Black bean protein combined modification method and application thereof
By using a combined modification method with Lactiplantibacillus plantarum ZHA-3 strain, the problem of poor modification effect of black soybean protein was solved, and its solubility, oil holding capacity and foam stability were significantly improved, making it suitable for the preparation of high-efficiency whole grain protein solid beverages.
Patent Information
- Application Number
- CN202511853092.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-10
- Publication Date
- 2026-02-10
AI Technical Summary
The modification effect of black soybean protein in the existing technology is poor, resulting in insufficient solubility, oil holding capacity and foam stability, which makes it difficult to meet the growing application needs.
A modified black soybean protein solid beverage was prepared by combining Lactiplantibacillus plantarum ZHA-3 strain with a mixture of mixed grain protein powder, dextran, and water, adjusting the pH, and then carrying out a glycosylation reaction. The mixture was then mixed with olive oil, xanthan gum, and maltodextrin to prepare a stable milk, which was then dried.
It significantly improved the solubility, oil holding capacity and foam stability of modified black soybean protein, reduced the vitality loss of Lactobacillus plantarum after freeze-drying, and improved the survival rate in simulated in vitro digestion.
Smart Images
Figure CN121495790A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of bio-food technology, and in particular to a method for the combined modification of black soybean protein and its application. Background Technology
[0002] With the increasing demand for protein, there is a significant opportunity to find high-quality and sustainably available protein sources. Plant protein, as a major dietary protein source, has attracted widespread attention in recent years. Black beans, in particular, are high in protein and are widely recognized as an excellent source of high-quality protein, possessing numerous potential biological benefits. However, due to the low sulfur amino acid content in black beans, their non-protein components can interact with dietary fiber, forming irreversible complexes with proteins. This affects the solubility of black bean protein, preventing its utilization from achieving the desired results.
[0003] Plant protein modification technology mainly employs physical, chemical, and biological methods to process plant proteins, altering their structure and properties to improve their functionality, stability, and applicability. Although some progress has been made in the research of plant protein modification technology, each single modification technique has its own limitations, such as limited and irreversible effects, causing severe hydrophobic aggregation of proteins, and reducing protein solubility, thus making it difficult to meet the growing application demands. Summary of the Invention
[0004] The purpose of this invention is to provide a method for combined modification of black soybean protein and its application, so as to solve the problems of poor modification effect and low quality of modified grain protein in the existing technology.
[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a plant lactobacillus, wherein the plant lactobacillus is *Lactobacillus plantarum*. Lactiplantibacillus plantarum The ZHA-3 strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is September 9, 2025, and the accession number is CGMCC No. 35850.
[0006] The present invention also provides the application of the aforementioned *Lactobacillus plantarum* in the preparation of solid beverages containing mixed grain protein.
[0007] The present invention also provides a solid beverage of mixed grain protein, comprising the following components in parts by weight: 8-16 parts of modified mixed grain protein, 30-55 parts of Lactobacillus plantarum, 5-15 parts of olive oil, 0.5-4 parts of xanthan gum, and 3-8 parts of maltodextrin.
[0008] Preferably, the initial viable bacterial concentration of the *Lactobacillus plantarum* is [value missing]. .
[0009] Preferably, the preparation method of the modified whole grain protein includes the following steps: mixing whole grain protein powder, dextran and water, adjusting the pH to 10-13, and then carrying out a glycosylation reaction at 90-100°C to obtain the modified whole grain protein.
[0010] Preferably, the volume ratio of the mixed grain protein powder and the dextran is 2 to 4:1; The mass-to-volume ratio of the mixed grain protein powder and water is 5-10g:100mL.
[0011] Preferably, the mixing time is 11 to 14 hours; The glycosylation reaction takes 2 to 4 hours.
[0012] The present invention also provides a method for preparing the aforementioned mixed grain protein solid beverage, comprising the following steps: mixing modified mixed grain protein, Lactobacillus plantarum, olive oil, xanthan gum and maltodextrin for 36-72 hours to obtain a stable milk, and drying the stable milk to obtain the mixed grain protein solid beverage.
[0013] Preferably, the mixing method is a short-time high-shear method.
