Yeast microcapsule collagen peptide oral delivery system as well as preparation method and application thereof

Abstract

The invention discloses a yeast microcapsule collagen peptide oral delivery system and a preparation method and application, and relates to the field of biological medicine, the preparation method comprises the following steps: S1, preparing yeast into a yeast microcapsule carrier; s2, dissolving collagen peptide and a conformation stabilizer in a solvent to prepare a co-load solution; s3, adding the yeast microcapsule carrier into the co-loading solution, and carrying out a constant-temperature oscillation reaction, so that the collagen peptide and the conformation stabilizer are jointly loaded in the yeast microcapsule carrier; and S4, centrifugally separating the product obtained in S3, collecting the precipitate, and drying to obtain YC-CP / S. The micro-capsule carrier is prepared by cooperatively treating yeast cells with ultrasound and biological enzyme, so that time-consuming steps can be avoided, the preparation period is remarkably shortened, impurity proteins and nucleic acids in the cells are effectively removed, cell wall pores are dredged, and the cell quality is improved. Therefore, a new way which is efficient, low in cost and easy for industrial production is provided for steady-state oral delivery of bioactive peptides such as collagen peptide.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more particularly to biomedical carriers and functional food processing, specifically a yeast microcapsule collagen peptide oral delivery system, its preparation method, and its application. Background Technology

[0002] Bioactive peptides, such as collagen peptides, have attracted much attention in the functional food and pharmaceutical fields due to their various physiological activities, including anti-inflammatory and antioxidant effects. However, oral administration of peptides faces significant challenges from gastric acid degradation and intestinal enzymatic breakdown, resulting in extremely low bioavailability. Therefore, developing oral delivery systems that can protect bioactive peptides and achieve targeted release into the intestine is crucial.

[0003] Yeast microcapsules (YCs) are empty cell walls obtained by removing intracellular protoplasm from yeast cells. Their natural cage-like structure and pH responsiveness make them ideal oral delivery carriers. Traditional yeast microcapsule preparation mainly employs a high-salt, high-temperature autolysis method, which typically requires treatment with a high-concentration salt solution at 55 °C for 48-72 hours. This method has significant drawbacks: firstly, the preparation cycle is too long, energy consumption is high, and production efficiency is low; secondly, relying solely on salt solution autolysis is insufficient to completely remove tightly bound intracellular proteins and nucleic acids, resulting in ineffective occupation of the drug-loading space within the microcapsule; finally, the natural pores of the cell wall are not fully opened, limiting the entry of large molecule drugs, making it difficult to meet the requirements for efficient delivery in terms of encapsulation efficiency and drug loading in the final product. Summary of the Invention

[0004] This invention overcomes the shortcomings of the prior art and provides a yeast microcapsule collagen peptide oral delivery system, its preparation method, and its application.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: Firstly, the present invention provides a method for preparing a yeast microcapsule collagen peptide oral delivery system, comprising the following steps:

[0006] S1. Yeast microcapsule carriers were prepared by a combination of ultrasound and enzymes.

[0007] S2. Collagen peptides and a conformation stabilizer are dissolved together in a solvent at a mass ratio of 2-6:1 to prepare a co-loaded solution; the conformation stabilizer is selected from cyclodextrins or polyphenols.

[0008] S3. Add the yeast microcapsule carrier to the co-loading solution and react with constant temperature and shaking to load the collagen peptides and conformation stabilizer into the yeast microcapsule carrier.

[0009] S4. Centrifuge the product from S3, collect the precipitate, and dry it to obtain YC-CP / S.

[0010] In a preferred embodiment of the present invention, the preparation of the yeast microcapsule carrier in step S1 includes the following steps:

[0011] S11. The yeast cells were suspended in a buffer solution, and protease was added. The mixture was treated with intermittent ultrasound at 45-60 ℃ and an ultrasonic power of 200-500 W and a frequency of 20-40 kHz for 4-6 h to obtain crude yeast microcapsules.

[0012] S12. Centrifuge, wash and dry the crude yeast microcapsule product to obtain the yeast microcapsule carrier.

[0013] In a preferred embodiment of the present invention, in step S11, the protease is one of alkaline protease, papain, or neutral protease; the amount of protease added is 0.5-3.0% of the dry weight of yeast; and the duration of each single operation and the interval in the intermittent ultrasound mode is 5 seconds.

[0014] In a preferred embodiment of the present invention, in step S12, the centrifugation speed is 7000~9000 rpm and the time is 3~8 min.

[0015] In a preferred embodiment of the present invention, in step S2, the cyclodextrin stabilizer is selected from β-cyclodextrin or hydroxypropyl-β-cyclodextrin; the polyphenol stabilizer is selected from epigallocatechin gallate, tea polyphenols or ferulic acid.

