Method for preparing high-solubility walnut protein powder through enzymolysis

Abstract

The invention discloses a method for preparing high-solubility walnut protein powder through enzymolysis, and belongs to the technical field of microbial materials. The invention provides a novel method for preparing high-solubility walnut protein powder. The novel method comprises the steps of multi-step enzymolysis, microbiological post-treatment, desalination, debitterization, purification, concentration, drying and the like. Compared with the traditional method, the walnut protein powder prepared by the method disclosed by the invention is remarkably improved in the aspects of solubility, flavor, stability, nutritional value and the like, and has a wide market application prospect. In the food industry, the protein additive can be used as a high-quality protein additive to be applied to products such as various drinks, dairy products, baked foods and the like, and the nutritional quality and the taste of the products are improved. In the field of health care products, the walnut protein powder with high solubility can be better absorbed and utilized by human bodies, and more effective nutritional supplement is provided for consumers.

Description

Technical Field

[0001] This invention belongs to the field of microbial materials technology, specifically relating to a method for preparing highly soluble walnut protein powder through enzymatic hydrolysis. Background Art

[0002] Walnuts, as a high-protein, high-fat food, possess significant nutritional, economic, and medicinal value. In terms of consumption, walnuts can be eaten directly or added as an ingredient to other nutritional products to enhance their functionality. However, in terms of deep processing, existing research mainly focuses on the preparation of walnut oil, while research on walnut protein powder is relatively limited, thus reducing the added value of walnuts.

[0003] Walnut protein mainly consists of albumin, globulin, prolamins, and glutenin, with a protein content as high as 24%, and is rich in essential amino acids. However, glutenin is insoluble in water, resulting in poor water solubility of walnut protein. During deep processing, the solubility of the protein decreases due to processing conditions, reducing its digestibility and absorption in the human body, and hindering the body's absorption and utilization of its nutritional value. Therefore, improving the solubility of walnut protein powder is crucial.

[0004] Enzymatic hydrolysis modification technology, as a type of biomodification, has advantages such as mild conditions, high specificity, and high safety, and is widely used in protein modification. Enzymatic hydrolysis modification technology mainly increases the solubility of proteins in water by breaking down peptide bonds, exposing the hydrophilic groups within the protein.

[0005] For example, Chinese patent application CN201410076807.3 discloses a method for preparing highly soluble walnut protein powder through moderate enzymatic hydrolysis, comprising the following steps: Step 1, mixing defatted walnut powder and purified water at a mass ratio of 1:6 to 1:16, and then stirring evenly to prepare a walnut defatted powder dispersion; Step 2, adding protease to the walnut defatted powder dispersion, wherein the amount of protease added per gram of defatted walnut powder is 250-3000U, and heating the walnut defatted powder dispersion to 30-70℃ for 10-40 minutes for enzymatic hydrolysis to obtain an enzymatic hydrolysate. The method of the present invention is simple to operate, has mild conditions, and a short production cycle for highly soluble walnut protein powder; the prepared highly soluble walnut protein powder has high nutritional value and good taste.

[0006] The main problems with existing technologies are: 1) Enzymatic hydrolysis schemes (with single or arbitrary enzyme selection and combinations) fail to effectively balance the relationship between hydrolysis degree, solubility improvement, and bitterness control; 2) Insufficient attention is paid to the thermal solubility (thermal stability) of the final product; 3) There is a lack of effective post-processing methods (such as targeted desalting and debittering, and drying protection) to maximize solubility; 4) It is difficult to simultaneously achieve ideal comprehensive performance indicators (high solubility + low bitterness + high thermal stability). Therefore, developing a new, efficient, controllable enzymatic hydrolysis method for preparing walnut protein powder that can simultaneously solve the problems of solubility, bitterness, and thermal stability is of great significance. Summary of the Invention

[0007] This invention addresses the problems of existing technologies by developing a novel method for preparing highly soluble walnut protein powder through enzymatic hydrolysis. This method, through a series of steps including multi-step enzymatic hydrolysis, microbial post-treatment, desalting and debittering purification, concentration, and drying, effectively solves the problems of imbalance between degree of hydrolysis, solubility improvement, and bitterness control; insufficient attention to thermal solubility; lack of effective post-treatment methods; and difficulty in achieving ideal overall performance indicators in existing technologies.

[0008] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: A method for preparing highly soluble walnut protein powder by enzymatic hydrolysis includes the following steps: (1) Raw material pretreatment: Mix defatted walnut powder with water, adjust the solid-liquid mass ratio to 1:10-20, adjust the pH to 6.0-8.0, and obtain a protein suspension; (2) First step of enzymatic hydrolysis: Add flavor protease to the protein suspension in step (1) at a concentration of 0.5%-2.0% of the substrate mass, and react for 0.5-2 hours at 45-55°C and pH 6.5-7.5. (3) Second step of enzymatic hydrolysis: Adjust the pH of the enzymatic hydrolysate obtained in step (2) to 7.0-9.0, add alkaline protease, the amount of which is 1.0%-3.0% of the mass of the enzymatic hydrolysate, and react at 50-60°C for 1-4 hours; (4) Third step of enzymatic hydrolysis: Adjust the pH of the enzymatic hydrolysate obtained in step (3) to 6.0-7.0, add transglutaminase, soybean peptides and active polysaccharides, react at 40-50°C for 0.5-2 hours, and after completion, add enzyme inactivation treatment at 80-95°C for 10 minutes. (5) Microbial post-treatment: Cool the enzymatic hydrolysate obtained in step (4) to room temperature, inoculate it with a compound beneficial bacteria freeze-dried agent for fermentation treatment. The amount of compound beneficial bacteria freeze-dried agent inoculated is 2%-5% of the mass of the enzymatic hydrolysate. Ferment at a constant temperature of 25-30°C for 8-12 hours. After fermentation, the fermentation broth is subjected to enzyme inactivation treatment by placing it in an environment of 80-95°C for 10-15 minutes to terminate enzyme activity. Then, impurities and insoluble substances in the fermentation broth are removed by centrifugation. The centrifugation speed is controlled at 4000-6000 rpm and the centrifugation time is 15-20 minutes. Collect the supernatant. (6) Desalting and debittering purification: The supernatant obtained in step (5) is ultrafiltered with an ultrafiltration membrane with a molecular weight cutoff of 3-10 kDa, and then nanofiltration is performed with a nanofiltration membrane with a molecular weight cutoff of 200-500 Da for further desalting and debittering. The nanofiltration retentate is collected. (7) Concentration and drying: The retentate obtained in step (6) is concentrated to a solid content of 20-30%, and 1-5% of maltodextrin by weight of the concentrate is added as a protective agent. The mixture is stirred evenly and then freeze-dried to obtain the highly soluble walnut protein powder.

