Protein / polypeptide sustained-release agent, sustained-release protein / polypeptide and preparation method and application thereof

Abstract

The invention belongs to the technical field of protein and polypeptide, and particularly relates to a protein / polypeptide sustained-release agent, sustained-release protein and polypeptide as well as a preparation method and application thereof. According to the specific technical scheme, the protein / polypeptide sustained-release agent is any one of benzoic acid debranched starch crystals, phosphorylated debranched starch crystals or OSA debranched starch crystals. On the basis of DBS, an anionic phosphate group, an amphiphilic OSA group and a cationic benzoic acid group are respectively introduced into hydroxyl sites of DBS through a debranched starch modification reaction, then low-humidity temperature-control induced crystallization is performed, and a protein / polypeptide loading strategy based on starch molecular chain restrictive recombination is provided for the first time and is suitable for various proteins / polypeptides. The chemically modified DBS can successfully load the protein / polypeptide, so that the protein / polypeptide is effectively protected, and the slow release of the protein / polypeptide is realized. Compared with unmodified DBS, the modified DBS has higher protein / polypeptide loading rate and longer release time.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of proteins and polypeptides, and particularly relates to a protein / polypeptide sustained-release agent, a sustained-release protein and polypeptide, and a preparation method and application. BACKGROUND

[0002] In various fields, the protection and sustained release of proteins / polypeptides are very important and necessary. The sustained-release agent can avoid the rapid degradation of proteins / polypeptides, prolong the action time, and enable the proteins / polypeptides to play a role again at the target position. Therefore, the protein / polypeptide sustained-release agent has been the research focus in various fields.

[0003] In the medical field, proteins and polypeptides have the problems of poor stability, short half-life, and difficult-to-meet targeting requirements, so it is necessary to load and package the proteins / polypeptides in a sustained-release agent / stabilizer / protective agent. (1) Stability: Proteins / polypeptides are easily inactivated due to enzymatic degradation, pH changes, and the influence of the in-vivo environment. For example, the oral bioavailability of insulin is only 0.1% to 2%, and direct injection requires frequent administration, which has low patient compliance. (2) Half-life: Most protein and polypeptide drugs have a half-life of only a few hours in the body, and need to be injected multiple times a day (such as interferon, which needs to be injected every other day). The sustained-release technology can prolong the action time and reduce the frequency of administration. (3) Targeting requirement: Through the sustained-release carrier (such as liposomes, exosomes), the drug can be enriched at the tumor or inflammation site, and the systemic toxicity is reduced. For example, exoIL-12 limits IL-12 to the tumor microenvironment, and the systemic exposure is reduced by 90%.

[0004] In the food and health care field, the protein / polypeptide sustained-release agent also plays an important role. Specifically, it includes: (1) functional activity maintenance: polypeptides (such as antihypertensive peptides, antioxidant peptides) are easily inactivated during processing. The sustained-release technology can protect the activity, for example, the moisturizing property and anti-wrinkle effect of collagen polypeptides are improved by 30% after being wrapped by microspheres. (2) Controlled release nutrition: the sustained-release dosage form can prolong the residence time of polypeptides in the intestinal tract and promote the absorption of minerals (such as calcium, iron). For example, the bioavailability of casein phosphopeptide (CPP) combined with calcium is improved by more than 40%. (3) Continuous regulation of physiological functions: polypeptides (such as immunomodulatory peptides, anti-fatigue peptides) need to be stably released for a long time to maintain the therapeutic effect. The sustained-release capsule can make the immunomodulatory peptides act in the body for 72 hours, and the effect is better than that of the ordinary dosage form.

[0005] In the agricultural field, part of the protein / polypeptide active ingredients are easily degraded, and the sustained-release agent can effectively prolong the action time, thereby reducing the use amount of pesticides and fertilizers by more than 50%.

[0006] Current protein / polypeptide sustained-release agents include biodegradable polymers, natural high molecular materials, etc. Among the biodegradable polymers, PLGA (poly-lactic-glycolic acid copolymer) is the mainstream carrier. Among the natural high molecular materials, silk fibroin, chitosan, etc. are used to prepare sustained-release hydrogels due to their excellent biocompatibility. However, the existing sustained-release agents have defects such as complex preparation process, poor universality (only applicable to some specific proteins / polypeptides), low loading rate, and contradiction between loading rate and stability. Especially, the contradiction between loading rate and stability limits the application of the sustained-release agent in industry. High loading capacity may lead to the destruction of the carrier structure and increase the leakage rate of the protein / polypeptide.

[0007] Therefore, if a new type of protein / polypeptide sustained-release agent with strong universality, high loading rate, stable properties, non-toxicity, and environmental friendliness can be provided, it will have important application prospects. SUMMARY

[0008] The purpose of the present application is to provide a polypeptide sustained-release agent, a sustained-release polypeptide, and a preparation method and application thereof.

[0009] To achieve the above-mentioned purpose of the application, the technical solution adopted by the present application is as follows: a protein / polypeptide sustained-release agent, wherein the sustained-release agent comprises a debranched starch crystal induced to crystallize under low humidity and temperature control after esterification modification. The sustained-release agent is any one of benzoic acid debranched starch crystal, phosphated debranched starch crystal, or OSA debranched starch crystal. In the benzoic acid debranched starch crystal, the ratio of benzoic acid group to debranched starch (molar ratio of benzoic acid group to glucose residue) is 1:10-3:1. In the phosphated debranched starch crystal, the ratio of phosphate group to debranched starch (molar ratio of phosphate group to glucose residue) is 1:10-3:1. In the OSA debranched starch crystal, the ratio of OSA to debranched starch (molar ratio of phosphate group to glucose residue) is 1:10-3:1.

