[0004] Existing technologies for enriching α-lactalbumin in whey protein typically employ techniques such as ion exchange chromatography, gel filtration chromatography, affinity chromatography, and isoelectric focusing. These technologies involve substantial initial equipment investment, extremely high consumable maintenance costs, and significant challenges for large-scale industrial production. Furthermore, the separation process requires the addition of large quantities of sodium hydroxide, dilute hydrochloric acid, citric acid, rennet, and other materials for chemical and biological reactions, making the process relatively complex.

[0005] The following are common separation methods:

[0006] Ion exchange chromatography

[0007] Ion exchange chromatography separates α-lactalbumin and β-lactoglobulin based on their difference in protein charge. Under specific pH conditions, α-lactalbumin and β-lactoglobulin have different surface charges, resulting in different binding forces to ion exchange resins. By controlling a salt concentration gradient, one protein is eluted first, followed by the other. α-lactalbumin and β-lactoglobulin are separated in different elution stages, thus achieving efficient purification.

[0008] Gel filtration chromatography (molecular sieve chromatography)

[0009] Gel filtration chromatography separates proteins based on differences in molecular size. α-lactalbumin has a molecular weight of approximately 14,000 Da, while β-lactoglobulin has a molecular weight of approximately 18,400 Da. This molecular weight difference results in different elution times for them on the gel chromatography column. This method allows for the further separation and purification of these two proteins.

[0010] Affinity chromatography

[0011] If the specific binding ligands for α-lactalbumin or β-lactoglobulin are known, affinity chromatography can be used for selective separation. For example, by using certain sugars or metal ions as ligands, one protein can bind to the ligand while the other is eluted. This method can provide very high purity, but requires the development of specific ligands for each protein.

[0012] Isoelectric focusing method

[0013] Isoelectric focusing separates different proteins based on their isoelectric point (pI). Alpha-lactalbumin and β-lactoglobulin have different isoelectric points, thus they focus in different pH regions. Under the influence of an electric field, α-lactalbumin and β-lactoglobulin will focus at their respective isoelectric points in the gel, achieving efficient separation.

[0014] SDS-PAGE electrophoresis

[0015] Electrophoresis (such as SDS-PAGE) is commonly used to analyze and validate purified proteins. Electrophoresis can confirm the purity and molecular weight of isolated α-lactalbumin and β-lactoglobulin.

[0016] Reference 1 discloses a new method for preparing compositions and related products enriched with α-lactalbumin, and their use in, for example, infant formula. α-lactalbumin can be enriched from crude whey protein solutions by selectively crystallizing β-lactoglobulin and removing β-lactoglobulin crystals.

[0017] Reference 2 discloses a method for preparing whey enriched with α-lactalbumin. It utilizes rennet to enzymatically hydrolyze casein to promote its cross-linking and aggregation. At the same time, it combines the property that most β-lactoglobulins are prone to agglomerate with casein while α-lactalbumin is less affected by it, to separate α-lactalbumin from β-lactoglobulins and casein, which belongs to the field of biological separation.

[0018] Reference 3 discloses a method for preparing α-lactalbumin using membrane technology combined with enzymatic methods. The method mainly involves centrifugation to recover milk fat, ultrafiltration to separate large protein molecules, concentration of the ultrafiltration permeate through a membrane followed by trypsin hydrolysis, and ultrafiltration to concentrate the α-lactalbumin.

[0019] Reference 4 discloses a method for separating and preparing high-purity α-lactalbumin from fresh milk. This method involves purification through a combination of microfiltration and anion exchange chromatography, using a DEAE Large Scale anion exchange chromatography column to adsorb α-lactalbumin.

[0020] Reference 5 discloses a method for concentrating and separating α-lactalbumin. The method mainly involves adjusting the pH value to 11.0-13.0 and 4.4-5.5 in stages, followed by membrane separation or centrifugation, reconstitution, drying, membrane separation or adsorption, and α-lactalbumin adsorbed in anion exchange resin to obtain the α-lactalbumin product.

[0021] Reference 6 discloses a method for separating and purifying α-lactalbumin from whey. The method mainly involves microfiltration separation of skim milk to obtain whey protein solution containing α-lactalbumin. The whey protein solution containing α-lactalbumin is then loaded into a equilibrated strong cation chromatography column. SP Purose 6XL packing material is used in combination with a specific 10-60mM pH4-6 acetate buffer. The flow-through is collected to obtain the separated and purified α-lactalbumin.

