Royal jelly active peptide composition with DPP-IV (dipeptidyl peptidase-IV) inhibition effect as well as preparation method and application thereof

Abstract

The invention discloses a royal jelly active peptide composition with a DPP-IV (dipeptidyl peptidase-IV) inhibiting effect as well as a preparation method and application thereof, and belongs to the technical field of foods and medicines. The royal jelly active peptide composition provided by the invention comprises at least one of five small molecule peptides; the amino acid sequences of the five small molecule peptides are respectively shown as SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5. The DPP-IV inhibitory peptide composition prepared by the invention is high in activity, large in yield and strong in operability, can be industrially produced, and provides an experimental basis for preparing the high-activity DPP-IV inhibitory peptide from royal jelly.

Description

Technical Field

[0001] The present invention relates to the technical field of food and medicine, and in particular to a royal jelly active peptide composition having a dipeptidyl peptidase IV (DPP-IV) inhibitory effect, and a preparation method and application thereof. Background Art

[0002] Diabetes is a worldwide epidemic disease. It is a progressive disease characterized by high blood sugar and complications. It is mainly manifested by the destruction of pancreatic β cells, leading to type 1 diabetes with insulin deficiency and type 2 diabetes with insulin resistance. DPP-IV (dipeptidyl peptidase IV) inhibitors are a class of drugs for the treatment of type 2 diabetes. They inhibit the inactivation of glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), increase the levels of endogenous GLP-1 and GIP, thereby promoting the release of insulin from pancreatic β cells, inhibiting the secretion of glucagon from pancreatic α cells, increasing the level of postprandial insulin, lowering blood sugar, and not easily inducing hypoglycemia and weight gain. Currently, there are more than 10 DPP-IV inhibitors on the market worldwide, including sitagliptin, vildagliptin, saxagliptin, etc., but these are synthetic drugs, which often cause more serious side effects, such as headaches, diarrhea, urinary tract infections, and kidney burden. In contrast, food-borne DPP-IV inhibitors have attracted attention due to their good stability and few side effects.

[0003] Royal jelly is a yellowish paste secreted by the hypopharyngeal gland and maxillary gland on the head of worker bees. The queen bee consumes royal jelly throughout her life, while the worker bees are only fed royal jelly for the first 3 days of the larval stage, after which they switch to food such as pollen and nectar. This different way of food supply has a profound impact on the development of female bees, leading to caste differentiation. Fresh royal jelly is composed of 60%~70% water and 30%~40% dry matter, of which the dry matter contains a variety of proteins, amino acids, fatty acids, carbohydrates, vitamins, minerals, nucleic acids, hormones and unknown components. Royal jelly has a variety of significant biological activities, such as antibacterial, anti-aging, anti-fatigue, anti-inflammatory, antioxidant, anti-tumor and anti-diabetic.

[0004] Although royal jelly has a variety of active effects, its stability is poor and requires cold chain transportation throughout the process, which significantly increases logistics and warehousing costs; it has poor water solubility and has unpleasant flavors such as sourness and spiciness; in addition, certain people may have allergic reactions to the ingredients in royal jelly. Studies have shown that the main allergens are the main proteins 1 and 2 in royal jelly. These defects significantly reduce the commercial value of royal jelly and limit its application potential in many fields. my country's royal jelly production accounts for more than 90% of the world's total production, but it is mainly exported in the form of raw materials with low added value. Therefore, it is particularly necessary to deeply process royal jelly and develop active peptide products with strong functions, good stability, excellent taste, removal of allergenic ingredients, and high added value.

[0005] The protein content in royal jelly is 9-18%, which is the main component that exhibits its special physiological activity. Enzymatic hydrolysis of proteins in royal jelly to prepare functional royal jelly peptides can not only significantly enrich functional activities, but also solve the inherent drawbacks of the above-mentioned fresh royal jelly products and increase added value. It is a very promising royal jelly deep processing technology. At present, some studies have been devoted to the development of royal jelly peptides with special functional activities, but there are few reports on royal jelly protein-derived DPP-IV inhibitory peptides. In addition, there is a lack of evaluation on whether royal jelly protein is suitable for the preparation of DPP-IV inhibitory peptides. Summary of the invention

[0006] In order to overcome the above-mentioned defects in the prior art, effectively develop royal jelly resources and improve their economic added value, the present invention provides a royal jelly active peptide composition with DPP-IV inhibitory effect and its preparation method and application based on the characteristics of royal jelly.

