Preparation method of donkey-hide gelatin peptide-metal ion chelate with high chelation rate

Abstract

The invention relates to the technical field of active peptide chelate preparation, and discloses a high-chelation-rate colla corii asini peptide-metal ion chelate preparation method, which comprises: preparing a colla corii asini protein solution; adding a metal ion solution into the colla corii asini protein solution to carry out pre-reaction so as to construct a metal ion template; then adding a core function protease with a self-speed-limiting characteristic on a product, and carrying out synchronous enzymolysis-chelation reaction under the guidance of a template; and finally terminating the reaction to obtain a product. According to the method, oriented cutting of protease hydrolysis sites is realized by pre-constructing a metal ion template, and excessive hydrolysis of the product is avoided by utilizing the self-speed-limiting characteristic of the core function protease. Enzymolysis and chelation reaction are carried out synchronously, so that the process flow is obviously simplified, and the loss of intermediate products is avoided. The colla corii asini peptide-metal ion chelate prepared by the method has the technical effects of high chelation rate, concentrated molecular weight distribution and high utilization rate of raw material protein.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of active peptide chelate preparation, in particular to a preparation method of high-chelation-rate donkey-hide gelatin peptide-metal ion chelate. BACKGROUND

[0002] Metal elements are essential trace elements for maintaining life activities and play a key physiological function in the body. Traditionally, metal elements are supplemented in the form of inorganic salts, which has the technical problems of low bioavailability and easy to cause gastrointestinal irritation. The peptide-metal ion chelate formed by chelating metal ions with polypeptides can significantly improve the absorption efficiency and biocompatibility of metal ions, and is the current technical direction of developing efficient and safe metal element supplements. As a traditional medicine and food homologous material, the main component of donkey-hide gelatin is collagen protein, which is a good raw material source for preparing bioactive peptides.

[0003] In the existing technology, the preparation of donkey-hide gelatin peptide-metal ion chelate usually adopts a step-by-step process. This process first hydrolyzes donkey-hide gelatin protein into a polypeptide mixture by enzymatic hydrolysis, then separates and purifies the polypeptide mixture by ultrafiltration to obtain a peptide segment with a specific molecular weight range, and finally mixes the purified peptide segment with a metal salt solution to perform a chelation reaction. However, this step-by-step process has inherent technical defects. The process flow is long and includes multiple independent operation units, resulting in complex operation and prolonged production cycle. More importantly, in the separation and purification step of the enzymatic hydrolysate, some effective peptide segments are inevitably lost, directly reducing the overall utilization rate of the raw material donkey-hide gelatin protein.

[0004] At the same time, the traditional enzymatic hydrolysis process itself lacks effective control mechanisms. The hydrolysis of the substrate by the conventional protease used is usually random and continuous, which results in a wide molecular weight distribution and complex composition of the enzymatic hydrolysate, and a large number of peptide segments with structures that are not conducive to forming stable chelate structures with metal ions, ultimately limiting the improvement of the chelation rate of the product. To simplify the process, some technical solutions attempt to synchronize the enzymatic hydrolysis and chelation reaction, but without the regulation of specific technical means, this simple synchronization reaction does not solve the randomness problem of the enzymatic hydrolysis process. If a conventional protease with high activity is used, excessive hydrolysis will easily occur, i.e., the target active peptide segments that have already been generated will be further degraded into short peptides or amino acids with smaller molecular weights and lower chelation activity, which will damage the quality of the final product.

[0005] Therefore, how to accurately regulate the hydrolysis process of donkey-hide gelatin protein in a simplified process, directionally generate target peptide segments with high chelation activity, and avoid material loss and product over-degradation, so as to simultaneously achieve high chelation rate, high protein utilization rate, and concentrated product molecular weight distribution, is a technical problem that needs to be solved in the current field. SUMMARY

[0006] In view of the deficiencies of the prior art, the application provides a preparation method of a high-chelating-rate Ejiao peptide-metal ion chelate, which solves the technical problems of the prior art, such as a complex process flow, low raw material utilization rate caused by loss of intermediate products, or low chelating rate and wide molecular weight distribution of the final product caused by uncontrollable enzymolysis process and easy over-hydrolysis in the process of the conventional synchronous method.