[0022] Reference 7 discloses a whey powder enriched with α-lactalbumin and its preparation method. The method includes the following steps: dissolving whey protein powder in water to obtain a solution, adjusting the pH of the solution to 3.6-4.2 with citric acid, stirring or letting stand at 50-60℃ for 0.5-2 hours, centrifuging to obtain a precipitate, washing the precipitate in a citric acid solution with a pH of 3.6-4.2, and then dissolving the precipitate with a salt solution with a pH of 6.5-8.5 to obtain a whey solution enriched with α-lactalbumin, which is then further processed to obtain the whey powder enriched with α-lactalbumin.

[0023] Reference 8 discloses a method for separating α-lactalbumin and β-lactoglobulin. This method primarily involves microfiltration of skim milk, with the permeate being whey containing α-lactalbumin, β-lactoglobulin components, lactose, and other components. The permeate is then subjected to hydrated calcium ion-assisted ultrafiltration to remove lactose and minerals from the whey, followed by aqueous two-phase extraction to achieve the separation of α-lactalbumin and β-lactoglobulin. Step five: The upper and lower phase solutions are sequentially subjected to hydrated calcium ion-assisted ultrafiltration and electrodialysis for desalination, concentrating α-lactalbumin and β-lactoglobulin.

[0024] References

[0025] Reference 1: CN112770637A

[0026] Reference 2: CN102090464A

[0027] Reference 3: CN114805547A

[0028] Reference 4: CN118271424A

[0029] Reference 5: CN114409762A

[0030] Reference 6: CN119320445A

[0031] Reference 7: CN106417613A

[0032] Reference 8: CN118909086A Summary of the Invention

[0033] The problem the invention aims to solve

[0034] Existing techniques for enriching α-lactalbumin in whey protein have the following problems:

[0035] 1) Techniques such as ion exchange chromatography, gel filtration chromatography, affinity chromatography, and isoelectric focusing are typically used, which are relatively complex and difficult to control.

[0036] 2) The separation and purification process requires the addition of large amounts of sodium hydroxide, dilute hydrochloric acid, citric acid, rennet and other materials for chemical and biological reaction treatment, which will introduce a large amount of exogenous components into the product.

[0037] 3) Existing technical solutions require huge initial equipment investment, have extremely high consumable maintenance costs, and are extremely difficult to scale up for industrial production.

[0038] In view of the problems of the prior art, the purpose of this invention is to provide a method for preparing whey protein by enriching α-lactalbumin through a purely physical method. The process is simple and achieves a high degree of enrichment of α-lactalbumin. At the same time, the entire preparation process does not introduce any exogenous components (such as biological enzymes or chemical substances), the materials are pure, and the hardware equipment can be used to achieve mass production based on existing conventional dairy processing equipment. The initial equipment investment is small, the consumable maintenance cost is low, and it is convenient for large-scale industrial production.

[0039] Solution for solving the problem

[0040] [1]. A method for preparing whey protein enriched with α-lactalbumin, characterized in that,

[0041] The method includes the following steps:

[0042] The process of defatting dairy feed to obtain skim milk;

[0043] The thermal aggregation reaction step involves heating the skim milk to 80-99°C for primary sterilization to obtain the processed product from the thermal aggregation reaction. The sterilization treatment is held for 10-60 seconds.

[0044] The whey separation step involves microfiltration to obtain the whey protein solution enriched with α-lactalbumin.

[0045] [2]. The method for preparing whey protein enriched with α-lactalbumin according to [1] is characterized in that,

[0046] In the thermal aggregation reaction step, after the main sterilization treatment, the treated material is further evaporated and concentrated to a solid content of 24-50%.

[0047] [3]. The method for preparing whey protein enriched with α-lactalbumin according to [1] or [2] is characterized in that,

[0048] In the whey separation step, before the microfiltration process, the whey is diluted to a solids content of 5-17%.

[0049] [4]. A method for preparing whey protein enriched with α-lactalbumin according to any one of [1] to [3], characterized in that,

[0050] The processed product obtained from the thermal aggregation reaction step is directly subjected to the whey separation step; or,

[0051] The processed material obtained from the thermal aggregation reaction step is dried to obtain skim milk powder, which is then reconstituted and followed by the whey separation step.

[0052] [5]. A method for preparing whey protein enriched with α-lactalbumin according to any one of [1] to [4], characterized in that,

[0053] Before the thermal aggregation reaction step, the skim milk is subjected to a pre-sterilization treatment, which involves heating the milk to 70-88°C and holding it at that temperature for 12-30 seconds. The temperature in the pre-sterilization treatment is lower than the temperature in the main sterilization treatment.

[0054] [6]. The preparation method according to any one of [1] to [5] is characterized in that the whey separation step does not use rennet or chemical coagulant.

[0055] [7]. The preparation method according to any one of [1] to [6] is characterized in that the milk source material is derived from cow's milk.