[0016] In a preferred embodiment of the present invention, in step S2, when the conformation stabilizer is a cyclodextrin, its mass ratio to collagen peptide is 1:4~6.5; when the conformation stabilizer is a polyphenol, its mass ratio to collagen peptide is 1:2.3~3.7.

[0017] In a preferred embodiment of the present invention, in step S3, the mass ratio of the yeast microcapsule carrier to the collagen peptide is 1-2:1-2; the temperature of the isothermal oscillation reaction is 30-40 °C, the pH value is 4.0-7.0, and the time is 2-4 h.

[0018] In a preferred embodiment of the present invention, in step S4, the centrifugation speed is 7000~9000 rpm and the time is 3~8 min.

[0019] In a second aspect, the present invention provides a yeast microcapsule collagen peptide oral delivery system, which is prepared by any one of the above-mentioned methods.

[0020] Thirdly, the present invention provides the application of the yeast microcapsule collagen peptide oral delivery system as described above in the preparation of anti-inflammatory, antioxidant, or beauty and health foods or drugs.

[0021] This invention addresses the shortcomings of the prior art and has the following beneficial effects:

[0022] (1) This invention provides a yeast microcapsule collagen peptide oral delivery system, preparation method and application. By using ultrasound and biological enzymes to synergistically treat yeast cells to prepare microcapsule carriers, the mechanical cavitation effect of ultrasound destroys the lipid bilayer structure of the cell membrane and accelerates the outflow of intracellular substances; the protease specifically hydrolyzes intracellular proteins and mannan proteins on the cell wall, thereby enabling the rapid preparation of yeast microcapsules. Compared with the traditional high-salt autolysis method which requires a long processing time, this method can avoid time-consuming steps, significantly shorten the preparation cycle, and effectively remove impurity proteins and nucleic acids in the cells and unclog cell wall pores, thus providing a new, efficient, low-cost and easy-to-industrialize oral delivery method for stable delivery of bioactive peptides such as collagen peptides.

[0023] (2) This invention introduces cyclodextrin conformation stabilizers when loading collagen peptides. The cyclodextrin molecules have hydrophobic cavities and can selectively encapsulate hydrophobic residues in the amino acid sequence of collagen peptides to form stable host-guest inclusion complexes. This can isolate the hydrophobic fragments of collagen peptides and effectively prevent the aggregation of peptide chains through hydrophobic interactions. Compared with the existing process where collagen peptides are loaded into yeast microcapsules by physical adsorption and electrostatic interaction, which easily leads to dissociation from the adsorption sites and the formation of aggregates under long-term immersion in the gastrointestinal environment, this invention restricts and anchors the conformational freedom of the encapsulated peptide segments, so that the collagen peptides maintain their natural conformation during delivery, thereby ensuring that the collagen peptides released at the terminal have higher biological activity.

[0024] (3) This invention constructs a novel oral delivery system by co-loading a conformational stabilizer and collagen peptides into yeast microcapsules. The stabilizer and collagen peptides interact to form a protective microenvironment inside the microcapsule, which continuously maintains the structural integrity of the collagen peptides throughout the gastrointestinal transit process. The collagen peptides released after simulated gastrointestinal digestion have a higher retention rate of secondary structures such as α-helices and β-sheets, and a more concentrated hydrodynamic diameter distribution, indicating that aggregation is effectively suppressed. Compared with traditional delivery systems that only focus on encapsulation rate and release rate and cannot solve the problem of conformational instability of active ingredients during delivery, which leads to impaired bioactivity, this invention not only achieves efficient delivery of collagen peptides, but also ensures that the functional activity of collagen peptides at the delivery endpoint is maximized, thereby improving the actual efficacy of oral products. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is a graph showing the encapsulation efficiency and drug loading of different addition ratios of YC-CP / β-CD in Example 1 of the present invention;

[0027] Figure 2 This refers to the drug loading and encapsulation efficiency of YC-CP / β-CD at different drug loading times in Example 1 of the present invention;

[0028] Figure 3 This is a diagram showing the Zeta potential (A) and particle size distribution (B) of yeast cells, yeast microcapsules, collagen peptides, and YC-CP / β-CD in Example 1 of the present invention.

[0029] Figure 4 These are SEM images of yeast cells (A), yeast microcapsules (B), and YC-CP / β-CD (C) from Example 1 of this invention.

[0030] Figure 5 The Fourier transform infrared spectra of yeast cells, yeast microcapsules, collagen peptides, and YC-CP / β-CD in Example 1 of this invention are shown.