[0014] The present invention has the following technical effects and advantages: The modified black soybean protein prepared by this invention has significantly improved processing properties such as solubility, oil holding capacity, foaming ability and foam stability. Compared with unmodified black soybean protein, the solubility is increased by 106.31%, the oil holding capacity is increased by 2.68 times, and the foaming ability and foam stability are increased by 4.72 times and 4.13 times, respectively. The technical solution of the present invention reduces the vitality loss of *Lactobacillus plantarum* after freeze-drying and improves the survival rate in simulated in vitro digestion processes. Attached Figure Description
[0015] Figure 1 The evaluation results of the black soybean protein co-modification method described in Example 1 are shown in the left figure, which shows the water-holding capacity result and the oil-holding capacity result. Figure 2 Two-dimensional contour plots and response surface plots of the solubility of modified black soybean protein as a function of glycosylation time and glycosylation ratio; Figure 3 Two-dimensional contour plots and response surface plots of the solubility of modified black soybean protein as a function of glycosylation time and pH; Figure 4 Two-dimensional contour plots and response surface plots of the solubility of modified black soybean protein as a function of glycosylation ratio and pH.
[0016] Preservation Instructions
[0017] Lactobacillus plantarum is Lactobacillus plantarum Lactiplantibacillus plantarum The ZHA-3 strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is September 9, 2025, and the accession number is CGMCC No. 35850. Detailed Implementation
[0018] This invention provides a plant lactobacillus, wherein the plant lactobacillus is *Lactobacillus plantarum*. Lactiplantibacillus plantarum The ZHA-3 strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is September 9, 2025, and the accession number is CGMCC No. 35850.
[0019] The present invention also provides the application of the aforementioned *Lactobacillus plantarum* in the preparation of solid beverages containing mixed grain protein.
[0020] The present invention also provides a solid beverage of mixed grain protein, comprising the following components in parts by weight: 8-16 parts of modified mixed grain protein, preferably 12 parts; 30-55 parts of Lactobacillus plantarum, preferably 35 parts; 5-15 parts of olive oil, preferably 10 parts; 0.5-4 parts of xanthan gum, preferably 2 parts; and 3-8 parts of maltodextrin, preferably 6 parts.
[0021] In this invention, the modified grain protein is preferably modified black soybean protein; The initial viable cell concentration of the *Lactobacillus plantarum* was: Preferred .
[0022] In this invention, the preparation method of the modified whole grain protein includes the following steps: mixing whole grain protein powder, dextran and water, adjusting the pH to 10-13, preferably 12, and then carrying out a glycosylation reaction at 90-100°C, preferably 100°C, to obtain the modified whole grain protein.
[0023] In this invention, the volume ratio of the mixed grain protein powder and dextran is 2 to 4:1, preferably 3:1; The mass-to-volume ratio of the mixed grain protein powder and water is 5-10g:100mL, preferably 7g:100mL.
[0024] In this invention, the mixing time is 11 to 14 hours, preferably 13 hours; The glycosylation reaction takes 2 to 4 hours, preferably 3.4 hours.
[0025] The present invention also provides a method for preparing the aforementioned mixed grain protein solid beverage, comprising the following steps: mixing modified mixed grain protein, Lactobacillus plantarum, olive oil, xanthan gum and maltodextrin for 36-72 hours, preferably 48 hours, to obtain a stable milk, and drying the stable milk to obtain the mixed grain protein solid beverage.
[0026] In this invention, the mixing method is a short-time high-shear method.
[0027] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0028] Example 1: Screening of methods for co-modification of black soybean protein
[0029] Black soybean protein was modified using three different methods: pH shift combined with TGase, pH shift combined with dextran glycosylation, and dextran glycosylation combined with TGase. (1) pH shift combined with TGase method: 7g of black soybean protein powder was dissolved in 100mL of deionized water and stirred at room temperature for 12h to prepare a PPI dispersion with a mass fraction of 7%. The pH was adjusted to 11 and maintained for 2h, and then the pH was adjusted to 7. 30U of TGase was added to 1g of PPI dispersion, incubated in a 45℃ water bath for 2h, and then heated in an 85℃ water bath for 10min to inactivate TGase. The modified black soybean protein was obtained by freeze drying. (2) pH shift combined with glucose glycosylation method: 7g black soybean protein powder and 1.05g glucose were dissolved in 100mL deionized water and stirred at room temperature for 12h to prepare PPI dispersion. After adjusting pH=11, stirring was continued for 1h. Then, the mixture was incubated in a water bath at 70℃ for 2h and freeze-dried to obtain modified black soybean protein. (3) Glucose glycosylation combined with TGase method: 7g black soybean protein powder and 1.05g glucose were dissolved in 100mL deionized water and stirred at room temperature for 12h to prepare PPI dispersion. After adjusting the pH to 9, stirring was continued for 1h. Then, the mixture was incubated in a 70℃ water bath for 2h. After cooling, 30U TGase was added to 1g PPI dispersion and incubated in a 45℃ water bath for 2h. Then, the mixture was heated in an 85℃ water bath for 10min to inactivate TGase. The modified black soybean protein was obtained by freeze drying.