[0007] To achieve the above object, the application is implemented by the following technical scheme: The application provides a preparation method of a high-chelating-rate Ejiao peptide-metal ion chelate, which comprises the following steps: S1, preparing an Ejiao protein solution: dissolving Ejiao in a 0.05-0.1M phosphate buffer solution to obtain an Ejiao protein solution with a mass concentration of 5.0%-15.0% (w / v), and adjusting the pH value of the Ejiao protein solution to 7.0-7.5; S2, pre-building a metal ion template: adding a metal salt to the Ejiao protein solution obtained in step S1, so that the mass ratio of Ejiao protein to metal ion is 10:1-20:1, and reacting at 45-55℃ for 20-40 minutes to obtain a reaction system containing a metal ion template; S3, performing self-limited synchronous enzymolysis-chelation reaction under the guidance of the template: adding a core functional protease to the reaction system obtained in step S2, and performing synchronous reaction at 48-52℃ and pH 7.0-7.5 for 4.0-7.0 hours to obtain a chelate mixture, wherein the mass ratio of the core functional protease to Ejiao protein is 1:150-1:250; S4, terminating the reaction and obtaining the product: heating the chelate mixture obtained in step S3 to 90-95℃ and maintaining for 15-20 minutes to inactivate the core functional protease, and then obtaining the Ejiao peptide-metal ion chelate through solid-liquid separation.

[0008] The technical mechanism of this invention is as follows: In step S2, metal ions pre-bind with amino acid residues (such as side chain groups of aspartic acid and glutamic acid) with high affinity on the long chain of donkey-hide gelatin protein, forming steric hindrance around these sites and constituting a metal ion template. In step S3, during hydrolysis, the core functional protease's cleavage of peptide bonds at sites with metal ion templates is hindered, thus preferentially cleaving peptide bonds at other sites. This process selectively releases peptides rich in high-affinity amino acid residues from the protein backbone. Once released, these peptides immediately form stable chelates with metal ions in the reaction system, achieving synchronization of enzymatic hydrolysis and chelation. As the reaction proceeds, the concentration of the generated donkey-hide gelatin peptide-metal ion chelate continuously increases, inhibiting the catalytic activity of the core functional protease and automatically reducing the hydrolysis rate, thereby avoiding over-cleavage of the generated target peptides.

[0009] As a specific embodiment of the technical solution of the present invention, in step S3, the preparation method of the core functional protease includes: selecting Bacillus subtilis, which is tolerant to the target metal ions, as the starting strain and fermenting it, and then centrifuging, ammonium sulfate precipitation and dialysis treatment of the obtained fermentation broth in sequence to obtain the core functional protease.

[0010] As a specific embodiment of the technical solution of the present invention, in step S3, the core functional protease is a neutral protease.

[0011] As a specific embodiment of the technical solution of the present invention, in step S3, the endpoint of the template-guided self-limiting synchronous enzymatic hydrolysis-chelation reaction is determined by the following criteria: monitoring the degree of hydrolysis of the reaction system, when the growth rate of the degree of hydrolysis is less than 1% within 0.5-1.5 hours, the reaction is considered to have reached the endpoint.

[0012] As a specific embodiment of the technical solution of the present invention, in step S2, the metal salt is a bio-acceptable organic acid salt that provides ferrous ions or zinc ions.

[0013] As a specific embodiment of the technical solution of the present invention, in step S2, the reaction of preconstructing the metal ion template is carried out at a stirring speed of 60-100 rpm.

[0014] As a specific embodiment of the technical solution of the present invention, after the endpoint determination in step S4, the method for monitoring the degree of hydrolysis is the online pH-stat method or the offline o-phthalaldehyde method.

[0015] As a specific embodiment of the technical solution of the present invention, in step S4, the solid-liquid separation method is centrifugal separation, the centrifugal force is set to 4000-6000×g, and the centrifugation time is set to 15-20 minutes.

[0016] As a specific embodiment of the technical scheme of the present application, the operation of dissolving the donkey-hide gelatin in the 0.05-0.1M phosphate buffer solution in step S1 is performed at a temperature of 55-65℃.