[0007] In order to achieve the above-mentioned invention object, this application provides the following technical solutions: On the one hand, the present application provides a royal jelly active peptide composition having a DPP-IV inhibitory effect, wherein the royal jelly active peptide composition comprises at least one of five small molecule peptides; The amino acid sequences of the five small molecule peptides are respectively SEQ ID NO.1: KQVEIPHDV, SEQ ID NO.2: IDKCDRLW, SEQ ID NO.3: HNSDDSFHRL, SEQ ID NO.4: VEIPHNIA, and SEQ ID NO.5: HGPPP.

[0008] In a second aspect, the present application provides a method for preparing the above royal jelly active peptide composition having DPP-IV inhibitory effect, comprising the following steps: (1) Raw material evaluation: Use bioinformatics to analyze the potential activity of royal jelly protein and evaluate the feasibility of using protein raw materials for the preparation of DPP-IV inhibitory peptides; (2) Enzymatic hydrolysis: mixing royal jelly water-soluble protein dry powder with water, adjusting the pH value, adding protease, stirring and mixing for enzymatic hydrolysis, inactivating the royal jelly protein, centrifuging and taking the supernatant, and freeze-drying to obtain a crude royal jelly protease hydrolysate with DPP-IV inhibitory activity; (3) Purification: The crude protein hydrolysate is purified by ultrafiltration, anion exchange chromatography, and reverse-phase high performance liquid chromatography in sequence to obtain the royal jelly active peptide composition with DPP-IV inhibitory effect.

[0009] Optionally, the bioinformatics method in step (1) includes: using the "PROTEINS", "ANALYSIS", "CALCULATIONS", "PROFILES OF POTENTIAL BIOLOGICAL ACTIVITY" functions in the BIOPEP database to predict the biological activities of Major Royal Jelly Proteins (MRJPs) 1~9.

[0010] Optionally, the royal jelly protein in step (1) includes MRJPs 1~9; among them, the protein number of MRJP1 in the NCBI database is ACS66836.1; the protein number of MRJP2 in the NCBI database is ACS66837.1, and the protein number of MRJP3 in the NCBI database is ADC55524.1; the protein number of MRJP4 in the NCBI database is ADB82660.1, the protein number of MRJP5 in the NCBI database is ADB96941.1, the protein number of MRJP6 in the NCBI database is AAQ82184.1, the protein number of MRJP7 in the NCBI database is DAA01512.1, the protein number of MRJP8 in the NCBI database is AAR83734.1, and the protein number of MRJP9 in the NCBI database is AAY21180.1.

[0011] Optionally, in step (2), the mixing mass ratio of the royal jelly water-soluble protein dry powder to water is 1:7-10; The component for adjusting pH value is edible alkali; The pH value is adjusted to 7-9; The protease is an alkaline protease, preferably an alkaline protease with an enzyme number of EC3.4.21.62; The added amount of the protease is 6000-9000 U / g protein, preferably 6000 U / g; The stirring speed is 100-300 rpm; The enzymatic hydrolysis temperature is 40-60°C, and the optimum temperature is 60°C; The enzymatic hydrolysis time is 2 to 4 hours; The centrifugal speed is 3000-5000 rpm, preferably 4500 rpm; The centrifugal temperature is 5-20°C; The centrifugation time is 10 to 15 minutes.

[0012] Optionally, the separation and purification operation in step (3) is to separate the royal jelly protein hydrolysate with DPP-IV inhibitory activity obtained in step (2) through 3kD and 1kD ultrafiltration tubes respectively, collect different components for freeze-drying, and then further separate and purify the component with the highest DPP-IV inhibitory activity using HiTrap DEAE-FF anion exchange; The conditions of the HiTrap DEAE-FF anion exchange purification are as follows: the mobile phase A of the anion exchange chromatography is 0.01 mol / L pH 7.0 phosphate buffer, the mobile phase B is 0.01 mol / L pH 7.0 phosphate buffer containing 1 mol / L NaCl, the elution conditions are 100% A for 35 min, the B solution is adjusted from 0 to 80% at 35 to 45 min, 80% B is maintained for 10 minutes, and the B is adjusted to 100% for the last 55 to 60 min and eluted for 10 min, the elution flow rate is 1 mL / min, and the absorbance is detected at 214 nm to collect the eluted components.

