A dpp-iv inhibitory peptide from fermented rohu skin and a preparation method and application thereof

Abstract

This invention discloses a DPP-IV inhibitory peptide from tilapia skin fermented with Aspergillus oryzae, its preparation method, and its applications, belonging to the field of food-derived bioactive peptide development technology. Using tilapia skin as raw material, this invention employs a combined process of acid-base pretreatment, warm water extraction, Aspergillus oryzae fermentation, gel column chromatography, and semi-preparative liquid chromatography to obtain tilapia skin bioactive peptides with high DPP-IV inhibitory activity. The amino acid sequences are AAGVGEF, GIADRMQ, and KPLRLPL, respectively. These three inhibitory peptides exhibit good DPP-IV inhibitory activity and can be used to develop health products or pharmaceuticals for regulating blood sugar.

Description

Technical Field

[0001] This invention belongs to the field of food-derived bioactive peptide development technology, specifically relating to a DPP-IV inhibitory peptide from tilapia skin fermented by Aspergillus oryzae, its preparation method, and its application. Background Technology

[0002] In recent years, the incidence of type 2 diabetes mellitus (T2DM) and its complications has risen significantly worldwide, becoming one of the most significant challenges in public health. In 2021, the disease caused approximately 6.7 million deaths, highlighting its serious threat to global health systems. If effective intervention is not implemented in the early stages of the disease, patients with T2DM often rely on medication to maintain stable blood sugar levels. However, chemically synthesized antidiabetic drugs are frequently associated with potential adverse reactions.

[0003] In type 2 diabetes mellitus (T2DM) management strategies, inhibiting dipeptidyl peptidase IV (DPP-IV) activity is considered an effective way to regulate blood glucose. DPP-IV is a metabolic enzyme widely expressed on the surface of various cells that degrades incretin hormones; inhibiting its activity can prolong the half-life of glucagon-like peptide-1 (GLP-1), thereby promoting insulin secretion and achieving blood glucose regulation. Therefore, food-derived hydrolysates or peptides with DPP-IV inhibitory properties hold promise for addressing the safety risks associated with long-term medication use.

[0004] Therefore, developing a novel DPP-IV inhibitor that is safe, non-toxic, has no side effects, and has good DPP-IV inhibitory effects has become a technical challenge that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] This invention provides a DPP-IV inhibitory peptide from tilapia skin fermented with Aspergillus oryzae, its preparation method, and its application. The invention uses tilapia skin gelatin protein as raw material and employs Aspergillus oryzae fermentation to obtain a short peptide with good DPP-IV inhibitory activity. The preparation method of this invention employs a combined process of acid-base pretreatment, warm water extraction, microbial fermentation, gel column chromatography, and semi-preparative liquid chromatography to finally obtain the tilapia skin DPP-IV inhibitory peptide.

[0006] The objective of this invention is achieved through the following technical solution: The objective of this invention is to provide a DPP-IV inhibitory peptide 1 for tilapia skin fermented by Aspergillus oryzae, wherein the amino acid sequence of the inhibitory peptide is AAGVGEF, as shown in SEQ ID NO:1.

[0007] The second objective of this invention is to provide a DPP-IV inhibitory peptide 2 from tilapia skin fermented with Aspergillus oryzae, wherein the amino acid sequence of the inhibitory peptide is GIADRMQ, as shown in SEQ ID NO:2.

[0008] The third objective of this invention is to provide a DPP-IV inhibitory peptide 3 from tilapia skin fermented with Aspergillus oryzae, wherein the amino acid sequence of the inhibitory peptide is: KPLRLPL, as shown in SEQ ID NO:3.

[0009] The present invention also provides the use of the above three inhibitory peptides or their complexes in the preparation of DPP-IV protease activity inhibitors.

[0010] Preferably, the DPP-IV protease activity inhibitor is a hypoglycemic drug or a health product with auxiliary hypoglycemic function.