[0017] As a specific embodiment of the technical scheme of the present application, in step S2, the operation of adding the metal salt is performed by pre-dissolving the metal salt in deionized water, and then slowly adding the obtained metal salt solution to the donkey-hide gelatin protein solution.

[0018] The present application provides a preparation method of a high-chelation-rate donkey-hide gelatin peptide-metal ion chelate. The following beneficial effects are achieved: 1. The present application pre-constructs a metal ion template to guide the selective hydrolysis of protease, preferentially releases specific peptide segments with high chelation activity, and realizes the synchronous performance of enzymolysis and chelation. This avoids the loss of active peptide segments in the traditional step-by-step method, thereby significantly improving the effective utilization rate of donkey-hide gelatin raw materials and the high chelation rate of the final product.

[0019] 2. The present application integrates the traditional steps of enzymolysis, separation, and chelation into a single reaction kettle to complete the synchronous reaction step, greatly simplifying the production process flow, eliminating the separation and transfer process of intermediate products, shortening the overall production cycle, reducing the requirements for types and quantities of equipment, and reducing production costs.

[0020] 3. The present application constructs a self-limiting reaction system by utilizing the activity inhibition of the product donkey-hide gelatin peptide-metal ion chelate on core functional protease. The system can automatically control the reaction endpoint at the stage where a large amount of target peptide segments are generated and not overcut, effectively preventing overhydrolysis, making the molecular weight distribution of the final product more concentrated and uniform, and ensuring the stability of product quality. DETAILED DESCRIPTION

[0021] The technical scheme in the embodiments of the present application will be described below in conjunction with examples, comparative examples, and test examples. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of the present application.

[0022] Raw materials and reagents: The main raw materials and reagents used in the following examples and comparative examples are as follows: the reagents not specifically mentioned are commercially available analytical pure or higher grade products.

[0023] L-ferrous lactate, CAS number: 5905-52-2; Zinc gluconate, CAS No.: 4468-02-4; Sodium phosphate, dibasic, CAS No.: 7558-79-4; Sodium phosphate, monobasic, CAS No.: 7558-80-7; Sodium hydroxide, CAS No.: 1310-73-2; Ammonium sulfate, CAS No.: 7783-20-2; Tryptone, CAS No.: 91079-40-2; Yeast extract, CAS No.: 8013-01-2; Sodium chloride, CAS No.: 7647-14-5.

[0024] Example 1-3: Example 1: The embodiment provides a preparation method of a high-chelation-rate colla peptide-ferrous ion chelate, and specific steps are as follows: Step S1, preparing a colla protein solution: 50g of colla is weighed and added into a 0.05M phosphate buffer solution, is dissolved by stirring at 55℃, and is diluted to 1L to obtain a colla protein solution with a mass concentration of 5.0% (w / v). The pH value of the colla protein solution is adjusted to 7.0 by using a 0.1M sodium hydroxide solution.

[0025] Step S2, pre-establishing a metal ion template: L-ferric lactate is dissolved in deionized water to form a solution. The L-ferric lactate solution is slowly added to the colla protein solution obtained in step S1 under the stirring speed of 60rpm, so that the final mass ratio of the colla protein to the ferrous ion is 20:1. After the addition is completed, the reaction system is reacted at 45℃ for 20 minutes.

[0026] Step S3, performing self-limited synchronous zymolysis-chelation reaction under template guidance: The core functional protease powder prepared in the preparation example 1 is added into the reaction system obtained in step S2, so that the mass ratio of the core functional protease to the colla protein is 1:250. The temperature of the reaction system is maintained at 48℃, the pH value is maintained at 7.0, and synchronous reaction is performed for 7.0 hours.

[0027] Step S4, terminating the reaction and obtaining the product: The chelate mixture obtained in step S3 is quickly heated to 90℃ and maintained at this temperature for 20 minutes to inactivate the core functional protease. The inactivated mixture is centrifuged at 4℃ and 4000xg for 15 minutes, and the supernatant is collected, which is a high-chelation-rate colla peptide-ferrous ion chelate solution.

[0028] Example 2: The embodiment provides a preparation method of a high-chelation-rate Ejiao peptide-zinc chelate, and specific steps are as follows: Step S1, preparation of an Ejiao protein solution: 100 g of Ejiao is weighed, added into a 0.075M phosphate buffer solution, dissolved by stirring at 60℃, and then diluted to 1L to obtain an Ejiao protein solution with a mass concentration of 10.0% (w / v). The pH value of the Ejiao protein solution is adjusted to 7.25 by using a 0.1M hydrochloric acid solution.