[0013] Optionally, in step (3), after the anion exchange purification, the collected fractions with the highest DPP-IV inhibitory activity are further purified by C18 semi-preparative liquid chromatography; The conditions for the C18 semi-preparative chromatography purification are as follows: mobile phase A is ultrapure water containing 0.1% TFA, mobile phase B is acetonitrile containing 0.1% TFA, the flow rate is 5 mL / min, and the gradient elution program is: ①0-5 min: B 10-20%, ②5-10 min: 20% B, ③10-20 min: B 20-30%; the absorbance is detected at 214 nm and the eluted components are collected, which are then freeze-dried and concentrated to obtain the royal jelly active peptide composition with DPP-IV inhibitory effect.

[0014] In the third aspect, the present application provides the use of the above royal jelly active peptide composition with DPP-IV inhibitory effect in the preparation of drugs that inhibit DPP-IV and / or auxiliary blood sugar-lowering health foods. The experimental results show that after intragastric administration of medium and high doses (200 mg / kg, 400 mg / kg) of royal jelly DPP-IV inhibitory peptide composition, the fasting blood glucose content of hyperglycemic mice can be reduced (P < 0.01), indicating that the royal jelly DPP-IV inhibitory peptide composition prepared in this experiment can effectively regulate the normal metabolism of blood glucose in hyperglycemic mice.

[0015] The main detection indicators of the royal jelly active peptide composition prepared by the present invention are as follows: (1) DPP-IV inhibition rate of the composition (2) Protein concentration of the composition Compared with the prior art, this application has the following beneficial effects: 1. Using bioinformatics as a tool to predict the potential biological activity of royal jelly water-soluble protein can quickly discover whether the protein raw material is suitable for developing peptides with specific functions, which has a good guiding role in obtaining specific functional peptides.

[0016] 2. The final step of separation and purification in this application was completed using a semi-preparative chromatographic column, and a total of 17 peptides were obtained, with large yield, strong operability, and suitable for industrial production. The amino acid sequences of the main active peptides in the obtained DPP-IV inhibitory peptide composition are KQVEIPHDV, IDKCDRLW, HNSDDSFHRL, VEIPHNIA, and HGPPP.

[0017] 3. The royal jelly protein source DPP-IV inhibitory peptide composition described in this application is safe, has no toxic side effects, and has a scientific and reasonable process. It can be developed as a health food, food additive, drug enhancer, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0019] Figure 1 The predicted frequencies of bioactive peptides that may be released from MRJPs 1 to 9 Figure 2 is the DPP-IV inhibition rate after different enzymes hydrolyzed royal jelly water-soluble protein; Figure 3 DPP-IV inhibition rate of each component obtained by ultrafiltration with different molecular weights; Figure 4 is the anion exchange chromatogram of the F3 component; Figure 5 is the DPP-IV inhibition rate of each component after anion exchange chromatography of the F3 component; Figure 6 This is a semi-preparative liquid chromatography purification chromatogram of component F3-1; Figure 7 It is the DPP-IV inhibition rate of each component after the F3-1 component was separated by semi-preparative liquid chromatography; Figure 8 It is the total ion current chromatogram of component F3-1-3; Fig. 9 This is the one-dimensional mass spectrometry analysis of the peptide KQVEIPHDV that has the highest score in the F3-1-3 component; Fig.10 This is the two-dimensional mass spectrometry analysis of the peptide KQVEIPHDV that has the highest score in the F3 1 3 component; Fig.11 This is a one-dimensional mass spectrum analysis of the peptide IDKCDRLW that has the second highest score in the F3-1-3 component; Fig.12 It is a two-dimensional mass spectrometry analysis diagram of the peptide IDKCDRLW with the second highest score in the F3-1-3 component; Fig.13 This is a one-dimensional mass spectrometry analysis of the peptide HNSDDSFHRL that has the third highest score in the F3-1-3 component; Fig.14 This is the two-dimensional mass spectrometry analysis of the peptide HNSDDSFHRL that has the third highest score in the F3-1-3 component; Fig.15 This is a one-dimensional mass spectrometry analysis of the peptide VEIPHNIA that ranks fourth in the F3-1-3 component; Fig.16 This is a two-dimensional mass spectrometry analysis of the peptide VEIPHNIA that ranks fourth in the F3-1-3 component; Fig.17 This is a one-dimensional mass spectrometry analysis of the peptide HGPPP that scored fifth in the F3-1-3 component; Fig.18 This is the two-dimensional mass spectrometry analysis of the fifth-ranking peptide HGPPP in the F3-1-3 component. DETAILED DESCRIPTION

[0020] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0021] Unless otherwise specified, the raw materials in the examples of the present application were purchased from commercial sources and used directly without any special treatment.

