Composite cordyceps protein peptide, preparation method and application thereof, and method for preparing iron chelating peptide

Abstract

The invention relates to the technical field of biology, and discloses a composite cordyceps protein peptide, a preparation method and application thereof, and a method for preparing an iron chelating peptide. The composite cordyceps sinensis protein peptide comprises one or more of cordyceps sinensis protein peptides with the amino acid sequences as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO.21. The invention further discloses a preparation method of the composite cordyceps sinensis protein peptide. The peptide can be efficiently chelated with iron ions, the composite cordyceps protein peptide is obtained from cordyceps mycelia through a microbial fermentation method and is chelated with iron salt, and the obtained iron chelating peptide has the capacity of promoting the absorption of the intestinal tract to the iron ions, so that the safe and efficient treatment effect is achieved, and the obvious prospect is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly relates to a compound cordyceps protein peptide, a preparation method and application thereof, and a method for preparing iron-chelated peptide. Background Art

[0002] Cordyceps is a precious medicinal fungus, which is widely used in traditional Chinese medicine and modern functional foods. Cordyceps contains rich bioactive substances, including polysaccharides, nucleosides, sterols, proteins, etc., and has various physiological activities such as antioxidant, anti-inflammatory, and immunomodulatory effects. In recent years, with the increasing demand for healthy foods and functional ingredients, the research and application of cordyceps protein and its derivatives have gradually become a hot topic in the fields of food science and biotechnology.

[0003] Iron is a key trace element for maintaining human physiological functions, but its traditional supplements (such as ferrous sulfate and ferrous fumarate) have problems such as low bioavailability and strong gastrointestinal irritation. In recent years, novel iron supplements based on peptide-iron chelates have become a research hotspot. Peptide-iron chelates can improve the absorption efficiency of iron and reduce side effects through the specific binding of amino acids or short peptides to iron ions. For example, chelated peptides from sources such as whey protein and soy protein have been partially applied. However, the existing technologies mainly rely on animal or plant proteins, and have limitations such as high raw material costs, potential allergen risks, or complex extraction processes.

[0004] Traditional methods for preparing iron-chelated peptides mainly rely on enzymatic hydrolysis technology, that is, specific proteases are used to hydrolyze proteins into small peptides, and then chelated with iron ions. However, enzymatic hydrolysis technology has some limitations, such as the selectivity of enzymes, the control of reaction conditions, and the purity of products. At present, there is no technical guidance to use cordyceps mycelium protein as a raw material to obtain a compound protein peptide that can chelate with iron ions with high efficiency through microbial fermentation, so that the compound protein peptide can chelate with iron ions with high efficiency and improve the iron absorption in the intestine. Summary of the Invention

[0005] The purpose of the present invention is to overcome the problem that there is no technical guidance to use cordyceps mycelium protein as a raw material to obtain a compound protein peptide that can chelate with iron ions with high efficiency through microbial fermentation in the prior art, and provide a compound cordyceps protein peptide, a preparation method and application thereof, and a method for preparing iron-chelated peptide. The compound cordyceps protein peptide has the characteristics of small molecular weight, high activity, and high safety, and can chelate with metallic iron with high efficiency to generate iron-chelated peptide. The method is safe and efficient, and the generated iron-chelated peptide can improve the iron absorption ability of the intestine and does not stimulate the intestine, and has significant industrial prospects.

[0006] To achieve the above object, a first aspect of the present invention provides a compound cordyceps protein peptide, which contains protein peptides shown by amino acid sequences of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO.21.

[0007] A second aspect of the present invention provides a preparation method of the compound cordyceps protein peptide, the method comprising: separating after mixing cordyceps protein and lactobacillus for fermentation.

[0008] The compound cordyceps protein peptide contains protein peptides shown by amino acid sequences of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO.21.

[0009] Preferably, the dosage of lactobacillus is 0.8×10 6 -3.6×10 6 CFU relative to 1 g of the cordyceps sinensis protein from Xinjiang.

[0010] Preferably, the cordyceps protein is a protein extracted from cordyceps sinensis from Xinjiang.

[0011] Preferably, the preparation process of the cordyceps protein comprises: drying and grinding the cordyceps sinensis mycelia from Xinjiang into a powder with a particle size of 20 - 30 mm, mixing the powder with a buffer solution, and then collecting supernatant I after homogenization and solid-liquid separation; filtering the supernatant I with a filter membrane of 0.4 - 0.5 μm, performing salting out and gradient elution, and then dialyzing with a dialysis membrane of less than 400 Da.

[0012] Preferably, the Lactobacillus is selected from one or more of Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus reuteri, and Lactobacillus delbrueckii.

[0013] Preferably, the Lactobacillus is Lactobacillus reuteri with the number CICC No. 6119.