[0010] Correspondingly, the preparation method of the sustained-release agent, when the sustained-release agent is the benzoic acid debranched starch crystal, comprises the following steps: suspending the debranched starch in water to form an alkalized starch solution, slowly adding benzoic acid chloride, performing esterification reaction at 20-30℃ for 1-2h, after the reaction is terminated, centrifuging to obtain a precipitate, and performing low humidity and temperature control induced crystallization on the precipitate to obtain the benzoic acid debranched starch crystal.

[0011] When the sustained-release agent is phosphorylated debranched starch crystals, the method includes the following steps: (1) preparing solution A: dissolving chloride salt (any one or a mixture of several of CaCl2, ZnCl2 or MgCl2) and alkaline phosphatase together in sodium pyrophosphate buffer, adjusting the pH to 5.0-7.0 to obtain solution A; (2) preparing suspension B: mixing debranched starch with sodium pyrophosphate solution, stirring at a constant temperature of 37°C to obtain suspension B; (3) adding solution A to suspension B, mixing evenly, and stirring at 37°C for 6-10 hours; (4) after the reaction is terminated, adjusting the pH to 5.0-6.0, centrifuging to obtain precipitate, and inducing crystallization of the precipitate under low humidity and temperature control to obtain the phosphorylated debranched starch crystals.

[0012] When the slow-release agent is OSA debranched starch crystals, the method includes the following steps: suspending debranched starch in water to obtain a starch suspension, adjusting the pH to 8.0±0.1, slowly adding octenyl succinic anhydride dropwise under constant stirring, reacting at 35℃ for 0.5–2 h, adjusting the pH to 6.5–7.0 after the reaction is terminated, centrifuging to obtain a precipitate, and inducing crystallization of the precipitate under low humidity and temperature control to obtain the OSA debranched starch crystals.

[0013] Accordingly, the sustained-release agent, or the sustained-release agent prepared by the preparation method, is used in non-disease diagnosis or treatment of sustained-release proteins or sustained-release peptides.

[0014] Accordingly, a sustained-release protein / peptide comprises a protein / peptide and the sustained-release agent.

[0015] This invention offers the following advantages: Based on starch-derived starch (DBS), this invention introduces anionic phosphate groups, amphiphilic OSA groups, and cationic benzoic acid groups into the hydroxyl sites of DBS through a debranched starch modification reaction, followed by induction of restricted recombination of the molecular chain. This invention proposes for the first time a protein / peptide loading strategy based on restricted recombination of starch molecular chains, applicable to various proteins / peptides. The chemically modified DBS can successfully load proteins / peptides, effectively protecting them while achieving slow release. Based on the restricted recombination loading strategy, stable modified starch-peptide crystals can be formed, with cavities within the crystals, allowing for the loading of more proteins / peptides. Therefore, compared to unmodified DBS, chemically modified DBS exhibits higher protein / peptide loading rates, longer release times, and better protection of proteins / peptides.

[0016] This invention has broad application prospects in agriculture, medicine, food, beauty and other fields. Attached Figure Description

[0017] Figure 1FTIR spectral characterizations of WS-Native and DBS;

[0018] Figure 2 FTIR spectra of various modified debranched starches;

[0019] Figure 3 Microscopic images of debranched starch crystals modified and crystallized under low humidity and temperature control under polarized light background;

[0020] Figure 4 Microscopic images of debranched starch (control group 1) modified and subjected to low humidity and temperature controlled crystallization under polarized light background;

[0021] Figure 5 Microscopic images of debranched starch (control group 2) modified and subjected to low humidity and temperature controlled crystallization under polarized light background;

[0022] Figure 6 FTIR spectra of the samples without SPT and with SPT.

[0023] Figure 7 Short-range order analysis plots for samples without SPT loading and those loaded with SPT;

[0024] Figure 8 Microscopic images of each sample against a polarized light background;

[0025] Figure 9 A schematic diagram showing the peptide loading rates of various modified debranched starches;

[0026] Figure 10 Schematic diagram of SPT release curves for each debranched starch load;

[0027] Figure 11 This is a schematic diagram of the release curves of agricultural protein peptides loaded with debranched starch. Detailed Implementation

[0028] I. This invention provides a protein / peptide sustained-release agent (stabilizer), the sustained-release agent comprising esterified debranched starch crystals: benzoic acid debranched starch crystals (BZDBS-P), phosphorylated debranched starch crystals (PDBS-P), or OSA debranched starch crystals (OSADBS-P), wherein each esterified debranched starch crystal is a starch crystal that has undergone esterification and low humidity temperature control crystallization.

[0029] In the benzoic acid-derived starch crystals, the ratio of benzoyl groups to debranched starch (molar ratio of benzoyl group to glucose residue) is 1:10 to 3:1. In the phosphorylated debranched starch crystals, the ratio of phosphate groups to debranched starch (molar ratio of phosphate group to glucose residue) is 1:10 to 3:1. In the OSA-derived starch crystals, the ratio of OSA to debranched starch (molar ratio of phosphate group to glucose residue) is 1:10 to 3:1.