[0011] This invention also provides a method for preparing the above three tilapia skin DPP-IV inhibitory peptides, comprising the following steps: S1. Tilapia skin pretreatment: After removing the scales and mincing the meat, tilapia skin is soaked in 4-7 times its volume of 0.1%-0.3% NaOH solution, 0.1%-0.3% H2SO4 solution and 0.5%-2.0% C6H8O7 solution for 2 hours each. S2. Preparation of tilapia skin supernatant: The fish skin processed in step S1 is stirred with a mixer to obtain fish skin fragments. Add 1-3 times the volume of warm water at 40-50℃, stir for 8-12 hours, centrifuge to collect the supernatant, sterilize the supernatant, and cool it to room temperature. S3. Preparation of tilapia skin fermentation broth: Activated Aspergillus oryzae seed liquid was inoculated into the supernatant obtained in step S2 at an inoculation amount of 10%. The resulting mixture was inoculated into a culture medium containing tilapia skin gelatin at a mass-volume ratio of 6%, and cultured at 28℃ and pH 6-8. Fermentation was carried out at a speed of 180 rpm to obtain the fermentation broth. S4. Concentration of tilapia skin fermentation broth: After inactivating the enzymes in the fermentation broth obtained in step S3, centrifuge and collect the supernatant. Concentrate the supernatant under reduced pressure to 1 / 4 of the original solution volume. S5. Tilapia skin fermentation broth separation and purification: After centrifugation, the fermentation broth obtained from step S4 was separated into three components: >10kDa, 3~10kDa, and <3kDa using ultrafiltration membrane pore size cutoff. DPP-IV inhibitory activity was measured. The obtained solution with a molecular weight less than 3kDa was loaded onto gel column chromatography, eluted, and the components were obtained. S6. Screening and synthesis of tilapia skin active peptides with DPP-IV inhibitory activity: The components obtained in step S5 were further eluted by semi-preparative high performance liquid chromatography, and the elution peaks at 28-30 minutes were collected to obtain the components. The obtained components were analyzed by liquid chromatography and mass spectrometry to screen out DPP-IV inhibitory peptides with higher activity, and the amino acid sequence of the specific peptides was determined. The selected peptides were synthesized by solid-phase chemical synthesis to obtain tilapia skin DPP-IV inhibitory peptides.

[0012] This invention utilizes Aspergillus oryzae fermentation of fish skin to prepare DPP-IV inhibitory peptides, the principle of which is as follows: 1. Fermentation using microorganisms under appropriate culture conditions produces desired compounds. During fermentation, microorganisms metabolize substrates (such as carbohydrates, nitrogen sources, etc.) to produce the target product.

[0013] 2. During microbial fermentation, the complex enzyme system produced catalyzes the hydrolysis of large protein molecules, converting them into smaller peptides. Furthermore, under the influence of microbial metabolic activities, these peptides undergo post-translational modifications and structural reconstruction, resulting in peptides with specific biological activities. Extracellular enzymes secreted during fermentation (such as proteases and peptidases) can systematically degrade protein substrates, generating short peptides and free amino acids. In addition, various enzymes in the fermentation system may also decompose residual lipids and odorous substances in the raw materials, and may generate certain physiologically active factors, thereby enhancing the nutritional value and functional properties of the peptide products.

[0014] 3. Aspergillus oryzae is widely used in the fermentation preparation of bioactive peptides because it can produce a variety of extracellular proteases during its growth process and has the characteristics of stable growth and strong adaptability to processing.

[0015] 4. During the fermentation of fish skin by Aspergillus oryzae, bioactive peptides are released through the action of microorganisms and endogenous proteolytic enzymes.

[0016] The effective effects of this invention are as follows: This invention uses tilapia skin as raw material, which is not only widely available but also inexpensive. Developing peptides with DPP-IV enzyme inhibitory activity based on tilapia skin can significantly increase its added value. The active peptides prepared by this invention exhibit excellent DPP-IV enzyme inhibitory effects, effectively suppressing postprandial blood glucose elevation in individuals in the pre-T2DM stage. Furthermore, the active peptides possess a reasonable amino acid composition, are non-allergenic, and safe, making them suitable for preparing hypoglycemic drugs or supplementary hypoglycemic health products, demonstrating promising application prospects and value. The Aspergillus oryzae fermentation technology employed is simple and feasible, offering advantages such as low production costs and readily available equipment, while maximizing the release of active ingredients from the fish skin. Attached Figure Description

[0017] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0018] Figure 1 This is a graph showing the DPP-IV inhibition rates of different molecular weight components in the fermentation broth of Example 1 of the present invention.

