Method for preparing polyglutamic acid chelated zinc, polyglutamic acid chelated zinc composition and application thereof

Abstract

This invention relates to the field of daily chemical products technology, and discloses a method for preparing polyglutamic acid chelated zinc, a polyglutamic acid chelated zinc composition, and its application. The method includes: dissolving an appropriate amount of sodium γ-polyglutamate powder in water to prepare a sodium γ-polyglutamate aqueous solution; adjusting the pH value to 3.5-4.0; heating to 80-90℃ and maintaining for 6-8 h; cooling to room temperature; and ultrafiltration to obtain a low molecular weight γ-polyglutamic acid aqueous solution; adjusting the pH value of the low molecular weight γ-polyglutamic acid aqueous solution to 4.0-4.5; adding ammonia and water-soluble zinc salt according to a preset ratio; stirring thoroughly to dissolve evenly; and then continuously heating in a water bath at 60-80℃ for 2-4 h to obtain a product solution; ultrafiltration of the product solution through a 0.8-1.2 kDa ultrafiltration membrane; and spray drying the obtained filtrate to obtain polyglutamic acid chelated zinc. The polyglutamic acid chelated zinc obtained by this method has a high chelation rate, very few free zinc ions, relatively stable physicochemical properties, and is produced under mild conditions, with a simple method and no organic solvents.

Description

Technical Field

[0001] This invention relates to the field of daily chemical products technology, and in particular to a method for preparing polyglutamic acid chelated zinc, a polyglutamic acid chelated zinc composition, and its applications. Background Technology

[0002] Zinc is one of the essential trace elements for the human body, playing an extremely important role in human growth and development. Its content in the human body is second only to iron. In skin care, zinc can improve acne. It participates in histone deacetylation, maintains the functional stability of macrophages, neutrophils and natural killer cells, regulates complement activity, stabilizes organelle membrane structure, reduces the production of inflammatory mediators, blocks ultraviolet rays, has antioxidant effects, and can promote the absorption of antibiotics.

[0003] Polyglutamic acid and its sodium salt are biocompatible natural polymers with excellent moisturizing and film-forming properties. They have a powerful hydrating function, improving skin hydration; they also have a highly effective water-locking and moisturizing function, leaving the skin feeling smooth and refreshed. Furthermore, they can replenish skin nutrients, enhancing skin plumpness and elasticity.

[0004] Polyglutamic acid chelated zinc can enhance the moisturizing properties of polyglutamic acid and its sodium salt by adding anti-inflammatory and whitening effects. Furthermore, compared to elemental zinc, zinc oxide, and zinc salts, the relative stability of its chelate bonds effectively improves the longevity of its effects. Existing polyglutamic acid chelated zinc is mostly used in feed or fertilizers, but the zinc chelation rate in the product is relatively low, and insoluble substances tend to precipitate out over long-term storage, resulting in poor stability and making it unsuitable as a cosmetic ingredient. Summary of the Invention

[0005] (a) Purpose of the invention The purpose of this invention is to provide a method for preparing polyglutamic acid chelated zinc, a polyglutamic acid chelated zinc composition and its application. The polyglutamic acid chelated zinc prepared by this method not only has a high zinc chelation rate and low free zinc ion content, but its chelate bonds also have good stability, which can effectively prolong the duration of efficacy.

[0006] (II) Technical Solution To address the above problems, a first aspect of the present invention provides a method for preparing polyglutamic acid chelated zinc, comprising: S1, dissolve sodium γ-polyglutamate powder in water to prepare a sodium γ-polyglutamate aqueous solution with a concentration of 5~35 wt%. After adjusting the pH value to 3.5~4.0, heat to 80~90℃ and maintain for 6~8 h, then cool to room temperature, and then ultrafilter through a 30~40 kDa ultrafiltration membrane. Take the supernatant and ultrafilter through an 8~12 kDa ultrafiltration membrane to obtain a low molecular weight sodium γ-polyglutamate aqueous solution. S2, adjust the pH of the low molecular weight γ-polyglutamic acid aqueous solution to 4.0~4.5, add ammonia and water-soluble zinc salt according to the preset ratio, stir thoroughly to dissolve evenly, and then continue in a water bath at 60~80℃ for 2~4 h to obtain the product solution; S3, the product solution is ultrafiltered through a 0.8~1.2 kDa ultrafiltration membrane, and the filtrate is spray-dried to obtain polyglutamic acid chelated zinc.

[0007] Preferably, the mass fraction of low molecular weight γ-polyglutamic acid in the aqueous solution of low molecular weight γ-polyglutamic acid is 3 wt% to 30 wt%.

[0008] Preferably, in step S2, the molar ratio between NH3 in the ammonia water and polyglutamic acid repeating units is 1:4~20, wherein the low molecular weight γ-polyglutamic acid is composed of polyglutamic acid repeating units.

[0009] Preferably, the low molecular weight γ-polyglutamic acid has an average molecular weight of 10 to 30 kDa.

[0010] Preferably, in S2, the molar ratio of zinc in the water-soluble zinc salt to polyglutamic acid repeating units is 0.01~0.1:1.

[0011] Preferably, the water-soluble zinc salt includes at least one of zinc chloride, zinc sulfate, zinc nitrate, zinc acetate, zinc lactate, zinc gluconate, and zinc aspartate.

[0012] Preferably, the zinc content in the polyglutamic acid chelated zinc is 3.0-5.0 wt%. If the zinc content in the polyglutamic acid chelated zinc is 3.0 wt%, and the polyglutamic acid content (i.e., soluble solids content) in the aqueous solution is 3.0 wt%, then the zinc content in the polyglutamic acid chelated zinc aqueous solution is 0.09 wt%. If the zinc content in the polyglutamic acid chelated zinc is 5.0 wt%, and the polyglutamic acid content (i.e., soluble solids content) in the aqueous solution is 30 wt%, then the zinc content in the polyglutamic acid chelated zinc aqueous solution is 1.5 wt%. Due to considerations of efficiency and viscosity in actual operation, the optimal value is 10 wt%. As shown in Table 6, the measured zinc content of the reaction product is in the range of 0.3-0.6 wt%.