[0014] Preferably, the conditions for the mixed fermentation include: the temperature is 20 - 50 °C, the rotation speed is 100 - 200 rpm, and the time is 10 - 55 h, preferably 24 - 48 h.

[0015] Preferably, the carbon-nitrogen ratio of the fermentation medium used for the mixed fermentation is 1:1 - 2.5, preferably 1:1 - 2, and the content of cordyceps protein in the fermentation medium is 5 - 12.5 mg / mL, preferably 5 - 10 mg / mL.

[0016] Preferably, the fermentation medium contains the cordyceps protein, glucose, dipotassium hydrogen phosphate

[0017] and distilled water.

[0018] Preferably, the separation process includes: subjecting the fermented broth obtained by fermentation to solid-liquid separation to obtain supernatant II, subjecting the supernatant II to membrane filtration, dialysis, and drying to obtain a protein hydrolysate, and subjecting the re-dissolved protein hydrolysate to ultrafiltration separation to obtain a fraction with a molecular weight less than 10 kDa as the composite cordyceps protein peptide.

[0019] Preferably, after the protein hydrolysate is re-dissolved, ultrafiltration separation is carried out to obtain a fraction with a molecular weight less than 3 kDa.

[0020] Preferably, the method further includes: subjecting the fermented broth obtained by fermentation to solid-liquid separation to obtain supernatant, subjecting the supernatant to membrane filtration, dialysis, and drying to obtain a protein hydrolysate, and subjecting the re-dissolved protein hydrolysate to ultrafiltration separation to obtain a fraction with a molecular weight less than 10 kDa.

[0021] More preferably, after the protein hydrolysate is re-dissolved, ultrafiltration separation is carried out to obtain a fraction with a molecular weight less than 3 kDa.

[0022] The present invention in a third aspect provides an application of the composite cordyceps protein peptide as described above and / or the composite cordyceps protein peptide prepared by the method as described above in the preparation of iron-chelating peptides.

[0023] The present invention in a fourth aspect provides a method for preparing an iron-chelating peptide, the method including: mixing the composite cordyceps protein peptide as described above and / or the composite cordyceps protein peptide prepared by the method as described above with an iron salt and a precipitant.

[0024] Preferably, the conditions for the mixing include: the time is 20 - 60 min, and the temperature is 37 - 50 °C.

[0025] Preferably, the iron salt is ferrous sulfate.

[0026] Preferably, the mass ratio of the compound cordyceps protein peptide to the iron salt is 10 - 20:1.

[0027] Preferably, the precipitant is selected from at least one of ethanol, acetone, polyethylene glycol, and ammonium sulfate.

[0028] The present invention provides, in a fifth aspect, the use of the iron-chelated peptide prepared by the method as described above in the preparation of a medicament for treating iron-deficiency anemia.

[0029] Through the above technical solution, the present invention hydrolyzes cordyceps mycelium protein by a microbial fermentation method to obtain a compound protein peptide that chelates with iron ions with high efficiency. This compound cordyceps protein peptide has a small molecular weight, high activity, high safety, and can chelate with metallic iron with high efficiency to generate an iron-chelated peptide. The generated iron-chelated peptide can improve the iron absorption ability of the intestine and does not stimulate the intestine, having significant industrial prospects. Description of the Drawings

[0030] Figure 1 is the SDS-PAGE electrophoresis pattern of Cordyceps sinensis protein in Example 1;

[0031] Figure 2 is the comparison diagram of the degree of protein hydrolysis of each strain fermentation in Example 2;

[0032] Figure 3 is the comparison diagram of the degree of hydrolysis of Cordyceps sinensis protein peptide with different fermentation parameters in Example 3;

[0033] Figure 4 is the standard curve diagram of serine concentration and absorbance in Example 4;

[0034] Figure 5 is the comparison diagram of the iron chelation efficiency of different components of Cordyceps sinensis protein peptide (OGP-1, OGP-2, OGP-3, and OGP-4) in Example 5;

[0035] Figure 6 is the chelation efficiency diagram of iron-chelated peptide under different chelation conditions (time, temperature, mass ratio of OGP-1 solution to FeSO4 solution) in Example 7;

[0036] Figure 7 is the ultraviolet-visible absorption spectrum diagram of OGP-1, OGP-1-Fe, and FeSO4 in Example 8. Detailed Embodiments

[0037] The endpoints and any values in the ranges disclosed herein are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0038] In one aspect, the present invention provides a compound cordyceps protein peptide, which contains protein peptides shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO.21 in terms of amino acid sequence. The inventors have found through research that the molecular weight of the compound cordyceps protein peptide containing the above amino acid sequence can chelate with metallic iron with high efficiency to generate iron-chelated peptide, and the generated iron-chelated peptide can significantly improve the iron absorption capacity of the intestine without stimulating the intestine.