[0009] Preferably, the amount of flavor protease added in step (2) is 1.0%-1.5% of the substrate protein mass, and the reaction conditions are 50°C, pH 7.0, and reaction time is 1 hour.

[0010] Preferably, the amount of alkaline protease added in step (3) is 1.5%-2.5% of the mass of the enzymatic hydrolysate, and the reaction conditions are 55°C, pH 8.0, and 2-3 hours.

[0011] Preferably, in step (4), the amount of glutamine transaminase added is 0.1%-1.0% of the mass of the hydrolysate, the amount of soybean peptide added is 1%-5% of the mass of the hydrolysate, and the active polysaccharide is trehalose, with an addition amount of 0.5%-3% of the mass of the hydrolysate.

[0012] Preferably, the preparation method of the compound beneficial bacteria freeze-dried agent in step (5) is as follows: 1) Lactococcus lactis strain numbered CGMCC No.1.15072 ( Lactococcus lactis ) and Saccharomyces cerevisiae strain numbered CGMCC No.2.3871 ( Saccharomyces cerevisiae After thawing and activation, Lactococcus lactis is inoculated into MRS liquid medium and cultured at 28-30℃ for 18-24 hours until the absorbance of the culture medium at a wavelength of 600 nm reaches OD. 600 The activated Saccharomyces cerevisiae was inoculated into YPD liquid medium and cultured at 23-25℃ for 18-24 hours until the absorbance value (OD) of the culture medium at 600 nm was reached.600 Reaching 0.8-1.2; 2) Collect Lactococcus lactis and Saccharomyces cerevisiae bacterial cultures separately, centrifuge at 4°C for 10-15 minutes, discard the supernatant, and obtain the corresponding wet cells; 3) Mix the obtained wet Lactococcus lactis cells and wet Saccharomyces cerevisiae cells at a live cell ratio of 1:1 to obtain a compound bacterial mud; 4) The composite bacterial mud is freeze-dried to obtain a composite beneficial bacteria freeze-dried agent, and the total number of effective viable bacteria in the freeze-dried agent is controlled to be not less than 1.0 × 10⁻⁶. 9 CFU / g.

[0013] Preferably, *Lactococcus lactis* strain CGMCC No. 1.15072 and *Saccharomyces cerevisiae* strain CGMCC No. 2.3871 were both purchased from the China General Microbiological Culture Collection Center (CGMCC) and are commercially available, requiring no further biological preservation. The original preservation date of *Lactococcus lactis* was January 25, 2015. The original preservation date of *Saccharomyces cerevisiae* was June 5, 2008.

[0014] Preferably, the molecular weight cutoff of the ultrafiltration membrane in step (6) is 5 kDa, and the molecular weight cutoff of the nanofiltration membrane is 300 Da.

[0015] Preferably, the amount of maltodextrin added as a protective agent in step (7) is 2-3% of the weight of the concentrate.

[0016] Preferably, the freeze-drying method in step (7) is: drying under the conditions of cold hydrazine temperature of -45℃ and vacuum degree of 0.07 MPa for 24 h.

[0017] This invention, through the innovative design of a three-step enzymatic hydrolysis-microbial fermentation synergistic system, combined with the addition of functional excipients and microbial post-processing, achieves a breakthrough improvement in the solubility, flavor, and functionality of walnut protein. Specific beneficial effects are reflected in the following aspects: (1) First, a gradient enzymatic hydrolysis is performed. Flavor protease pretreatment (pH 6.5–7.5) selectively cleaves the terminal hydrophobic groups, eliminating bitter structural sites. Alkaline protease deeply hydrolyzes the protein, exposing hydrophilic amino acid residues (such as aspartic acid and glutamic acid), causing the isoelectric point of the protein to shift and widening the solubility pH range. Meanwhile, glutamine transaminase catalyzes the cross-linking of glutamine and lysine under pH 6.0–7.0 conditions, forming a flexible hydrophilic network structure, further enhancing solubility. At the same time, soybean peptides and trehalose are added as auxiliary agents in the third enzymatic hydrolysis step. Soybean peptides, as a high-quality acyl acceptor of glutamine transaminase (TGase), accelerate the cross-linking reaction rate and form more hydrophilic ε-(γ-glutamyl) lysine bonds. Trehalose stabilizes the hydrophilic domains of the protein through hydrogen bonds, inhibits denaturation and aggregation during the drying process, and shortens the rehydration time of the freeze-dried product.

[0018] (2) After enzymatic hydrolysis, the post-fermentation process involves microbial fermentation. The compound beneficial bacteria freeze-dried agent plays multiple positive roles in the fermentation process. Lactococcus lactis produces lactic acid and other organic acids during metabolism, which lowers the pH of the fermentation broth, creating a suitable acid-base environment for subsequent processing. Furthermore, organic acids can interact with the surface charge of proteins, further enhancing protein solubility. Saccharomyces cerevisiae generates various enzymes and bioactive substances during fermentation, which help to further decompose the complex structure of proteins, making protein molecules more dispersed, thereby improving protein solubility and stability. Using both in equal proportions produces a significant synergistic effect, greatly improving protein solubility and stability.

[0019] (3) In addition, the desalting and debittering purification steps employ ultrafiltration and nanofiltration membranes with specific molecular weight cutoffs, which can precisely remove salt and bitter substances from the enzymatic hydrolysate. Ultrafiltration effectively retains larger molecular impurities and incompletely hydrolyzed protein fragments, while nanofiltration further removes smaller molecular salts and bitter components, resulting in a purer, non-bitter final product. Simultaneously, this refined purification method preserves the effective components of walnut protein, ensuring that the product's nutritional value is not compromised.

[0020] (4) In the concentration and drying stage, after concentrating the retentate to a suitable solid content, maltodextrin is added as a protective agent. Maltodextrin can form a protective film around the protein molecules, preventing the protein from being damaged by the formation of ice crystals during freeze-drying, thereby maintaining the protein's natural structure and solubility. After freeze-drying, the resulting highly soluble walnut protein powder is in powder form and has good flowability and rehydration properties. At room temperature, with simple stirring, the protein powder can quickly dissolve in water to form a uniform and stable solution.