[0030] The solubility, water-holding capacity, and oil-holding capacity of the modified black soybean protein prepared by the above three combined modification methods were measured and compared with those obtained by single heat treatment (100℃ and 150℃), ultrasound, microwave, glucose, and dextran. The results are as follows: Figure 1 As shown.
[0031] The results showed that the modified black soybean protein prepared by pH shift combined with glucose glycosylation significantly improved in terms of water retention and oil retention, indicating that the suitable combined modification method for black soybean protein is pH shift combined with glucose glycosylation.
[0032] Example 2: Single-factor optimization of the co-modification method of black soybean protein
[0033] (1) Glycosylation ratio: Black soybean protein and dextran were mixed in 100 mL of deionized water at mass ratios of 2:1, 3:1, 4:1, 5:1, 6:1 and 7:1, respectively, and incubated in a water bath at 70°C for 2 h to carry out glycosylation reaction. Modified black soybean protein was obtained after freeze drying. (2) Glycosylation time: Black soybean protein and dextran were mixed in a mass ratio of 3:1 and dissolved in 100 mL of deionized water. The mixture was incubated in a 70°C water bath for 1 h, 2 h, 3 h and 4 h respectively to carry out the glycosylation reaction. After freeze drying, modified black soybean protein was obtained. (3) Glycosylation temperature: Black soybean protein and dextran were mixed in 100 mL of deionized water at a mass ratio of 3:1 and incubated in a water bath at 75℃, 80℃, 85℃, 90℃, 95℃ and 100℃ for 3 h to carry out glycosylation reaction. After freeze drying, modified black soybean protein was obtained. (4) pH: Black soybean protein and dextran were mixed in a mass ratio of 3:1 and dissolved in 100 mL of deionized water. The pH was adjusted to 9, 10, 11, 12 and 13 respectively. Then, the mixture was incubated in a water bath at 100℃ for 3 h to carry out the glycosylation reaction. After freeze drying, modified black soybean protein was obtained.
[0034] The results showed that as the glycosylation ratio increased, the solubility of modified black soybean protein first increased and then decreased, with the highest solubility at a black soybean protein:glucan ratio of 3:1. The solubility of modified black soybean protein was highest after 3 hours of glycosylation reaction, and then decreased with the extension of time. The solubility of modified black soybean protein was greatest at a glycosylation temperature of 100℃ and a pH of 12.
[0035] Example 3: Response surface optimization of a co-modification method for black soybean protein
[0036] Using the central composite experimental design principle, three factors were selected as independent variables: glycosylation time (2, 3, 4 h), pH (11, 12, 13), and glycosylation ratio (2:1, 3:1, 4:1). Response surface methodology was performed with the solubility of modified black soybean protein as the response value. Each experiment was repeated three times, and the average value was taken. The factor levels of the response surface methodology design are shown in Table 1, and the optimization results of the preparation conditions of modified black soybean protein are shown in Table 2.
[0037] Table 1. Response Surface Analysis Factor Level Table
[0038] Table 2 Results of Response Surface Analysis
[0039] Using Design-Expert 13 software, with deglycosylation time, glycosylation ratio, and pH as independent variables, and the solubility of modified black soybean protein as the dependent variable, the regression equation was established as: Solubility (%) = The results of the analysis of variance for the regression model are shown in Table 3. The interaction effects of AB, AC, and BC were analyzed based on the regression equation, and the results are as follows: Figures 2-4 As shown.