[0039] In another aspect, the present invention provides a preparation method of a compound cordyceps protein peptide, and the method includes: separating after mixing and fermenting cordyceps protein with lactobacillus;

[0040] The compound cordyceps protein peptide contains protein peptides shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO.21 in terms of amino acid sequence.

[0041] The separation method used in the present invention is membrane filtration method.

[0042] According to the present invention, preferably, the dosage of Lactobacillus relative to 1 g of the Cordyceps sinensis protein from Xinjiang is 0.8×10 6 -3.6×10 6 CFU. Further preferably, the dosage of the Lactobacillus relative to 1 g of the Cordyceps sinensis protein from Xinjiang is 1×10 6 -3×10 6 CFU.

[0043] According to the present invention, preferably, the Cordyceps sinensis protein is the protein extracted from Cordyceps sinensis in Xinjiang. Further preferably, the preparation process of the Cordyceps sinensis protein includes: drying and grinding the Cordyceps sinensis mycelium into a powder with a particle size of 20 - 30 mm, mixing the powder with a buffer solution, followed by homogenization and solid-liquid separation to collect supernatant I; filtering the supernatant I through a 0.4 - 0.5 μm filter membrane, followed by salting out and gradient elution, and then dialysis using a dialysis membrane with a molecular weight cut-off less than 400 Da. The drying can use freeze-drying method, vacuum drying method, heat drying method, etc., and the drying method used in the present invention is freeze-drying. The buffer solution can be any one of phosphate buffer solution, physiological saline, and Tris buffer solution. The salting out and gradient elution can use (NH4)2SO4 solution.

[0044] Exemplarily, the preparation method of the composite Cordyceps sinensis protein peptide includes: freeze-drying the Cordyceps sinensis mycelium obtained by liquid fermentation in the laboratory and grinding it into a powder with a particle size of 20 - 30 mm. The powder is mixed with phosphate buffered saline (PBS, pH 7.4) at a ratio of 1:20 (w / v), homogenized on ice, centrifuged (6000 g, 15 min, 4°C) and the supernatant is collected. The supernatant is filtered through a 0.45 μm filter membrane, and fractionated precipitation is carried out by sequentially standing in 30 wt%, 70 wt%, and 100 wt% (NH4)2SO4 solutions at 4°C for 1 day. The precipitate is dissolved in gradient with 80 wt%, 40 wt%, 20 wt%, 10 wt%, and 5 wt% (NH4)2SO4 solutions, and then dialyzed against ultrapure water at 4°C for 2 days using a 400 Da dialysis membrane, changing the water 10 - 12 times, and freeze-dried to obtain the crude extract of Cordyceps sinensis protein, which is stored at -80°C to -70°C.

[0045] According to the present invention, preferably, the Lactobacillus is selected from one or more of Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus reuteri, and Lactobacillus delbrueckii. Further preferably, the Lactobacillus is Lactobacillus reuteri, with the number CICC No. 6119.

[0046] According to the present invention, preferably, the conditions for the mixed fermentation include: the temperature is 20 - 50 °C, specifically it can be 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, or any value between the above two values; the rotation speed is 100 - 200 rpm, specifically it can be 100 rpm, 120 rpm, 140 rpm, 160 rpm, 180 rpm, 200 rpm, or any value between the above two values; the time is 10 - 55 h, specifically it can be 10 h, 15 h, 20 h, 25 h, 30 h, 35 h, 40 h, 45 h, 50 h, 55 h, or any value between the above two values; more preferably it is 24 - 48 h; the carbon-nitrogen ratio of the fermentation medium used for the mixed fermentation is 1:1 - 2.5, specifically it can be 1:1, 1:1.5, 1:2, 1:2.5, or any value between the above two values; more preferably it is 1:1 - 2.

[0047] According to the present invention, preferably, the content of cordyceps sinensis protein in the fermentation medium is 5 - 12.5 g / L, specifically it can be 5 mg / mL, 8 mg / mL, 10 mg / mL, 12 mg / mL, 12.5 mg / mL, or any value between the above two values; more preferably it is 5 - 10 mg / mL.

[0048] According to the present invention, preferably, the fermentation medium contains the cordyceps sinensis protein, glucose, dipotassium hydrogen phosphate and distilled water. The present invention controls the carbon-nitrogen ratio by adjusting the quality of the cordyceps sinensis protein and glucose. Among them, the cordyceps sinensis is added to the fermentation medium in the form of freeze-dried powder; the content of dipotassium hydrogen phosphate in the fermentation medium is 1 - 3 g / L.

[0049] According to the present invention, preferably, the separation process includes: subjecting the fermented fermentation broth to solid-liquid separation to obtain supernatant II, subjecting the supernatant II to membrane filtration, dialysis, and drying to obtain a protein hydrolysate, and after redissolving the protein hydrolysate, performing ultrafiltration separation using a molecular weight cut-off (MWCO) membrane to obtain a fraction with a molecular weight less than 10 kDa as the composite cordyceps sinensis protein peptide. More preferably, after redissolving the protein hydrolysate, ultrafiltration separation is performed to obtain a fraction with a molecular weight less than 3 kDa. The

[0050] The inventors' research found that by using the above preferred implementation method, the prepared composite cordyceps sinensis protein peptide has high stability, is safer, and has a higher efficiency of chelating with metallic iron to form iron-chelated peptides.