[0021] (5) In summary, this invention organically combines multiple steps such as multi-step enzymatic hydrolysis, microbial post-processing, desalting, debittering purification, and concentration and drying to form a complete and efficient preparation system. Compared with traditional methods, the walnut protein powder prepared by this invention has significantly improved solubility, flavor, stability, and nutritional value, and has broad market application prospects. In the food industry, it can be used as a high-quality protein additive in various beverages, dairy products, baked goods, and other products to improve the nutritional quality and taste of the products. In the health product field, highly soluble walnut protein powder can be better absorbed and utilized by the human body, providing consumers with more effective nutritional supplementation. Attached Figure Description

[0022] Figure 1 This is a microstructure diagram of the protein powder obtained in Example 1 of the present invention; Figure 2 The images show the microstructures of the protein powders obtained in Comparative Examples 1-7, where (A) is Comparative Example 1, (B) is Comparative Example 2, (C) is Comparative Example 3, (D) is Comparative Example 4, (E) is Comparative Example 5, (F) is Comparative Example 6, and (G) is Comparative Example 7. Detailed Implementation

[0023] The technical solution of the present invention will be further described below with reference to specific embodiments, but it is not limited thereto.

[0024] Example 1 A method for preparing highly soluble walnut protein powder by enzymatic hydrolysis includes the following steps: (1) Raw material pretreatment: Mix defatted walnut powder with water, adjust the solid-liquid mass ratio to 1:20, adjust the pH to 6.0-8.0, and obtain a protein suspension; (2) First step of enzymatic hydrolysis: Add flavor protease to the protein suspension in step (1) at a concentration of 0.5% of the substrate mass, and react for 0.5 hours at 45-55°C and pH 6.5-7.5. (3) Second step of enzymatic hydrolysis: Adjust the pH of the enzymatic hydrolysate obtained in step (2) to 7.0-9.0, add alkaline protease, the amount added is 1.0% of the mass of the enzymatic hydrolysate, and react at 50-60°C for 1 hour; (4) Third step of enzymatic hydrolysis: Adjust the pH of the enzymatic hydrolysate obtained in step (3) to 6.0-7.0, add transglutaminase, soybean peptides and active polysaccharides, react at 40-50°C for 0.5 hours, and after completion, add enzyme inactivation treatment at 80-95°C for 10 minutes. (5) Microbial post-treatment: Cool the enzymatic hydrolysate obtained in step (4) to room temperature, inoculate it with a compound beneficial bacteria freeze-dried agent for fermentation treatment. The amount of compound beneficial bacteria freeze-dried agent inoculated is 2% of the mass of the enzymatic hydrolysate. Ferment for 8 hours at a constant temperature of 25-30°C. After fermentation, the fermentation broth is subjected to enzyme inactivation treatment by placing it in an environment of 80-95°C for 10-15 minutes to terminate enzyme activity. Then, impurities and insoluble substances in the fermentation broth are removed by centrifugation. The centrifugation speed is controlled at 4000-6000 rpm and the centrifugation time is 15-20 minutes. Collect the supernatant. (6) Desalting and debittering purification: The supernatant obtained in step (5) is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, and then further desalted and debittered using a nanofiltration membrane with a molecular weight cutoff of 500 Da. The nanofiltration retentate is collected. (7) Concentration and drying: The retentate obtained in step (6) is concentrated to a solid content of 20%, and 1% of maltodextrin by weight of the concentrate is added as a protective agent. The mixture is stirred evenly and then freeze-dried. The freeze-drying method is as follows: the mixture is placed in a cold hydrazine temperature of -45℃ and a vacuum degree of 0.07 MPa and dried for 24 h to obtain the highly soluble walnut protein powder.

[0025] In step (4), the amount of glutamine transaminase added is 0.1% of the mass of the enzymatic hydrolysate, the amount of soybean peptide added is 1% of the mass of the enzymatic hydrolysate, and the active polysaccharide is trehalose, which is added at 0.5% of the mass of the enzymatic hydrolysate.

[0026] The preparation method of the compound beneficial bacteria freeze-dried agent in step (5) is as follows: 1) Lactococcus lactis strain numbered CGMCC No.1.15072 ( Lactococcus lactis ) and Saccharomyces cerevisiae strain numbered CGMCC No.2.3871 ( Saccharomyces cerevisiae After thawing and activation, Lactococcus lactis is inoculated into MRS liquid medium and cultured at 28-30℃ for 18-24 hours until the absorbance of the culture medium at a wavelength of 600 nm reaches OD. 600 The activated Saccharomyces cerevisiae was inoculated into YPD liquid medium and cultured at 23-25℃ for 18-24 hours until the absorbance value (OD) of the culture medium at 600 nm was reached. 600 Reaching 0.8-1.2; 2) Collect Lactococcus lactis and Saccharomyces cerevisiae bacterial cultures separately, centrifuge at 4°C for 10-15 minutes, discard the supernatant, and obtain the corresponding wet cells; 3) Mix the obtained wet Lactococcus lactis cells and wet Saccharomyces cerevisiae cells at a live cell ratio of 1:1 to obtain a compound bacterial mud; 4) The composite bacterial mud is freeze-dried to obtain a composite beneficial bacteria freeze-dried agent, and the total number of effective viable bacteria in the freeze-dried agent is controlled to be not less than 1.0 × 10⁻⁶. 9 CFU / g.