[0019] Figure 2This is a graph showing the DPP-IV inhibition rates of the three DPP-IV inhibitory peptides in Example 2 of the present invention. Detailed Implementation

[0020] To better understand the present invention, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. However, those skilled in the art will understand that the following embodiments are not intended to limit the scope of protection of the present invention, and any changes and variations made on the basis of the present invention are within the scope of protection of the present invention.

[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0022] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0023] The strain used in this invention is Aspergillus oryzae CGMCC 3.951.

[0024] Methods for determining the DPP-IV inhibition rate: The lyophilized sample was reconstituted in ultrapure water at a concentration of 10 mg / mL. 40 µL of 0.5 mM Gly-Pro-pNA dissolved in 100 mM Tris-HCl buffer, 50 µL of the sample, and 10 µL of DPP-IV enzyme dissolved in 100 mM Tris-HCl buffer at a concentration of 0.025 U / mL were added to each well in a 96-well plate. The plates were incubated at 37°C in the dark for 60 min. The absorbance was measured at 405 nm, and the DPP-IV inhibition rate was calculated using the formula shown in formula (1).

[0025] Example 1: Preparation of DPP-IV inhibitory peptide This embodiment describes a method for preparing tilapia skin DPP-IV inhibitory peptides using Aspergillus oryzae fermentation, comprising the following steps: S1. Weigh 500g of tilapia skin, remove scales and minced meat, rinse three times with clean water, add 4-7 times the volume of 0.1%-0.3% NaOH solution, soak for two hours; then soak for two hours with 4-7 times the volume of 0.1%-0.3% H2SO4 solution; finally soak for two hours with 4-7 times the volume of 0.5%-2% C6H8O7 solution; rinse with clean water until the pH value is 7.0 before each solvent change. S2. Place the processed fish skin in a mixer and stir to obtain fish skin fragments. Add 1-3 times the volume of warm water at 40-50℃ and stir for 8-12 hours. Centrifuge and collect the supernatant. Add the obtained supernatant to C6H42-H2O solution with a solid-liquid ratio of 13-16%. 12After adding O6, 0.3~0.6% MgCl2, 0.5~0.8% KCl, and 0.5~0.8% K2HPO4, sterilize at 121℃ for 20 min, then cool to room temperature; S3. Add activated Aspergillus oryzae CGMCC 3.951 seed culture to the supernatant obtained in step S2 at an inoculation ratio of 10%; inoculate the resulting mixture into tilapia skin gelatin medium (6%, w / v, fish skin gelatin / PBS), and culture at 28℃ and pH 6-8. After fermentation at 180 rpm for 24 h, the fermentation broth is obtained. S4. Heat the fermentation broth obtained in step S3 to 90℃ for 5 minutes to inactivate the enzyme. After inactivation, centrifuge the fermentation broth at 5000 rpm for 15 minutes and carefully collect the supernatant. Concentrate the supernatant under reduced pressure until the solution volume is 1 / 4 of the original solution volume. S5. Filter the fermentation broth obtained in step S4 through a 0.45µM water membrane, centrifuge at 4℃ and 8000rpm for 15min, and separate the DPP-IV inhibitory peptide into three fractions: >10kDa, 3~10kDa, and <3kDa. Measure the DPP-IV inhibition rate of the three fractions. Figure 1 As shown. The resulting solution with a molecular weight less than 3 kDa was loaded onto a gel column for chromatography, eluted, and the fractions were obtained. The gel chromatography column was packed with G-25 gel, with a column size of 2.8×80cm. The injection volume was 2mL, and distilled water was used for elution at a rate of 1.5mL / min. The elution peak was detected at a wavelength of 220nm, and the fraction eluent was collected after 15-30 minutes.

[0026] Depend on Figure 1 It can be seen that the DPP-IV inhibition rate of components with a molecular weight of less than 3kDa is higher than that of other components, indicating that water membrane filtration can screen out components with weak DPP-IV inhibitory activity.

[0027] S6. The components obtained in step S5 are further eluted using semi-preparative high-performance liquid chromatography (HPLC). The elution peaks at 28-30 minutes are collected to obtain the components. The obtained components are analyzed using liquid chromatography and mass spectrometry (LC-MS) to screen out DPP-IV inhibitory peptides with higher activity. The amino acid sequences of specific peptide segments are determined. The selected peptides are synthesized using solid-phase chemical synthesis to obtain tilapia skin DPP-IV inhibitory peptides.