[0051] The present invention in a third aspect provides an application of the compound cordyceps protein peptide as described above and / or the compound cordyceps protein peptide prepared by the method as described above in the preparation of iron-chelating peptides. The inventors have found through research that the iron-chelating peptides prepared from the compound cordyceps protein peptide as described above and / or the compound cordyceps protein peptide prepared by the method as described above can improve the iron absorption ability of the intestine and do not stimulate the intestine.

[0052] The present invention in a fourth aspect provides a method for preparing iron-chelating peptides, the method comprising: mixing the compound cordyceps protein peptide as described above and / or the compound cordyceps protein peptide prepared by the method as described above with an iron salt and a precipitant. There are no particular limitations on the method of mixing, as long as the compound cordyceps protein peptide can be evenly mixed with the iron salt and the precipitant, and specifically, methods such as stirring and shaking can be used.

[0053] According to the present invention, preferably, the conditions for the mixing include: the time is 20 - 60 min, more preferably 40 - 80 min; the temperature is 37 - 50 °C, more preferably 45 - 55 °C.

[0054] According to the present invention, preferably, the iron salt is ferrous sulfate.

[0055] According to the present invention, preferably, the mass ratio of the compound cordyceps protein peptide to the iron salt is 10 - 20:1, more preferably 10 - 17:1.

[0056] According to the present invention, preferably, the precipitant is selected from at least one of ethanol, acetone, polyethylene glycol, and ammonium sulfate.

[0057] The inventors have found through research that by adopting the above preferred embodiments, the chelation efficiency of metallic iron with the compound cordyceps protein peptide can be further improved, and the generated iron-chelating peptides can further improve the iron absorption ability of the intestine.

[0058] The present invention in a fifth aspect provides an application of the iron-chelating peptides prepared by the method as described above in the preparation of drugs for treating iron deficiency anemia.

[0059] The present invention will be described in detail below through examples.

[0060] In the following examples, the mycelium of Cordyceps sinensis var. xingjiangensis was obtained by liquid fermentation from the laboratory of Academician Huang He of the School of Food and Pharmaceutical Engineering, Nanjing Normal University. This strain has been disclosed in CN114456947A; the nutrient broth (MRS) medium was purchased from Beijing Solarbio Science & Technology Co., Ltd.; Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus helveticus, and Lactobacillus delbrueckii were purchased from the China Center for Industrial Culture Collection; the rest of the reagents were obtained through conventional channels.

[0061] The fermentation medium used in the following examples was a self-made fermentation medium in the laboratory. The carbon source was glucose, and the nitrogen source was Cordyceps protein (in the form of freeze-dried powder). The carbon-nitrogen ratio (C / N) was controlled by adjusting the masses of glucose and Cordyceps protein. The content of K2HPO4 in the fermentation medium was 2 g / L, and the rest was distilled water.

[0062] The determination of the iron chelation rate in the following examples was carried out by the ferrozine method, and the steps were as follows:

[0063] The protein peptide sample was diluted with distilled water to a final concentration of 5 mg / mL. 500 μL of the diluted protein peptide sample solution was mixed with 50 μL of FeSO4 (2 mM), and then incubated at 37 °C for 20 min. After the incubation, 250 μL of a ferrozine solution with a concentration of 2 mM was added to terminate the reaction. Subsequently, the absorbance of the iron-Fe 2+ complex was measured at 562 nm.

[0064] The iron chelation rate was calculated according to the formula [(Ab - As) / Ab] × 100%, where Ab was the blank absorbance, that is, the absorbance of the solution containing only FeSO4 and ferrozine without adding the sample, which reflected the absorbance generated by the reaction of ferrozine with ferrous ions without sample interference; As was the sample absorbance, representing the absorbance of the reaction system after adding the sample. By multiplying the ratio of the difference between the two and the blank absorbance by 100%, the chelation efficiency of the sample for ferrous ions could be obtained.