[0027] Example 2 A method for preparing highly soluble walnut protein powder by enzymatic hydrolysis includes the following steps: (1) Raw material pretreatment: Mix defatted walnut powder with water, adjust the solid-liquid mass ratio to 1:15, adjust the pH to 6.0-8.0, and obtain a protein suspension; (2) First step of enzymatic hydrolysis: Add flavor protease to the protein suspension in step (1) at a rate of 1% of the substrate mass, and react for 1 hour at 45-55°C and pH 6.5-7.5. (3) Second step of enzymatic hydrolysis: Adjust the pH of the enzymatic hydrolysate obtained in step (2) to 7.0-9.0, add alkaline protease, the amount added is 2% of the mass of the enzymatic hydrolysate, and react at 50-60°C for 2 hours. (4) Third step of enzymatic hydrolysis: Adjust the pH of the enzymatic hydrolysate obtained in step (3) to 6.0-7.0, add transglutaminase, soybean peptides and active polysaccharides, react at 40-50°C for 1.5 hours, and after completion, add enzyme inactivation treatment at 80-95°C for 10 minutes. (5) Microbial post-treatment: Cool the enzymatic hydrolysate obtained in step (4) to room temperature, inoculate it with a compound beneficial bacteria freeze-dried agent for fermentation treatment. The amount of compound beneficial bacteria freeze-dried agent inoculated is 3.5% of the mass of the enzymatic hydrolysate. Ferment for 10 hours at a constant temperature of 25-30°C. After fermentation, the fermentation broth is subjected to enzyme inactivation treatment. It is placed in an environment of 80-95°C for 10-15 minutes to terminate the enzyme activity. Then, impurities and insoluble substances in the fermentation broth are removed by centrifugation. The centrifugation speed is controlled at 4000-6000 rpm and the centrifugation time is 15-20 minutes. The supernatant is collected. (6) Desalting and debittering purification: The supernatant obtained in step (5) is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 5 kDa, and then nanofiltration is performed using a nanofiltration membrane with a molecular weight cutoff of 300 Da for further desalting and debittering. The nanofiltration retentate is collected. (7) Concentration and drying: The retentate obtained in step (6) is concentrated to a solid content of 25%, and 3% by weight of maltodextrin is added as a protective agent. The mixture is stirred evenly and then freeze-dried. The freeze-drying method is as follows: the mixture is placed in a cold hydrazine temperature of -45℃ and a vacuum degree of 0.07 MPa and dried for 24 h to obtain the highly soluble walnut protein powder.

[0028] In step (4), the amount of glutamine transaminase added is 0.5% of the mass of the enzymatic hydrolysate, the amount of soybean peptide added is 3% of the mass of the enzymatic hydrolysate, and the active polysaccharide is trehalose, which is added at 1.5% of the mass of the enzymatic hydrolysate.

[0029] The preparation method of the compound beneficial bacteria freeze-dried agent in step (5) is as follows: 1) Lactococcus lactis strain numbered CGMCC No.1.15072 ( Lactococcus lactis ) and Saccharomyces cerevisiae strain numbered CGMCC No.2.3871 ( Saccharomyces cerevisiae After thawing and activation, Lactococcus lactis is inoculated into MRS liquid medium and cultured at 28-30℃ for 18-24 hours until the absorbance of the culture medium at a wavelength of 600 nm reaches OD. 600 The activated Saccharomyces cerevisiae was inoculated into YPD liquid medium and cultured at 23-25℃ for 18-24 hours until the absorbance value (OD) of the culture medium at 600 nm was reached. 600 Reaching 0.8-1.2; 2) Collect Lactococcus lactis and Saccharomyces cerevisiae bacterial cultures separately, centrifuge at 4°C for 10-15 minutes, discard the supernatant, and obtain the corresponding wet cells; 3) Mix the obtained wet Lactococcus lactis cells and wet Saccharomyces cerevisiae cells at a live cell ratio of 1:1 to obtain a compound bacterial mud; 4) The composite bacterial mud is freeze-dried to obtain a composite beneficial bacteria freeze-dried agent, and the total number of effective viable bacteria in the freeze-dried agent is controlled to be not less than 1.0 × 10⁻⁶. 9 CFU / g.

[0030] Example 3 A method for preparing highly soluble walnut protein powder by enzymatic hydrolysis includes the following steps: (1) Raw material pretreatment: Mix defatted walnut powder with water, adjust the solid-liquid mass ratio to 1:10, adjust the pH to 6.0-8.0, and obtain a protein suspension; (2) First step of enzymatic hydrolysis: Add flavor protease to the protein suspension in step (1) at an amount of 2.0% of the substrate mass, and react for 2 hours at 45-55°C and pH 6.5-7.5; (3) Second step of enzymatic hydrolysis: Adjust the pH of the enzymatic hydrolysate obtained in step (2) to 7.0-9.0, add alkaline protease, the amount added is 3.0% of the mass of the enzymatic hydrolysate, and react at 50-60°C for 4 hours; (4) Third step of enzymatic hydrolysis: Adjust the pH of the enzymatic hydrolysate obtained in step (3) to 6.0-7.0, add transglutaminase, soybean peptides and active polysaccharides, react at 40-50°C for 2 hours, and after completion, add enzyme inactivation treatment at 80-95°C for 10 minutes. (5) Microbial post-treatment: Cool the enzymatic hydrolysate obtained in step (4) to room temperature, inoculate it with a compound beneficial bacteria freeze-dried agent for fermentation treatment. The amount of compound beneficial bacteria freeze-dried agent inoculated is 5% of the mass of the enzymatic hydrolysate. Ferment for 12 hours at a constant temperature of 25-30°C. After fermentation, the fermentation broth is subjected to enzyme inactivation treatment by placing it in an environment of 80-95°C for 10-15 minutes to terminate enzyme activity. Then, impurities and insoluble substances in the fermentation broth are removed by centrifugation. The centrifugation speed is controlled at 4000-6000 rpm and the centrifugation time is 15-20 minutes. Collect the supernatant. (6) Desalting and debittering purification: The supernatant obtained in step (5) is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3kDa, and then further desalted and debittered using a nanofiltration membrane with a molecular weight cutoff of 200Da. The nanofiltration retentate is collected. (7) Concentration and drying: The retentate obtained in step (6) is concentrated to a solid content of 30%, and 5% by weight of maltodextrin is added as a protective agent. The mixture is stirred evenly and then freeze-dried. The freeze-drying method is as follows: the mixture is placed in a cold hydrazine temperature of -45℃ and a vacuum degree of 0.07 MPa and dried for 24 h to obtain the highly soluble walnut protein powder.

[0031] In step (4), the amount of transglutaminase added is 1.0% of the mass of the hydrolysate, the amount of soybean peptide added is 5% of the mass of the hydrolysate, and the active polysaccharide is trehalose, which is added at 3% of the mass of the hydrolysate.