[0030] II. The present invention also provides a method for preparing the polypeptide sustained-release agent, comprising the following steps:

[0031] (1) Preparation of debranched starch (DBS): Weigh WS-Native (waxy corn starch) and suspend it in DMSO solvent at a mass ratio of WS-Native:DMSO = 1:10-15, preferably 1:15. Mix the suspension in an 80°C water bath for 1-3 hours to ensure sufficient dispersion of starch particles. Then, centrifuge at 4000 rpm for 10 minutes to precipitate undissolved starch particles and collect the supernatant. Precipitate the starch in the supernatant with 20 mL of anhydrous ethanol, centrifuge again at 4000 rpm for 10 minutes, and discard the supernatant. Wash the precipitate with 10 mL of anhydrous ethanol to remove residual DMSO, repeat the centrifugation step, and discard the supernatant. Then, disperse the obtained precipitate in hot distilled water (95-100°C) and heat continuously in a boiling water bath, maintaining a boiling state, until all precipitates are completely dispersed to form a clear solution. Next, the dispersion was cooled to room temperature, and 0.1M acetate buffer containing pullulanase (2.5 U / g starch) was added to maintain the pH of the system between 4.5 and 5.5 (i.e., the optimal pH range for pullulanase). It was then placed in a 37°C water bath and stirred for 3 hours to obtain a debranched starch suspension. The pH of the debranched starch suspension was then adjusted to neutral with 0.1M NaOH solution, and after cooling to room temperature, it was freeze-dried to obtain DBS.

[0032] (2-1) Preparation of benzoic acid debranched starch crystals (BZDBS-P): DBS was suspended in a 15% (v / v) aqueous ethanol solution, DBS: ethanol aqueous solution = 1:12, g / mL. Then, 5M NaOH aqueous solution was added, the amount added being 1 / 4 to 1 / 5 of the reaction system volume. The mixture was magnetically stirred at 300 rpm for 20 min at 25℃ to form an alkalized starch solution. Subsequently, benzoyl chloride was slowly added dropwise. The esterification reaction was carried out at 20–30℃ for 1–2 h. After the reaction was terminated, the mixture was centrifuged at 5000 rpm for 10 min. The precipitate was washed three times with ethanol and distilled water, freeze-dried, and the product was subjected to low-humidity temperature-controlled induced crystallization (restricted recombination) to obtain starch BZDBS-P.

[0033] (2-2) Preparation of phosphorylated debranched starch crystals (PDBS-P): Solution A preparation: Dissolve chloride salt (any one or a mixture of CaCl2, ZnCl2, or MgCl2) and alkaline phosphatase in sodium pyrophosphate buffer, adjust the pH to 6.5, and obtain solution A. Suspension B preparation: Mix DBS with 0.25M sodium pyrophosphate solution, stir at 37℃ for 30 min, and obtain suspension B. Add solution A to all of suspension B, mix thoroughly, and react at 37℃ and 600 rpm for 6–10 h. After the reaction is terminated, adjust the pH to 5.0, centrifuge at 5000 rpm for 10 min, wash the precipitate three times with ethanol and distilled water, freeze-dry, and subject the product to low-humidity, temperature-controlled induced crystallization (restricted recombination) to obtain starch as PDBS-P.

[0034] (2-3) Preparation of OSA-debranched starch crystals (OSADBS-P): DBS was suspended in distilled water to prepare a 30% (w / v) starch suspension. The pH of the system was adjusted to 8.0 ± 0.1. OSA (octenyl succinic anhydride) was slowly added dropwise under constant stirring. The reaction system was continuously reacted in a 35℃ water bath for 1 h. After the reaction was terminated, the pH was adjusted to 6.5, and the mixture was centrifuged for 10 min. The precipitate was washed three times with ethanol and distilled water, then freeze-dried. The product was subjected to low-humidity temperature-controlled induced crystallization (restricted recombination) to obtain the starch, which was OSADBS-P.

[0035] In steps (2-1), (2-2), and (2-3), when performing low-humidity temperature-controlled induced crystallization on the esterified debranched starch (modified starch), the method includes: mixing the modified starch with pure water, controlling the water content to be below 50-80%, heating the modified starch at 120-140°C for 20-40 minutes while maintaining a constant water content, and then controlling the gradient cooling to room temperature or the stable temperature of the protein / peptide to be loaded. If protein / peptide loading is to be performed directly after starch crystallization, the temperature can be lowered to the protein / peptide stable temperature, for example, 70-80°C; if protein / peptide loading is not to be performed temporarily, the temperature can be lowered to room temperature for storage first.

[0036] Third, the present invention also provides a sustained-release protein / peptide based on the aforementioned sustained-release peptide agent, wherein the sustained-release protein / peptide comprises a protein / peptide and a sustained-release peptide agent, and the sustained-release peptide agent is loaded onto the protein / peptide. Preferably, the mass ratio of protein / peptide:peptide sustained-release agent is 1:1 to 10.

[0037] IV. This invention also provides a method for preparing the sustained-release peptide, specifically comprising the following steps: adding the protein / peptide to be modified for sustained release to a peptide sustained-release agent (aqueous solution) and shaking for 1 hour. Preferably, the peptide sustained-release agent (aqueous solution) is heated to above room temperature but below the stability temperature of the protein / peptide to be added before adding the protein / peptide. The temperature gradient is then lowered to room temperature (if carried out at room temperature, the cooling step is unnecessary), and the mixture is allowed to stand for 24 hours to promote recombination. The sample after standing is placed in an oven and dried at a low temperature overnight (the drying temperature should be below the stability temperature of the protein / peptide, for example, 40–50°C). After drying, the mixture is gently ground into a uniform powder to obtain the sustained-release formulation loaded with protein / peptide.

[0038] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the data obtained are all average values ​​obtained after at least three repetitions, and each repetition yields valid data.