[0029] Step S2, pre-construction of a metal ion template: Zinc gluconate is dissolved in deionized water to form a solution. The zinc gluconate solution is slowly added to the Ejiao protein solution obtained in step S1 under the stirring speed of 80rpm, so that the final mass ratio of Ejiao protein to zinc ions is 15:1. After the addition is completed, the reaction system is reacted at 50℃ for 30 minutes.

[0030] Step S3, template-directed self-limited synchronous enzymolysis-chelation reaction: The core functional protease powder prepared in Preparation Example 1 is added to the reaction system obtained in step S2, so that the mass ratio of the core functional protease to the Ejiao protein is 1:200. The temperature of the reaction system is maintained at 50℃, the pH value is maintained at 7.25, and synchronous reaction is performed for 5.5 hours.

[0031] Step S4, termination of the reaction and obtaining of the product: The chelate mixture obtained in step S3 is quickly heated to 92.5℃, and maintained at this temperature for 17.5 minutes to inactivate the core functional protease. The inactivated mixture is centrifuged at 4℃ and 5000xg for 17.5 minutes, and the supernatant is collected, which is a high-chelation-rate Ejiao peptide-zinc chelate solution.

[0032] Example 3: The embodiment provides a preparation method of a high-chelation-rate Ejiao peptide-zinc chelate, and specific steps are as follows: Step S1, preparation of an Ejiao protein solution: 150 g of Ejiao is weighed, added into a 0.1M phosphate buffer solution, dissolved by stirring at 65℃, and then diluted to 1L to obtain an Ejiao protein solution with a mass concentration of 15.0% (w / v). The pH value of the Ejiao protein solution is adjusted to 7.5 by using a 0.1M hydrochloric acid solution.

[0033] Step S2, pre-construction of a metal ion template: L-ferric lactate was dissolved in deionized water to form a solution. The L-ferric lactate solution was slowly added to the colla-gen solution obtained in step S1 under stirring at a speed of 100 rpm, so that the final mass ratio of collagen to ferrous ions was 10:1. After the addition was completed, the reaction system was reacted at 55℃ for 40 minutes.

[0034] Step S3, performing template-directed self-limited synchronous enzymatic hydrolysis-chelation reaction: To the reaction system obtained in step S2, the core functional protease powder prepared in Preparation Example 1 was added, so that the mass ratio of core functional protease to collagen was 1:150. The temperature of the reaction system was maintained at 52℃, and the pH value was maintained at 7.5, and the synchronous reaction was performed for 4.0 hours.

[0035] Step S4, terminating the reaction and obtaining the product: The chelate mixture obtained in step S3 was quickly heated to 95℃ and maintained at this temperature for 15 minutes to inactivate the core functional protease. The inactivated mixture was centrifuged at 4℃ and 6000xg for 20 minutes, and the supernatant was collected, which was the high-chelation-rate collagen peptide-ferrous chelate solution.

[0036] Comparative Examples 1-3: Comparative Example 1: Compared with Example 2, the difference is that the present comparative example uses the traditional step-by-step method: first, the collagen solution is reacted with the commercially available ordinary neutral protease to perform sufficient enzymatic hydrolysis, then the enzymatic hydrolysate is separated by ultrafiltration, and finally the separated peptide component is chelated with zinc gluconate, and the rest is the same.

[0037] Comparative Example 2: Compared with Example 2, the difference is that the present comparative example omits the step of pre-building a metal ion template, and instead mixes the collagen solution, zinc gluconate and core functional protease at the beginning of the reaction to perform synchronous reaction, and the rest is the same.

[0038] Comparative Example 3: Compared with Example 2, the difference is that the core functional protease used in step S3 of the present comparative example is replaced by an equal-activity commercially available ordinary neutral protease, and the rest is the same.

[0039] Test Examples 1-3: Test Example 1: Chelation rate determination: The present test example is used to determine the metal ion chelation rate of the products prepared in Examples 1-3 and Comparative Examples 1-3.