[0013] Preferably, the molecular weight of the polyglutamic acid chelated zinc, calculated based on the average molecular weight of γ-polyglutamic acid, ranges from 10 to 30 kDa.

[0014] A second aspect of the present invention provides a chelated zinc composition for use in cosmetics, the chelated zinc composition comprising polyglutamic acid chelated zinc obtained by any of the preparation methods described above, wherein the zinc mass fraction in the chelated zinc composition is between 0.03 wt% and 0.4 wt%.

[0015] Preferably, the soluble solids content in the chelated zinc composition is 5 wt% to 30 wt%.

[0016] Preferably, the content of free zinc ions in the chelated zinc composition does not exceed 1% of the total zinc content.

[0017] A third aspect of the present invention provides the application of the chelated zinc composition as described above in any of the above-described rinse-out shampoo and conditioner products, the rinse-out shampoo and conditioner products comprising the following components by mass percentage: 84%~96% main cleaning component, 2.0%~5.0% conditioning and thickening component, 1.0%~10% functional preservative component, and 0.3%~4.5% post-treatment component, the sum of all components being 100%, wherein in the functional preservative component, the chelated zinc composition accounts for 0.3%~1.0% by mass in the rinse-out shampoo and conditioner products.

[0018] (III) Beneficial Effects The above-mentioned technical solution of the present invention has the following beneficial technical effects: The present invention provides a method for preparing polyglutamic acid chelated zinc, a polyglutamic acid chelated zinc composition and its application. In the preparation method, the degradation and acidification of sodium polyglutamate are completed in one step by acid hydrolysis to obtain a low molecular weight γ-polyglutamic acid aqueous solution suitable for chelation reaction; then ammonia water and water-soluble zinc salt are added, and the mixture is continuously and thoroughly stirred under water bath conditions. After ultrafiltration, the obtained solution is spray dried to obtain polyglutamic acid chelated zinc. Compared to existing processes, the advantages are as follows: 1) The preparation method of this invention uses water as a solvent for homogeneous reaction, without violent acid-base neutralization and exothermic processes, making the process simple and under mild conditions; 2) In the method of this invention, acid hydrolysis is used to complete the degradation and acidification processes in one step, eliminating the need for re-acidification compared to existing enzymatic degradation methods, thus simplifying the process route; 3) The polyglutamic acid chelated zinc obtained by the preparation method of this invention has a chelation rate of over 99% based on elemental zinc, with very few free zinc ions and stable physicochemical properties; 4) Polyglutamic acid is obtained through microbial fermentation, and compared to chemically polymerized polymers, it has better biocompatibility and skin affinity, making it suitable for use as an active ingredient in cosmetics. The polyglutamic acid chelated zinc prepared by this invention, when applied to cosmetics, can effectively exert the dual effects of polyglutamic acid chelated zinc in moisturizing, whitening, oil control, and anti-inflammation. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the polyglutamic acid chelated zinc molecule structure of the present invention; Figure 2 This is a schematic diagram of the reaction process of the present invention; Figure 3 This is a detailed process route diagram of the preparation method of polyglutamic acid chelated zinc of the present invention. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and the accompanying drawings. It should be understood that these descriptions are merely exemplary and not intended to limit the scope of the invention. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the invention.

[0021] The term "water" as used in this invention, unless otherwise specified, refers to deionized water, ultrapure water, or distilled water. The terms "polyglutamic acid," "γ-polyglutamic acid," "gamma-polyglutamic acid," and "γ-PGA" used in this invention have the same meaning, and the terms "polyglutamic acid chelated zinc," "γ-polyglutamic acid chelated zinc," "gamma-polyglutamic acid chelated zinc," and "PGA chelated zinc" can also be used interchangeably.

[0022] In this invention, the total zinc content and free zinc ion content were determined using atomic absorption spectrophotometry and EDTA titration, respectively, according to the group standard T / CFIAS 6007-2024. This invention also employed cell experiments to analyze and compare the efficacy of polyglutamic acid chelated zinc. The molecular structure of the product, polyglutamic acid chelated zinc, is shown below. Figure 1 The reaction process is shown in Figure 2 For the specific process route, please refer to Figure 3 .

[0023] I. Acid hydrolysis of polyglutamic acid The preparation method of polyglutamic acid chelated zinc described in this invention mainly includes two major steps: acid hydrolysis and chelation, which are described below.

[0024] For the optimization of the acid hydrolysis process, based on the existing reference process conditions, a three-factor, three-level orthogonal experimental design was carried out with the three most important parameters as variables: pH value (3.5, 3.8, 4.0), reaction temperature (80℃, 85℃, 90℃), and reaction time (6 h, 7 h, 8 h), as shown in Table 1. Specifically, 5.0 g of sodium γ-polyglutamate powder (average molecular weight 40 kDa) was accurately weighed and added to a 50 mL centrifuge tube, along with 10 mL of deionized water. The solution was then completely dissolved by shaking on a shaker. The pH of the solution was adjusted to the set value using citric acid solution, and the shaker was turned on at the set temperature for acid hydrolysis, which was maintained for the set time. After acid hydrolysis, the reaction solution was transferred to 50 mL ultrafiltration tubes with molecular weight cutoffs of 10 kDa and 30 kDa, and ultrafiltered at 8000 rpm for 15 min. The 10 kDa-30 kDa fraction was freeze-dried and weighed to obtain the proportion of polyglutamic acid with the target molecular weight (10-30 kDa) (i.e., the yield). Since a parameter was needed to determine the results of the orthogonal experiment, the experimental product was dried to obtain the yield data. In actual production, drying is not required before proceeding to the next step. The yield was used as the criterion for determination, and range (R) analysis was performed to determine the primary and secondary influences of each factor on the molecular weight distribution. The results are shown in Table 2.