[0056] [8]. A method for preparing a formula milk powder, characterized in that,

[0057] The method includes the whey protein preparation method enriched with α-lactalbumin according to [1]~[7].

[0058] [9]. The preparation method according to [8] is characterized in that,

[0059] The method further includes concentrating the whey protein solution enriched with α-lactalbumin by ultrafiltration to obtain a concentrated whey protein solution.

[0060]

[10] . The preparation method according to [9] is characterized in that,

[0061] The method further includes drying the concentrated whey protein solution.

[0062] Technical effects of the invention

[0063] Based on the inventors' research, after undergoing a series of specific treatments in this invention, the proportion of α-lactalbumin in the whey of skim milk is significantly higher than that of skim milk whey that has been directly microfiltered without treatment. This phenomenon is mainly due to the different response characteristics of whey proteins to heat treatment and the interaction between casein and whey proteins.

[0064] First, whey protein is one of the most heat-sensitive proteins in milk, and it denatures during heating. β-lactoglobulin, in particular, is more thermally unstable than α-lactalbumin. During heat treatment, whey protein denatures. Denatured β-lactoglobulin readily forms covalent or non-covalent complexes with casein micelles or κ-casein, thus integrating into the casein micelles. This means that during subsequent microfiltration, this portion of denatured whey protein is retained along with the casein and does not enter the permeate (i.e., whey liquor) of the microfiltration membrane.

[0065] In contrast, α-lactalbumin exhibits relatively high thermal stability. Under certain heat treatment conditions, its denaturation is less than that of β-lactoglobulin, and it binds less to casein micelles. Therefore, after heat treatments such as pasteurization and direct steam sterilization, most β-lactoglobulin may have denatured and bound to casein or aggregated into large molecules, while α-lactalbumin can better maintain its solubility or exist in the form of smaller aggregates. This phenomenon is even more pronounced under high concentration conditions.

[0066] When skim milk that has undergone thermal aggregation and dried skim milk powder are reconstituted, their protein structure and solubility change. Denatured β-lactoglobulin and its complex with casein may be difficult to dissolve completely during reconstitution, or may form large aggregates. These aggregates are more easily retained by the membrane during microfiltration. Therefore, microfiltration membranes are more effective at removing these large molecular aggregates, while relatively smaller, undenatured or slightly denatured α-lactalbumin can pass through the microfiltration membrane into the permeate, resulting in an increase in the relative content of α-lactalbumin in the whey.

[0067] In summary, the proportion of α-lactalbumin in the microfiltered whey of skim milk after heat treatment, evaporation concentration, spray drying and resolidification increased significantly. This is because heat treatment caused a large amount of denaturation of other whey proteins such as β-lactoglobulin, which then combined with casein or formed large molecular aggregates. These aggregates were removed during the microfiltration process, while α-lactalbumin, which has relatively high thermal stability, was retained in the whey, thus increasing its relative content.

[0068] This invention specifically provides a method for preparing whey protein enriched with α-lactalbumin. This method utilizes the thermal aggregation effect of β-lactoglobulin and relies on a purely physical method to prepare whey protein enriched with α-lactalbumin. This achieves the following beneficial effects:

[0069] 1) High purity: The technical solution of this invention can achieve a protein ratio of α-lactalbumin to β-lactoglobulin of 3-5 in whey protein, which is a significant improvement compared to existing technologies, greatly enriching α-lactalbumin. If the raw material is further processed by chromatographic chromatography, the protein ratio of α-lactalbumin to β-lactoglobulin in whey protein can be increased to 9-10.

[0070] 2) The process is simple. This technical solution makes full use of the thermal aggregation effect of β-lactoglobulin and relies solely on pure physical methods to prepare whey protein enriched with α-lactalbumin. The process is very simple and can be achieved through simple processes such as centrifugation, heat sterilization, evaporation concentration, and microfiltration.

[0071] 3) The materials are pure, and the entire preparation process does not introduce exogenous components such as sodium hydroxide, dilute hydrochloric acid, citric acid, or rennet for chemical or biological reaction treatment.

[0072] 4) It facilitates large-scale industrial production. The hardware equipment can be based on existing conventional dairy processing equipment such as separators, pasteurizers, evaporators, and microfiltration membrane separation systems to achieve mass production. The initial equipment investment is small and the consumable maintenance cost is low. Attached Figure Description

[0073] Figure 1 Images of sweet whey protein enriched with α-lactalbumin and sodium lauryl sulfate-polyacrylamide gel.