[0040] Experimental steps: Sample preparation: Accurately measure 5.0 mL of the supernatant prepared in Examples 1-3 and Comparative Examples 1-3.

[0041] Gel filtration chromatography separation: A Sephadex G-25 column (1.6cm × 40cm) was used with deionized water as the mobile phase at a flow rate of 0.5mL / min. The sample from step (1) was loaded onto the column, and the eluent was collected using a fraction collector. Based on the absorption peak at 280nm detected by the UV detector, the eluents of the macromolecular component (donkey-hide gelatin peptide-metal ion chelate) and the small molecule component (free metal ions) were collected separately.

[0042] Determination of metal ion content: The collected eluents of macromolecular and small molecule components were respectively diluted to 50 mL. The concentration of metal ions in the two component solutions was determined by atomic absorption spectrometry (AAS) according to the standard curve of the corresponding metal element (iron or zinc).

[0043] Chelation rate calculation: The chelation rate is calculated according to the following formula: Chelation rate (%) = [Total mass of metal ions in macromolecular components / (Total mass of metal ions in macromolecular components + Total mass of metal ions in small molecule components)] × 100%.

[0044] The experimental results were measured three times for the products of each example and comparative example, and the average value of the results was recorded in Table 1.

[0045] Table 1. Chelation rate determination results of products from each example and comparative example

[0046] The results in Table 1 show that the chelation rates of the products prepared in Examples 1-3 are significantly higher than those in Comparative Examples 1-3. The chelation rates of the examples are all above 90.0%, while the chelation rates of all comparative examples are below 70.0%, indicating that the products obtained by this technical solution have a higher level of metal ion binding.

[0047] The above results are attributed to the fact that this technical solution first constructs a metal ion-donkey-hide gelatin protein template in step S2. The formation of the metal ion-donkey-hide gelatin protein template allows the metal ion to initially interact with the protein at specific sites, thereby exposing sites in the spatial conformation that are favorable for subsequent enzymatic cleavage. When the core functional protease is added, it can perform directed hydrolysis based on the conformation of the metal ion-donkey-hide gelatin protein template, preferentially cleaving peptide bonds at adjacent metal ion interaction sites, generating specific peptides with high metal ion binding activity. Because the enzymatic hydrolysis and chelation reactions occur simultaneously, the newly generated active peptides can immediately and effectively chelate with free metal ions in the system, avoiding potential loss or conformational changes of the active peptides in subsequent separation steps.

[0048] The step-by-step method of hydrolysis followed by chelation in Comparative Example 1 randomly produces a large number of peptide segments, and the proportion of high-activity peptide segments is low. In addition, there is a loss of activity in the separation process, resulting in a low final chelation rate. Although Comparative Example 2 uses a synchronous method, it lacks the pre-construction of a metal ion template, and the hydrolysis of the protease is not directional, so it cannot preferentially generate high-activity peptide segments. Therefore, its chelation rate is significantly lower than that of the examples. Although Comparative Example 3 also uses a synchronous method, the commercially available ordinary neutral protease used does not have the self-limiting property of the product, and the hydrolysis specificity is insufficient, so it cannot achieve efficient cutting at specific sites. Therefore, its chelation rate is also lower than that of the examples of the present technical solution.

[0049] Test Example 2: Determination of product molecular weight distribution: This test example is used to determine the molecular weight distribution of the products prepared in Examples 1-3 and Comparative Examples 1-3.

[0050] Experimental steps: Sample preparation: The supernatant prepared in Examples 1-3 and Comparative Examples 1-3 was freeze-dried into powder. Each sample powder was accurately weighed, dissolved and diluted to 2.0 mg / mL with the mobile phase. The solution was filtered through a 0.22 μm microporous filter membrane, and the filtrate was used as the sample to be tested.

[0051] Chromatographic conditions: A high-performance liquid chromatography (HPLC) system was used for analysis. The chromatographic column was TSK-gel G2000SWXL (300 mm x 7.8 mm); the mobile phase was 0.1 M phosphate buffer solution (containing 0.15 M NaCl, pH 7.0); the flow rate was 0.5 mL / min; the column temperature was maintained at 30°C; the detection wavelength was 220 nm; and the injection volume was 10 μL.