[0031] Figure 6 The standard curves of collagen peptides in simulated gastric juice (A), simulated small intestinal juice (B), and simulated colonic juice (C) of Example 1 of the present invention, and the release diagrams of collagen peptides and YC-CP / β-CD in simulated gastric juice (D), simulated small intestinal juice (E), and simulated colonic juice (F);

[0032] The symbols and materials in the diagram are as follows: Yeast represents yeast cells; YC represents yeast microcapsules; CP represents collagen peptides; and YC-CP / β-CD represents the final delivery system. Detailed Implementation

[0033] 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. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein. Therefore, the scope of protection of the invention is not limited to the specific embodiments disclosed below.

[0035] Application Overview:

[0036] The applicant's in-depth research revealed that the loading of collagen peptides onto yeast microcapsules primarily relies on physical adsorption and electrostatic interactions. During prolonged immersion in a simulated gastrointestinal environment, the collagen peptide molecules loaded within the microcapsule are subjected to constantly changing pH, ionic strength, and potential permeation by digestive enzymes. This continuous dynamic immersion may cause some collagen peptide molecules that have dissociated from the adsorption sites to aggregate within the microcapsule cavity, forming insoluble aggregates.

[0037] More importantly, the characteristic triple helix or disordered coil structure essential for collagen peptides to maintain their bioactivity can undergo irreversible unwinding or misfolding due to environmental disturbances. These microscopic structural changes are difficult to detect in conventional encapsulation and release rate tests, but they can severely impair the final bioactivity of collagen peptides, significantly reducing the actual efficacy of the delivery system. Existing technologies often focus only on whether and how much is delivered, neglecting whether the active ingredient retains its natural and efficient conformational state upon delivery.

[0038] To address the aforementioned issues, this invention proposes a yeast microcapsule collagen peptide oral delivery system, its preparation method, and its application. During the loading process, collagen peptides are co-encapsulated with a specific conformation stabilizer within the yeast microcapsule carrier, allowing it to specifically interact with the collagen peptide molecules. This stabilizes the active conformation of the collagen peptides like a molecular chaperone throughout the gastrointestinal transit process, effectively inhibiting their aggregation and misfolding, thereby maintaining their natural conformation and biological activity.

[0039] It should be noted that the raw materials, equipment and reagents used in this invention can all be purchased from the market or obtained through existing preparation methods.

[0040] A method for preparing a yeast microcapsule collagen peptide oral delivery system includes the following steps:

[0041] S1. Yeast microcapsule carriers (YC) were prepared by using ultrasound and enzyme synergy.

[0042] S2. Collagen peptides and a conformation stabilizer are dissolved together in a solvent at a mass ratio of 2-6:1 to prepare a co-loaded solution; the conformation stabilizer is selected from cyclodextrins or polyphenols.

[0043] S3. Add the yeast microcapsule carrier to the co-loading solution and react with constant temperature and shaking to load the collagen peptides and conformation stabilizer into the yeast microcapsule carrier.

[0044] S4. Centrifuge the product from S3, collect the precipitate, and dry it to obtain YC-CP / S.

[0045] In some specific embodiments, the preparation of the yeast microcapsule carrier in step S1 includes the following steps:

[0046] S11. The yeast cells were suspended in a buffer solution, and protease was added. The mixture was treated with intermittent ultrasound at 45-60 ℃ and an ultrasonic power of 200-500 W and a frequency of 20-40 kHz for 4-6 h to obtain crude yeast microcapsules.

[0047] S12. Centrifuge, wash and dry the crude yeast microcapsule product to obtain the yeast microcapsule carrier.

[0048] In some specific embodiments, in step S11, the protease is one of alkaline protease, papain, or neutral protease; the amount of protease added is 0.5-3.0% of the dry weight of yeast; and the duration of each single operation and the interval in the intermittent ultrasound mode is 5 seconds.

[0049] In some specific implementations, in step S12, the centrifugation speed is 7000~9000 rpm and the time is 3~8 min.

[0050] In some specific embodiments, in step S2, the cyclodextrin stabilizer is selected from β-cyclodextrin or hydroxypropyl-β-cyclodextrin; the polyphenol stabilizer is selected from epigallocatechin gallate (EGCG), tea polyphenols or ferulic acid.

[0051] In some specific embodiments, in step S2, when the conformation stabilizer is a cyclodextrin, its mass ratio to collagen peptide is 1:4 to 6.5; when the conformation stabilizer is a polyphenol, its mass ratio to collagen peptide is 1:2.3 to 3.7.

[0052] In some specific embodiments, in step S3, the mass ratio of yeast microcapsule carrier to collagen peptide is 1-2:1-2; the isothermal shaking reaction temperature is 30-40 °C, the pH value is 4.0-7.0, and the time is 2-4 h.

[0053] In some specific implementations, in step S4, the centrifugation speed is 7000~9000 rpm and the time is 3~8 min.

[0054] This invention provides an oral delivery system for yeast microcapsule collagen peptides, prepared by any of the above-mentioned methods.

[0055] This invention provides the application of the yeast microcapsule collagen peptide oral delivery system as described above in the preparation of anti-inflammatory, antioxidant, or beauty and health foods or drugs.

