Polypeptide product containing osteogenic growth peptide as well as preparation method and application thereof

Abstract

The invention provides a preparation method of a polypeptide product containing osteogenic growth peptide, which comprises the following steps: sterilizing bone marrow or blood of an animal as a raw material; after sterilization, adjusting the pH value to 6-8, inoculating 1-3% of micrococcus luteus (1-8 * 10 < 9 > cfu / mL), inoculating 2-5% of lactobacillus curvatus (1-8 * 10 < 10 > cfu / mL), inoculating 0.5-4% of bacillus coagulans (1-8 * 10 < 8 > cfu / g), and fermenting at 30-42 DEG C for 2-6 hours; adjusting the temperature to 20-30 DEG C and the pH value to 6-8, inoculating 1-8% of geotrichum candidum (1-8 * 10 < 6 > cfu / mL), fermenting for 1-5 days, and sterilizing for 20-40 minutes under the microwave of 200-500 W to obtain fermentation liquor; adjusting the pH value of the fermentation liquor to 5-9, adding 0.5-5% of kiwi fruit protease, bromelain and alkaline protease according to a ratio of 1: (1-4): (2-5), carrying out enzymolysis at 45-65 DEG C for 2-8 hours, heating to 85-95 DEG C, and carrying out heat preservation for 20-40 minutes to obtain enzymatic hydrolysate; and separating from the enzymatic hydrolysate to obtain the polypeptide product containing the osteogenic growth peptide. The invention provides a brand-new method for preparing the polypeptide product containing the osteogenic growth peptide, the process is relatively simple, and the yield of the osteogenic growth peptide is relatively high, so that the cost is reduced.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a polypeptide product containing an osteogenic growth peptide, a preparation method thereof, and an application thereof. Background Art

[0002] Osteogenic growth peptide (OGP) is a peptide isolated from healing bone marrow cells by Israeli researchers Bab et al. in 1989. It was found that it can activate the hematopoietic system in vivo and promote bone anabolism. It can also stimulate the proliferation and differentiation of osteoblasts and bone marrow stromal cells in vitro. It has the effects of promoting bone growth and bone healing, preventing osteoporosis, and alleviating bone marrow suppression after radiotherapy and chemotherapy.

[0003] Both children and the middle-aged and elderly population have huge demands for bone health, but the current preparation process of osteogenic growth peptides is complex, the yield is low, the cost is high, and it is difficult to popularize and apply. Summary of the Invention

[0004] To at least partially solve the above problems, the embodiments of the present application use animal bone marrow or blood as raw materials and adopt fermentation, enzymatic hydrolysis, purification and other processes to prepare polypeptide products containing osteogenic growth peptides.

[0005] According to the first aspect of the present application, the present application provides a method for preparing a polypeptide product containing osteogenic growth peptide, comprising:

[0006] Step S1: using animal bone marrow or blood as raw material and sterilizing the raw material;

[0007] Step S2: After sterilization, adjust the pH to 6-8, inoculate 1-3% by volume, and the initial concentration is 1-8×10 9 cfu / mL of Micrococcus luteus, inoculated with 2-5% by volume, with an initial concentration of 1-8×10 10 cfu / mL of Lactobacillus curvatus, inoculated with 0.5-4% by weight at an initial concentration of 1-8×10 8 cfu / g of Bacillus coagulans, 30-42°C, fermentation for 2-6h; adjust the temperature to 20-30°C, pH 6-8, inoculate 1-8% by volume, and the initial concentration is 1-8×10 6 cfu / mL of Geotrichum candidum, ferment for 1-5 days to obtain the fermentation broth;

[0008] Step S3, adjusting the pH value of the fermentation liquid to 5-9, adding 0.5-5% of kiwifruit protease, bromelain, and alkaline protease in a ratio of 1:1-4:2-5, enzymolysis temperature 45-65° C., enzymolysis for 2-8 hours, heating to 85-95° C., and keeping warm for 20-40 minutes to obtain an enzymolysis solution;

[0009] Step S4: Separate and obtain a polypeptide product containing osteogenic growth peptide from the enzymatic hydrolyzate.