[0032] The preparation method of the compound beneficial bacteria freeze-dried agent in step (5) is as follows: 1) Lactococcus lactis strain numbered CGMCC No.1.15072 ( Lactococcus lactis ) and Saccharomyces cerevisiae strain numbered CGMCC No.2.3871 ( Saccharomyces cerevisiae After thawing and activation, Lactococcus lactis is inoculated into MRS liquid medium and cultured at 28-30℃ for 18-24 hours until the absorbance of the culture medium at a wavelength of 600 nm reaches OD. 600 The activated Saccharomyces cerevisiae was inoculated into YPD liquid medium and cultured at 23-25℃ for 18-24 hours until the absorbance value (OD) of the culture medium at 600 nm was reached. 600 Reaching 0.8-1.2; 2) Collect Lactococcus lactis and Saccharomyces cerevisiae bacterial cultures separately, centrifuge at 4°C for 10-15 minutes, discard the supernatant, and obtain the corresponding wet cells; 3) Mix the obtained wet Lactococcus lactis cells and wet Saccharomyces cerevisiae cells at a live cell ratio of 1:1 to obtain a compound bacterial mud; 4) The composite bacterial mud is freeze-dried to obtain a composite beneficial bacteria freeze-dried agent, and the total number of effective viable bacteria in the freeze-dried agent is controlled to be not less than 1.0 × 10⁻⁶. 9 CFU / g.

[0033] Comparative Example 1 This comparative example, except for the absence of sequential enzymatic hydrolysis, uses only a one-step enzymatic hydrolysis process; the raw materials and process steps are the same as in Example 1. That is: A method for preparing highly soluble walnut protein powder by enzymatic hydrolysis includes the following steps: (1) Raw material pretreatment: Mix defatted walnut powder with water, adjust the solid-liquid mass ratio to 1:20, adjust the pH to 6.0-8.0, and obtain a protein suspension; (2) Enzymatic hydrolysis: Add flavor protease, alkaline protease and transglutaminase to the protein suspension in step (1). The amount of flavor protease added is 0.5% of the substrate mass, the amount of alkaline protease added is 1.0% of the hydrolysate mass, and the amount of transglutaminase added is 0.1% of the hydrolysate mass. First, react at 45-55°C and pH 6.5-7.5 for 0.5 hours. Adjust the pH to 7.0-9.0 and react at 50-60°C for 1 hour. Add soybean peptides and active polysaccharides, adjust the pH to 6.0-7.0, and react at 50-60°C for 1 hour. After completion, add enzyme inactivation treatment at 80-95°C for 10 minutes. (3) Microbial post-treatment: Cool the obtained enzymatic hydrolysate to room temperature, inoculate it with a compound beneficial bacteria freeze-dried agent for fermentation treatment. The amount of compound beneficial bacteria freeze-dried agent inoculated is 2% of the mass of the enzymatic hydrolysate. Ferment at a constant temperature of 25-30°C for 8 hours. After fermentation, the fermentation broth is subjected to enzyme inactivation treatment by placing it in an environment of 80-95°C for 10-15 minutes to terminate enzyme activity. Then, impurities and insoluble substances in the fermentation broth are removed by centrifugation. The centrifugation speed is controlled at 4000-6000 rpm and the centrifugation time is 15-20 minutes. Collect the supernatant. (4) Desalting and debittering purification: The supernatant obtained in step (5) is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, and then further desalted and debittered using a nanofiltration membrane with a molecular weight cutoff of 500 Da. The nanofiltration retentate is collected. (5) Concentration and drying: The retentate obtained in step (6) is concentrated to a solid content of 20%, and 1% of maltodextrin by weight of the concentrate is added as a protective agent. The mixture is stirred evenly and then freeze-dried. The freeze-drying method is as follows: the mixture is placed in a cold hydrazine temperature of -45℃ and a vacuum degree of 0.07 MPa and dried for 24 h to obtain the highly soluble walnut protein powder.

[0034] The amount of soybean peptide added is 1% of the mass of the enzymatic hydrolysate, and the active polysaccharide is trehalose, which is added at 0.5% of the mass of the enzymatic hydrolysate.

[0035] Comparative Example 2 In this comparative example, except for the absence of microbial post-treatment, the raw materials and process steps are the same as in Example 1. That is: A method for preparing highly soluble walnut protein powder by enzymatic hydrolysis includes the following steps: (1) Raw material pretreatment: Mix defatted walnut powder with water, adjust the solid-liquid mass ratio to 1:20, adjust the pH to 6.0-8.0, and obtain a protein suspension; (2) First step of enzymatic hydrolysis: Add flavor protease to the protein suspension in step (1) at a concentration of 0.5% of the substrate mass, and react for 0.5 hours at 45-55°C and pH 6.5-7.5. (3) Second step of enzymatic hydrolysis: Adjust the pH of the enzymatic hydrolysate obtained in step (2) to 7.0-9.0, add alkaline protease, the amount added is 1.0% of the mass of the enzymatic hydrolysate, and react at 50-60°C for 1 hour; (4) Third step of enzymatic hydrolysis: Adjust the pH of the enzymatic hydrolysate obtained in step (3) to 6.0-7.0, add transglutaminase, soybean peptides and active polysaccharides, and react at 40-50°C for 0.5 hours; (5) Post-treatment: The enzyme hydrolysate is subjected to enzyme inactivation treatment by placing it in an environment of 80-95°C for 10-15 minutes to terminate enzyme activity; then, impurities and insoluble substances in the fermentation broth are removed by centrifugation, with the centrifugation speed controlled at 4000-6000 rpm and the centrifugation time at 15-20 minutes, and the supernatant is collected. (6) Desalting and debittering purification: The supernatant obtained in step (5) is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, and then further desalted and debittered using a nanofiltration membrane with a molecular weight cutoff of 500 Da. The nanofiltration retentate is collected. (7) Concentration and drying: The retentate obtained in step (6) is concentrated to a solid content of 20%, and 1% of maltodextrin by weight of the concentrate is added as a protective agent. The mixture is stirred evenly and then freeze-dried. The freeze-drying method is as follows: the mixture is placed in a cold hydrazine temperature of -45℃ and a vacuum degree of 0.07 MPa and dried for 24 h to obtain the highly soluble walnut protein powder.

[0036] In step (4), the amount of glutamine transaminase added is 0.1% of the mass of the enzymatic hydrolysate, the amount of soybean peptide added is 1% of the mass of the enzymatic hydrolysate, and the active polysaccharide is trehalose, which is added at 0.5% of the mass of the enzymatic hydrolysate.

[0037] Comparative Example 3 In this comparative example, except for changing the strain composition of the compound beneficial bacteria freeze-dried agent, the raw materials and process steps are the same as in Example 1. That is, only Lactococcus lactis is used: The preparation method of the beneficial bacteria freeze-dried agent in step (5) is as follows: 1) After thawing and activating Lactococcus lactis strain CGMCC No.1.15072, the strain was inoculated into MRS liquid medium and cultured at 28-30℃ for 18-24 hours until the absorbance of the culture medium at 600 nm reached an OD value. 600 Reaching 0.8-1.0; 2) Collect the Lactococcus lactis culture, centrifuge at 4℃ for 10-15 minutes, discard the supernatant, and obtain the corresponding wet cells; 3) Freeze-dry the bacterial cells to obtain a lyophilized beneficial bacteria agent, ensuring that the total number of viable bacteria in the lyophilized agent is not less than 1.0 × 10⁻⁶. 9 CFU / g.