[0065] Example 1

[0066] Extraction and analysis of Cordyceps sinensis var. xingjiangensis protein

[0067] S1. Protein extraction: The mycelia of Cordyceps sinensis var. heteropoda obtained by liquid fermentation in the laboratory were freeze-dried, ground into powder and passed through a 120-mesh sieve. The powder was mixed with phosphate buffered saline (PBS, pH 7.4) at a ratio of 1:20 (w / v), homogenized on ice, centrifuged (6000g, 15 min, 4 °C) and the supernatant was collected. The supernatant was filtered through a 0.45 μm filter membrane, and fractionated precipitation was carried out with 30 wt%, 70 wt% and 100 wt% (NH4)2SO4 solutions at 4 °C for 24 h. The precipitate was dissolved in a gradient of 80 wt%, 40 wt%, 20 wt%, 10 wt%, 5 wt% (NH4)2SO4 solutions, dialyzed against ultrapure water at 4 °C for 2 days using a 400 Da dialysis membrane, with water changed 12 times, and freeze-dried to obtain the crude extract of Cordyceps sinensis var. heteropoda protein, stored at -80 °C, and then analyzed.

[0068] S2. SDS-PAGE electrophoresis analysis of Cordyceps sinensis var. heteropoda protein: The crude extract of Cordyceps sinensis var. heteropoda protein prepared in step S1 was formulated into a Cordyceps sinensis var. heteropoda protein solution with a final concentration of 2 mg / mL, and 5× protein loading buffer was added, followed by boiling water bath for 10 min. A 15 wt% separating gel and a 5 wt% stacking gel were prepared, and the glass plates were checked for leaks before gel preparation. The vertical plate electrophoresis apparatus was assembled, Tris-Glycine-SDS buffer was added, and 5 μL of protein Maker and 10 μL of 2 mg / mL Cordyceps sinensis var. heteropoda protein solution were loaded respectively. The constant voltage of the stacking gel was set at 80 V for 30 min, and the constant voltage of the separating gel was set at 120 V for 90 min for electrophoresis. After completion, the gel was taken out, ultrapure water was added, heated to boiling and then discarded; Coomassie Brilliant Blue rapid staining solution was added, heated to boiling and maintained for 1 minute, shaken for 10 minutes and then discarded; finally, ultrapure water was added, heated to boiling and maintained for 1 minute, shaken for 10 minutes and then discarded, and analyzed by photographing with a gel imaging system as Figure 1 shown. Marker lane bands: Presented clear and orderly spaced bands, indicating that the electrophoresis process was normal, and the molecular weight standards were well separated, which could be used as an accurate molecular weight reference. Protein lane bands: Multiple protein bands could be seen in the sample, indicating that Cordyceps sinensis var. heteropoda protein was composed of multiple proteins with different molecular weights. By comparing with the Marker lane, the molecular weight ranges corresponding to the protein bands in the sample could be roughly estimated. For example, there were obvious bands at positions close to 35 kDa and slightly higher than 25 kDa, indicating that Cordyceps sinensis var. heteropoda protein contained protein components in these molecular weight ranges. This helped researchers understand the compositional characteristics of Cordyceps sinensis var. heteropoda protein and provided basic data for subsequent in-depth studies on its structure and function.

[0069] Example 2

[0070] Screening of Fermentation Strains for Cordyceps sinensis var. heteropoda Protein Peptide

[0071] The following Lactobacillus plantarum, numbered CICC NO.25024; Lactobacillus reuteri, numbered CICC NO.6119; Lactobacillus helveticus, numbered CICC NO.6024; Lactobacillus delbrueckii, numbered CICC NO.6256; all of the above strains were purchased from the China Center of Industrial Culture Collection.

[0072] The screening steps are as follows:

[0073] Incubate the Lactobacillus plantarum strain in a nutrient broth (MRS) medium at 37°C with shaking.

[0074] Incubate the Lactobacillus helveticus strain in a nutrient broth (MRS) medium at 30°C with shaking.

[0075] Incubate the Lactobacillus reuteri strain in a nutrient broth (MRS) medium at 37°C with shaking.

[0076] Incubate the Lactobacillus delbrueckii strain in a nutrient broth (MRS) medium at 42°C with shaking.

[0077] Subsequently, inoculate with 1 mL / 100 mL inoculum (10 6 viable bacteria / mL medium) into the fermentation medium as described above (containing 1 g of Cordyceps sinensis protein prepared in Example 1, pH 7.0), ferment at 37°C with shaking (150 rpm) for 48 h to obtain a fermentation broth, and centrifuge the fermentation broth at 10000×g for 25 min at 4°C to obtain a supernatant. Filter the supernatant through a 0.45 μm membrane to remove bacterial cells in the fermentation broth, and dialyze and freeze-dry the filtrate to obtain a protein hydrolysate.

[0078] By comparing the degree of hydrolysis of proteins fermented by each strain, the results are as Figure 2 shown, and it can be Figure 2 seen that the strain that best hydrolyzes Cordyceps sinensis protein is Lactobacillus reuteri.