[0022] Unless otherwise specified, the analysis methods in the examples all adopt conventional settings and conventional analysis methods of instruments or equipment.

[0023] Example 1 Prediction of potential biological activities of royal jelly major proteins MRJPs 1-9 (1) The amino acid sequences of MRJP1 (ACS66836.1), MRJP2 (ACS66837.1), MRJP3 (ADC55524.1), MRJP4 (ADB82660.1), MRJP5 (ADB96941.1), MRJP6 (AAQ82184.1), MRJP7 (DAA01512.1), MRJP8 (AAR83734.1), and MRJP9 (AAY21180.1) were downloaded from the NCBI database (https: / / www.ncbi.nlm.nih.gov).

[0024] (2) The “PROTEINS”, “ANALYSIS”, “CALCULATIONS”, and “PROFILES OF POTENTIAL BIOLOGICALACTIVITY” functions in the BIOPEP database (http: / / biochemia.uwm.edu.pl / biopep-uwm / ) were used to predict the biological activities of MRJPs 1–9 and explore the possible activities of royal jelly proteins after enzymatic hydrolysis. The results are shown in Figure 1 .Depend on Figure 1 It can be seen that among the nine major royal jelly proteins, the frequency of DPP-IV inhibitory peptide release is the highest, indicating that it is completely feasible to generate DPP-IV inhibitory peptides by enzymatic hydrolysis of royal jelly major proteins MRJPs 1~9.

[0025] Note: Figure 1 AE in the table represents the release frequency of the possible biologically active fragments of the nine main proteins.

[0026] In the formula, is the frequency of occurrence of active peptides, is the number of active peptides, and is the total number of amino acid residues in the selected royal jelly main protein sequence.

[0027] Example 2 Preparation of royal jelly protein hydrolysate with DPP-IV inhibition: Royal jelly water-soluble protein powder was dissolved in deionized water at a mass ratio of 10%, and protease was added at an amount of 6000 U / g protein. The optimal reaction conditions of each enzyme were adjusted for enzymatic hydrolysis for 4 hours to obtain an enzymatic solution. After the enzymatic hydrolysis was completed, the enzymatic solution was placed at 90°C to inactivate the enzyme for 15 minutes, cooled to room temperature, centrifuged at 20°C, 10000r / min for 5 minutes, the supernatant was collected, and the DPP-IV inhibition rate was determined by freeze-drying. The results are as follows: Figure 2 The results show that the enzymatic hydrolysate obtained by alkaline protease hydrolysis has the highest DPP-IV inhibition rate.

[0028] Protein content determination: according to the biuret method.

[0029] Determination of DPP-IV inhibitory activity: The substrate Gly-Pro-pNA is degraded under the action of DPP-IV, and the generated p-nitroaniline has strong light absorption at a wavelength of 405 nm. Therefore, the rate of the enzymatic reaction can be expressed based on the degree of change in absorbance per unit time.

[0030] Take a 96-well plate and use a micropipette to add 25 μL of 1.6 mmol / L substrate Gly to the reaction well, then add 25 μL of the sample to be tested (add 0.1 mol / L PBS, pH 8 for the control, use sitagliptin as the positive control group, react at 37 °C for 10 min, add 450 μL of DPP-IV, react at 37 °C for 1 h, and finally add 100 μL of 0.1 mol / L Na 2 CO 3 The reaction was terminated and the absorbance of the samples was detected at a wavelength of 405 nm using an enzyme-labeled instrument. Three parallels were set up for each group.