[0010] Preferably, in step S1, the raw material is one of bovine bone marrow, bovine blood, sheep bone marrow, sheep blood or duck blood.

[0011] Preferably, in step S1, the raw materials are sterilized, comprising: weighing the raw materials, adding 8-20 times of water, adjusting the pH value to 6-8, heating to 75-95° C., and keeping warm for 20-40 min.

[0012] Preferably, in step S2, the pH value is adjusted to 6-7 after sterilization, and the initial concentration of 2-3 volume % inoculated is 4-8×10 9 cfu / mL of Micrococcus luteus, inoculated with 3-5% by volume, with an initial concentration of 4-8×10 10 cfu / mL of Lactobacillus curvatus, inoculated with 2-4% by weight, with an initial concentration of 4-8×10 8 cfu / g of Bacillus coagulans, 30-37°C, fermentation for 3-6h; adjust the temperature to 25-30°C, pH 6.5-7.5, inoculate 4-8% by volume, and the initial concentration is 3-8×10 6 cfu / mL of Geotrichum candidum, and ferment for 2-4 days to obtain fermentation liquid.

[0013] Preferably, in step S2, after obtaining the fermentation liquid, the fermentation liquid is sterilized by microwave at 200-500W for 20-40 min.

[0014] Preferably, in step S3, the pH value of the fermentation liquid is adjusted to 6-8, 1-3% of kiwi protease, bromelain, and alkaline protease are added in a ratio of 1:1-4:2-5, the enzymatic hydrolysis temperature is 50-60°C, the enzymatic hydrolysis is carried out for 4-6 hours, the temperature is raised to 85-95°C, and the temperature is kept warm for 25-35 minutes.

[0015] Preferably, in step S3, the ratio of kiwi protease, bromelain and alkaline protease is 1:2-3:3-4.

[0016] Preferably, the polypeptide product containing osteogenic growth peptide is separated and obtained from the enzymatic hydrolysate, comprising:

[0017] Step S41, decolorizing the enzymatic hydrolyzate to obtain a decolorized solution;

[0018] Step S42: adding a decolorizing solution to the adsorptive macroporous resin at a flow rate of 1-5 SV, and collecting the effluent between 10-40 min as the refined solution;

[0019] Step S43: nanofiltration is performed on the refined liquid to remove inorganic salts and water-soluble free amino acids, and spray drying is performed to obtain a polypeptide product containing osteogenic growth peptide.

[0020] Preferably, in step S41, the enzymatic hydrolyzate is decolorized to obtain a decolorized solution, comprising: adding 0.5-5% hydrogen peroxide to the enzymatic hydrolyzate for 20-40 minutes, then adding 0.5-5% of a mixed decolorizing agent of coconut shell activated carbon, activated clay, and diatomaceous earth in a ratio of 10-2:1:1-5, at a temperature of 55-65° C., decolorizing for 30-90 minutes, and filtering by plate and frame;

[0021] Preferably, in step S41, 2-5% hydrogen peroxide is added to the enzymatic hydrolyzate for decolorization for 20-40 minutes, and then 1-3% of a mixed decolorizing agent of coconut shell activated carbon, activated clay and diatomaceous earth is added in a ratio of 6-3:1:2-4, the temperature is 55-65°C, the decolorization is carried out for 40-60 minutes, and plate and frame filtration is performed.

[0022] Preferably, in step S42, a decolorizing solution is added to the adsorptive macroporous resin at a flow rate of 1-3 SV, and the effluent is collected for 15-30 min.

[0023] According to the second aspect of the present application, the present application provides a polypeptide preparation containing osteogenic growth peptide prepared by the above preparation method.

[0024] Preferably, the polypeptide product is in the form of tablets, powder or oral solution.

[0025] According to the third aspect of the present application, the present application provides the use of the above-mentioned polypeptide preparation containing osteogenic growth peptide in products for promoting bone growth, preventing and improving osteoporosis.