[0025] Table 1. Orthogonal experimental design for the acid hydrolysis of polyglutamic acid. Table 2 Results of orthogonal experiments on the acid hydrolysis of polyglutamic acid As shown in Table 2, the proportion of the target molecular weight exhibits a significant "first increase, then decrease" trend with pH, ​​reaching a peak (K2=68.93%) at pH=3.8 (level 2). Excessive acidity (pH=3.5) leads to excessive degradation of the polyglutamic acid molecular chain, generating a large number of small molecular fragments below 10 kDa; excessive alkalinity (pH=4.0) results in insufficient degradation of the molecular chain, increasing the proportion of large molecules greater than 30 kDa, both leading to a decrease in the proportion of the target range.

[0026] Meanwhile, the proportion of the target molecular weight showed a "first increase, then stabilize" trend with increasing temperature, reaching its highest proportion (K2=61.60%) at 85℃ (level 2). At 80℃, the degradation reaction rate was slow, and the macromolecules were not fully broken; at 90℃, the molecular chains degraded excessively, resulting in a slightly lower target proportion than at 85℃.

[0027] Furthermore, the proportion of the target molecular weight showed a trend of "slowly increasing and then leveling off" with the extension of reaction time. The proportion was slightly higher at 6h (level 3) (K=59.27%), but the difference between 7h and 8h was only 0.2%. This indicates that the degradation reaction was close to equilibrium at 5h, and the proportion of molecules in the target molecular weight range reached a relatively high level. Further extending the time would not have a significant effect on increasing the target proportion, but might instead increase energy consumption.

[0028] As shown in Table 2, the optimal process conditions for polyglutamic acid hydrolysis are: A2B2C3, i.e., reaction pH=3.8, reaction temperature 85℃, and reaction time 8 h. The pH value is the most influential factor. Since the reaction time has a very small impact, 7 h can be used in actual production to save time and reduce costs.

[0029] II. Scale-up production of polyglutamic acid hydrolysis process Based on the process conditions of Example 5 above, 5.0 kg of polyglutamic acid raw material was accurately weighed and placed in a 50 L reaction vessel containing 10 L of deionized water for acid hydrolysis. The hydrolysis product was purified by ultrafiltration through a MWCO (molecular weight cutoff) membrane of 30 kDa, and then purified by ultrafiltration through a MWCO (molecular weight cutoff) membrane of 10 kDa to obtain a concentrated solution, yielding 8.12 kg of colorless solution. 1 kg of the colorless product solution was accurately measured, freeze-dried, and yielded 0.49 kg of white solid. That is, the colorless product solution contained 3.98 kg of low molecular weight (10 kDa-30 kDa) polyglutamic acid, with a yield of 79.58%, consistent with the results of the orthogonal experiments above. The obtained colorless product solution was stored in a sealed container at low temperature for later use.

[0030] III. Chelation reaction of low molecular weight polyglutamic acid with zinc Using the dried low molecular weight polyglutamic acid (low molecular weight γ-polyglutamic acid) obtained from the above-mentioned scaled-up production (process conditions of Example 5) as raw material, zinc sulfate as zinc source, and three-factor, three-level orthogonal experimental design with the three most important parameters as variables, pH value (4.0, 4.3, 4.5), reaction temperature (60℃, 70℃, 80℃) and reaction time (2 h, 3 h, 4 h), as shown in Table 3. Accurately weigh 3.0 g of the aforementioned low molecular weight polyglutamic acid and add it to a 50 mL beaker pre-filled with 30.0 mL of deionized water. After complete dissolution under magnetic stirring, add 0.17 mL of ammonia (NH3 molar ratio of polyglutamic acid repeating unit to ammonia is 10:1) and 0.19 g of zinc sulfate (molar ratio of zinc to polyglutamic acid repeating unit is 0.05:1). After thorough stirring and complete dissolution, adjust the pH of the solution to the set value and adjust the water bath to the set temperature. After the chelation reaction is carried out for the set time, allow it to cool naturally to room temperature. Transfer the reaction solution to a 50 mL ultrafiltration tube with a molecular weight cutoff of 3 kDa and centrifuge at 8000 rpm for 15 min to remove unchelated free zinc ions, sulfate ions, and other small molecules. The total zinc content of the resulting solution is determined by ICP method. Using the total zinc content as the criterion, range (R) analysis is performed to determine the primary and secondary effects of each factor on the molecular weight distribution. The results are shown in Table 4. Each experiment is repeated three times.

[0031] Table 3 Orthogonal experimental design for the polyglutamic acid chelate zinc reaction Table 4. Results of the orthogonal experiment on the polyglutamic acid chelation reaction with zinc. As shown in Table 4, based on the magnitude of the range R: R(A)=0.28>R(B)=0.15>R(C)=0.02, it can be concluded that the order of influence of each factor on the total zinc content of polyglutamic acid chelated zinc is: factor A (pH value)> factor B (temperature)> factor C (time), that is, pH value is the key influencing factor.

[0032] Furthermore, the influence of various factor levels on total zinc content follows these patterns: (1) Key factor A: The effect of pH value Trend: When the pH value increased from 4.0 (A1) to 4.3 (A2), the average total zinc content increased significantly from 0.35 wt% to 0.44 wt% (k1→k2, an increase of 25.7%, equivalent to a zinc mass percentage of 3.39 wt% in polyglutamic acid chelated zinc); when the pH value continued to increase to 4.5 (A3), the average content decreased slightly to 0.41 wt% (k2→k3, a decrease of 6.8%).