[0039] Example 1: Preparation and Analysis of Peptide Sustained-Release Agents

[0040] 1. Preparation and analysis of DBS and DBS-P

[0041] Weigh 2.0 g of WS-Native and suspend it in 30 mL of DMSO solvent. Mix the suspension in an 80 °C water bath for 2 h to ensure sufficient dispersion of starch granules. Then, centrifuge at 4000 rpm for 10 min to precipitate undissolved starch granules and collect the supernatant. Precipitate the starch in the supernatant with 20 mL of anhydrous ethanol, centrifuge again at 4000 rpm for 10 min, and discard the supernatant. Wash the precipitate with 10 mL of anhydrous ethanol to remove residual DMSO, repeat the centrifugation step, and discard the supernatant. Disperse the obtained precipitate in hot distilled water and heat in a boiling water bath until all precipitates are completely dispersed to form a clear solution. Cool the dispersion to room temperature and add 3 mL of 0.1 M acetate buffer containing pullulanase (2.5 U / g starch), place it in a 37 °C water bath and stir for 3 h. Finally, the pH of the debranched starch suspension was adjusted to neutral with 0.1M NaOH solution, cooled to room temperature, and then freeze-dried to obtain the DBS sample.

[0042] Take 20 mg of modified starch and mix it with 80 μL of distilled water to prepare a starch suspension. Place the suspension in a high-temperature resistant glass tube and place it in a 135 °C oven to dry it until the sample moisture content is 80%. Continue heating for 30 min and then transfer it to a 95 °C metal shaker to cool it down to 75 °C to obtain modified debranched starch crystals DBS-P with low humidity and temperature control crystallization.

[0043] FTIR spectroscopic characterization was performed on WS-Native and DBS respectively, and the results are as follows: Figure 1 As shown in the figure. The results show that the FTIR spectra of DBS and WS-Native are basically the same, indicating that the debranching treatment did not induce the formation of new functional groups in starch molecules. Further evaluation of the short-range order of starch using R1047 / 1022 revealed that the R1047 / 1022 of DBS was significantly lower than that of WS-Native. This may be because after pullulanase specifically hydrolyzes the α-1,6 glycosidic bonds of starch molecules, the proportion of short starch chains increases. Since the intermolecular forces of short starch chains are weakened, they are less likely to form a stable double helix structure, leading to a decrease in the short-range order of DBS. This result is consistent with the trend that pullulanase modification reduces the short-range order of starch.

[0044] The molecular weight distribution of DBS is as follows Figure 1 As shown in Figure B, the peak in the molecular weight distribution curve shifts to the right, indicating that waxy starch is gradually hydrolyzed into short-chain glucans by pullulanase. The α-1,6 glycosidic bonds present at the branch points can be selectively hydrolyzed by pullulanase, resulting in the production of lower molecular weight short-chain amylose. Compared to WS-Native, DBS contains more short-chain amylose, which can endow starch molecules with stronger mobility, enabling them to arrange and aggregate in an orderly manner. This property plays an important role in molecular recombination and resistance to amylases.

[0045] 2. Preparation and analysis of polypeptide sustained-release agent (modified debranched starch)

[0046] (1) Preparation of PDBS and PDBS-P

[0047] Preparation of solution A: Dissolve 1 mmol ZnCl2 (0.136 g), 1 mmol MgCl2 (0.095 g) and 0.1 mg / mL alkaline phosphatase (final concentration) in 2 mL of 0.25 M sodium pyrophosphate buffer, bring the volume to 10 mL with distilled water, and adjust the pH to 6.5 with 0.1 M HCl or NaOH to obtain solution A.

[0048] Preparation of suspension B: Mix 3.0 g DBS with 2 mL of 0.25 M sodium pyrophosphate solution (starch:solution mass-volume ratio of 1.5:1, g / mL) and stir at 37 °C and 600 rpm for 30 min to obtain suspension B.

[0049] Add 8 mL of solution A to all of suspension B, mix well, and react at 37 °C and 600 rpm for 8 h. After the reaction is terminated, adjust the pH to 5.0 with 0.1 M HCl, centrifuge at 5000 rpm for 10 min, wash the precipitate three times with ethanol and distilled water, and freeze-dry to obtain modified starch PDBS.

[0050] Take 20 mg of modified starch PDBS and mix it with 80 μL of distilled water to prepare a starch suspension. Place the suspension in a high-temperature resistant glass sealed tube and heat it in a 135℃ oven until the sample moisture content is 80%. Continue heating in a completely sealed oven for 30 min (keeping the moisture content constant). Then transfer it to a metal shaker at 95℃ to cool it down to 75℃ to obtain modified debranched starch crystals PDBS-P that have undergone low humidity temperature-controlled recrystallization.

[0051] (2) Preparation of OSADBS and OSADBS-P

[0052] 1.0 g of DBS was suspended in distilled water to prepare a 30% (w / v) starch suspension. The pH of the system was adjusted to 8.0 ± 0.1 using 4% (w / v) NaOH solution. 14.5 mmol g of OSA (octenyl succinic anhydride) was slowly added dropwise over 2 hours with constant stirring (200 rpm). The reaction system was incubated in a 35°C water bath for 1 hour. After the reaction was terminated, the pH was adjusted to 6.5 with 2% (w / v) HCl solution, followed by centrifugation at 5000 rpm for 10 minutes. The precipitate was washed three times with ethanol and distilled water, and then freeze-dried to obtain modified starch OSADBS.