[0026] The present application provides a method for preparing a polypeptide product containing osteogenic growth peptide, which uses Micrococcus luteus, Lactobacillus curvatus, Bacillus coagulans and Geotrichum candidum to ferment the bone marrow or blood of an animal, and simultaneously uses kiwifruit protease, bromelain and alkaline protease to enzymatically hydrolyze the fermentation broth, and finally separates and obtains the polypeptide product containing osteogenic growth peptide from the enzymatic hydrolyzate. This method provides a new method for preparing a polypeptide product containing osteogenic growth peptide, which has a relatively simple process and a high yield of osteogenic growth peptide, thereby reducing costs. The polypeptide product containing osteogenic growth peptide can promote bone growth, prevent and improve osteoporosis, and is acid-resistant, high-temperature resistant, has a small molecular weight, is easily absorbed, can be processed into tablets, powders and oral liquids, and can also be used in ordinary foods to meet the needs of children's growth promotion and bone health of middle-aged and elderly people. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the HPLC profile of the osteogenic growth peptide standard;

[0028] Figure 2 is the HPLC spectrum of the polypeptide preparation obtained in Example 1;

[0029] Figure 3This is the HPLC profile of the polypeptide preparation obtained in Example 2;

[0030] Figure 4 This is the HPLC profile of the polypeptide preparation obtained in Example 3;

[0031] Figure 5 This is the HPLC profile of the polypeptide preparation obtained in Example 4;

[0032] Figure 6 This is the HPLC spectrum of the polypeptide preparation obtained in Comparative Example 1;

[0033] Figure 7 This is the HPLC spectrum of the polypeptide preparation obtained in Comparative Example 2. DETAILED DESCRIPTION

[0034] The technical solutions of the preferred embodiments of the present application will be clearly and completely described below. Obviously, the embodiments described are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection claimed in this application. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment and materials used in each embodiment are the same.

[0035] In the examples and comparative examples of the present application, Micrococcus luteus, Geotrichum candidum and Lactobacillus curvatum were purchased from Shanghai Chen Microbiology Technology Co., Ltd., Bacillus coagulans was purchased from Xi'an Xitai Biotechnology Co., Ltd., kiwifruit protease was purchased from Shanghai Yimiao Chemical Technology Co., Ltd., bromelain and alkaline protease were both purchased from Nanning Pangbo Bioengineering Co., Ltd., hydrogen peroxide was purchased from Henan Huize Bioengineering Co., Ltd., diatomaceous earth was purchased from Hebei Runhuabang New Materials Technology Co., Ltd., activated carbon was purchased from Zhengzhou Yongkun Environmental Protection Technology Co., Ltd., activated white clay was purchased from Jinan Shanhai Chemical Technology Co., Ltd., macroporous resin was purchased from Bengbu Sanyi Technology Co., Ltd., and osteogenic growth peptide standards were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0036] Example 1

[0037] A polypeptide product containing osteogenic growth peptide is prepared by the following steps:

[0038] Step S1, sterilization of raw materials: weigh 100 g of bovine bone marrow powder, add 10 times water, adjust the pH value to 7, heat to 90° C., and keep warm for 30 min.

[0039] Step S2, preparation of fermentation broth: after sterilization, adjust the pH value to 6.5, inoculate 3% by volume with an initial concentration of 6×10 9 cfu / mL of Micrococcus luteus, with an initial inoculation concentration of 4% by volume of 5 × 1010 cfu / mL of Lactobacillus curvatus, inoculated with 3% by weight, with an initial concentration of 5×10 8 cfu / g of Bacillus coagulans, fermented at 35℃ for 4h; the temperature was adjusted to 28℃, the pH value was 7, and the initial concentration of 5% by volume inoculation was 7×10 6 cfu / mL of Geotrichum candidum was fermented for 3 days. The fermentation broth was sterilized by microwave at 400W for 30 min to obtain the fermentation broth.