[0079] Example 3

[0080] Preparation of Cordyceps sinensis protein peptide

[0081] The best strain, Lactobacillus reuteri, screened in Example 2 was incubated with shaking in a nutrient broth (MRS) medium at 37°C. Subsequently, it was inoculated into 100 mL of fermentation medium (containing 1 g of Cordyceps sinensis protein from Xinjiang prepared in Example 1, pH 7.0) with an inoculum of 1 mL / 100 mL (10 6 viable bacteria / mL of medium), and then incubated with shaking (150 rpm) at 37°C. After 48 h of fermentation, the fermentation broth was centrifuged at 10,000×g for 25 min at 4°C. The supernatant was filtered through a 0.45 μm membrane to remove bacterial cells in the fermentation broth, and the filtrate was dialyzed and freeze-dried to obtain a protein hydrolysate.

[0082] The fermentation parameters were optimized by single-factor analysis, including fermentation time (12 h, 24 h, 36 h, 48 h), C / N ratio (weight ratio of carbon source to nitrogen source) (1, 1.5, 2, 2.5), concentration of Cordyceps sinensis protein from Xinjiang prepared in Example 1 (5 mg / mL, 7.5 mg / mL, 10 mg / mL, 12.5 mg / mL), and strain inoculum amount (0.8×10 6 CFU, 1×10 6 CFU, 3.6×10 6 CFU).

[0083] By comparing the degree of hydrolysis of proteins under different fermentation parameters, the optimal fermentation conditions were obtained. The results are as Figure 3 shown. It can be Figure 3 seen that the preferred fermentation conditions are a fermentation time of 24 - 48 h, a substrate concentration of 5 - 10 mg / mL, a C / N ratio of 1 - 2, and an inoculum amount of 1×10 6 -3×10 6 CFU / mL.

[0084] Example 4

[0085] Determination of the degree of hydrolysis of Cordyceps sinensis protein peptides

[0086] The degree of hydrolysis was determined by the o-phthalaldehyde method. The newly prepared Cordyceps sinensis protein sample in Example 1 was diluted with distilled water to a final concentration of 1 mg / mL. Then, 400 μL aliquots of the sample were dispensed into 3 mL aliquots of OPA solution and vortex-mixed. After 2 min, the absorbance was measured at 340 nm. Distilled water and serine (0.9516 mM) were used as the control and standard, respectively. o-Phthalaldehyde (OPA) can react with the free amino groups generated by protein hydrolysis under alkaline conditions to form products with fluorescence or absorption peaks at specific wavelengths. By measuring the absorbance of the product at a specific wavelength (here 340 nm), the degree of protein hydrolysis can be indirectly reflected. Because the higher the degree of protein hydrolysis, the more free amino groups are produced, the more products are generated by reacting with OPA, and the higher the absorbance. Distilled water was used as a blank control to correct the instrument absorbance and exclude errors caused by factors such as the solution itself and the instrument. Serine was used as a standard because it is an amino acid with a free amino group. By establishing a standard curve of serine concentration vs. absorbance (the standard curve graph is as shown in Figure 4 ), the content of free amino groups in the sample can be calculated based on the absorbance of the sample, and then the degree of protein hydrolysis can be converted.

[0087] Example 5

[0088] Ultrafiltration fractionation of Xinjiang Cordyceps sinensis protein peptides

[0089] The Xinjiang Cordyceps sinensis peptides were redissolved with ultrapure water and ultrafiltered using 3 kDa, 5 kDa, and 10 kDa molecular weight cut-off (MWCO) membranes (6000 g, 15 min, 4 °C). Four fractions, namely OGP-1 (<3 kDa) (>10 kDa), OGP-2 (3 - 5 kDa), OGP-3 (5 - 10 kDa), and OGP-4 (>10 kDa), were collected. The iron chelating efficiency of each fraction was determined. The determination of the iron chelating rate was carried out using the ferrozine method as described above. The samples of Xinjiang Cordyceps sinensis protein peptides OGP-1, OGP-2, OGP-3, and OGP-4 were diluted with distilled water to a final concentration of 5 mg / mL. 500 μL of each diluted solution was mixed with 50 μL of FeSO4 (2 mM), and then incubated at 37 °C for 20 min. Ferrozine (250 μL, 2 mM) was added to terminate the reaction. The absorbance of the iron-Fe 2+ complex was measured at 562 nm; the formula for calculating the iron chelating rate is [(Ab - As) / Ab] * 100%, where Ab is the blank absorbance and As is the sample absorbance.

[0090] The measurement results are as shown in Figure 5 shown, and from Figure 5It can be seen that the component with the highest iron chelating efficiency is the OGP-1 (<3 kDa) group. The component with the highest iron chelating efficiency (OGP-1) was lyophilized and stored at -20 °C for use in the following examples.