[0053] Take 20 mg of modified starch OSADBS and mix it with 80 μL of distilled water to prepare a starch suspension. Place the suspension in a high-temperature resistant glass sealed tube and heat it in an oven at 120–140 °C until the sample moisture content reaches 80%. Continue heating in a completely sealed environment for 30 min (keeping the moisture content constant). Then transfer the suspension to a metal vibrator at 95 °C to cool it down to 75 °C to obtain modified debranched starch crystals OSADBS-P that have undergone low-humidity temperature-controlled recrystallization.

[0054] (3) Preparation of BZDBS and BZDBS-P

[0055] 1.0 g of DBS was suspended in 12 mL of 15% (v / v) ethanol aqueous solution, followed by the addition of 2.7 mL of 5M NaOH aqueous solution. The mixture was magnetically stirred at 300 rpm for 20 min at 25 °C to form an alkalized starch solution. Subsequently, 6 mmol of benzoyl chloride was slowly added dropwise, and the esterification reaction was carried out at 25 °C for 1.5 h. After the reaction was terminated, the mixture was centrifuged at 5000 rpm for 10 min. The precipitate was washed three times with ethanol and distilled water, and then freeze-dried to obtain modified starch BZDBS.

[0056] Take 20 mg of modified starch BZDBS and mix it with 80 μL of distilled water to prepare a starch suspension. Place the suspension in a high-temperature resistant glass sealed tube and heat it in a 135℃ oven until the sample moisture content is 80%. Continue heating in a completely sealed oven for 30 min (keeping the moisture content constant). Then transfer the suspension to a metal shaker at 95℃ to cool it down to 75℃ to obtain modified debranched starch crystals BZDBS-P that have undergone low-humidity temperature-controlled recrystallization.

[0057] (4) The modified debranched starches were characterized by FTIR spectroscopy, and the results are as follows: Figure 2 As shown. The results show that PDBS is at 1134 cm. -1 A P=O stretching vibration peak and a peak at 1052 cm⁻¹ appeared at this location. -1 The characteristic peaks at the POC stretching vibration point indicate the successful preparation of phosphorylated starch. OSADBS at 1576 cm⁻¹... -1 and 1724cm -1 Two new characteristic peaks appeared at 1698 cm⁻¹, mainly caused by the stretching vibrations of the asymmetric stretching vibration of the carboxyl group and the stretching vibration of the C=O stretching vibration of the ester carbonyl group. These changes indicate that during the esterification reaction, the octenyl succinic anhydride ring opens, generating new characteristic peaks for carboxylic acid and ester bonds in DBS, respectively. In the BZDBS sample, a peak was observed at 1698 cm⁻¹. -1 C=O stretching vibration peak at 1583 cm⁻¹ -1 C=C at the location and 1448cm -1 The C=N bending vibration peaks at 1299 cm⁻¹ indicate the presence of benzoic acid groups. Furthermore, the peak at 1299 cm⁻¹... -1 The COC stretching vibration peak at the point further confirms the binding of the benzoic acid group to the starch skeleton.

[0058] (5) Microscopic imaging of DBS-P, PDBS-P, OSADBS-P, and BZDBS-P was performed under a polarized light background, and the results are as follows: Figure 3 As shown in the figure. The results showed that no obvious crystallization was observed in DBS-P, while polarized light phenomena were observed in each modified debranched starch after restricted recrystallization. This may be because the starch molecular chains are arranged in an orderly manner to a certain extent, forming a crystalline structure, that is, they have undergone a process of restricted recrystallization or recrystallization.

[0059] Control group 1: Under the same conditions, distilled water was added during the preparation of PDBS-P using PDBS, OSADBS-P using OSADBS, and BZDBS-P using BZDBS. The samples were heated to 200% water content at a high temperature (135℃) and then heated in a completely sealed environment for 30 minutes (maintaining constant water content) to obtain PDBS-P1, OSADBS-P1, and BZDBS-P1. Microscopic imaging was performed under a polarized light background, and the results are as follows: Figure 4 As shown (from top to bottom: PDBS-P1, OSADBS-P1, and BZDBS-P1). The results show that modified starch cannot crystallize when the moisture content is higher than 80%.

[0060] Control Group 2: Under the same conditions, the oven temperatures for preparing PDBS-P from PDBS, OSADBS-P from OSADBS, and BZDBS-P from BZDBS were changed from 135℃ to 110℃ and 150℃, respectively. The results showed that modified starch could not crystallize at temperatures below 120℃ or above 140℃. The results at 110℃ are as follows... Figure 5 As shown (from top to bottom: PDBS-P2, OSADBS-P2 and BZDBS-P2).

[0061] Example 2: Preparation and Analysis of Slow-Release Soybean Peptides

[0062] 1. Weigh out the modified debranched starch crystallization solutions prepared in Example 1 under low humidity and temperature control (starch crystallization solutions prepared from 20 mg of modified starch), and add 4 mg of SPT (soybean peptide, purchased from Shanghai Maclean Biochemical Technology Co., Ltd., S916495) at 75°C, and continue shaking for 1 hour. Then lower the temperature to 25°C and let stand for 24 hours to promote recombination. Place the samples after standing in a 45°C oven to dry overnight. After drying, gently grind into a uniform powder using a mortar and pestle. Use the sample group without SPT loading as a blank control. The low humidity and temperature control recrystallized esterified modified starch samples without SPT loading are named DBS-P, PDBS-P, OSADBS-P, and BZDBS-P; the recrystallized esterified modified starch samples loaded with SPT are named DBS-SPT, PDBS-SPT, OSADBS-SPT, and BZDBS-SPT, respectively.