[0074] Figure 2 Images of sweet whey protein enriched with α-lactalbumin and sodium lauryl sulfate-polyacrylamide gel. Detailed Implementation

[0075] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0076] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0077] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0078] In this invention, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0079] In this invention, the range of values ​​represented by "value A to value B" refers to the range that includes the endpoint values ​​A and B.

[0080] In this invention, the term "about" is used to define that the numerical ranges and parameters of this invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. Unless otherwise explicitly stated, it should be understood that all ranges, quantities, values, and percentages used in this invention are modified by the term "about". Here, "about" generally means that the actual value is within ±3%, ±1%, ±0.5%, or ±0.1% of a specific value or range. Furthermore, the values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0081] In this invention, terms such as "some specific / preferred embodiments," "other specific / preferred embodiments," and "implementation" refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to a particular embodiment that are included in at least one of the embodiments described herein, and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0082] In this invention, all unit names used are international standard unit names, and unless otherwise stated, the "%" used refers to weight or mass percentage content.

[0083] In this invention, "infants and toddlers" refers to the human group under the age of 3 years, including infants aged 0-6 months, older infants aged 6-12 months, and toddlers aged 12-36 months.

[0084] In this invention, "children" refers to a group of human beings who are older than 3 years old and younger than 12 years old and are in the growth and development stage.

[0085] In this invention, "adolescents" refers to a group of people aged 12 years or older but under 18 years old who are in the growth and development stage.

[0086] In this invention, "middle-aged and elderly" refers to the human population aged 45 and above.

[0087] In this invention, "animal milk" refers to the fluid obtained from the mammary glands of a mammal in the process of lactation. The term "animal milk" should be interpreted broadly and encompasses both raw milk (i.e., the fluid obtained directly from the mammary glands) and standardized dairy products (such as skim milk or whole milk).

[0088] <Methods for preparing whey protein enriched with α-lactalbumin>

[0089] This invention provides a method for preparing whey protein enriched with α-lactalbumin. The method includes the following steps: a defatting step of the milk source to obtain skim milk; a thermal aggregation reaction step, in which the skim milk is heated to 80-99°C for primary sterilization treatment to obtain the processed product of the thermal aggregation reaction, the sterilization treatment holding time being 10-60 seconds; and a whey separation step, in which a whey protein solution enriched with α-lactalbumin is obtained by microfiltration. The resulting whey protein solution is enriched with α-lactalbumin.

[0090] There are no particular restrictions on the milk raw materials used in this invention. For example, they can be fresh milk derived from various animal milks, such as cow milk, buffalo milk, sheep milk, horse milk, camel milk, etc. Preferably, they can be derived from cow milk.

[0091] There are no particular restrictions on the methods for defatting raw milk; it can be done by methods such as centrifugation. For example, the temperature during centrifugation can be controlled at 48-55℃, and the centrifuge speed can be controlled at 4000-4900 r / min.

[0092] Through defatting treatment, the fat content in skim milk is controlled to be below 3% by mass, preferably below 2%, and more preferably below 1%.

[0093] In some specific implementations, the skim milk may be pre-sterilized before the thermal aggregation reaction step. The pre-sterilization treatment may be, for example, pasteurization. The temperature of the pre-sterilization treatment is lower than the temperature of the main sterilization treatment, for example, 70-88°C, and the holding time is, for example, 12-30 seconds.

[0094] In some specific implementation schemes, the material is cooled to 10-60°C after pre-sterilization treatment.

[0095] The method of performing the primary sterilization treatment on the skim milk in the thermal aggregation reaction is not particularly limited as long as the required temperature and holding time are reached to achieve the protein thermal coagulation reaction. For example, direct steam injection sterilization is preferred.

[0096] From the perspective of facilitating the denaturation of most β-lactoglobulin and its binding or aggregation into large molecules with casein, thereby enabling α-lactalbumin to better maintain its solubility or exist in smaller aggregates, in some preferred embodiments, the temperature for the primary sterilization treatment during the thermal aggregation reaction is 80-99°C, preferably 85-98°C, for example, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, or 98°C; the holding time for the primary sterilization treatment during the thermal aggregation reaction is 10-60 seconds, preferably... The duration is 12-60 seconds, for example, it can be 12s, 13s, 14s, 15s, 16s, 17s, 18s, 19s, 20s, 21s, 22s, 23s, 24s, 25s, 26s, 27s, 28s, 29s, 30s, 31s, 32s, 33s, 34s, 35s, 36s, 37s, 38s, 39s, 40s, 41s, 42s, 43s, 44s, 45s, 46s, 47s, 48s, 49s, 50s, 51s, 52s, 53s, 54s, 55s, 56s, 57s, 58s, 59s, 60s. By controlling the temperature and holding time of the main sterilization treatment, most of the β-lactoglobulin can be denatured and bound to casein or aggregate into large molecules. As a result, during the subsequent microfiltration process, this denatured whey protein will be retained along with the casein and will not enter the permeate (i.e., whey) of the microfiltration membrane. Meanwhile, α-lactalbumin binds less to casein micelles and can better maintain its solubility or exist in the form of smaller aggregates.