[0091] Example 6

[0092] Amino acid sequence identification and activity screening of Cordyceps sinensis (Berk.) Sacc. var. xinjiangensis iron chelating peptides

[0093] The Easy-nLC 1200 system was connected to a Q Exactive TM hybrid quadrupole-orbitrap TM mass spectrometer (Thermo Fisher Scientific, USA). The OGP-1 sample prepared in Example 5 was reduced with 10 mM dithiothreitol (DTT) at 56 °C for 1 hour, then alkylated with 50 mM iodoacetamide (IAA) at room temperature in the dark for 40 minutes, desalted, and the solvent was evaporated to dryness in a vacuum centrifuge concentrator at 45 °C. Before LC-MS / MS analysis, the peptide was resuspended in 20 μL of 0.1 wt% formic acid. The reversed-phase column was an Acclaim PepMap RPLC C18 nanochromatographic column (150 μm × 15 cm) (1.9 μm, Dr. Maisch GmbH, Germany), and the sample loading volume was 5 μL. The mobile phase consisted of 0.1 wt% formic acid aqueous solution (A) and 20 wt%, 0.1 wt% formic acid aqueous solution - 80 wt% acetonitrile (B), and the flow rate was 600 nL / min. The analysis time for each component was 66 minutes. The gradient elution was from 4 wt% to 8 wt% B for 2 minutes; from 8 wt% to 28 wt% B for 43 minutes; from 28 wt% to 40 wt% B for 10 minutes; from 40 wt% to 95 wt% B for 1 minute; from 95 wt% to 95 wt% B for 10 minutes. The capillary temperature was set at 270 °C, and the spray voltage was set at 2.2 kV.

[0094] First-level mass spectrometry parameters: Resolution is 70000, AGC target is 3e6, maximum IT is 100 ms, Scanrange is 300 - 1800 m / z; Second-level mass spectrometry parameters: Resolution is 17500, AGC target is 1e5, Maximum IT is 50 ms, TopN is 20; NCE / steppedNCE is 28. De novo analysis was performed on the original MS file using PEAKS Studio 8.5. The peptide mass tolerance of the first-level mass spectrometry is 20 ppm, and the fragment mass tolerance of the second-level mass spectrometry is 0.02 Da.

[0095] Computer prediction analysis of potential biological activities was performed on the identified high-confidence peptides using Peptide Ranker. Peptides with a score greater than 0.5 were considered to have biological activities. The analysis results showed that the scores of 21 peptides were above 0.5 as shown in Table 1, indicating that OGP-1 has potential high biological activities;

[0096] Table 1

[0097]

[0098]

[0099] Example 7

[0100] Chelation of Cordyceps sinensis (Berk.) Sacc. protein peptide from Xinjiang with ferrous ions

[0101] The OGP-1 solution screened in Example 5 was mixed with FeSO4 at different ratios (OGP:FeSO4 is 10:1, 15:1, 20:1, 25:1, w / w). The solution was oscillated and reacted at different temperatures (37 °C, 45 °C, 50 °C, 55 °C) for different times (20 min, 40 min, 60 min, 80 min), and 5 volumes of ethanol were added to the mixture to obtain a precipitate of the OGP-Fe chelate. Then, the solution was centrifuged at 10000×g at 4 °C for 10 min, and the precipitate was freeze-dried to obtain the OGP-Fe powder.

[0102] The iron chelation rate was measured according to the ferrozine method. The determination of the iron chelation rate was carried out using the ferrozine method as described above. The OGP-Fe samples obtained from the chelation reaction at different temperatures (37 °C, 45 °C, 50 °C, 55 °C) and different times (20 min, 40 min, 60 min, 80 min) were diluted with distilled water to a final concentration of 5 mg / mL. 500 μL of each diluted solution was mixed with 50 μL of FeSO4 (2 mM), and then incubated at 37 °C for 20 min. The reaction was terminated by adding ferrozine (250 μL, 2 mM). The absorbance of the iron-Fe2+ complex was measured at 562 nm; the calculation formula for the iron chelation rate was [(Ab - As) / Ab]*100%, where Ab was the blank absorbance and As was the sample absorbance. The optimal chelation conditions were obtained by comparing the chelation rates, and the results were as Figure 6 shown, and it can be seen from Figure 6 that the preferred chelation conditions for the OGP-1 solution and FeSO4 include: the time is 40 - 80 min, the temperature is 45 - 55 °C, and the mass ratio of the OGP-1 solution to the FeSO4 solution is 10 - 17:1.

[0103] Example 8

[0104] Morphological Analysis and Structural Characterization of Cordyceps sinensis (Berk.) Sacc. var. thibetica (Hook.f.) Secc. Iron Chelating Peptide

[0105] The structure was characterized by ultraviolet spectrophotometer (UV-Vis) technology. The powders of Cordyceps sinensis (Berk.) Sacc. var. thibetica (Hook.f.) Secc. protein peptide OGP-1 prepared in Example 5 and iron chelating peptide OGP-1-Fe prepared in Example 6 were dispersed in distilled water at a concentration of 1 mg / mL. The ultraviolet-visible absorption spectrum was measured by a UV absorption spectrophotometer (UV 2550, Shimadzu Ltd., Tokyo, Japan). The wavelength range was 200 - 400 nm, and the absorbance of deionized water was used as the blank control. The measurement of the ultraviolet-visible absorption spectrum was as Figure 7 shown.