[0056] To further simplify and make the present invention achieve its objectives and effects, the present invention will be further illustrated in conjunction with the following specific embodiments and comparative examples, but the present invention is not limited to the scope of the embodiments described herein.

[0057] Example 1:

[0058] S1. Commercially available active dry yeast was washed three times with water to remove surface culture medium and other impurities. It was then suspended in phosphate buffer at pH 8.0. Alkaline protease at 1.5% of the yeast dry weight was added. The mixture was treated at 55 ℃ and with an ultrasonic power of 300 W and a frequency of 40 kHz for 5 h using an ultrasonic mode of 5 s working and 5 s intermittent. After the reaction was completed, the mixture was centrifuged at 8000 rpm for 5 min. The supernatant was discarded, and the precipitate was washed five times with water until no protein was detected in the supernatant. The mixture was then freeze-dried to obtain yeast microcapsules (YC).

[0059] S2. Weigh out blackfish skin collagen peptides (GPSGPQG sequence, purity 95.8%) and β-cyclodextrin in a mass ratio of 5:1, and dissolve them together in phosphate buffer (PBS, pH 6.0) to prepare a 1 mg / mL co-loading solution.

[0060] S3. Weigh the yeast microcapsule carrier at a mass ratio of 1:1 and add it to the co-loading solution. Place it in a constant temperature shaker at 37 ℃ and shake at 200 rpm for 3 h for adsorption.

[0061] S4. Centrifuge the product from S3 at 8000 rpm for 10 min, collect the precipitate, wash the precipitate twice with water, freeze-dry, and obtain YC-CP / β-CD.

[0062] Results Analysis: Refer to Figure 1 As shown in the mass ratio effect, the experiment shows that as the proportion of carrier increases, the encapsulation efficiency increases but the drug loading decreases. At a mass ratio of 1:1, the benefits are maximized.

[0063] Reference Figure 2 As shown, the adsorption equilibrium was reached in 4 hours, with an encapsulation efficiency of 81.60% ± 0.11% and a drug loading of 15.54% ± 0.08%. Compared with traditional methods, the microcapsules prepared by this invention have better pore permeability and a faster adsorption equilibrium time.

[0064] Reference Figure 3The zeta potential and particle size are shown. The zeta potential of the empty microcapsule YC is -19.79 mV (due to enzymatic hydrolysis removing some protein and exposing more negatively charged groups). The zeta potential of the collagen peptide CP is +16.31 mV. After complexation, the YC-CP / β-CD potential becomes -6.78 mV, and the particle size increases from 2.5 μm to 4.4 μm. The neutralization of the potential and the increase in particle size confirm that electrostatic adsorption is the main loading mechanism. The result of the particle size change can also be seen in... Figure 4 The SEM images of yeast before and after treatment show this.

[0065] Reference Figure 5 The FTIR analysis shown shows that in the YC-CP / β-CD spectrum, the characteristic amide peak of collagen peptides (1657 cm⁻¹) is present. -1 and 1537 cm -1 The change in intensity and positional shift indicate a strong interaction between the carboxyl groups of the peptide chain and the amino / hydroxyl groups of the microcapsule wall.

[0066] Reference Figure 6 In vitro release experiments were conducted in simulated gastrointestinal environments: Simulated gastric juice (pH 1.2): The release rate of free collagen peptides reached as high as 57.07% within 2 hours, indicating their high susceptibility to loss; while the cumulative release rate of the YC-CP / β-CD system of this invention was only 43.63% within 2 hours. This indicates that the yeast microcapsules, under strong acid conditions, have a dense structure, effectively blocking gastric acid from contacting the internal peptides and inhibiting burst release. Simulated intestinal juice (pH 6.8 / 7.4): In the small and colonic environments, YC-CP / β-CD exhibited significant sustained-release characteristics. The cumulative release rate after 54 hours was approximately 48%, significantly lower than the 62% of free peptides. This is because the microcapsules swell at the pH of the intestinal juice, opening the pores and allowing the internal drug to be slowly released through diffusion.

[0067] Example 2:

[0068] This embodiment is basically the same as Example 1, except that the type of conformation stabilizer is different. The specific steps of S2 are as follows: weigh black fish skin collagen peptide (GPSGPQG sequence, purity 95.8%) and EGCG in a mass ratio of 3:1, dissolve them together in water, and prepare a 1 mg / mL co-loaded solution.

[0069] Example 3:

[0070] This embodiment is basically the same as Example 1, except that the amount of β-cyclodextrin used is different. The specific steps of S2 are as follows: weigh black fish skin collagen peptide (GPSGPQG sequence, purity 95.8%) and β-cyclodextrin in a mass ratio of 6.5:1, and dissolve them together in phosphate buffer (PBS, pH 6.0) to prepare a 1 mg / mL co-loaded solution.