[0033] Analysis of the cause: The chelating ability of polyglutamic acid is closely related to the dissociation state of the carboxyl group (-COOH) in its molecular structure. At pH=4.0, the carboxyl group is incompletely dissociated (high proportion of -COOH), providing few negative charge sites and resulting in weak complexation ability with Zn²⁺. At pH=4.3, a large number of carboxyl groups dissociate into carboxylate (-COO⁻), increasing the number of negative charge sites. This allows for the formation of stable five- or six-membered chelate rings with Zn²⁺ through the "carboxyl-O" group, achieving the highest chelation efficiency. When pH>4.3, the concentration of OH⁻ in the solution increases, and Zn²⁺ easily combines with OH⁻ to form Zn(OH)₂ precipitate, which reduces the chance of complexation with polyglutamic acid, leading to a decrease in total zinc content.

[0034] (2) Secondary factor B: The effect of temperature Trend: When the temperature rises from 60℃ (B1) to 70℃ (B2), the average total zinc content increases from 0.38wt% to 0.43wt% (k1→k2, an increase of 13.2%); when the temperature continues to rise to 80℃ (B3), the average content decreases to 0.40wt% (k2→k3, a decrease of 7.0%).

[0035] Analysis of the cause: Increased temperature can increase the rate of molecular thermal motion, accelerate the collision frequency of polyglutamic acid and Zn²⁺, and promote the chelation reaction. Therefore, the reaction efficiency is better at 70℃ than at 60℃. However, excessively high temperature (80℃) will cause thermal degradation of polyglutamic acid molecular chains, destroy some carboxyl groups, and may also make the structure of the formed chelate unstable, resulting in a decrease in total zinc content.

[0036] (3) Secondary factor C: The effect of time Trend: When the time was extended from 2h (C1) to 4h (C3), the average total zinc content remained at around 0.40wt%, and the differences between k1, k2 and k3 were very small (R=0.02), indicating that the time within 2-4h had almost no significant effect on the experimental results.

[0037] Analysis of the cause: The chelation reaction between polyglutamic acid and Zn²⁺ is a rapid complexation reaction, which basically reaches the reaction equilibrium within 2 hours. If the reaction time is extended further, the concentration of unreacted Zn²⁺ will be extremely low, and the chelation amount cannot be further increased. Therefore, the time factor is not a limiting condition within this range.

[0038] Based on the k-values ​​of each factor level (higher k-values ​​are better), combined with the primary and secondary relationships of the factors, and considering actual production conditions, the theoretically optimal reaction conditions are selected as: A2B2C. 3（ The reaction time is pH=4.3, temperature 70℃, and time 4h. However, since the effect of reaction time is very small, 2h can be used in actual production to save time.

[0039] IV. Optimization of other parameters in the polyglutamic acid chelation reaction with zinc Based on the above orthogonal experimental results, with pH=4.3, temperature 70℃, and time 2h as the reaction conditions, and other relevant parameters changed, the experimental design scheme is detailed in Table 5. Table 6 shows the product test results corresponding to each example in Table 5. The total zinc content was measured by ICP (atomic absorption spectrometry), and the free zinc content was determined by EDTA titration according to "TCFIAS 6007-2024 Determination of Total Zinc and Free Zinc in Chelated Zinc Additives".

[0040] Table 5. Preparation process parameters of polyglutamic acid chelated zinc Table 6 Detection results for each embodiment As can be seen from Table 6: (1) Examples 19, 21, and 34: The molecular weight of the raw material polyglutamic acid is different, but other conditions are the same.

[0041] If the molecular weight is large (≥30kDa), the high molecular weight chain segments are severely entangled, and the carboxyl groups are not sufficiently exposed, which affects the chelation reaction rate and causes the total zinc content to decrease. If the molecular weight is low (≤10kDa), the entanglement between polyglutamic acid molecular chains becomes stronger, which also affects the chelation reaction rate and causes the total zinc content to decrease.

[0042] (2) Examples 20, 21, and 22: The amount of ammonia added is different, while other conditions are the same.

[0043] When the amount of ammonia added is low, the number of amino or ammonium ions is insufficient, resulting in fewer coordinated zinc ions. As the amount of ammonia added increases, the compatibility between carboxyl group density and zinc ion concentration gradually improves, coordination efficiency increases, and zinc content increases. However, when the amount of ammonia added is too high, the pH change caused by the alkalinity of the ammonia itself, and the reaction of NH3 with the side chain -COOH of polyglutamic acid to generate NH4, will lead to problems. + COO - This, in turn, affects the efficiency of the chelation reaction and reduces the total zinc content.

[0044] (3) Examples 21, 23, 24, and 25: The amount of water added is different, but other conditions are the same.

[0045] In Examples 21, 23, 24, and 25, the mass fractions of low molecular weight γ-polyglutamic acid were 10 wt%, 3 wt%, 20 wt%, and 33.3 wt%, respectively. The results show that appropriately increasing the amount of water added can increase the total zinc content. This is because: firstly, the zinc salt dissolves more fully, optimizing the coordination environment; and secondly, the polyglutamic acid chains extend more fully, which is more conducive to the exposure of carboxyl groups, thus increasing the probability of chelation reaction. However, excessive water reduces the concentration of reactants in the reaction system, inevitably affecting the reaction rate.

[0046] (4) Examples 21, 26, and 27: The amount of zinc salt added is different, while other conditions are the same.

[0047] Appropriately increasing the amount of zinc salt added can increase the concentration of zinc ions and increase the chance of coordination; however, if too much zinc is added to the reaction system, the zinc ions will become oversaturated, some will form precipitates or cannot coordinate effectively, and the total zinc content will decrease.