[0040] Table 3. Analysis of Variance for Regression Models
[0041] The results show that the regression model P <0.01 indicates that the regression model has reached a highly significant level; lack of fit term P =0.1724( P A value greater than 0.05 indicates that the difference in the lack-of-fit terms is not significant. (Regression model coefficient of determination) This indicates that 99.22% of the changes in response values are related to the selected factors, while the adjusted coefficient of determination of the regression model... This indicates that the regression model can represent 97.83% of the response value changes. The order of influence of each factor on the solubility of modified black soybean protein is C > A > B, i.e., pH > glycosylation time > glycosylation ratio; the interaction term BC showed a significant difference. P <0.05), the difference in the interaction term AC reached a highly significant level ( P <0.01); With the extension of glycosylation time and the increase of glycosylation ratio, the solubility of modified black soybean protein showed a trend of first increasing and then decreasing. The interaction between glycosylation time (A) and pH (C) had a significant effect, which is consistent with the results of analysis of variance. In the effect of glycosylation ratio (B) and pH (C) on the solubility of modified black soybean protein, the contour lines showed an elliptical shape and the slope of the three-dimensional response surface plot was large, indicating that the interaction between these two factors was significant, which is consistent with the results of analysis of variance. According to the calculation results of Design-expert13 software, the optimal reaction conditions for black soybean protein modification are: glycosylation time of 3.4 h, glycosylation ratio of 3:1, and pH of 12. Under these conditions, the predicted solubility of modified black soybean protein is 42.044%.
[0042] Lactobacillus plantarum Lactiplantibacillus plantarum ZHA-3 strain was activated and enriched in MRS medium. After centrifugation to collect the bacterial sludge, it was resuspended in sterile physiological saline to obtain a concentration of [missing value]. The plant lactobacillus resuspension was prepared. 35g of plant lactobacillus resuspension, 10g of olive oil, 12g of modified black soybean protein, 2g of xanthan gum, and 6g of maltodextrin were mixed for 48h using a short-time high-shear method to obtain a stable milk, which was then freeze-dried to obtain a black soybean protein solid beverage.
[0043] As can be seen from the above embodiments, the present invention provides a method for combined modification of black soybean protein and its application. The modified black soybean protein prepared by the present invention has significantly improved processing characteristics such as solubility, oil holding capacity, foaming ability, and foam stability. The technical solution of the present invention reduces the activity loss of *Lactobacillus plantarum* after freeze-drying and improves the survival rate in simulated in vitro digestion.
[0044] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A plant lactobacillus, characterized in that, The plant lactobacillus is *Lactobacillus plantarum*. Lactiplantibacillus plantarum The ZHA-3 strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is September 9, 2025, and the accession number is CGMCC No. 35850.
2. The application of *Lactobacillus plantarum* as described in claim 1 in the preparation of solid beverages containing mixed grain protein.
3. A solid beverage containing mixed grain protein, characterized in that, It includes the following components in parts by weight: 8-16 parts modified whole grain protein, 30-55 parts Lactobacillus plantarum, 5-15 parts olive oil, 0.5-4 parts xanthan gum, and 3-8 parts maltodextrin; The *Lactobacillus plantarum* is the *Lactobacillus plantarum* as described in claim 1.
4. The mixed grain protein solid beverage according to claim 3, characterized in that, The initial viable cell concentration of the *Lactobacillus plantarum* was: .
5. The mixed grain protein solid beverage according to claim 4, characterized in that, The preparation method of the modified whole grain protein includes the following steps: mixing whole grain protein powder, dextran and water, adjusting the pH to 10-13, and then carrying out a glycosylation reaction at 90-100℃ to obtain the modified whole grain protein.
6. The mixed grain protein solid beverage according to claim 5, characterized in that, The volume ratio of the mixed grain protein powder and dextran is 2 to 4:1; The mass-to-volume ratio of the mixed grain protein powder and water is 5-10g:100mL.
7. The mixed grain protein solid beverage according to claim 6, characterized in that, The mixing time is 11–14 hours; The glycosylation reaction takes 2 to 4 hours.
8. The method for preparing the mixed grain protein solid beverage according to any one of claims 3 to 7, characterized in that, The process includes the following steps: mixing modified whole grain protein, Lactobacillus plantarum, olive oil, xanthan gum and maltodextrin for 36-72 hours to obtain a stable milk, and drying the stable milk to obtain a whole grain protein solid beverage.
9. The preparation method according to claim 8, characterized in that, The mixing method is the short-time high-shear method.