[0071] Example 4:

[0072] This embodiment is basically the same as Example 1, except that the amount of β-cyclodextrin used is different. The specific steps of S2 are as follows: weigh black fish skin collagen peptide (GPSGPQG sequence, purity 95.8%) and β-cyclodextrin in a mass ratio of 4:1, and dissolve them together in phosphate buffer (PBS, pH 6.0) to prepare a 1 mg / mL co-loaded solution.

[0073] Example 5:

[0074] This embodiment is basically the same as embodiment 2, except that the amount of EGCG used is different. The specific steps of S2 are as follows: weigh black fish skin collagen peptide (GPSGPQG sequence, purity 95.8%) and EGCG in a mass ratio of 3.7:1, dissolve them together in water, and prepare a 1 mg / mL co-loaded solution.

[0075] Example 6:

[0076] This embodiment is basically the same as embodiment 2, except that the amount of EGCG used is different. The specific steps of S2 are as follows: weigh black fish skin collagen peptide (GPSGPQG sequence, purity 95.8%) and EGCG in a mass ratio of 2.3:1, dissolve them together in water, and prepare a 1 mg / mL co-loaded solution.

[0077] Comparative Example 1:

[0078] This embodiment is basically the same as Example 1, except that no conformation stabilizer was added. The specific steps of S2 are as follows: weigh the blackfish skin collagen peptides, dissolve them in phosphate buffer (PBS, pH 6.0), and prepare a 1 mg / mL co-loaded solution.

[0079] Comparative Example 2:

[0080] This embodiment is basically the same as that of embodiment 1, except that β-cyclodextrin is replaced with an equal mass of EGCG. The specific steps of S2 are as follows: weigh blackfish skin collagen peptide (GPSGPQG sequence, purity 95.8%) and EGCG in a mass ratio of 5:1, dissolve them together in phosphate buffer (PBS, pH 6.0) to prepare a 1 mg / mL co-loaded solution.

[0081] Comparative Example 3:

[0082] This embodiment is basically the same as embodiment 2, except that the amount of EGCG used is different. The specific steps of S2 are as follows: weigh black fish skin collagen peptide (GPSGPQG sequence, purity 95.8%) and EGCG in a mass ratio of 2:1, dissolve them together in water, and prepare a 1 mg / mL co-loaded solution.

[0083] Comparative Example 4:

[0084] This embodiment is basically the same as Example 2, except that EGCG is replaced with an equal mass of β-cyclodextrin. The specific steps of S2 are as follows: weigh black fish skin collagen peptide (GPSGPQG sequence, purity 95.8%) and β-cyclodextrin in a mass ratio of 3:1, dissolve them together in water, and prepare a 1 mg / mL co-loaded solution.

[0085] Comparative Example 5:

[0086] This embodiment is basically the same as Example 1, except that the amount of β-cyclodextrin used is different. The specific steps of S2 are as follows: weigh black fish skin collagen peptide (GPSGPQG sequence, purity 95.8%) and β-cyclodextrin in a mass ratio of 7:1, dissolve them together in phosphate buffer (PBS, pH 6.0) to prepare a 1 mg / mL co-loaded solution.

[0087] Comparative Example 6:

[0088] This embodiment is basically the same as Embodiment 1, except that the common loading step is omitted and a stepwise loading is adopted. The specific steps of S2 are as follows: weigh blackfish skin collagen peptide (GPSGPQG sequence, purity 95.8%), dissolve it in phosphate buffer (PBS, pH 6.0) to prepare a 1 mg / mL CP loading solution; weigh β-cyclodextrin, dissolve it in phosphate buffer (PBS, pH 6.0) to prepare a 1 mg / mL β-cyclodextrin loading solution;

[0089] The specific steps for S3 are as follows: Weigh the yeast microcapsule carrier at a mass ratio of 1:1 and add it to the CP loading solution. Place it in a constant temperature shaker at 37 ℃ and shake at 200 rpm for 3 h for adsorption.

[0090] The specific steps for S4 are as follows: the product of S3 is centrifuged at 8000 rpm for 10 min, the precipitate is collected, the precipitate is washed twice with water, and then freeze-dried to obtain YC-CP; YC-CP is weighed at a mass ratio of 1:1 and added to the β-cyclodextrin loading solution, placed in a constant temperature shaker at 37 ℃, and adsorbed by shaking at 200 rpm for 3 h, centrifuged at 8000 rpm for 10 min, the precipitate is collected, the precipitate is washed twice with water, and then freeze-dried to obtain YC-CP / β-CD.