[0052] Data analysis: A molecular weight standard (cytochrome C, 12384 Da; aprotinin, 6511 Da; bacitracin, 1422 Da) was used to prepare a standard curve. According to the standard curve and the retention time of the sample, the peak area corresponding to the molecular weight of the peptide segment in the range of 1000-3000 Da in each product was calculated as a percentage of the total peak area.

[0053] Experimental results: The products of each example and comparative example were analyzed, and the results of the peak area ratio in a specific molecular weight range are recorded in Table 2.

[0054] Table 2. Peak area ratio of products of each example and comparative example in a specific molecular weight range

[0055] The data in Table 2 show that the peak area proportions of the products of Examples 1-3 in the molecular weight range of 1000-3000 Da are significantly higher than those of Comparative Examples 1-3. The proportions of components in the molecular weight range of 1000-3000 Da in the products of Examples are all more than 85.0%, while the proportions of components in the range in all the products of Comparative Examples are all less than 50.0%, which confirms that the products prepared by the technical solution have more concentrated molecular weight distribution.

[0056] The technical reason for this result is that the technical solution pre-constructs a metal ion template, so that the core functional protease can hydrolyze specific sites in subsequent steps to generate peptide segments in the target molecular weight range. Furthermore, the core functional protease has a self-limiting property, that is, its enzymatic reaction activity is inhibited by the product Ejiao peptide-metal ion chelate. As the reaction proceeds, the concentration of the target product increases, and the inhibition of the protease also increases, eventually making the enzymolysis reaction automatically slow down or stop when a large amount of target peptides are generated and not excessively hydrolyzed. This mechanism avoids excessive hydrolysis, so that the molecular weight of the final product can be concentrated in the pre-set target range.

[0057] In contrast, Comparative Example 1 uses the traditional enzymatic hydrolysis method, and the hydrolysis process is not selective, resulting in a wide distribution of product molecular weight. Comparative Example 2 lacks pre-construction of a metal ion template, and the enzymatic hydrolysis site is not clear, so it cannot generate peptide segments in the target molecular weight range. Comparative Example 3 uses a commercially available ordinary neutral protease that does not have a self-limiting property, and continuously hydrolyzes proteins and target peptides generated during the reaction, resulting in excessive hydrolysis, so that the final product contains a large amount of small peptides and amino acids with a molecular weight less than 1000 Da, and therefore the proportion of components in the target molecular weight range is low.

[0058] Test Example 3: Determination of Ejiao protein utilization rate: This test example is used to determine the utilization rate of the starting material Ejiao protein in Examples 1-3 and Comparative Examples 1-3.

[0059] Experimental steps: Sample preparation: The supernatant of the final product obtained in Examples 1-3 and Comparative Examples 1-3 was freeze-dried to obtain a powdery product. The mass of the raw material Ejiao and each powdery product was accurately weighed.

[0060] Protein content determination: A certain amount of raw material Ejiao and each powdery product was weighed, and the total nitrogen content was determined by the Kjeldahl method. The protein content was converted according to the following formula: Protein content (%) = total nitrogen content (%) x 5.55. Among them, 5.55 is the nitrogen-protein conversion coefficient of Ejiao protein.

[0061] Colla protein utilization rate calculation: Colla protein utilization rate (%) = [(total mass of the final powder product x its protein content) / (total mass of the raw material colla input x its protein content)] x 100%.

[0062] The experimental results were measured for the products of each example and comparative example, and the calculated colla protein utilization rate results are recorded in Table 3.

[0063] Table 3. Colla protein utilization rate of each example and comparative example

[0064] The experimental results of Table 3 show that the colla protein utilization rates of Examples 1-3 are all higher than 93.0%, while the colla protein utilization rates of Comparative Examples 1-3 are all lower than 89.0%. This data confirms that the technical solution can convert a higher proportion of starting colla protein into the final product compared to the technical path of the comparative examples.

[0065] The high protein utilization rate achieved by the technical solution is determined by the technical features of its process flow. In this solution, enzyme hydrolysis and chelation reaction are integrated in a reaction system and carried out simultaneously, and after the reaction is completed, only one step of heat treatment and one step of centrifugation are needed to obtain the final product solution, and the process does not include any material separation or purification steps. This process design avoids the physical loss of proteins or peptide segments that occurs in additional operation units such as ultrafiltration, dialysis or chromatographic separation, so that most of the proteins in the starting material are retained in the final product.