[0048] (5) Examples 21, 28-33: Different types of zinc salts, but other conditions are the same.

[0049] As can be seen, the total zinc content is not differentiated by whether it is organic or inorganic zinc. Therefore, to improve efficiency, inorganic zinc salts with good solubility and fast dissolution rate (such as zinc sulfate with a high total zinc content in the product) can be preferred as the zinc source.

[0050] V. Scale-up production of low molecular weight polyglutamic acid chelated zinc Based on the process conditions of Example 21 above, the production of low molecular weight polyglutamic acid chelated zinc was scaled up.

[0051] To simplify production, the low molecular weight polyglutamic acid obtained from the acid hydrolysis process of Example 5 was prepared into aqueous solutions of different mass concentrations. The soluble solids content of each solution was measured using a saccharimeter, and the results are shown in Table 7. The results were plotted and fitted with a curve equation. The fitted equation had an r-value of 0.99977, indicating a very good fit. Therefore, in production, soluble solids data can be measured quickly and easily and converted into mass percentage concentration without the need for the cumbersome drying and weighing method.

[0052] Table 7. Mass percentage concentration and measured soluble solids content of low molecular weight polyglutamic acid Data Comparison Table According to the above scheme, the remaining 7.12 kg of low molecular weight polyglutamic acid solution (soluble solids content 36.3%) from the scale-up production using the acid hydrolysis process in Example 5 was transferred to a 50 L reaction vessel, and 24.28 kg of deionized water was added and stirred until homogeneous. The soluble solids content was measured to be 9.08%, or approximately 9.34% by mass. Then, according to the formulation in Example 21, appropriate amounts of ammonia and zinc sulfate were added, and the pH was adjusted to 4.3. The mixture was then stirred at 70°C for 2 hours. Afterward, the product was ultrafiltered through an ultrafiltration membrane with an MWCO=1 kDa, and the filtrate was spray-dried to obtain 3.51 kg of white powder. The total zinc content was measured to be 0.53 wt% by ICP, and no free zinc was detected by EDTA titration. This result is basically consistent with that of Example 21. This indicates that the process is suitable for scale-up production.

[0053] VI. Preparation of Polyglutamic Acid Chelated Zinc Composition Take an appropriate amount of the polyglutamic acid chelated zinc powder prepared in Example 21 above, dissolve it in an appropriate amount of deionized water according to the proportions in Table 8, and prepare polyglutamic acid chelated zinc compositions with different proportions.

[0054] Table 8. Formulation of Polyglutamic Acid Chelated Zinc Composition VII. Efficacy Evaluation Since polyglutamic acid has moisturizing and whitening effects, and zinc compounds have anti-inflammatory and oil-controlling effects, this invention characterizes its whitening, anti-inflammatory and oil-controlling effects by investigating the effects of polyglutamic acid chelated zinc on the inhibition of hyaluronidase, tyrosinase and 5-α reductase.

[0055] 1. Evaluation of anti-inflammatory efficacy (hyaluronidase inhibition rate) Hyaluronidase is involved in type I hypersensitivity reactions. Hyaluronidase is strongly correlated with inflammation and allergies. Studies have reported that various drugs that release histamine from mast cells can regulate hyaluronidase activity. Some anti-allergy drugs have strong inhibitory effects on hyaluronidase activity. Therefore, the inhibition of hyaluronidase activity is used as an indicator for studying anti-inflammatory effects.

[0056] The polyglutamic acid chelated zinc compositions prepared according to this invention (Examples 35-38) were subjected to hyaluronidase inhibition rate testing. The testing methods included: (1) Prepare the test solution according to the components shown in Table 9.

[0057] Table 9 Components of the solution to be tested (2) After keeping each solution obtained in step (1) at 37°C for 20 min, add 0.1 mL of 2.5 mol / L CaCl2 solution to each solution. After keeping the solution at 37°C for another 20 min, add 0.5 mL of 0.5 mg / mL sodium hyaluronate solution to solutions A and C respectively, and add 0.5 mL of pH 5.6 acetate buffer solution to solutions B and D respectively.

[0058] (3) After keeping the solutions obtained in step (2) at 37°C for 40 min, place them at room temperature (25°C) for 10 min, and add 0.5 mL of pure water and 0.1 mL of 5 mol / L NaOH solution respectively; at the same time, add 0.5 mL of mixed solution respectively. The mixed solution is prepared by mixing 50 mL of 10 mol / L sodium carbonate solution and 3.5 mL of acetylacetone evenly to form a mixed solution.

[0059] (4) Place each solution obtained in step (3) above in a boiling water bath for 15 min, then in an ice bath for 10 min, and then let it stand at room temperature (25℃) for 10 min; dissolve 0.8 g of p-dimethylaminobenzaldehyde in 15 ml of concentrated hydrochloric acid and 15 ml of anhydrous ethanol to prepare P-DAB colorimetric reagent; add 1 mL of P-DAB colorimetric reagent to each solution obtained in step (3) above.

[0060] (5) After thoroughly shaking the solution obtained in step (4) above, add anhydrous ethanol to make up the solution volume to 8 mL; after standing at room temperature (25℃) for 30 min, use a UV spectrophotometer to detect the absorbance at 530 nm.

[0061] The method for calculating the hyaluronidase inhibition rate is as follows: ; Where: A is the UV absorbance of solution A (i.e., a mixture of the sample, hyaluronidase, and sodium hyaluronate); B is the UV absorbance of solution B (i.e., a mixture of the sample and acetate buffer); C is the UV absorbance of solution C (i.e., a mixture of hyaluronidase and sodium hyaluronate); and D is the UV absorbance of solution D (i.e., acetate buffer).