[0091] Comparative Example 7:

[0092] This embodiment is basically the same as Example 1, except that the carrier is prepared by the traditional high-salt autolysis method. The specific steps of S1 are as follows: commercially available active dry yeast is washed three times with water to remove impurities such as surface culture medium, suspended in phosphate buffer at pH 8.0, and soaked at 55 °C for 48 h with intermittent stirring. After the reaction is completed, it is centrifuged at 8000 rpm for 5 min, the supernatant is discarded, the precipitate is washed five times with water until no protein is detected in the supernatant, and then freeze-dried to obtain yeast microcapsules (YC).

[0093] Performance testing: The delivery systems obtained in Examples 1-6 and Comparative Examples 1-7 were subjected to performance tests on conformational stability, aggregation state and bioactivity, respectively. The results are shown in Table 1.

[0094] Conformational stability: 2 g NaCl and 3.2 g pepsin (activity ≥2500 U / mg) were dissolved in about 800 mL of water, the pH was adjusted to 1.2 with 1 M HCl, and the volume was brought to 1000 mL to prepare simulated gastric juice (SGF); 6.8 g KH2PO4 was dissolved in about 500 mL of water, 10 g trypsin was added, the pH was adjusted to 6.8 with 0.1 M NaOH, and the volume was brought to 1000 mL to prepare simulated intestinal juice (SIF).

[0095] Digestion Procedure: Weigh 50 mg of the sample to be tested and disperse it in 25 mL of SGF. Incubate the mixture in a constant temperature shaking water bath at 37 ℃ and 100 rpm for 2 h. Take out half of the digest (12.5 mL) and immediately place it on ice for gastric stage analysis. Quickly add an equal volume (12.5 mL) of preheated 0.2 M NaHCO3 solution to the remaining digest to neutralize it; then add 25 mL of preheated SIF to bring the total volume to approximately 50 mL, maintaining the pH at around 6.8. Continue incubation at 37 ℃ and 100 rpm for 4 h with shaking. After the intestinal stage is completed, place all the digest in an ice bath to terminate the reaction.

[0096] Sample pretreatment and peptide separation: The digestion solutions from each stage were centrifuged at 4 ℃ and 12000×g for 15 min. The supernatant was collected and centrifuged at 4 ℃ and 4000×g for 30 min using an ultrafiltration centrifuge tube with a molecular weight cutoff of 3 kDa to remove macromolecular carrier fragments, undigested proteins, and enzymes. The filtrate (i.e., the digestion product containing free peptides) was collected and stored at -80 ℃ for analysis.

[0097] Circular dichroism (CD) analysis: The above filtrate was appropriately diluted with 10 mM phosphate buffer (pH 7.4) to achieve a collagen peptide concentration in the range of 0.1-0.2 mg / mL. Each sample was scanned three times using a circular dichroism spectrometer, and the average was taken. Using CDNN analysis software, the obtained CD spectra were deconvolved, and the percentage of secondary structure components of collagen peptides in the sample was calculated: α-helix, β-sheet, β-turn, and random coil. The percentage of each ordered structure in the sample after simulated digestion was compared with the percentage of ordered structure in the undigested original collagen peptide control solution (treated in the same way but without digestion), and the percentage retention rate was calculated.

[0098] Aggregation status: The final filtrate (containing free peptides) prepared after simulated whole gastrointestinal digestion (SGF+SIF) in the conformational stability test was used, without further dilution or treatment. Simultaneously, an undigested original collagen peptide solution (same buffer, same concentration) was prepared as a control. A dynamic light scattering particle size analyzer equipped with a 633 nm laser and a 173° detection angle was used. Approximately 1 mL of sample solution was injected into a clean plastic or quartz cuvette, avoiding air bubbles. The measurement temperature was set to 25 °C, and the equilibration time was 60 s. Each sample was automatically measured three times, with each measurement lasting 13 cycles. The instrument software reported the polydispersity index (PDI) of the sample based on cumulative analysis or intensity distribution.

[0099] Bioactivity: Equal volumes of 7.4 mM ABTS diammonium salt solution and 2.6 mM K2S2O8 solution were mixed and allowed to stand for 12 h at room temperature in the dark to generate stable ABTS. + • Free radical stock solution. Before use, dilute this stock solution with anhydrous ethanol or phosphate buffer (pH 7.4) to achieve an absorbance (Abs) of 0.70 at 734 nm. Add 100 μL of the sample solution to be tested (i.e., the digested filtrate obtained from the conformational stability test) to a test tube. Quickly add 2.0 mL of diluted ABTS. + • Mix the working solution by vortexing. React precisely for 6 min at room temperature in the dark. Immediately measure the absorbance (As) of the reaction mixture at 734 nm. Use the buffer solution instead of the sample as a blank control and measure the absorbance (Ac). Construct a standard curve using different concentrations of Trolox (a water-soluble vitamin E analog).