[0097] In some specific embodiments, during the thermal aggregation reaction step, after the primary sterilization treatment, the material is cooled to 40-70°C for temporary storage and then concentrated using an evaporator. The evaporation and concentration temperatures can be, for example, a first-effect temperature of 65-85°C, a second-effect temperature of 60-80°C, and a third-effect temperature of 55-75°C. In some preferred embodiments, the evaporation and concentration are carried out until the solid content is 24-50%, followed by cooling to 10-50°C for temporary storage.

[0098] In some specific implementations, the processed product obtained from the thermal aggregation reaction step is subjected to the whey separation step.

[0099] In other specific embodiments, the processed product obtained from the thermal aggregation reaction step is dried to obtain skim milk powder, which is then reconstituted before the whey separation step is performed. The reconstitution of the skim milk powder involves simply re-dissolving it, for example, by dissolving it in water at 40-55°C. The method for obtaining the skim milk powder is, for example, spray drying or freeze drying. In some specific embodiments, the skim milk powder is a moderately heated skim milk powder.

[0100] In some specific implementations, during the whey separation step, dilution is performed before the microfiltration process to achieve a solids content of 5-17%.

[0101] In the whey separation step of this invention, the microfiltration treatment is used to remove most of the denatured β-lactoglobulin and the large molecules bound to or aggregated by casein. In some specific embodiments, considering both efficiency and separation effect, the microfiltration separation conditions include: a microfiltration membrane pore size of 0.05–0.2 μm, preferably 0.1–0.2 μm; an operating temperature of 5–50°C, preferably 10–50°C; and an operating pressure of 0.1–2 bar, preferably 0.5–1.5 bar. There are no particular restrictions on the material and form of the microfiltration membrane in principle. In some preferred embodiments, the microfiltration membrane can be made of polyethersulfone; the microfiltration membrane can be further preferably a spiral wound membrane. In some specific embodiments, the ratio (L / h) of the dialysate (whey side) flow rate to the concentrate (casein side) flow rate in the microfiltration separation can be, for example, 3.0–10.0, preferably 4.0–10.0. The dialysate (whey side) obtained after microfiltration is a whey protein solution enriched with α-lactalbumin.

[0102] In some specific implementations, the whey separation step does not use rennet or chemical coagulants, so that no exogenous components are introduced into the entire preparation process, the materials are pure, and since α-lactalbumin is enriched solely by physical methods, the process is simple, the cost of consumables is low, and it is easy to carry out large-scale industrial production.

[0103] In some specific embodiments, the mass ratio of α-lactalbumin to β-lactoglobulin in the whey protein solution enriched with α-lactalbumin is 1.50-10.00, for example, 2.00-8.00. In some preferred embodiments, the mass ratio of α-lactalbumin to β-lactoglobulin in the whey protein solution enriched with α-lactalbumin is 3.00-6.00, for example, 3.00, 3.50, 4.00, 4.50, 5.00, 5.50, or 6.00. In some preferred embodiments, the mass ratio of α-lactalbumin to total protein in the whey protein solution enriched with α-lactalbumin is 0.50 or more, more preferably 0.60 or more, further preferably 0.70 or more, and even more preferably 0.80 or more.

[0104] <Preparation Method of Formula Milk Powder>

[0105] This invention provides a method for preparing formula milk powder. Since the whey protein product obtained by the above-mentioned method for preparing whey protein enriched with α-lactalbumin is used in this preparation method, this preparation method also includes the above-mentioned method for preparing whey protein enriched with α-lactalbumin of this invention.

[0106] In some specific embodiments, the method further includes concentrating the whey protein solution enriched with α-lactalbumin by ultrafiltration to obtain a concentrated whey protein solution. In some specific embodiments, the ultrafiltration conditions may be, for example, using a 10-30 kDa ultrafiltration system at 10-25°C.

[0107] In some specific embodiments, the method further includes drying the concentrated whey protein solution. The drying method may include, for example, spray drying or freeze drying. In some specific embodiments, the inlet air temperature of the spray dryer is, for example, 160-180°C, and the outlet air temperature is, for example, 85-90°C.

[0108] In addition to the whey protein products obtained by the above-described method for preparing whey protein by enriching α-lactalbumin, the formula milk powder of the present invention may also contain any one or more of other components, such as other protein components, carbohydrate components, oil components, mineral components, vitamin components, nucleotide components, and probiotic components.