[0062] The hyaluronidase inhibition rate was tested according to the above method. The composition samples of Examples 35-38 were diluted 20 times as samples. A 1 wt% sodium polyglutamate aqueous solution was used as a negative control, a 2.48 wt% zinc sulfate aqueous solution was used as a positive control, and acetate buffer was used as a blank. The results are shown in Table 10.

[0063] Table 10 Results of Hyaluronidase Inhibition Rate Detection As shown in Table 10, the hyaluronidase inhibition rate of the polyglutamic acid chelated zinc composition was significantly higher than that of the sodium polyglutamate aqueous solution, indicating that zinc has a significant promoting effect. Furthermore, the hyaluronidase inhibition rate of chelated zinc is higher than that of free zinc ions, indicating that the chelation structure can improve the utilization rate of zinc. Moreover, the ratio of the polyglutamic acid chelated zinc composition (i.e., the soluble solids content and zinc mass fraction) also affects the hyaluronidase inhibition rate. These results indicate that the composition ratio in Example 37 showed the best hyaluronidase inhibition rate. Therefore, it can be concluded that a suitable ratio of polyglutamic acid chelated zinc composition significantly enhances the hyaluronidase inhibition rate and possesses good anti-inflammatory effects.

[0064] 2. Evaluation of skin whitening efficacy (tyrosinase inhibition rate) Tyrosinase is the rate-limiting enzyme in melanin synthesis. The depth of skin pigmentation and pigmentation disorders are related to the activity of tyrosinase in the body; higher activity and higher content lead to easier melanin formation. During metabolism, L-tyrosine can be converted to dopa by tyrosinase, and dopa can then be further catalyzed by tyrosinase to form dopaquinone. Since dopaquinone has a maximum absorption peak at 475 nm, measuring the absorbance of the product dopaquinone can characterize the sample's inhibition efficiency against tyrosine and demonstrate its whitening effect. Test methods for the whitening effect of polyglutamic acid chelated zinc include: (1) Prepare the test solution according to the components shown in Table 11.

[0065] Table 11. Proportions of the solution to be tested (2) After heating each solution in a water bath at 37°C for 10 min, the absorbance at 475 nm was measured using a UV spectrophotometer.

[0066] The method for calculating the tyrosinase inhibition rate is as follows: ; Where: C1 is the absorbance of the tyrosine solution; C2 is the absorbance of the mixed solution of tyrosinase and tyrosine; T1 is the absorbance of the test sample and the tyrosine solution; T2 is the absorbance of the test sample, the mixed solution of tyrosinase and tyrosine.

[0067] The tyrosinase inhibition rate was tested using the method described above. The composition of Example 37 was diluted 20 times as a sample. A 1 wt% sodium polyglutamate (molecular weight 8 kDa) aqueous solution was used as a negative control, a 2.48 wt% zinc sulfate aqueous solution was used as a positive control, and phosphate buffer was used as a blank. The results are shown in Table 12.

[0068] Table 12 Tyrosinase Inhibition Rate As shown in Table 12, all sample solutions exhibited tyrosinase inhibition, similar to the hyaluronidase inhibition pattern described above. Specifically, the tyrosinase inhibition rate of polyglutamic acid chelated zinc was significantly higher than that of sodium polyglutamate, indicating that zinc plays a significant promoting role. Furthermore, the tyrosinase inhibition rate of chelated zinc was higher than that of free zinc ions, suggesting that the chelation structure improves the utilization rate of zinc. Therefore, polyglutamic acid chelated zinc significantly enhances the tyrosinase inhibition rate and possesses excellent whitening effects.

[0069] 3. Evaluation of oil-controlling efficacy (5α-reductase inhibition rate) 5α-reductase is a membrane protease dependent on reduced coenzyme II (NADPH) and an important enzyme in the skin that metabolizes androgens. It irreversibly converts testosterone to dihydrotestosterone (DHT), the most potent androgen, which can induce excessive sebum secretion from the sebaceous glands. Inhibiting 5α-reductase activity to reduce DHT levels can effectively alleviate excessive sebum secretion. Therefore, inhibiting 5α-reductase activity is used as an indicator in research on oil-controlling effects.

[0070] The test methods for the inhibition rate of polyglutamic acid chelated zinc 5α-reductase include: (1) Take out the Omnimabs Human Steroid 5 Alpha Reductase (SRD5a) kit and place it at room temperature (20-25℃) for 30 min.

[0071] (2) Follow the kit operation procedure strictly and finally measure the UV absorption at 450nm.

[0072] (3) The content of 5α-reductase was obtained from the standard curve, and the inhibition rate of 5α-reductase was calculated.

[0073] The method for calculating the 5α-reductase inhibition rate is as follows: ; Where: C is the 5α-reductase content of the negative control; T is the 5α-reductase content of the sample solution.

[0074] The tyrosinase inhibition rate was tested according to the above method. The composition of Example 37 was diluted 20 times as a sample. 1 wt% sodium polyglutamate (molecular weight 8 kDa) aqueous solution and 2.48 wt% zinc sulfate aqueous solution were used as controls. Deionized water was used as a negative control and 0.5 μmol dutasteride was used as a positive control. The results are shown in Table 13.

[0075] Table 13 5α-Reductase Inhibition Rate As shown in Table 13, all sample solutions exhibited 5α-reductase inhibition, similar to the hyaluronidase inhibition pattern described above. Specifically, the tyrosinase inhibition rate of polyglutamic acid chelated zinc was significantly higher than that of sodium polyglutamate, indicating that zinc plays a significant promoting role. Furthermore, the tyrosinase inhibition rate of chelated zinc was higher than that of free zinc ions, suggesting that the chelation structure improves the utilization rate of zinc. Therefore, polyglutamic acid chelated zinc significantly enhances the 5α-reductase inhibition rate and possesses excellent oil-controlling effects.