[0063] 2. The FTIR spectra of each sample, both unloaded and SPT-loaded, are as follows: Figure 6 As shown, short-range order analysis is as follows: Figure 7As shown in the figure. The results showed that among the unloaded soybean peptides, PDBS-P exhibited the highest short-range order, significantly higher than BZDBS-P, OSADBS-P, and DBS-P. This indicates that phosphorylation modification enhances the intermolecular interactions of starch by introducing charged groups, promoting higher short-range order of starch; while benzoic acid esterification may lead to a decrease in crystallinity due to steric hindrance. After loading with SPT, the short-range order of PDBS-SPT, OSADBS-SPT, and BZDBS-SPT decreased by 1.04%, 1.24%, and 4.55% respectively compared with the unloaded system, indicating that peptide molecules reduced the short-range order of starch through hydrophobic interactions or hydrogen bonding. The short-range order of DBS-SPT Crystal increased by 0.54%, possibly because the peptide promoted the restricted recombination of debranched starch chains, enhancing the short-range order of starch through intermolecular forces.

[0064] In the FTIR spectrum of the SPT-loaded sample, 1660–1650 -1 cm, 1471cm -1 (BZDBS-SPT) and 1390cm -1 New absorption peaks appear at this point, and these peaks are similar to the characteristic peak of SPT (1664 cm⁻¹). -1 1471cm -1 and 1390cm -1 The close proximity of the two samples indicates that SPT was successfully loaded into the starch matrix. This likely signifies an electrostatic interaction between the starch sample and SPT. Since SPT exists in an amorphous form in solution, its interaction with starch is primarily attributed to starch chain reorganization. Compared to DBS-P, the OH tensile vibration of DBS-SPT decreased from 3400 cm⁻¹. -1 Offset to 3406cm -1 The OH tensile vibration of PDBS-SPT, OSADBS-SPT and BZDBS-SPT ranged from 3402 cm⁻¹. -1 Offset to 3406cm -1 The peak shift indicates that hydrogen bonds may have formed between SPT and starch molecules. These changes may be influenced by the presence of free hydrogen bonds, intramolecular hydrogen bonds, and intermolecular hydrogen bonds during starch recrystallization.

[0065] In summary, after loading with SPT, the hydroxyl characteristic peak of starch gel particles shifted slightly to a higher wavenumber, indicating that the interaction between SPT and starch during the molecular chain-restricted recombination process affected the intermolecular hydrogen bonds of the recombinant starch.

[0066] 3. Microscopic imaging was performed on each SPT-loaded sample under a polarized light background. The results are as follows: Figure 8 As shown. Simultaneously combined with Figure 3Comparative observations were made on recrystallized starch without SPT loading. The results showed that all modified debranched starches without SPT loading exhibited birefringence under polarized light after restricted recrystallization. The modified debranched starches loaded with SPT still showed birefringence under polarized light, indicating that even with the introduction of SPT, the ordered arrangement and crystalline structure of the starch molecular chains were not completely destroyed, and spherulitic structures could still be formed through restricted recrystallization. This phenomenon reflects that the support still maintains a certain degree of crystallinity after SPT loading.

[0067] Further observation of the DBS-SPT and BZDBS-SPT carriers revealed that some small spherulites fused into larger aggregates. This significant aggregation of small spherulites indicates surface adsorption. In contrast, the crystals formed by PDBS-SPT and OSADBS-SPT were more dispersed, suggesting that the introduction of phosphate and OSA groups may have weakened the restricted recombination capacity of starch, resulting in a looser crystalline structure. BZDBS-SPT, on the other hand, may have formed cavities with greater space, allowing for the loading of more proteins or peptides and enabling the rapid release of more proteins or peptides within the same timeframe.

[0068] 4. SPT Loading Rate Analysis. The SPT standard curve was determined using an enzyme-linked immunosorbent assay (ELISA) reader, and the SPT content was calculated based on the standard curve. 2 mg of each modified debranched starch sample loaded with peptides was dissolved in DMSO and subjected to heating and sonication to ensure complete sample decomposition. The sample was centrifuged at 5000 g for 10 min, and the optical density (OD) value of the supernatant was detected using the enzyme-labeled immunosorbent assay (ELISA) marker. The loading rate was calculated using the following formula:

[0069]

[0070] The theoretical maximum possible SPT loading is the actual amount of SPT added during the preparation process. The results are as follows: Figure 9 As shown in the figure. The results show that there are significant differences in the peptide loading rates of different chemically modified debranched starches. The loading rates of all modified debranched starches are higher than those of DBS. BZDBS-SPT has the highest loading rate, close to 80%. This may be because BZDBS, as a cationic compound, has a strong electrostatic interaction with SPT, thus forming a more stable bond and creating cavities inside, which significantly improves the loading rate. PDBS-SPT has the second highest loading rate, at about 60%. This may be because phosphorylated starch can effectively inhibit the restrictive recombination of starch molecular chains, providing effective cavities for loading SPT. OSADBS-SPT has a loading rate of about 50%; although the hydrophilicity of OSA may help the dissolution of peptides, its amphiphilicity may also cause the interaction between peptides and OSADBS starch molecules to be less stable than that of cationic or anionic starches.