[0100] Free radical scavenging rate (%) = [(Ac-As) / Ac] × 100%. Calculate the required sample concentration (IC50) to achieve a 50% scavenging rate based on the standard curve. 50 IC 50 The lower the value, the stronger the antioxidant activity.

[0101] Table 1:

[0102] project Conformation retention rate (%) PDI <![CDATA[IC 50 (μg / mL)]]> Example 1 89.2 0.18 21.5 Example 2 87.6 0.19 19.8 Example 3 86.5 0.23 22.3 Example 4 88.1 0.20 24.9 Example 5 85.8 0.25 22.1 Example 6 86.9 0.22 19.3 Comparative Example 1 62.3 0.45 48.7 Comparative Example 2 71.2 0.32 35.6 Comparative Example 3 73.5 0.30 33.2 Comparative Example 4 70.8 0.34 36.1 Comparative Example 5 72.1 0.31 34.5 Comparative Example 6 75.6 0.28 30.8 Comparative Example 7 68.9 0.37 41.2

[0103] As shown in Table 1:

[0104] A comparison of Examples 1-2 with Comparative Example 1 reveals that, in Comparative Example 1 without the addition of a conformational stabilizer, the collagen peptides lose their protective mechanism during simulated gastrointestinal digestion. The hydrophobic amino acid residues in the collagen peptide molecules are exposed, making them prone to intermolecular aggregation through hydrophobic interactions, forming insoluble aggregates. Furthermore, the lack of molecular chaperone effects from stabilizers such as β-cyclodextrin or EGCG increases the conformational freedom of the collagen peptides, making them more susceptible to misfolding under pH fluctuations and digestive enzyme activity. Ultimately, this results in a conformational retention rate of only 62.3%, a PDI increase to 0.45, and a low IC50 for antioxidant activity. 50 At a concentration as high as 48.7 μg / mL, the biological activity decreased significantly.

[0105] A comparison of Examples 2 and 5-6 with Comparative Examples 2-3 reveals that a suitable amount of EGCG can stabilize the conformation of collagen peptides through multiple molecular interactions: the phenolic hydroxyl groups of EGCG form a hydrogen bond network with the amide and carboxyl groups of collagen peptides; the phenolic ring structure and the aromatic amino acids of the peptide chain undergo π-π stacking; and hydrophobic interactions further enhance the binding force, acting like molecular chaperones to lock the active conformation of collagen peptides. In Comparative Example 2, when the amount of EGCG is too low, the intermolecular interaction sites are not adequately covered, failing to effectively inhibit the hydrophobic aggregation and conformational drift of collagen peptides; in Comparative Example 3, when the amount is too high, excessive EGCG molecules easily form self-aggregates, not only occupying the loading space inside the yeast microcapsules but also potentially interfering with the native conformation of collagen peptides through non-specific binding, resulting in a decrease in conformational retention to 71.2-73.5%, an increase in PDI to 0.30-0.32, and a significant reduction in antioxidant activity.

[0106] A comparison of Examples 1 and 3-4 with Comparative Examples 4-5 reveals that a suitable amount of β-cyclodextrin can stabilize collagen peptides through host-guest inclusion. The hydrophobic lumen of β-cyclodextrin selectively includes hydrophobic residues in the collagen peptide sequence, isolating them from the external environment and preventing aggregation between peptide chains through hydrophobic interactions. Simultaneously, it restricts the conformational freedom of the included peptide segments, achieving a molecular anchoring effect. In Comparative Example 4, when the amount of β-cyclodextrin was too low, there were insufficient inclusion sites, exposing the hydrophobic residues of the collagen peptides, making aggregation more likely. In Comparative Example 5, when the amount was too high, excessive β-cyclodextrin molecules easily formed complexes, occupying the pores of yeast microcapsules, reducing the effective loading sites for collagen peptides. It may also non-specifically bind to collagen peptides, disrupting their native conformation, resulting in a decrease in conformational retention to 70.8-72.1%, an increase in the PDI to 0.31-0.34, and a decrease in the antioxidant activity IC50. 50It increased to 34.5-36.1 μg / mL.

[0107] A comparison of Examples 1-2 and Comparative Example 6 reveals that when the co-loading step is omitted and a stepwise loading method is used, the conformational stabilizer and collagen peptide cannot form a synergistic protective mechanism within the yeast microcapsules. During the stepwise loading process, the collagen peptide first binds to the microcapsule wall through physical adsorption and electrostatic interactions. The subsequently added stabilizer struggles to penetrate deep into the microcapsule and fully interact with the key active sites of the collagen peptide, failing to effectively form host-guest inclusion complexes or hydrogen bond networks. Furthermore, stepwise loading may cause the stabilizer to preferentially occupy adsorption sites on the microcapsule surface, reducing the effective loading of collagen peptide. In the gastrointestinal environment, the stabilizer and collagen peptide are prone to dissociation, failing to continuously stabilize the collagen peptide conformation. Ultimately, the conformational retention rate is only 75.6%, the PDI is 0.28, and the antioxidant activity IC50 is low. 50 The concentration was 30.8 μg / mL, which is significantly lower than the performance of the example with the same load.