[0109] The present invention does not make any particular limitation on the target audience of the formula milk powder. For example, the formula milk powder can be infant formula milk powder, children's formula milk powder, adolescent formula milk powder, or middle-aged and elderly formula milk powder, etc.

[0110] Example

[0111] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0112] Experimental materials: Fresh milk (Flyhe Original Ecological Farming Company, fresh milk within 12 hours of arrival at the factory)

[0113] Example 1:

[0114] 1. Centrifugal skimming: Fresh milk is heated to 53°C and then separated from cream using a separator at a speed of 4500 r / min. The fat content in the skimmed milk is controlled to be less than 1% by mass.

[0115] 2. Pasteurization: Heat the skim milk to 85°C and keep it at that temperature for 24 seconds for pasteurization, then cool it down to 55°C for temporary storage.

[0116] 3. Thermal aggregation reaction: The skim milk is steam-injected again for sterilization and heated to 95°C. The temperature is maintained for 24 seconds to carry out the protein thermal aggregation reaction. The temperature is then cooled to 60°C for temporary storage. After sterilization, the skim milk enters the evaporator for concentration. The first effect temperature is 80°C, the second effect temperature is 75°C, and the third effect temperature is 70°C. The concentration is carried out until the solid content is 36%. The temperature is then cooled to 15°C for temporary storage.

[0117] 4. Whey separation: After the thermal aggregation reaction, the skim milk is diluted to a solids content of 9.5% and then enters a 0.1μm microfiltration system at 15℃. The ratio of dialysate (whey side) flow rate to concentrate (casein side) flow rate (L / h) is 5.8. The dialysate (whey side) is the whey protein solution enriched with α-lactalbumin.

[0118] 5. Whey Concentration: The whey protein solution from step 4 is introduced into a 30Kd ultrafiltration system at 15℃ to remove lactose and ash. The ratio of the flow rate of the dialysate (lactose side) to the flow rate of the concentrate (whey side) (L / h) is 7.3. The dialysate (lactose side) is the concentrated whey protein solution enriched with α-lactalbumin.

[0119] 6. Drying: After concentration, the whey protein solution is spray-dried to obtain whey protein powder enriched with α-lactalbumin.

[0120] Example 2:

[0121] 1. Centrifugal skimming: Fresh milk is heated to 48°C and then separated from cream using a separator. The separator rotates at 4900 r / min, and the fat content in the skimmed milk is controlled to be less than 1% by mass.

[0122] 2. Pasteurization: Heat the skim milk to 70°C and keep it at that temperature for 30 seconds for pasteurization. Then cool it down to 60°C and store it temporarily.

[0123] 3. Thermal aggregation reaction: The skim milk is steam-injected again for sterilization and heated to 85°C. It is kept at this temperature for 60 seconds to carry out the protein thermal aggregation reaction. It is then cooled to 70°C for temporary storage. After sterilization, the skim milk enters the evaporator for concentration. The first effect temperature is 85°C, the second effect temperature is 80°C, and the third effect temperature is 75°C. It is concentrated until the solid content is 50%, and then cooled to 50°C for temporary storage.

[0124] 4. Whey separation: After the thermal aggregation reaction, the skim milk is diluted to a solid content of 17% and then enters a 0.1μm microfiltration system at 50℃. The ratio of the flow rate of the dialysate (whey side) to the flow rate of the concentrate (casein side) (L / h) is 10. The dialysate (whey side) is the whey protein solution enriched with α-lactalbumin.

[0125] 5. Whey Concentration: The whey protein solution from step 4 is introduced into a 30Kd ultrafiltration system at 25°C to remove lactose and ash. The ratio of the flow rate of the dialysate (lactose side) to the flow rate of the concentrate (whey side) (L / h) is 10. The dialysate (lactose side) is the concentrated whey protein solution enriched with α-lactalbumin.

[0126] 6. Drying: After concentration, the whey protein solution is spray-dried to obtain whey protein powder enriched with α-lactalbumin.

[0127] Example 3:

[0128] 1. Centrifugal skimming: Fresh milk is heated to 55°C and then separated from cream using a separator. The separator rotates at 4000 r / min, and the fat content in the skimmed milk is controlled to be less than 1% by mass.

[0129] 2. Pasteurization: Heat the skim milk to 88°C and keep it at that temperature for 12 seconds for pasteurization. Then cool it down to 10°C and store it temporarily.