[0071] 5. Analysis of in vitro release effect

[0072] To investigate the effects of different modified DBS on the sustained release of SPT, an in vitro release experiment was conducted in simulated gastric and intestinal fluids. The specific method was as follows: A starch matrix was ground, and 10 mg of starch was mixed with 1 mL of distilled water to obtain a starch solution. 0.5 mL of a simulated gastric digestive environment (pH=3, containing pepsin, 8 units / mg starch, SGF, according to Minekus et al. 2014) was added to the starch solution and kept for 30 minutes. After this, before adding 0.5 mL of a simulated intestinal digestive environment buffer (pH=7, containing trypsin, 1.6 units / mg starch, SIF, according to Minekus et al. 2014), aliquots were collected at "0 h". Subsequently, the enzymatic digest was placed in a 37°C water bath and mixed at a constant speed of 200 rpm using a magnetic stirrer. At fixed time points, aliquots (50 μL) were collected from the enzymatic hydrolysis process, and 450 μL of Na2CO3 (0.5 M) solution was added to terminate starch hydrolysis. The samples were then mixed and centrifuged (2000 r / min, 10 min).

[0073] At predetermined time intervals, a quantitative solution was aspirated and equal amounts of SGF and SIF were replenished, respectively. The SPT release percentage was calculated based on a standard curve to assess the cumulative release rate of SPT.

[0074] SPT release curves for different modified debranched starch loadings are shown below. Figure 10 As shown in the figure. The results showed that all SPT-loaded starches exhibited time-dependent release characteristics. In the simulated stomach stage, SPT was rapidly released within the first 30 minutes, with a cumulative release of more than 15%, followed by a slow release phase. This phenomenon is mainly because some of the SPT attached to the debranched starch surface and not effectively bound to it was more easily dissolved in the release medium, resulting in rapid release in the early stage. From 60 to 480 minutes, SPT continued to be released, and the release of the complex also increased to varying degrees during this period, indicating that the encapsulation effect of DBS and modified DBS can protect SPT to a certain extent, reduce its release in the stomach, and inhibit its degradation in highly acidic gastric juice. The cumulative release of soybean peptides from all four complexes reached more than 45% after 8 hours; however, there were differences in the release characteristics among the different chemically modified DBS-SPT complexes, which may be related to the degradation characteristics of different modified DBS.

[0075] Specifically, PDBS-SPT exhibits a sustained, slow release characteristic in SIF, with an 8-hour cumulative release rate lower than the DBS-SPT control group. This sustained-release characteristic may be due to phosphorylation modification enhancing the cross-linking density and chain entanglement of starch, thereby improving its swelling capacity and effectively controlling the release rate of the active ingredient to achieve a sustained-release effect. Alternatively, phosphorylation modification may increase the starch's resistance to enzymatic hydrolysis, thus promoting its sustained-release properties.

[0076] The release rate of OSADBS-SPT was higher than that of DBS-SPT before 2 hours, but lower after 2 hours. This may be due to the good water solubility and dispersibility of OSADBS, which allows OSADBS-SPT to disperse rapidly in the aquatic environment. The good dispersibility of OSA starch allows OSADBS-SPT to be released gradually as needed, thus exhibiting a release trend of rapid initial release followed by slower release.

[0077] BZDBS-SPT crystals achieved a release rate of 61% within 480 minutes, significantly higher than other modified DBS. This is mainly due to the ability of BZDBS to load more SPT. BZDBS-SPT exhibited a unique two-stage release pattern, with rapid release in the initial 2 hours followed by a steady-state release.

[0078] Furthermore, the percentage of SPT release in SIF was significantly higher for all complexes than in SGF. This indicates that the encapsulation effect of DBS allows for more precise regulation of SPT release. This regulation can, to some extent, protect SPT from gastric acid, enabling it to reach the small intestine smoothly and be fully absorbed, thereby improving its bioavailability.

[0079] 6. Release kinetics analysis

[0080] The release behavior of SPT was fitted using a first-order kinetic model, the Higuchi model, and the Korsmeyer-Peppas model. The first-order kinetic model showed a good fit (R²) for all starch groups. 2 All values ​​were above 0.95, indicating that SPT release follows first-order kinetics, meaning the cumulative release amount exhibits a non-linear relationship with time. To further explore the release mechanism, the Higuchi model was fitted. The fitting results are shown in Table 1. In Table 1, R... 2 The value represents the goodness of fit between the release curve and the fitted model. When n≤0.45, the release mechanism of the active ingredient is attributed to Fickian.

[0081] Table 1. Fitting analysis of SPT release results by different kinetic models.

[0082]

[0083] The results show that R under the Higuchi model2 The value is lower than that of the first-order kinetic model, indicating that in some matrices, the release behavior of SPT is mainly influenced by the diffusion mechanism. However, the simple diffusion model is insufficient to explain this release behavior. A comprehensive analysis of the entire release process requires combining multiple mechanism models (such as the Korsmeyer-Peppas model) involving dissolution and swelling. The Korsmeyer-Peppas model has a fitting R-value of [missing value]. 2 The results are superior to the Higuchi model, indicating that SPT release is regulated by a synergistic effect of diffusion and dissolution. Looking at the release index n values, the n values ​​for the four groups are 0.26, 0.30, 0.24, and 0.16, respectively, all less than 0.45, consistent with the Fickian diffusion mechanism.

[0084] In summary, different starch-type sustained-release agents affect the in vitro release behavior of SPT by altering release kinetic parameters. The high goodness of fit (R²) of the first-order kinetic models for all groups was observed. 2 The value >0.95 provides a theoretical basis for the design of sustained-release systems, thus the starch-based carrier exhibits good controlled-release performance during in vitro simulation of SPT release. Parameter analysis (n-value) of the Korsmeyer-Peppas model reveals diffusion effects. SPT release is not only controlled by diffusion but also involves multiple mechanisms such as dissolution and swelling. A slow and controlled release mode is highly advantageous in active ingredient delivery systems, ensuring sustained release in vivo and better efficacy control.