[0031]

[0032] Where: A is Gly + sample + DPP-IV + Na 2 CO 3 ; B is Gly + sample + PBS + Na 2 CO 3 ; C is Gly+PBS+DPP-IV+Na 2 CO 3 ; D is Gly+PBS+PBS+Na 2 CO 3。

[0033] Example 3 Isolation and purification of a royal jelly active peptide composition with DPP-IV inhibitory effect Using the lyophilized powder of the enzymatic hydrolysate prepared by the optimal enzymatic cleavage process selected in Example 2 as a raw material, purifying the DPP-IV inhibitory peptide comprises the following steps: (1) Ultrafiltration separation The lyophilized powder was dissolved in pure water to prepare a solution with a concentration of 100 mg / mL, and separated by 3 kD and 1 kD ultrafiltration tubes, and the different components were collected and freeze-dried, and the DPP-IV inhibitory activity of each component was detected (see Figure 3 ), the fraction smaller than 1 kD has the best inhibitory activity, and this step is repeated continuously to collect the fraction smaller than 1 kD, concentrate it, and freeze-dry it.

[0034] (2) Ion exchange chromatography column separation HiTrap DEAE-FF anion exchange chromatography column (25mm×150mm) was used to separate the components less than 1kD. The mobile phase A was 0.01mol / L pH 7.0 phosphate buffer, and the mobile phase B was 0.01mol / L pH 7.0 phosphate buffer containing 1mol / L NaCl. The elution conditions were 100% A for 35min, and the B solution was adjusted from 0 to 80% at 35-45min, and 80% B was maintained for 10min. Finally, the B was adjusted to 100% for 55min-60min and eluted for 10min. The elution flow rate was 1mL / min, and the absorbance was detected at 214nm to collect the eluted components. From the spectrum (see Figure 4 ) It can be seen that there are chromatographic peaks F3-1 and F3-2. The components corresponding to each chromatographic peak are collected and the DPP-IV inhibitory activity of each component is detected (see Figure 5 ), the component corresponding to the chromatographic peak F3-1 has the best DPP-IV inhibitory activity, and this step is repeated continuously to collect the fractions corresponding to the chromatographic peak F3-1, concentrate them, and freeze-dry them.

[0035] (3) Semi-preparative reverse phase column chromatography separation The components corresponding to the chromatographic peak F3-1 were further separated using a semi-preparative chromatographic column Shim-pack GIS C18 (10 μm, 250 mm × 20 mm). The sample volume was 100 μL, mobile phase A was ultrapure water (containing 0.1% trifluoroacetic acid), mobile phase B was acetonitrile (containing 0.1% trifluoroacetic acid), the flow rate was 5 mL / min, and the gradient elution program was: ①0-5 min: B10-20%, ②5-10 min: 20% B, ③10-20 min: B 20-30%. Detection was performed at 214 nm. From the spectrum (see Figure 6 ) It can be seen that there are chromatographic peaks F3-1-1, F3-1-2, F3-1-3, and F3-1-4. The fractions corresponding to each chromatographic peak are collected and the DPP-IV inhibitory activity of each fraction is detected (see Figure 7 ), the DPP-IV inhibitory activity of the fraction corresponding to the chromatographic peak F3-1-3 is the best, and this step is repeated continuously to collect the fractions corresponding to the chromatographic peak F3-1-3, concentrate them, and freeze-dry them. Example 4

[0036] Mass spectrometry identification of a royal jelly active peptide composition with DPP-IV inhibitory effect: (1) Sample pretreatment Weigh 1 mg of sample (F3-1-3) in a centrifuge tube and dissolve it in 100 μL pure water. Accurately pipette 1 μL 1 mol / L DTT solution into the sample, reduce it in a 56℃ water bath for 1 hour, accurately pipette 4 μL 0.5 mol / L IAM solution into the sample, make the final concentration of IAM 20 mmol / L, react at room temperature in the dark for 40 minutes, accurately pipette 1 μL 1 mol / L DTT solution into the sample, make the final concentration of DTT 10 mmol / L, to neutralize the unreacted IAM. Desalt with a C18 stage-tip and dry in a vacuum at 45℃.