[0028] Liquid chromatography separation conditions: A 0.1% formic acid aqueous solution was used as mobile phase A, and a mixed solution of 0.1% formic acid and 80% acetonitrile was used as mobile phase B. Gradient elution was set as follows: 0-2min, 4%-8%B; 2-45min, 8%-28%B; 45-55min, 28%-40%B; 55-56min, 40%-95%B; 56-66min, 95%-95%B, The flow rate is 600 nL / min.

[0029] Mass spectrometry analysis was performed using Orbitrap for a single-stage full scan, with an m / z scan range of 100–1500, a resolution of 70,000, a maximum ion introduction time of 100 ms, and automatic gain control (AGC) of 3 × 10⁻⁶. 6 Subsequently, secondary mass spectrometry (MS / MS) analysis was performed using a high-energy collisional dissociation method, with a collision energy of 28%, a resolution of 17500, and an AGC of 1×10⁻⁶. 5 The maximum ion introduction time is 50ms, and the peptide fragmentation collision energy is set to 28%.

[0030] Example 2: Determination of DPP-IV inhibitory peptide activity The fraction with the highest DPP-IV inhibition rate from the fermentation broth was used to sequence the highly active tilapia skin peptide using Nano LC MS / MS, and the DPP-IV inhibitory peptide sequence was determined using the BLAST tool in NCBI. Based on the sequencing results, the DPP-IV inhibitory peptide was chemically synthesized, and its activity was measured. The specific method for determining the DPP-IV inhibitory peptide activity was as follows: The lyophilized sample was reconstituted in ultrapure water at an initial concentration of 10 mg / mL. In a 96-well plate, 40 µL of 0.5 mM Gly-Pro-pNA dissolved in 100 mM Tris-HCl buffer, 50 µL of the sample, and 10 µL of DPP-IV enzyme dissolved in 100 mM Tris-HCl buffer at a concentration of 0.025 U / mL were added, respectively. The plate was incubated at 37°C in the dark for 60 min. The absorbance was measured at 405 nm. For peptide inhibition assays, the inhibition rate of a series of peptide concentrations (0.001–10 mg / mL) was determined according to the above procedure. Subsequently, the IC50 was calculated using GraphPad Prism software 8 based on an S-shaped dose-response plot of peptide concentration (µM) versus inhibitory activity (%). 50 Values. The logarithm of the DPP-IV inhibitory peptide concentration is plotted on the x-axis, along with the DPP-IV IC50 value. 50 The value is the ordinate, and the result is as follows: Figure 2 As shown.

[0031] The sequencing results of the three isolated DPP-IV repressor peptides are shown in Table 1. Table 1. Inhibitory activity and sequencing results of three DPP-IV inhibitory peptides.

[0032] Depend on Figure 2 As shown in Table 1, the IC50 values ​​of peptides 1, 2, and 3 are... 50 The values ​​were 998±103µM, 1075±188µM, and 1103±112µM, respectively, and the DPP-IV inhibition rates were 80.48±2.34, 81.37±3.67, and 76.04±3.90, respectively, indicating that these three peptides have a strong inhibitory effect on DPP-IV enzyme.

[0033] In summary, the method of this invention first utilizes microorganisms to ferment under appropriate culture conditions to produce the desired inhibitory peptides. During fermentation, microorganisms can metabolize substrates (such as carbohydrates, nitrogen sources, etc.) to produce the target product. Secondly, the complex enzyme system produced during microbial fermentation hydrolyzes large protein molecules into smaller polypeptides. Under the action of microorganisms, peptide groups undergo modification and recombination to obtain bioactive peptides. Extracellular enzymes produced during microbial fermentation can systematically degrade large protein molecules into shorter polypeptides and amino acids. Furthermore, various enzymes produced during microbial fermentation may decompose small amounts of fat and odorous substances in the fermentation raw materials and may also produce certain physiologically active factors, thereby improving the nutritional value and health benefits of the polypeptide product. Aspergillus oryzae is widely used in the fermentation preparation of bioactive peptides due to its ability to produce various extracellular proteases during growth, its stable growth, and its strong adaptability to processing. During the fermentation of fish skin by Aspergillus oryzae, bioactive peptides are released through the action of microorganisms and endogenous proteolytic enzymes.