[0066] Comparative Example 1 uses a step-by-step method, and the ultrafiltration separation step included in its process is the direct cause of its significant reduction in protein utilization rate. Some peptides that do not meet the molecular weight requirements of the membrane cut-off are removed in this step, resulting in material loss. Although Comparative Examples 2 and 3 also use simultaneous reactions, due to the non-directed nature or excessive hydrolysis of their enzyme hydrolysis processes, some proteins or peptides form insoluble precipitates, or are degraded into small molecules that cannot stably exist in the final chelate system. These substances are removed in the final centrifugation step, also causing loss of proteins, so their protein utilization rates are lower than those of the examples of the technical solution.

Claims

1. A preparation method of a high chelation rate Ejiao peptide-metal ion chelate, characterized in that, The method comprises the following steps: S1, preparing an ass's hooves protein solution: dissolving ass's hooves in a 0.05-0.1M phosphate buffer solution to obtain an ass's hooves protein solution with a mass concentration of 5.0%-15.0%(w / v), and adjusting the pH value of the ass's hooves protein solution to 7.0-7.5; S2, pre-building a metal ion template: adding a metal salt to the ass's hooves protein solution obtained in step S1, with a mass ratio of ass's hooves protein to metal ion of 10:1-20:1, and reacting at 45-55°C for 20-40 minutes to obtain a reaction system containing a metal ion template; S3, performing template-directed self-limited synchronous enzymatic hydrolysis-chelation reaction: adding a core functional protease to the reaction system obtained in step S2, and performing synchronous reaction at 48-52°C and pH 7.0-7.5 for 4.0-7.0 hours to obtain a chelate mixture, with a mass ratio of the core functional protease to ass's hooves protein of 1:150-1:250; S4, terminating the reaction and obtaining the product: heating the chelate mixture obtained in step S3 to 90-95°C and maintaining for 15-20 minutes to inactivate the core functional protease, and then obtaining the ass's hooves peptide-metal ion chelate through solid-liquid separation.

2. The method according to claim 1, wherein the method is characterized in that, The preparation method of the core functional protease in step S3 comprises: selecting Bacillus subtilis with tolerance to the target metal ion as the starting strain and performing fermentation, and sequentially performing centrifugation, ammonium sulfate precipitation and dialysis treatment on the obtained fermentation liquor to obtain the core functional protease.

3. The method according to claim 1, wherein the method is characterized in that, The core functional protease in step S3 is a neutral protease.

4. The method according to claim 1, wherein the method is characterized in that, The end point of the template-directed self-limited synchronous enzymatic hydrolysis-chelation reaction in step S3 is judged by the following standard: monitoring the degree of hydrolysis of the reaction system, and when the growth rate of the degree of hydrolysis within 0.5-1.5 consecutive hours is less than 1%, the reaction is considered to have reached the end point.

5. The method according to claim 1, wherein the method is characterized in that, The metal salt in step S2 is a biologically acceptable organic acid salt that provides ferrous ions or zinc ions.

6. The method according to claim 1, wherein the method is characterized in that, The reaction for pre-building the metal ion template in step S2 is performed at a stirring speed of 60-100 rpm.

7. The method for preparing a high-chelation-rate donkey-hide gelatin peptide-metal ion chelate according to claim 4, characterized in that, The degree of hydrolysis is monitored by an online pH-stat method or an offline o-phthaldehyde method.

8. The method according to claim 1, wherein the method is characterized in that, The solid-liquid separation in step S4 is centrifugal separation, with a centrifugal force of 4000-6000xg and a centrifugal time of 15-20 minutes.

9. The method according to claim 1, wherein the method is characterized in that, The operation of dissolving ass's hooves in a 0.05-0.1M phosphate buffer solution in step S1 is performed at a temperature of 55-65°C.

10. The method according to claim 1, wherein the method is characterized by, The operation mode of adding the metal salt in step S2 is: pre-dissolving the metal salt in deionized water, and then slowly adding the obtained metal salt solution to the ass's hooves protein solution.

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