[0038] Comparative Example 4 In this comparative example, except for the change in the strain composition of the compound beneficial bacteria freeze-dried agent, the raw materials and process steps are the same as in Example 1. That is, only brewer's yeast is used: The preparation method of the compound beneficial bacteria freeze-dried agent in step (5) is as follows: 1) After thawing and activating the Saccharomyces cerevisiae strain CGMCC No. 2.3871, inoculate it into YPD liquid medium and incubate at 23-25℃ for 18-24 hours until the absorbance of the culture medium at a wavelength of 600 nm reaches OD. 600 Reaching 0.8-1.2; 2) Collect the brewing yeast culture, centrifuge at 4℃ for 10-15 minutes, discard the supernatant, and obtain the corresponding wet cells; 3) Freeze-dry the bacterial cells to obtain a lyophilized beneficial bacteria agent, ensuring that the total number of viable bacteria in the lyophilized agent is not less than 1.0 × 10⁻⁶. 9 CFU / g.

[0039] Comparative Example 5 This comparative example, except for changing the strain composition in the compound beneficial bacteria freeze-dried agent, uses the same raw materials and process steps as Example 1. Specifically, it combines *Lactococcus lactis* wet cells and *Saccharomyces cerevisiae* wet cells at a live cell ratio of 2:1. The preparation method of the compound beneficial bacteria freeze-dried agent in step (5) is as follows: 1) After thawing and activating Lactococcus lactis strain CGMCC No. 1.15072 and Saccharomyces cerevisiae strain CGMCC No. 2.3871, Lactococcus lactis was inoculated into MRS liquid medium and cultured at 28-30℃ for 18-24 hours until the absorbance of the culture medium at 600 nm reached OD. 600 The activated Saccharomyces cerevisiae was inoculated into YPD liquid medium and cultured at 23-25℃ for 18-24 hours until the absorbance value (OD) of the culture medium at 600 nm was reached. 600 Reaching 0.8-1.2; 2) Collect Lactococcus lactis and Saccharomyces cerevisiae bacterial cultures separately, centrifuge at 4°C for 10-15 minutes, discard the supernatant, and obtain the corresponding wet cells; 3) Mix the obtained wet Lactococcus lactis cells and wet Saccharomyces cerevisiae cells at a live cell ratio of 2:1 to obtain a compound bacterial mud; 4) The composite bacterial mud is freeze-dried to obtain a composite beneficial bacteria freeze-dried agent, and the total number of effective viable bacteria in the freeze-dried agent is controlled to be not less than 1.0 × 10⁻⁶. 9 CFU / g.

[0040] Comparative Example 6 This comparative example, except for changing the strain composition in the compound beneficial bacteria freeze-dried agent, uses the same raw materials and process steps as Example 1. Specifically, it combines *Lactococcus lactis* wet cells and *Saccharomyces cerevisiae* wet cells at a live cell ratio of 1:2. Comparative Example 7 The existing technology is walnut protein powder, with application number 201410076807.3.

[0041] The preparation method of the compound beneficial bacteria freeze-dried agent in step (5) is as follows: 1) After thawing and activating Lactococcus lactis strain CGMCC No. 1.15072 and Saccharomyces cerevisiae strain CGMCC No. 2.3871, Lactococcus lactis was inoculated into MRS liquid medium and cultured at 28-30℃ for 18-24 hours until the absorbance of the culture medium at 600 nm reached OD. 600 The activated Saccharomyces cerevisiae was inoculated into YPD liquid medium and cultured at 23-25℃ for 18-24 hours until the absorbance value (OD) of the culture medium at 600 nm was reached. 600 Reaching 0.8-1.2; 2) Collect Lactococcus lactis and Saccharomyces cerevisiae bacterial cultures separately, centrifuge at 4°C for 10-15 minutes, discard the supernatant, and obtain the corresponding wet cells; 3) Mix the obtained wet Lactococcus lactis cells and wet Saccharomyces cerevisiae cells at a live cell ratio of 1:2 to obtain a compound bacterial mud; 4) The composite bacterial mud is freeze-dried to obtain a composite beneficial bacteria freeze-dried agent, and the total number of effective viable bacteria in the freeze-dried agent is controlled to be not less than 1.0 × 10⁻⁶. 9 CFU / g.

[0042] Performance testing Basic nutritional index determination The embodiments of the present invention and the comparative sample were tested using the following methods: Moisture content was determined by the direct drying method in GB 5009.3—2016 "National Food Safety Standard - Determination of Moisture in Food"; protein content was determined by the Kjeldahl method in GB 5009.5—2016 "National Food Safety Standard - Determination of Protein in Food"; ash content was determined by the direct ashing method in GB 5009.4—2016 "National Food Safety Standard - Determination of Ash in Food". Amino acid content was determined by the hydrolysis method in GB 5009.124—2016 "National Food Safety Standard - Determination of Amino Acids in Food". All indicators for each sample group were measured three times, and the average value was taken.

[0043] Wettability test: Add 100 mL of distilled water to a 250 mL beaker, weigh 0.5 g of the protein powder sample to be tested, and spread it evenly on the water surface. Measure the time (in seconds) required for the sample to completely settle after being added to characterize the wettability. Repeat the test 3 times for each sample and take the average value.

[0044] The test results are shown in Table 1: Table 1. Test of basic nutritional indicators of samples As shown in Table 1, the basic nutritional indicators of the protein powder in this invention embodiment are better than those in the comparative examples, with higher protein content and relatively lower ash content. This may be due to the enzymatic hydrolysis and microbial post-treatment process used in this invention, which allows for more complete hydrolysis and modification of walnut protein, reducing residual impurities. During enzymatic hydrolysis, the synergistic effect of multiple enzymes can decompose walnut protein into small molecule peptides and amino acids, improving protein solubility and bioavailability. In the microbial post-treatment stage, the synergistic fermentation of lactic acid bacteria and yeast further improves the quality of the protein powder, decomposes some large molecule impurities, and reduces ash content. In contrast, the enzymatic hydrolysis effect of Comparative Example 1, which uses mixed enzymes in a one-step process, is far less significant than that of the step-by-step sequential enzymatic hydrolysis of this invention. In some of the comparative examples, microbial post-treatment was not performed, resulting in incomplete removal of impurities (Comparative Example 2). Comparative Examples 3-6 altered the strain composition in the compound beneficial bacteria freeze-dried inoculant, disrupting the synergistic balance between strains and resulting in poor fermentation effects, failing to fully leverage the beneficial bacteria's role in improving the quality of the protein powder. We further observed the microstructure of the particles.