[0034] The bioactive peptides prepared by this invention exhibit excellent inhibitory activity against DPP-IV enzyme activity, effectively suppressing postprandial blood glucose elevation in patients in the pre-T2DM stage. They possess a reasonable amino acid composition, are non-allergenic, and safe and reliable, making them suitable for preparing hypoglycemic drugs or supplementary hypoglycemic health products, demonstrating significant application value. This invention utilizes Aspergillus oryzae fermentation technology, which is simple, feasible, low-cost, and requires readily available equipment, while maximizing the release of active ingredients from fish skin.

[0035] While specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and not intended to limit the scope of the present invention. Equivalent modifications and variations made by those skilled in the art in accordance with the spirit of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A DPP-IV inhibitory peptide 1 from tilapia skin fermented by Aspergillus oryzae, characterized in that, The amino acid sequence of the inhibitory peptide 1 is as shown in SEQ ID NO:

1.

2. A DPP-IV inhibitory peptide 2 from tilapia skin fermented by Aspergillus oryzae, characterized in that, The amino acid sequence of the inhibitory peptide 2 is as shown in SEQ ID NO:

2.

3. A DPP-IV inhibitory peptide 3 from tilapia skin fermented by Aspergillus oryzae, characterized in that, The amino acid sequence of the inhibitory peptide 3 is as shown in SEQ ID NO:

3.

4. The use of the inhibitory peptide 1 or its complex as described in claim 1 in the preparation of DPP-IV protease activity inhibitors.

5. The application according to claim 4, characterized in that, The DPP-IV protease activity inhibitor is a hypoglycemic drug or a health product that helps lower blood sugar.

6. The use of the inhibitory peptide 2 or its complex as described in claim 2 in the preparation of DPP-IV protease activity inhibitors.

7. The application according to claim 6, characterized in that, The DPP-IV protease activity inhibitor is a hypoglycemic drug or a health product that helps lower blood sugar.

8. The use of the inhibitory peptide 3 or its complex as described in claim 3 in the preparation of DPP-IV protease activity inhibitors.

9. The application according to claim 8, characterized in that, The DPP-IV protease activity inhibitor is a hypoglycemic drug or a health product that helps lower blood sugar.

10. A method for preparing tilapia skin DPP-IV inhibitory peptides 1-3 as described in claims 1-3, comprising the following steps: S1. Tilapia skin pretreatment: After removing the scales and mincing the meat, tilapia skin is soaked in 4-7 times its volume of 0.1%-0.3% NaOH solution, 0.1%-0.3% H2SO4 solution and 0.5%-2.0% C6H8O7 solution for 2 hours each. S2. Preparation of tilapia skin supernatant: The fish skin processed in step S1 is stirred with a mixer to obtain fish skin fragments. Add 1-3 times the volume of warm water at 40-50℃, stir for 8-12 hours, centrifuge to collect the supernatant, sterilize the supernatant, and cool it to room temperature. S3. Preparation of tilapia skin fermentation broth: Activated Aspergillus oryzae seed liquid was inoculated into the supernatant obtained in step S2 at an inoculation amount of 10%. The resulting mixture was inoculated into a culture medium containing tilapia skin gelatin at a mass-volume ratio of 6%, and cultured at 28℃ and pH 6-8. Fermentation was carried out at a speed of 180 rpm to obtain the fermentation broth. S4. Concentration of tilapia skin fermentation broth: After inactivating the enzymes in the fermentation broth obtained in step S3, centrifuge and collect the supernatant. Concentrate the supernatant under reduced pressure to 1 / 4 of the original solution volume. S5. Tilapia skin fermentation broth separation and purification: After centrifugation, the fermentation broth obtained from step S4 was separated into three components: >10kDa, 3~10kDa, and <3kDa using ultrafiltration membrane pore size cutoff. DPP-IV inhibitory activity was measured. The obtained solution with a molecular weight less than 3kDa was loaded onto gel column chromatography, eluted, and the components were obtained. S6. Screening and synthesis of tilapia skin active peptides with DPP-IV inhibitory activity: The components obtained in step S5 were further eluted by semi-preparative high performance liquid chromatography, and the elution peaks at 28-30 minutes were collected to obtain the components. The obtained components were analyzed by liquid chromatography and mass spectrometry to screen out DPP-IV inhibitory peptides with higher activity, and the amino acid sequence of the specific peptides was determined. The selected peptides were synthesized by solid-phase chemical synthesis to obtain tilapia skin DPP-IV inhibitory peptides.

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