[0106] It can be seen from Figure 7 that there were differences in absorption peaks: OGP-1 had an obvious absorption peak at about 223.85 nm. After chelation with iron, the OGP-Fe chelate had an obvious absorption peak at 208.71 nm, and the absorption peak showed a blue shift. This indicates that after the formation of the chelate, the molecular structure changed, resulting in the change of the absorption spectrum. This change proves the successful preparation of Cordyceps sinensis (Berk.) Sacc. var. thibetica (Hook.f.) Secc. chelating peptide OGP-1-Fe, because if chelation did not occur, the spectrum of OGP-1-Fe should be similar to the simple superposition of OGP and FeSO4, rather than showing new absorption spectral characteristics. By comparing the absorption spectral differences among OGP-1, OGP-1-Fe, and FeSO4, it strongly supports the conclusion that Cordyceps sinensis (Berk.) Sacc. var. thibetica (Hook.f.) Secc. protein peptide was successfully chelated with ferrous ions.

[0107] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. A compound cordyceps protein peptide, characterized in that, The composite cordyceps protein peptide contains protein peptides with amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO.

21.

2. A preparation method of a compound cordyceps protein peptide, characterized in that, The method includes: mixing cordyceps protein with Lactobacillus for mixed fermentation and then separating; The composite cordyceps protein peptide contains protein peptides with amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO.

21.

3. The method according to claim 2, wherein The dosage of the Lactobacillus is 0.8×10 6 -3.6×10 6 CFU with respect to 1 g of the Cordyceps sinensis protein from Xinjiang; Preferably, the cordyceps protein is the protein extracted from Cordyceps sinensis (Berk.) Sacc. var. xinjiangensis. Preferably, the preparation process of the cordyceps protein includes: drying and grinding the mycelia of Cordyceps sinensis (Berk.) Sacc. var. xinjiangensis into a powder with a particle size of 20 - 30 mm, mixing the powder with a buffer solution, followed by homogenization and solid-liquid separation to collect supernatant I; filtering the supernatant I through a 0.4 - 0.5 μm filter membrane, then performing salting out and gradient elution, and further dialyzing with a dialysis membrane with a molecular weight cut-off less than 400 Da.

4. The method according to claim 2 or 3, characterized in that The Lactobacillus is selected from one or more of Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus reuteri and Lactobacillus delbrueckii. Preferably, the Lactobacillus is Lactobacillus reuteri with the number CICC No.6119.

5. The method according to claim 2 or 3, characterized in that, The conditions for the mixed fermentation include: temperature of 20 - 50 °C, rotation speed of 100 - 200 rpm, time of 10 - 55 h, preferably 24 - 48 h; The carbon-nitrogen ratio of the fermentation medium used for the mixed fermentation is 1:1 - 2.5, preferably 1:1 - 2, and the content of cordyceps protein in the fermentation medium is 5 - 12.5 mg / mL, preferably 5 - 10 mg / mL; Preferably, the fermentation medium contains the cordyceps protein, glucose, dipotassium hydrogen phosphate and distilled water. Preferably, the separation process includes: subjecting the fermented broth obtained by fermentation to solid-liquid separation to obtain supernatant II, subjecting the supernatant II to membrane filtration, dialysis, and drying to obtain a protein hydrolysate, and re-dissolving the protein hydrolysate and then performing ultrafiltration separation to obtain a component with a molecular weight less than 10 kDa as the composite cordyceps protein peptide; Preferably, after re-dissolving the protein hydrolysate, ultrafiltration separation is performed to obtain a component with a molecular weight less than 3 kDa.

6. Use of the composite cordyceps protein peptide as claimed in claim 1 and / or the composite cordyceps protein peptide prepared by the method as claimed in any one of claims 2 to 5 in the preparation of iron-chelating peptides.

7. A method for preparing an iron-chelating peptide, characterized in that, The method includes: mixing the composite cordyceps protein peptide as claimed in claim 1 and / or the composite cordyceps protein peptide prepared by the method as claimed in any one of claims 2 to 5 with an iron salt and a precipitant.

8. The method according to claim 7, wherein The conditions for the mixing include: the time is 20 - 60 min, and the temperature is 37 - 50 °C; Preferably, the iron salt is ferrous sulfate; Preferably, the mass ratio of the composite cordyceps protein peptide to the iron salt is 10 - 20:

1.

9. The method according to claim 7 or 8, characterized in that The precipitant is selected from at least one of ethanol, acetone, polyethylene glycol, and ammonium sulfate.

10. Use of the iron-chelating peptide prepared by the method as claimed in any one of claims 7 to 9 in the preparation of a medicament for treating iron-deficiency anemia.

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