[0045] Microstructure observation: The surface morphology of the protein powder was observed using scanning electron microscopy (SEM). Double-sided tape was attached to the sample stage, and a small amount of sample was evenly placed on the tape. Excess sample was blown away, and the sample was then sputter-coated with gold. SEM was then used for scanning observation and imaging. Figure 1-2 Looking at the microscopic particle morphology of the protein powders obtained in Example 1 and Comparative Examples 1-7, the protein powders obtained in the examples exhibit a relatively regular and uniform morphology, with a relatively smooth surface and good dispersibility between particles. This indicates that the enzymatic hydrolysis and microbial post-processing technology of the present invention enables walnut protein to have good structural stability and dispersibility at the microscopic level, which is beneficial to the dissolution and absorption of the protein.

[0046] In contrast, the protein powder particles obtained from the one-step enzymatic hydrolysis in Comparative Example 1 exhibited irregular morphology, significant size differences, and some particles showed aggregation. This indicates that the one-step enzymatic hydrolysis failed to fully hydrolyze the walnut protein, resulting in complex interactions between protein molecules and thus affecting the microstructure of the particles.

[0047] Comparative Example 2, which did not undergo microbial post-treatment, had rough protein powder particles with numerous impurities attached, and the particles were tightly connected, forming large aggregates. This further demonstrates the important role of microbial post-treatment in removing impurities and improving the microstructure of protein powder.

[0048] Comparative Examples 3-6 altered the strain composition of the compound beneficial bacteria freeze-dried inoculants, resulting in varying degrees of changes in the microstructure of the particles. While some particles showed improvement compared to Comparative Example 2, they were still not as regular and uniform as those in the examples; others exhibited voids and cracks. This may be due to changes in the metabolites and mechanisms of action produced during fermentation after the strain composition was altered, affecting the spatial structure of proteins and particle formation.

[0049] Comparative Example 7, representing existing walnut protein powder, exhibits the most irregular particle morphology, severe agglomeration, and numerous impurities and protrusions on its surface. This reflects a significant deficiency in the traditional process for processing walnut protein, failing to achieve the microstructural quality of the protein powder prepared by the process of this invention.

[0050] The microstructure observations further confirm that the stepwise sequential enzymatic hydrolysis and microbial post-processing technology employed in this invention has significant advantages in improving the quality of walnut protein powder. It not only performs excellently in basic nutritional indicators but also exhibits better stability and dispersibility in its microstructure, thus providing more favorable conditions for its application in food and other fields. Next, we conducted an in-depth analysis of the wettability of the protein powder.

[0051] Determination of nitrogen solubility index: Accurately weigh 2g of the sample to be tested, disperse it in deionized water, and bring the volume to 50 mL. Shake at room temperature for 2 h, centrifuge at 4500 r / min for 20 min, and measure 5 mL of the supernatant to determine the protein content using a Kjeldahl nitrogen analyzer. The nitrogen solubility index is calculated using the following formula: NSI = (10×N1 / N2)×100%(I), where NSI is the nitrogen solubility index of the enzymatic hydrolysis product (%), N1 is the soluble nitrogen in 5 mL of supernatant (mg), and N2 is the total nitrogen in 2 g of sample (mg). Each sample was measured three times, and the average value was taken.

[0052] Determination of thermal nitrogen solubility index: Accurately weigh 2g of the sample to be tested, disperse it in deionized water, and make up to 50 mL. Heat in a constant temperature water bath at 90±0.5℃ for 15 min, cool and centrifuge at 4500 r / min for 20 min, take 5 mL of the supernatant and determine the protein content using a Kjeldahl nitrogen analyzer. Calculate the nitrogen solubility index according to formula (I).

[0053] Bitterness value: Accurately weigh 1.0 g of protein powder (to a precision of 0.0001 g), dissolve it in 100 mL of distilled water, and stir magnetically for 1 h. Adjust the pH to 4.5 ± 0.1 (simulating the acidic environment of the oral cavity) with 0.1 M HCl. Centrifuge for 15 min (25℃) and collect the supernatant.

[0054] Pour the supernatant into a 1 cm quartz cuvette. Use distilled water (pH 4.5) as a blank control. Measure the absorbance at 420 nm. Small molecule bitter peptides aggregate under weakly acidic conditions, producing turbidity; the absorbance at 420 nm is positively correlated with the intensity of bitterness.

[0055] Bitterness value = A 420 × Dilution factor, Note: Dilution factor = 100 (because 1 g / 100 mL = 1% solution), if A is measured 420 =0.0008, then the bitterness value = 0.0008 × 100 = 0.08.

[0056] Stability test: The protein powders prepared in the examples and comparative examples were sealed and stored for 6 months at a temperature of 25±1℃ and a relative humidity of 60±5% before testing. The emulsion stability coefficient was tested according to section 1.2.3.1 of the article "Comparison of the Stability of Soy Protein Isolate and Concentrated Protein Emulsion Systems" in the Journal of Henan University of Technology (Natural Science Edition): 2 mL of emulsion was accurately added to a 2 mL round-bottom centrifuge tube, centrifuged at 400 rpm for 30 min, and a sample was taken 1 cm from the bottom of the tube. The absorbance (A500) was determined using the turbidimetric method. The emulsion stability coefficient (%) was calculated as: At / A0 × 100, where A0 is the absorbance of the emulsion before centrifugation, and At is the absorbance of the emulsion after centrifugation. Five parallel tests were conducted, and the average value was taken.

[0057] Table 2 Performance Test Results From the data above, we can see that the walnut protein powder prepared in this embodiment of the invention is significantly superior to the comparative example in all performance indicators. In the nitrogen solubility index and thermal nitrogen solubility index tests, the values ​​of the embodiment are also much higher than those of the comparative example, indicating that the protein powder of the embodiment has better solubility under both room temperature and heating conditions.