[0040] Step S3, preparation of enzymatic hydrolysate: adjusting the pH of the fermentation broth to 7.5, adding 3% kiwifruit protease, bromelain, and alkaline protease in a ratio of 1:3:3, enzymatic hydrolysis temperature of 55°C, enzymatic hydrolysis for 6 hours, raising the temperature to 90°C, and keeping warm for 30 minutes.

[0041] Step S41, decolorization of the fermentation hydrolyzate: 3% hydrogen peroxide was added to the fermentation hydrolyzate for 30 minutes, and then 3% of a mixed decolorizing agent of coconut shell activated carbon, activated clay and diatomaceous earth (ratio 5:1:3) was added, the temperature was 60°C, the decolorization time was 60 minutes, and plate and frame filtration was performed.

[0042] Step S42, purification of osteogenic growth peptide: add decolorizing solution to the adsorptive macroporous resin at a flow rate of 3 SV, and collect the effluent between 15-30 minutes.

[0043] Step S43: The liquid after purification is nanofiltered to remove inorganic salts and water-soluble free amino acids, and spray-dried to obtain a polypeptide product containing osteogenic growth peptide.

[0044] Example 2

[0045] A polypeptide product containing osteogenic growth peptide is prepared by the following steps:

[0046] Step S1, sterilization of raw materials: weigh 100 g of bovine blood powder, add 10 times water, adjust the pH value to 7, heat to 90° C., and keep warm for 30 min.

[0047] Step S2, preparation of fermentation broth: after sterilization, adjust the pH value to 6.5, inoculate 3% by volume with an initial concentration of 6×10 9 cfu / mL of Micrococcus luteus, with an initial inoculation concentration of 4% by volume of 5 × 10 10 cfu / mL of Lactobacillus curvatus, inoculated with 3% by weight, with an initial concentration of 5×10 8 cfu / g of Bacillus coagulans, fermented at 35℃ for 4h; the temperature was adjusted to 28℃, the pH value was 7, and the initial concentration of 5% by volume inoculation was 7×10 6 cfu / mL of Geotrichum candidum was fermented for 3 days and sterilized by microwave at 400W for 30 min to obtain the fermentation broth.

[0048] Step S3, preparation of enzymatic hydrolysate: adjusting the pH of the fermentation broth to 7.5, adding 3% kiwifruit protease, bromelain, and alkaline protease in a ratio of 1:3:3, enzymatic hydrolysis temperature of 55°C, enzymatic hydrolysis for 6 hours, raising the temperature to 90°C, and keeping warm for 30 minutes.

[0049] Step S41: Decolorization of the fermentation hydrolysate: 3% hydrogen peroxide was added to the fermentation hydrolysate for 30 minutes. Then, 3% of a mixed decolorizer consisting of coconut shell activated carbon, activated clay, and diatomaceous earth (in a ratio of 5:1:3) was added. The mixture was decolorized at 60°C for 60 minutes and then filtered on a plate and frame.

[0050] Step S42, purification of osteogenic growth peptide: add decolorizing solution to the adsorptive macroporous resin at a flow rate of 3 SV, and collect the effluent between 15-30 minutes.

[0051] Step S43: The liquid after purification is nanofiltered to remove inorganic salts and water-soluble free amino acids, and spray-dried to obtain a polypeptide product containing osteogenic growth peptide.

[0052] Example 3

[0053] A polypeptide product containing osteogenic growth peptide is prepared by the following steps:

[0054] Step S1, sterilization of raw materials: weigh 100 g of bovine blood powder, add 10 times water, adjust the pH value to 7, heat to 90° C., and keep warm for 30 min.

[0055] Step S2, preparation of fermentation broth: after sterilization, adjust the pH value to 6.5, inoculate 1% by volume with an initial concentration of 1×10 9 cfu / mL of Micrococcus luteus, with an initial concentration of 1×10 10 cfu / mL of Lactobacillus curvatus, inoculated with 0.5 wt% of the initial concentration of 1 × 10 8 cfu / g of Bacillus coagulans, fermented at 35℃ for 4h; the temperature was adjusted to 28℃, the pH value was 7, and the initial concentration of 1 volume% inoculated was 1×10 6 cfu / mL of Geotrichum candidum was fermented for 3 days and sterilized by microwave at 400W for 30 min to obtain the fermentation broth.