[0037] (2) Capillary liquid chromatography conditions Analytical column: 75 μm id× 150 mm, NanoViper C18 3 μm, 100A; mobile phase A: 0.1% formic acid; mobile phase B: 0.1% formic acid, 80% ACN; flow rate: 400 nL / min; analysis time for each component: 60 min. Specific chromatographic conditions: 0min-B1%, 4min-B8%, 53min-B28%, 57min-B99%, 60min-B99%, 60.1min-B1% Mass spectrometry conditions The full scan range of the mass spectrometer was 100-1500 m / z, the primary mass spectrometer resolution was set to 60,000, AGC was Custom, MaximumIT: Custom; the parent ion with an ion intensity of 20 in the full scan was selected and fragmented using the high-energy collision fragmentation (HCD) method for secondary mass spectrometry detection. The secondary mass spectrometer resolution was set to Resolution: 15,000, AGC was Custom, MaximumIT: Auto, and the peptide fragmentation collision energy was set to 30 to generate mass spectrometry detection raw data (.raw).

[0038] Search conditions The mass spectrometry raw file uses software to search the target protein database, and the search parameters are as follows: 1) Fixed modifications: Carbamidomethyl (C).

[0039] 2) Variable modifications: Oxidation (M), Acetyl (Peptide N-term).

[0040] 3) Enzyme: Non specific.

[0041] 4) Database: uniprotkb_taxonomy_id_7460_2024_08_20.

[0042] 5) Peptide Mass Tolerance: 20 ppm 6) Fragment Mass Tolerance: 0.02 Da Collecting the raw data of mass spectrometry results can generate a total ion current chromatogram ( Figure 8 ). The mass spectrometry results showed that the peptides with high scores and high contents were five small molecule peptides: KQVEIPHDV, IDKCDRLW, HNSDDSFHRL, VEIPHNIA, and HGPPP (Table 1). It can be seen that the significance scores of the five small peptides in the DPP-IV inhibitory peptide composition were all above 32, and these five peptides accounted for 73.21% of the total number of peptides. The primary and secondary mass spectrometry test results of these small peptides are shown in Figure 9~Figure 18 .

[0043]

[0044] Example 5 In vivo activity identification of a royal jelly active peptide composition with DPP-IV inhibitory effect: (1) Experimental animals Thirty 8-week-old SPF male db / db diabetic mice and six healthy male non-diabetic db / m mice from the same litter were selected. All mice were fed with normal feed, and the temperature of the breeding environment was (25±1)℃, the relative humidity was 50%~60%RH, and the indoor lighting was controlled at a 12h / 12h light-dark cycle rhythm.

[0045] (2) Grouping and intervention methods Thirty db / db mice were randomly divided into five groups according to their fasting blood glucose levels, with six mice in each group. The experimental groups were: model control group (M), positive control group of metformin hydrochloride (Y), experimental group (LR) with low-dose royal jelly DPP-IV inhibitory peptide composition, experimental group (MR) with medium-dose royal jelly DPP-IV inhibitory peptide composition, and experimental group (HR) with high-dose royal jelly DPP-IV inhibitory peptide composition. The metformin hydrochloride control group was given an intragastric dose of 200 mg / kg; the low, medium, and high doses of royal jelly-derived DPP-IV inhibitory peptide composition were 100 mg / kg, 200 mg / kg, and 400 mg / kg, respectively; the normal group (N) and model group (M) were given an intragastric dose of distilled water of equal volume. Each experimental group was given an intragastric dose of 0.2 mL once a day, and the intragastric dose remained unchanged during the intragastric dose. After 4 weeks of continuous intragastric administration, the tail vein blood of the mice was taken after fasting for 5 hours, and the fasting blood glucose was measured using a microglucometer.

[0046] As shown in Table 2, the fasting blood glucose of db / db hyperglycemic mice was significantly higher than that of mice in group N (P < 0.01). Compared with mice in group M, the fasting blood glucose of mice in group Y and groups MR and HR that were gavaged with the royal jelly DPP-IV inhibitory peptide composition was significantly reduced (P < 0.01), which indicates that the royal jelly DPP-IV inhibitory peptide composition can control the blood glucose level of db / db mice.

[0047]

[0048] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A royal jelly active peptide composition having DPP-IV inhibitory effect, characterized in that: The royal jelly active peptide composition includes at least one of five small molecule peptides; The amino acid sequences of the five small molecule peptides are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, respectively.