[0076] 7. Formulation of rinse-off shampoo and conditioner products using polyglutamic acid chelated zinc compositions Using the composition of Example 37 as raw material, different rinse-off shampoo and conditioner products were formulated according to the formulations in Tables 14-20.

[0077] Table 14 Example 39: Formulation of an anti-allergic and oil-controlling shampoo containing polyglutamic acid chelated zinc Table 15 Example 40: Oil-controlling and anti-dandruff shampoo formula containing polyglutamic acid chelated zinc Table 16 Example 41: Oil-controlling cleansing mousse formula containing polyglutamic acid chelated zinc VIII. Effects of using rinse-out shampoo and conditioner products with polyglutamic acid chelated zinc composition (1) Anti-allergy and oil-controlling shampoo Based on the formula in Table 14, the types and amounts of zinc-containing components were appropriately adjusted according to Table 17, and the amount of deionized water was adjusted accordingly. The remaining components and their amounts remained unchanged, resulting in several different shampoos. Thirty male volunteers aged 35-50 with short hair were selected and divided into 5 groups. Each group strictly followed the methods described below to use the 5 shampoos listed in Table 17. The trial lasted for 2 weeks, and all volunteers participated after signing informed consent forms.

[0078] 1) Preliminary preparation: Enter the test environment and adapt to the temperature of 18-22℃ and humidity of 40%-60%, sit quietly for 15-30 minutes; moisten the scalp with warm water at 38-40℃ for 1 minute; complete the index testing, including the following core index tests: scalp oil content change rate (scalp oil content is measured by a sebum meter, and the ratio of scalp oil content at the end of the test to that at the beginning of the test is used as the change rate), fluffiness (visual score, score 1-10, the higher the score, the better the fluffiness), freshness (double-blind score by the subject, score 1-10, the higher the score, the better the freshness), and irritation (double-blind score by the subject, score 1-10, the higher the score, the more obvious the irritation).

[0079] 2) Product usage: Pipette 2ml of shampoo; after lathering in your palm, apply and rub for 5 seconds until rich foam is formed, evenly covering the scalp and hair; massage properly, massaging the scalp in circular motions with your fingertips for 1 minute, and gently massaging the hair for 30 seconds (without scratching with nails); leave the foam on the hair and scalp for 3 minutes; then rinse thoroughly with warm water at 38-40℃ for 1 minute, leaving no slippery residue.

[0080] 3) Post-use treatment: Use a towel to absorb water. Gently press the towel of the same size to absorb the water until it no longer drips, without rubbing; then let it air dry naturally.

[0081] 4) Product usage requirements during the period: Wash your hair once every morning before noon until the end of the trial; use only the designated shampoo and discontinue the use of other hair care / styling products; abide by the lifestyle restrictions, do not dye or perm your hair, and avoid washing your hair immediately after sun exposure or strenuous exercise; report any discomfort, and stop using the product immediately and report any discomfort such as redness, swelling, or itching.

[0082] Table 17 Types and Amounts of Zinc-Containing Components The efficacy evaluation results are shown in Table 18. It is evident that when the amount of the polyglutamic acid chelated zinc composition added is less than a certain value, the oil-controlling effect of the shampoo significantly decreases. Furthermore, for different zinc-containing compositions with the same added amount, the comfort level, as characterized by the volume and freshness scores, is worse compared to the polyglutamic acid chelated zinc composition. The irritation score also shows that the polyglutamic acid chelated zinc composition has a relatively lower irritation level.

[0083] Table 18. Efficacy evaluation results (mean) of different anti-allergy and oil-control shampoos (2) Oil-controlling and anti-dandruff shampoo Based on the formula in Table 15, the types and amounts of zinc-containing components were appropriately adjusted according to Table 19, and the amount of deionized water was adjusted accordingly. The remaining components and their amounts remained unchanged, resulting in several different shampoos. The volunteers and testing methods were the same as for the signed anti-allergy and oil-control shampoo. The only difference was that the scalp oil change rate was adjusted to the dandruff quantity change rate (using a scalp tester, magnified 100 times, to take a picture of the scalp at the center of the top of the head, and the data was recorded per centimeter). 2 The amount of dandruff within the area, and the ratio of the result at the end of the test to that at the beginning of the test as the rate of change.

[0084] Table 19 Types and Amounts of Zinc-Containing Components The efficacy evaluation results are shown in Table 20. It is evident that when the amount of the polyglutamic acid chelated zinc composition added is less than a certain value, the dandruff-removing effect of the shampoo significantly decreases. Furthermore, for different zinc-containing compositions with the same added amount, the comfort level, as characterized by the volume and freshness scores, is worse compared to the polyglutamic acid chelated zinc composition. The irritation score also shows that the polyglutamic acid chelated zinc composition has a relatively lower irritation level.

[0085] Table 20 Efficacy evaluation results (mean) of different oil-control and anti-dandruff shampoos (3) Oil-controlling cleansing mousse Based on the formula in Table 16, the types and amounts of zinc-containing components were appropriately adjusted according to Table 21, and the amount of deionized water was adjusted accordingly. The remaining components and their amounts remained unchanged, resulting in several different cleansing mousses. Thirty male volunteers aged 35-50 without allergic dermatitis, acne, or other skin diseases, and without facial lesions, inflammation, or scars, were selected and divided into 5 groups. Each group strictly followed the methods described below to use one of the 5 oil-controlling cleansing mousses listed in Table 12. The trial lasted for 4 weeks, and all volunteers participated after signing informed consent forms.

[0086] 1) Preliminary preparation: Enter the test environment and adapt to the temperature of 18-22℃ and humidity of 40%-60%, and sit quietly for 15-30 minutes.