[0056] Step S3, preparation of enzymatic hydrolysate: adjusting the pH of the fermentation broth to 7.5, adding 0.5% kiwifruit protease, bromelain, and alkaline protease in a ratio of 1:2:3, enzymatic hydrolysis temperature of 55°C, enzymatic hydrolysis for 6 hours, raising the temperature to 90°C, and keeping warm for 30 minutes.

[0057] Step S41, decolorization of the fermentation hydrolyzate: 3% hydrogen peroxide was added to the fermentation hydrolyzate for 30 minutes, and then 3% of a mixed decolorizing agent of coconut shell activated carbon, activated clay and diatomaceous earth (ratio 5:1:3) was added, the temperature was 60°C, the decolorization time was 60 minutes, and plate and frame filtration was performed.

[0058] Step S42, purification of osteogenic growth peptide: add decolorizing solution to the adsorptive macroporous resin at a flow rate of 1 SV, and collect the effluent between 15-30 minutes.

[0059] Step S43: The liquid after purification is nanofiltered to remove inorganic salts and water-soluble free amino acids, and spray-dried to obtain a polypeptide product containing osteogenic growth peptide.

[0060] Example 4

[0061] A polypeptide product containing osteogenic growth peptide is prepared by the following steps:

[0062] Step S1, sterilization of raw materials: weigh 100 g of bovine blood powder, add 10 times water, adjust the pH value to 7, heat to 90° C., and keep warm for 30 min.

[0063] Step S2, preparation of fermentation broth: after sterilization, adjust the pH value to 6.5, inoculate 3% by volume with an initial concentration of 8×10 9 cfu / mL of Micrococcus luteus, the initial concentration of 5% by volume was 8×10 10 cfu / mL of Lactobacillus curvatus, with an initial inoculation concentration of 4% by weight and 8 × 10 8 cfu / g of Bacillus coagulans, fermented at 35℃ for 4h; the temperature was adjusted to 28℃, the pH value was 7, and the initial concentration of 8% by volume was 8×10 6 cfu / mL of Geotrichum candidum was fermented for 3 days and sterilized by microwave at 400W for 30 min to obtain the fermentation broth.

[0064] Step S3, preparation of enzymatic hydrolysate: adjusting the pH of the fermentation broth to 7.5, adding 5% kiwifruit protease, bromelain, and alkaline protease in a ratio of 1:3:4, enzymatic hydrolysis temperature of 55°C, enzymatic hydrolysis for 6 hours, raising the temperature to 90°C, and keeping warm for 30 minutes.

[0065] Step S41, decolorization of the fermentation hydrolyzate: 3% hydrogen peroxide was added to the fermentation hydrolyzate for 30 minutes, and then 3% of a mixed decolorizing agent of coconut shell activated carbon, activated clay and diatomaceous earth (ratio 5:1:3) was added, the temperature was 60°C, the decolorization time was 60 minutes, and plate and frame filtration was performed.

[0066] Step S42, purification of osteogenic growth peptide: add decolorizing solution to the adsorptive macroporous resin at a flow rate of 5 SV, and collect the effluent between 15-30 minutes.

[0067] Step S43: The liquid after purification is nanofiltered to remove inorganic salts and water-soluble free amino acids, and spray-dried to obtain a polypeptide product containing osteogenic growth peptide.