2. The method for preparing the royal jelly active peptide composition having DPP-IV inhibitory effect according to claim 1, characterized in that: The steps include: (1) Raw material evaluation: Use bioinformatics to analyze the potential activity of royal jelly protein and evaluate the feasibility of using protein raw materials for the preparation of DPP-IV inhibitory peptides; (2) Enzymatic hydrolysis: mixing royal jelly water-soluble protein dry powder with water, adjusting the pH value, adding protease, stirring and mixing for enzymatic hydrolysis, inactivating the royal jelly protein, centrifuging and taking the supernatant, and freeze-drying to obtain a crude royal jelly protease hydrolysate with DPP-IV inhibitory activity; (3) Purification: The crude protein hydrolysate is purified by ultrafiltration, anion exchange chromatography, and reverse-phase high performance liquid chromatography in sequence to obtain the royal jelly active peptide composition with DPP-IV inhibitory effect.

3. The preparation method of the royal jelly active peptide composition having DPP-IV inhibitory effect according to claim 2, characterized in that: The royal jelly proteins described in step (1) include MRJP1~MRJP9; their protein numbers in the NCBI database are ACS66836.1, ACS66837.1, ADC55524.1, ADB82660.1, ADB96941.1, AAQ82184.1, DAA01512.1, AAR83734.1, and AAY21180.1, respectively.

4. The method for preparing the royal jelly active peptide composition having DPP-IV inhibitory effect according to claim 2, characterized in that: The bioinformatics method in step (1) includes: using the "PROTEINS", "ANALYSIS", "CALCULATIONS", and "PROFILES OF POTENTIAL BIOLOGICAL ACTIVITY" functions in the BIOPEP database to predict the biological activity of MRJP1~MRJP9.

5. The method for preparing the royal jelly active peptide composition having DPP-IV inhibitory effect according to claim 2, characterized in that: In step (2), the mixing mass ratio of the royal jelly water-soluble protein dry powder to water is 1:7-10; The component for adjusting pH value is edible alkali; The pH value is adjusted to 7-9; The protease is alkaline protease; The added amount of the protease is 6000-9000U / g protein; The stirring speed is 100-300 rpm; The temperature of the enzymatic hydrolysis is 40-60°C; The enzymolysis time is 2 to 4 hours; The centrifugal speed is 3000-5000 rpm; The centrifugal temperature is 5-20°C; The centrifugation time is 10 to 15 minutes.

6. The method for preparing the royal jelly active peptide composition having DPP-IV inhibitory effect according to claim 2, characterized in that: The separation and purification operation in step (3) is to separate the crude royal jelly protease hydrolysate with DPP-IV inhibitory activity obtained in step (2) through 3kD and 1kD ultrafiltration tubes, collect different components for freeze-drying, and then further purify the component with the highest DPP-IV inhibitory activity using HiTrap DEAE-FF anion exchange; The conditions of the HiTrap DEAE-FF anion exchange purification are as follows: the mobile phase A of the anion exchange chromatography is 0.01 mol / L pH 7.0 phosphate buffer, the mobile phase B is 0.01 mol / L pH 7.0 phosphate buffer containing 1 mol / L NaCl, the elution conditions are 100% A for 35 min, the B solution is adjusted from 0 to 80% at 35-45 min, 80% B is maintained for 10 min, and finally B is adjusted to 100% for 55-60 min and eluted for 10 min, the elution flow rate is 1 mL / min, and the absorbance is detected at 214 nm to collect the eluted components. The fraction with the highest DPP-IV inhibitory activity was further purified using C18 semi-preparative liquid chromatography; The conditions for the C18 semi-preparative liquid chromatography purification are as follows: mobile phase A is ultrapure water containing 0.1% TFA, mobile phase B is acetonitrile containing 0.1% TFA, the flow rate is 5 mL / min, and the gradient elution program is: ①0~5min: B 10-20%, ②5~10min: 20%B, ③10~20min: B 20~30%; the absorbance is detected at 214nm and the eluted components are collected, and the components with the highest DPP-IV inhibitory activity are freeze-dried and concentrated to obtain the royal jelly active peptide composition with DPP-IV inhibitory effect.

7. Use of the royal jelly active peptide composition with DPP-IV inhibitory effect as claimed in claim 1 or the royal jelly active peptide composition with DPP-IV inhibitory effect prepared as claimed in claims 2 to 6 in the preparation of drugs for inhibiting DPP-IV and / or auxiliary blood sugar-lowering health foods.

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