[0087] 2) Product usage: Pipette 2ml of cleansing mousse → wet face → apply mousse → massage in circular motions for 1 minute → rinse with 38-40℃ warm water for 1 minute → gently pat dry with a towel.

[0088] 3) Product usage requirements during the period: Use the cleansing mousse once in the morning and once in the evening every day until the end of the trial; use only the designated cleansing product and discontinue the use of other cleansing / cleaning products; abide by the taboos in life, and avoid hair dyeing, perming, and sun exposure; report any discomfort, and stop immediately and report any discomfort such as redness, swelling, or itching.

[0089] 4) After using the cleansing mousse for 4 weeks, the following indicators were tested: oil control efficacy, facial skin oil content (the skin oil content of the forehead and left and right cheeks was measured with a skin oil tester and the average value was taken), facial skin oil content change rate (the change rate of facial skin oil content before and after the test); 2-hour skin oil content reduction rate (the change rate of skin oil content before and 2 hours after use); refreshing feeling (double-blind score by the subjects, with a score of 1-10, the higher the score, the better the refreshing feeling); irritation (double-blind score by the subjects, with a score of 1-10, the higher the score, the more obvious the irritation).

[0090] Table 21 Types and Amounts of Zinc-Containing Components The efficacy evaluation results are shown in Table 22. It can be seen that when the amount of polyglutamic acid chelated zinc composition added is less than a certain value, the oil control effect of the cleansing mousse decreases significantly. Furthermore, for different zinc-containing compositions with the same amount of added zinc, the comfort level, characterized by refreshingness and irritation scores, is worse than that of the polyglutamic acid chelated zinc composition.

[0091] Table 22 Efficacy evaluation results (mean) of different oil-controlling cleansing mousses It should be understood that the specific embodiments described above are merely illustrative or explanatory of the principles of the invention and do not constitute a limitation thereof. Therefore, any modifications, equivalent substitutions, improvements, etc., made without departing from the spirit and scope of the invention should be included within the protection scope of the invention. Furthermore, the appended claims are intended to cover all variations and modifications falling within the scope and boundaries of the appended claims, or equivalent forms of such scope and boundaries.

Claims

1. A method for preparing polyglutamic acid chelated zinc, characterized in that, include: S1, dissolve sodium γ-polyglutamate powder in water to prepare sodium γ-polyglutamate aqueous solution, adjust the pH value to 3.5~4.0, heat to 80~90℃ and maintain for 6~8 h, cool to room temperature, and obtain low molecular weight sodium γ-polyglutamate aqueous solution after ultrafiltration; S2, adjust the pH of the low molecular weight γ-polyglutamic acid aqueous solution to 4.0~4.5, add ammonia and water-soluble zinc salt according to the preset ratio, stir thoroughly to dissolve evenly, and then continue in a water bath at 60~80℃ for 2~4 h to obtain the product solution; S3, the product solution is ultrafiltered through a 0.8~1.2 kDa ultrafiltration membrane, and the filtrate is spray-dried to obtain polyglutamic acid chelated zinc.

2. The method for preparing polyglutamic acid chelated zinc according to claim 1, characterized in that, The low molecular weight γ-polyglutamic acid aqueous solution contains 3 wt% to 30 wt% of low molecular weight γ-polyglutamic acid.

3. The method for preparing polyglutamic acid chelated zinc according to claim 2, characterized in that, In S2, the molar ratio between NH3 in the ammonia water and polyglutamic acid repeating units is 1:4~20, wherein the low molecular weight γ-polyglutamic acid is composed of polyglutamic acid repeating units.

4. The method for preparing polyglutamic acid chelated zinc according to claim 2, characterized in that, The low molecular weight γ-polyglutamic acid has an average molecular weight of 10–30 kDa.

5. The method for preparing polyglutamic acid chelated zinc according to claim 3, characterized in that, In S2, the molar ratio of zinc in the water-soluble zinc salt to polyglutamic acid repeating units is 0.01~0.1:

1.

6. The method for preparing polyglutamic acid chelated zinc according to claim 5, characterized in that, The water-soluble zinc salt includes at least one of zinc chloride, zinc sulfate, zinc nitrate, zinc acetate, zinc lactate, zinc gluconate, and zinc aspartate.

7. The method for preparing polyglutamic acid chelated zinc according to claim 1, characterized in that, The zinc content in the polyglutamic acid chelated zinc is 3.0-5.0 wt%.

8. The method for preparing polyglutamic acid chelated zinc according to claim 7, characterized in that, The molecular weight of the polyglutamic acid chelated zinc is calculated based on the average molecular weight of γ-polyglutamic acid, and its range is 10~30 kDa.

9. A chelated zinc composition for use in cosmetics, characterized in that, The chelated zinc composition comprises polyglutamic acid chelated zinc obtained by the preparation method according to any one of claims 1-8, wherein the zinc mass fraction in the chelated zinc composition is 0.03wt% to 0.4wt%.

10. The chelated zinc composition for cosmetics according to claim 9, characterized in that, The soluble solids content in the chelated zinc composition is 5% to 30%.

11. The chelated zinc composition for cosmetics according to claim 9, characterized in that, The content of free zinc ions in the chelated zinc composition does not exceed 1% of the total zinc content.

12. The application of the chelated zinc composition as described in any one of claims 9-11 in rinse-out hair care products, characterized in that, The rinse-out type of shampoo and conditioner comprises the following components by weight percentage: 84%~96% main cleaning component, 2.0%~5.0% conditioning and thickening component, 1.0%~10% functional preservative component, and 0.3%~4.5% post-treatment component, with the sum of all components being 100%. Among the functional preservative component, the chelated zinc composition accounts for 0.3%~1.0% by weight in the rinse-out type of shampoo and conditioner.

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