[0130] 3. Thermal aggregation reaction: The skim milk is sterilized again by direct steam injection and heated to 98°C. The temperature is maintained for 60 seconds to carry out the protein thermal aggregation reaction. The temperature is then cooled to 40°C for temporary storage. After sterilization, the skim milk enters the evaporator for concentration. The first effect temperature is 65°C, the second effect temperature is 60°C, and the third effect temperature is 55°C. The concentration is carried out until the solid content is 24%. The temperature is then cooled to 10°C for temporary storage.

[0131] 4. Whey separation: After the thermal aggregation reaction, the skim milk is diluted to a solid content of 5% and then enters a 0.1μm microfiltration system at 10℃. The ratio of the flow rate of the dialysate (whey side) to the flow rate of the concentrate (casein side) (L / h) is 4. The dialysate (whey side) is the whey protein solution enriched with α-lactalbumin.

[0132] 5. Whey Concentration: The whey protein solution from step 4 is introduced into a 10Kd ultrafiltration system at 10℃ to remove lactose and ash. The ratio of the flow rate of the dialysate (lactose side) to the flow rate of the concentrate (whey side) (L / h) is 5. The dialysate (lactose side) is the concentrated whey protein solution enriched with α-lactalbumin.

[0133] 6. Drying: After concentration, the whey protein solution is spray-dried to obtain whey protein powder enriched with α-lactalbumin.

[0134] Example 4:

[0135] After performing the same steps 1 to 3 as in Example 1, the product is dried to obtain skim milk powder for temporary storage.

[0136] After the skim milk powder is dissolved in water at 40-55°C to reconstitute it, the same steps 4-6 as in Example 1 are performed.

[0137] Comparative Example 1:

[0138] 1. Centrifugal skimming: Fresh milk is heated to 53°C and then separated from cream using a separator at a speed of 4500 r / min. The fat content in the skimmed milk is controlled to be less than 1% by mass.

[0139] 2. Pasteurization: Heat the skim milk to 85°C and keep it at that temperature for 24 seconds for pasteurization, then cool it down to 55°C for temporary storage.

[0140] 3. Thermal aggregation reaction: The skim milk is steam-injected again for sterilization and heated to 125°C. The temperature is maintained for 4 seconds to carry out the protein thermal aggregation reaction. The temperature is then cooled to 60°C for temporary storage. After sterilization, the skim milk enters the evaporator for concentration. The first effect temperature is 80°C, the second effect temperature is 75°C, and the third effect temperature is 70°C. The concentration is carried out until the solid content is 36%. The temperature is then cooled to 15°C for temporary storage.

[0141] 4. Whey separation: After the thermal aggregation reaction, the skim milk is diluted to a solids content of 9.5% and then enters a 0.1μm microfiltration system at 15℃. The ratio of dialysate (whey side) flow rate to concentrate (casein side) flow rate (L / h) is 5.8. The dialysate (whey side) is the whey protein solution enriched with α-lactalbumin.

[0142] 5. Whey Concentration: The whey protein solution from step 4 is introduced into a 30Kd ultrafiltration system at 15℃ to remove lactose and ash. The ratio of the flow rate of the dialysate (lactose side) to the flow rate of the concentrate (whey side) (L / h) is 7.3. The dialysate (lactose side) is the concentrated whey protein solution enriched with α-lactalbumin.

[0143] 6. Drying: After concentration, the whey protein solution is spray-dried to obtain whey protein powder enriched with α-lactalbumin.

[0144] Comparative Example 2:

[0145] 1. Centrifugal skimming: Fresh milk is heated to 53°C and then separated from cream using a separator at a speed of 4500 r / min. The fat content in the skimmed milk is controlled to be less than 1% by mass.

[0146] 2. Pasteurization: Heat the skim milk to 72°C and keep it at that temperature for 24 seconds for pasteurization. Then cool it down to 15°C and store it temporarily.

[0147] 3. Whey separation: After pasteurization, skim milk enters a 0.1μm microfiltration system, with a flow rate ratio (L / h) of dialysate (whey side) to concentrate (casein side) of 5.8.

[0148] 4. Whey concentration: The whey protein solution from step 3 is introduced into a 30Kd ultrafiltration system at 15°C to remove lactose and ash. The ratio of dialysate (lactose side) flow rate to concentrate (whey side) flow rate (L / h) is 7.3.

[0149] 5. Drying: After concentration, the whey protein solution is spray-dried to obtain whey protein powder enriched with α-lactalbumin.

[0150] <Evaluation of Sweet Whey Protein Products>

[0151] For the whey protein solutions obtained in each specific embodiment and comparative example (i.e., the whey protein solutions obtained in the "whey separation" step of each embodiment and comparative example), Qingdao Huace Testing Technology Co., Ltd. was commissioned to conduct testing and verification of the technical solution in the patent. The specific testing methods are as follows:

[0152] Protein content was determined using Method I of GB 5009.5-2016. Non-protein nitrogen content was determined using GB / T 21704-2008.