[0085] Example 3: Preparation and Analysis of Other Sustained-Release Peptides

[0086] 20 mg of each modified debranched starch sample prepared in Example 1 was weighed and mixed with 80 μL of distilled water to prepare starch suspensions, which were then placed in high-temperature resistant glass tubes. Each starch suspension was placed in a 135°C oven and heated until the sample moisture content reached 80%, then heated for another 30 min under sealed conditions. The suspensions were then transferred to a metal shaker at 95°C and cooled to 45°C. At this temperature, 4 mg of agricultural microalgae protein peptide was added to each suspension, and shaking was continued for 1 h. The temperature was then lowered to 25°C and allowed to stand for 24 h to promote recombination. The stood samples were then dried overnight in a 45°C oven. After drying, the samples were gently ground into a uniform powder using a mortar and pestle.

[0087] Add 0.5 mL of pH 6.8 PBS (containing 0.1 μL α-amylase) to a starch solution containing 10 mg of dry starch and 1 mL of distilled water, and stir in a 25°C water bath for 30 minutes. Stop the reaction by placing 500 μL of the reaction solution sample in an ice bath at 15, 30, and 60 minutes after the start of the reaction. Centrifuge the sample at 4°C (2000 r / min, 10 minutes), and measure the absorbance of the supernatant at 280 nm.

[0088] The results of the sustained-release effect are as follows Figure 11 As shown. According to Figure 11 It can be seen that there are significant differences in the in vitro release behavior of different starch recombinant crystals. Debranched starch recombinant crystals and OSA starch recombinant crystals release rapidly within the first 30 minutes, with cumulative release rates reaching 17% and 19%, respectively; subsequently, they enter a slow release phase. Phosphorylated starch recombinant crystals release relatively slowly in the first 30 minutes, but the release rate accelerates between 30 and 60 minutes, reaching a cumulative release rate of 25% by 60 minutes, higher than the release levels of debranched starch and OSA starch recombinant crystals during the same period. Benzoic acid starch recombinant crystals have the fastest release rate and the highest release extent, with a cumulative release rate as high as 33% during the rapid release period of 0–30 minutes, further increasing to 36% by 60 minutes, significantly higher than the other three starch recombinant crystals.

[0089] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, alterations, substitutions, or variations made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.

Claims

1. A protein / peptide sustained-release agent, characterized in that: The sustained-release agent comprises debranched starch crystals that have been esterified and then induced to crystallize under low humidity and temperature control.

2. The sustained-release agent according to claim 1, characterized in that: The sustained-release agent is any one of benzoic acid debranched starch crystals, phosphorylated debranched starch crystals, or OSA debranched starch crystals.

3. The sustained-release agent according to claim 2, characterized in that: In the defatted starch benzoate crystals, the molar ratio of benzoyl group to glucose residue is 1:10 to 3:

1.

4. The sustained-release agent according to claim 2, characterized in that: In the phosphorylated debranched starch crystals, the molar ratio of phosphate to glucose residues is 1:10 to 3:

1.

5. The sustained-release agent according to claim 2, characterized in that: In the OSA debranched starch crystals, the molar ratio of phosphate to glucose residues is 1:10 to 3:

1.

6. The method for preparing the sustained-release agent according to claim 2, characterized in that: The slow-release agent is benzoic acid debranched starch crystals. The method includes the following steps: suspending debranched starch in water to form an alkalized starch solution, slowly adding benzoyl chloride, and performing an esterification reaction at 20-30°C for 1-2 hours. After the reaction is terminated, centrifuging is used to obtain a precipitate, and the precipitate is subjected to low humidity and temperature-controlled induced crystallization to obtain the benzoic acid debranched starch crystals.

7. The method for preparing the sustained-release agent according to claim 2, characterized in that: The sustained-release agent is phosphorylated debranched starch crystals, and the method includes the following steps: (1) Preparation of solution A: Dissolve chloride salt and alkaline phosphatase in sodium pyrophosphate buffer, adjust the pH to 6.5, and obtain solution A; (2) Preparation of suspension B: Debranched starch was mixed with sodium pyrophosphate solution and stirred at a constant temperature of 37°C to obtain suspension B; (3) Add solution A to suspension B, mix well, and stir at 37°C for 6-10 hours; (4) After the reaction is terminated, adjust the pH to 5.0 and centrifuge to obtain the precipitate; (5) The precipitate is subjected to low humidity and temperature controlled induced crystallization to obtain the phosphorylated debranched starch crystals.

8. The method for preparing the sustained-release agent according to claim 2, characterized in that: The slow-release agent is OSA debranched starch crystals. The method includes the following steps: suspending debranched starch in water to obtain a starch suspension, adjusting the pH to 8.0±0.1, slowly adding octenyl succinic anhydride dropwise under constant stirring, reacting at 35℃ for 0.5-2 hours, adjusting the pH to 6.5 after the reaction is terminated, centrifuging to obtain a precipitate, and inducing crystallization of the precipitate under low humidity and temperature control to obtain the OSA debranched starch crystals.

9. The use of the sustained-release agent according to any one of claims 1 to 5, or the sustained-release agent prepared by the preparation method according to any one of claims 6 to 8, in the non-disease diagnosis or treatment of sustained-release proteins or sustained-release peptides.

10. A sustained-release protein / sustained-release polypeptide, characterized in that: The sustained-release protein / peptide comprises a protein / peptide and a sustained-release agent, wherein the sustained-release agent is the sustained-release agent according to any one of claims 1 to 5, or the sustained-release agent prepared by the preparation method according to any one of claims 6 to 8.

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