[0108] A comparison of Examples 1-2 and Comparative Example 7 reveals that yeast microcapsule carriers prepared using the traditional high-salt autolysis method suffer from drawbacks such as numerous internal impurities and poor pore permeability. The high-salt autolysis method cannot completely remove intracellular proteins and nucleic acids, which occupy the internal space of the microcapsules, reducing the loading sites for collagen peptides and stabilizers. Simultaneously, the cell wall pores are not sufficiently cleared, making it difficult for collagen peptides and stabilizers to enter the microcapsule interior; they can only adsorb onto the surface and are prone to rapid dissociation in the gastrointestinal environment. Furthermore, the microcapsules prepared by the traditional method exhibit poor structural stability and are prone to rupture during simulated gastrointestinal digestion, leading to direct exposure of collagen peptides to digestive enzymes and an acidic environment. This results in rapid conformational instability, ultimately reducing the conformational retention rate to 68.9%, increasing the PDI to 0.37, and decreasing the antioxidant activity IC50. 50 With a concentration as high as 41.2 μg / mL, its performance is far lower than that of the carrier system prepared by the ultrasound-enzyme synergistic method.

[0109] The above description is based on the preferred embodiments of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects. The scope of the invention is defined by the appended claims rather than the foregoing description, and all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0110] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing a yeast microcapsule collagen peptide oral delivery system, characterized in that, Includes the following steps: S1. Yeast microcapsule carriers were prepared by a combination of ultrasound and enzymes. S2. Collagen peptides and a conformation stabilizer are dissolved together in a solvent at a mass ratio of 2-6:1 to prepare a co-loaded solution; the conformation stabilizer is selected from cyclodextrins or polyphenols. S3. Add the yeast microcapsule carrier to the co-loading solution and react with constant temperature and shaking to load the collagen peptides and conformation stabilizer into the yeast microcapsule carrier. S4. Centrifuge the product from S3, collect the precipitate, and dry it to obtain YC-CP / S.

2. The preparation method of the yeast microcapsule collagen peptide oral delivery system according to claim 1, characterized in that: In step S1, the preparation of the yeast microcapsule carrier includes the following steps: S11. The yeast cells were suspended in a buffer solution, and protease was added. The mixture was treated with intermittent ultrasound at 45-60 ℃ and an ultrasonic power of 200-500 W and a frequency of 20-40 kHz for 4-6 h to obtain crude yeast microcapsules. S12. Centrifuge, wash and dry the crude yeast microcapsule product to obtain the yeast microcapsule carrier.

3. The method for preparing a yeast microcapsule collagen peptide oral delivery system according to claim 2, characterized in that: In step S11, the protease is one of alkaline protease, papain, or neutral protease; the amount of protease added is 0.5-3.0% of the dry weight of yeast; and the duration of each single operation and interval in the intermittent ultrasound mode is 5 seconds.

4. The preparation method of the yeast microcapsule collagen peptide oral delivery system according to claim 2, characterized in that: In step S12, the centrifugation speed is 7000~9000 rpm and the time is 3~8 min.

5. The method for preparing a yeast microcapsule collagen peptide oral delivery system according to claim 1, characterized in that: In step S2, the cyclodextrin stabilizer is selected from β-cyclodextrin or hydroxypropyl-β-cyclodextrin; the polyphenol stabilizer is selected from epigallocatechin gallate, tea polyphenols or ferulic acid.

6. The method for preparing a yeast microcapsule collagen peptide oral delivery system according to claim 1, characterized in that: In step S2, when the conformation stabilizer is a cyclodextrin, its mass ratio to collagen peptide is 1:4~6.5; when the conformation stabilizer is a polyphenol, its mass ratio to collagen peptide is 1:2.3~3.

7.

7. The method for preparing a yeast microcapsule collagen peptide oral delivery system according to claim 1, characterized in that: In step S3, the mass ratio of the yeast microcapsule carrier to the collagen peptide is 1-2:1-2; the temperature of the isothermal oscillation reaction is 30-40 °C, the pH value is 4.0-7.0, and the time is 2-4 h.

8. The method for preparing a yeast microcapsule collagen peptide oral delivery system according to claim 1, characterized in that: In step S4, the centrifugation speed is 7000~9000 rpm and the time is 3~8 min.

9. A yeast microcapsule collagen peptide oral delivery system, characterized in that, It is prepared by any one of claims 1-8.

10. The use of the yeast microcapsule collagen peptide oral delivery system as described in claim 9 in the preparation of anti-inflammatory, antioxidant, or beauty and health foods or pharmaceuticals.

Images