[0058] Regarding bitterness, the examples showed extremely low bitterness values, all not exceeding 0.06, while the comparative example had the highest bitterness value reaching 0.42. This indicates that the protein powder prepared in the examples has significantly reduced bitterness and a better taste. In stability tests, the emulsion stability coefficients of the examples were all above 92%, far higher than those of the comparative example, indicating that the protein powder of the examples can better maintain its stability during storage. Therefore, the design of the sequential enzymatic hydrolysis steps and the microbial post-processing of this invention play a crucial role in achieving high performance in walnut protein powder; the absence of either step weakens the overall effect.

[0059] In summary, the stepwise sequential enzymatic hydrolysis and microbial post-processing method adopted in this invention is an effective way to prepare walnut protein powder with high solubility, low bitterness, and high stability. It has broad application prospects and significant market value, and is expected to provide new technical support and product options for the development of the walnut protein industry.

[0060] It should be noted that the above embodiments are merely some preferred embodiments of the present invention, and not all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

Claims

1. A method for preparing highly soluble walnut protein powder via enzymatic hydrolysis, characterized in that, Includes the following steps: (1) Raw material pretreatment: Mix defatted walnut powder with water, adjust the solid-liquid mass ratio to 1:10-20, adjust the pH to 6.0-8.0, and obtain a protein suspension; (2) First step of enzymatic hydrolysis: Add flavor protease to the protein suspension in step (1) at a concentration of 0.5%-2.0% of the substrate mass, and react for 0.5-2 hours at 45-55°C and pH 6.5-7.

5. (3) Second step of enzymatic hydrolysis: Adjust the pH of the enzymatic hydrolysate obtained in step (2) to 7.0-9.0, add alkaline protease, the amount of which is 1.0%-3.0% of the mass of the enzymatic hydrolysate, and react at 50-60°C for 1-4 hours; (4) Third step of enzymatic hydrolysis: Adjust the pH of the enzymatic hydrolysate obtained in step (3) to 6.0-7.0, add transglutaminase, soybean peptides and active polysaccharides, react at 40-50°C for 0.5-2 hours, and after completion, add enzyme inactivation treatment at 80-95°C for 10 minutes. (5) Microbial post-treatment: Cool the enzymatic hydrolysate obtained in step (4) to room temperature, inoculate it with a compound beneficial bacteria freeze-dried agent for fermentation treatment. The amount of compound beneficial bacteria freeze-dried agent inoculated is 2%-5% of the mass of the enzymatic hydrolysate. Ferment at a constant temperature of 25-30°C for 8-12 hours. After fermentation, the fermentation broth is subjected to enzyme inactivation treatment by placing it in an environment of 80-95°C for 10-15 minutes to terminate enzyme activity. Then, impurities and insoluble substances in the fermentation broth are removed by centrifugation. The centrifugation speed is controlled at 4000-6000 rpm and the centrifugation time is 15-20 minutes. Collect the supernatant. (6) Desalting and debittering purification: The supernatant obtained in step (5) is ultrafiltered with an ultrafiltration membrane with a molecular weight cutoff of 3-10 kDa, and then nanofiltration is performed with a nanofiltration membrane with a molecular weight cutoff of 200-500 Da for further desalting and debittering. The nanofiltration retentate is collected. (7) Concentration and drying: The retentate obtained in step (6) is concentrated to a solid content of 20-30%, and 1-5% of maltodextrin by weight of the concentrate is added as a protective agent. The mixture is stirred evenly and then freeze-dried to obtain the highly soluble walnut protein powder.

2. The method for preparing highly soluble walnut protein powder by enzymatic hydrolysis according to claim 1, characterized in that, The amount of flavor protease added in step (2) is 1.0%-1.5% of the substrate protein mass, and the reaction conditions are 50°C, pH 7.0, and reaction time is 1 hour.

3. The method for preparing highly soluble walnut protein powder by enzymatic hydrolysis according to claim 1 or 2, characterized in that, The amount of alkaline protease added in step (3) is 1.5%-2.5% of the mass of the enzyme hydrolysate, and the reaction conditions are 55°C, pH 8.0, and 2-3 hours.

4. The method for preparing highly soluble walnut protein powder by enzymatic hydrolysis according to claim 1 or 2, characterized in that, In step (4), the amount of glutamine transaminase added is 0.1%-1.0% of the mass of the enzymatic hydrolysate, the amount of soybean peptide added is 1%-5% of the mass of the enzymatic hydrolysate, and the active polysaccharide is trehalose, with an addition amount of 0.5%-3% of the mass of the enzymatic hydrolysate.

5. The method for preparing highly soluble walnut protein powder by enzymatic hydrolysis according to claim 1 or 2, characterized in that, The preparation method of the compound beneficial bacteria freeze-dried agent in step (5) is as follows: 1) Lactococcus lactis strain numbered CGMCC No.1.15072 ( Lactococcus lactis ) and Saccharomyces cerevisiae strain CGMCC No. 2.3871 ( Saccharomyces cerevisiae After thawing and activation, Lactococcus lactis is inoculated into MRS liquid medium and cultured at 28-30℃ for 18-24 hours until the absorbance of the culture medium at a wavelength of 600 nm reaches OD. 600 The activated Saccharomyces cerevisiae was inoculated into YPD liquid medium and cultured at 23-25℃ for 18-24 hours until the absorbance value (OD) of the culture medium at 600 nm reached 0.8-1.

0. 600 Reaching 0.8-1.2; 2) Collect Lactococcus lactis and Saccharomyces cerevisiae bacterial cultures separately, centrifuge at 4°C for 10-15 minutes, discard the supernatant, and obtain the corresponding wet cells; 3) The obtained wet Lactococcus lactis cells and wet Saccharomyces cerevisiae cells are mixed evenly at a live cell ratio of 1:1 to obtain a composite bacterial sludge; the composite bacterial sludge is freeze-dried to obtain a composite beneficial bacteria freeze-dried agent, and the total effective live cell count in the freeze-dried agent is controlled to be not less than 1.0 × 10⁻⁶. 9 CFU / g.

6. The method for preparing highly soluble walnut protein powder by enzymatic hydrolysis according to claim 1 or 2, characterized in that, The molecular weight cutoff of the ultrafiltration membrane in step (6) is 5 kDa, and the molecular weight cutoff of the nanofiltration membrane is 300 Da.

7. The method for preparing highly soluble walnut protein powder by enzymatic hydrolysis according to claim 1 or 2, characterized in that, The amount of maltodextrin added as the protective agent in step (7) is 2-3% of the weight of the concentrate.

Images