[0153] The content of α-lactalbumin and β-lactoglobulin was determined using CTI's internal detection method, specifically as follows: After trypsin digestion into specific peptides, the specific peptides of the target protein were determined by stable isotope dilution liquid chromatography-tandem mass spectrometry, with quantification using the internal standard method. Based on the principle that 1 mole of target protein digestion generates 1 mole of specific peptide, the content of α-lactalbumin and β-lactoglobulin in the sample was measured.

[0154] The component analysis results of the whey protein solutions obtained in each specific embodiment and comparative example are shown in Table 1.

[0155] Table 1. Composition content of whey protein solution

[0156]

[0157] This invention selectively alters the aggregation state of β-lactoglobulin by controlling the conditions in the thermal aggregation reaction, thereby creating conditions for subsequent microfiltration separation and ultimately achieving efficient enrichment of α-lactalbumin in whey. As shown in Table 1, the whey protein solution preparation method according to this invention stably achieves a significant increase in the ratio of α-lactalbumin to β-lactoglobulin, greatly maximizing the enrichment of α-lactalbumin. The results from the examples and comparative examples demonstrate that controlling the conditions in the thermal aggregation reaction is crucial; failure to meet the specific conditions described in this invention will prevent the achievement of its technical effects.

[0158] For example, in Comparative Example 1, the temperature of the thermal aggregation reaction was too high. It is speculated that due to the irreversible denaturation of α-lactalbumin, it could not be fully separated from β-lactoglobulin in the whey separation step. Therefore, the proportion of α-lactalbumin in the resulting whey protein solution decreased, and α-lactalbumin enrichment could not be achieved. In Comparative Example 2, the sample was only pasteurized at a lower temperature. Because this was insufficient to fully denature and aggregate β-lactoglobulin, it could not be fully separated from α-lactalbumin in the whey separation step. Therefore, the proportion of α-lactalbumin in the resulting whey protein solution decreased, and α-lactalbumin enrichment could not be achieved.

[0159] The whey protein solutions obtained in Examples 1-4 achieved efficient enrichment of α-lactalbumin in whey, stabilizing the mass ratio of α-lactalbumin to β-lactoglobulin in whey protein between 3 and 5.

Claims

1. A method for preparing whey protein enriched with α-lactalbumin, characterized in that, The method includes the following steps: The process of defatting dairy feed to obtain skim milk; The thermal aggregation reaction step involves heating the skim milk to 80-99°C for primary sterilization to obtain the processed product from the thermal aggregation reaction. The primary sterilization treatment is held for 10-60 seconds. The whey separation step involves microfiltration to obtain the whey protein solution enriched with α-lactalbumin.

2. The method for preparing whey protein enriched with α-lactalbumin according to claim 1, characterized in that, In the thermal aggregation reaction step, after the main sterilization treatment, the treated material is further evaporated and concentrated to a solid content of 24-50%.

3. The method for preparing whey protein enriched with α-lactalbumin according to claim 1 or 2, characterized in that, In the whey separation step, before the microfiltration process, the whey is diluted to a solids content of 5-17%.

4. The method for preparing whey protein enriched with α-lactalbumin according to any one of claims 1 to 3, characterized in that, The processed product obtained from the thermal aggregation reaction step is directly subjected to the whey separation step; or, The processed material obtained from the thermal aggregation reaction step is dried to obtain skim milk powder, which is then reconstituted and followed by the whey separation step.

5. The method for preparing whey protein enriched with α-lactalbumin according to any one of claims 1 to 4, characterized in that, Before the thermal aggregation reaction step, the skim milk is subjected to a pre-sterilization treatment, which involves heating the milk to 70-88°C and holding it at that temperature for 12-30 seconds. The temperature in the pre-sterilization treatment is lower than the temperature in the main sterilization treatment.

6. The preparation method according to any one of claims 1 to 5, characterized in that, The whey separation step does not use rennet or chemical coagulants.

7. The preparation method according to any one of claims 1 to 6, characterized in that, The milk source is derived from cow's milk.

8. A method for preparing formula milk powder, characterized in that, The method includes the whey protein preparation method enriched with α-lactalbumin according to claims 1 to 7.

9. The preparation method according to claim 8, characterized in that, The method further includes concentrating the whey protein solution enriched with α-lactalbumin by ultrafiltration to obtain a concentrated whey protein solution.

10. The preparation method according to claim 9, characterized in that, The method further includes drying the concentrated whey protein solution.

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