[0068] Comparative Example 1

[0069] A polypeptide product is prepared by the following steps:

[0070] Step S1, sterilization of raw materials: weigh 100 g of bovine bone marrow powder, add 10 times water, adjust the pH value to 7, heat to 90°C, and keep warm for 30 min;

[0071] Step S2, preparation of fermentation broth: after sterilization, adjust the pH value to 6.5, inoculate 3% by volume with an initial concentration of 6×10 9 cfu / mL of Micrococcus mutans, the initial concentration of 4% by volume was 5×10 10 cfu / mL of sake Lactobacillus, inoculated with 3% by weight, with an initial concentration of 5×10 8 cfu / g of Bacillus subtilis, fermented at 35℃ for 4h; the temperature was adjusted to 28℃, the pH value was 7, and the initial concentration of 5% by volume inoculation was 7×10 6 cfu / mL of Mucor, fermented for 3 days, and sterilized by microwave at 400W for 30 min to obtain the fermentation broth.

[0072] Step S3, preparation of enzymatic hydrolysate: adjusting the pH of the fermentation broth to 7.5, adding 3% kiwifruit protease, bromelain, and alkaline protease in a ratio of 1:3:3, enzymatic hydrolysis temperature of 55°C, enzymatic hydrolysis for 6 hours, raising the temperature to 90°C, and keeping warm for 30 minutes.

[0073] Step S41, decolorization of the fermentation hydrolyzate: 3% hydrogen peroxide was added to the fermentation hydrolyzate for 30 minutes, and then 3% of a mixed decolorizing agent of coconut shell activated carbon, activated clay and diatomaceous earth (ratio 5:1:3) was added, the temperature was 60°C, the decolorization time was 60 minutes, and plate and frame filtration was performed.

[0074] Step S42, purification of osteogenic growth peptide: add decolorizing solution to the adsorptive macroporous resin at a flow rate of 3 SV, and collect the effluent between 15-30 minutes.

[0075] Step S43: The liquid after purification is nanofiltered to remove inorganic salts and water-soluble free amino acids, and then spray-dried to obtain a polypeptide product.

[0076] Micrococcus variants was obtained from Guangdong Institute of Microbiology, Lactobacillus sakei was purchased from Shaanxi Shuoyang Biotechnology Co., Ltd., Bacillus subtilis was purchased from Shaanxi Shuoyang Biotechnology Co., Ltd., and Mucor was purchased from Shanghai Collection Biotechnology Center.

[0077] Comparative Example 2

[0078] A polypeptide product is prepared by the following steps:

[0079] Step S1, sterilization of raw materials: weigh 100 g of bovine bone marrow powder, add 10 times water, adjust the pH value to 7, heat to 90°C, and keep warm for 30 min;

[0080] Step S2, preparation of fermentation broth: after sterilization, adjust the pH value to 6.5, inoculate 3% by volume with an initial concentration of 6×10 9 cfu / mL of Micrococcus luteus, with an initial inoculation concentration of 4% by volume of 5 × 10 10 cfu / mL of Lactobacillus curvatus, inoculated with 3% by weight, with an initial concentration of 5×10 8 cfu / g of Bacillus coagulans, fermented at 35℃ for 4h; the temperature was adjusted to 28℃, the pH value was 7, and the initial concentration of 5% by volume inoculation was 7×10 6 cfu / mL of Geotrichum candidum was fermented for 3 days and sterilized by microwave at 400W for 30 min to obtain the fermentation broth.

[0081] Step S3, preparation of enzymatic hydrolysate: adjusting the pH of the fermentation broth to 7.5, adding 3% papain, trypsin, and alkaline protease in a ratio of 1:3:3, enzymatic hydrolysis temperature of 55°C, enzymatic hydrolysis for 6 hours, raising the temperature to 90°C, and keeping warm for 30 minutes.

[0082] Step S41, decolorization of the fermentation hydrolyzate: 3% hydrogen peroxide was added to the fermentation hydrolyzate for 30 minutes, and then 3% of a mixed decolorizing agent of coconut shell activated carbon, activated clay and diatomaceous earth (ratio 5:1:3) was added, the temperature was 60°C, the decolorization time was 60 minutes, and plate and frame filtration was performed.

[0083] Step S42, purification of osteogenic growth peptide: add decolorizing solution to the adsorptive macroporous resin at a flow rate of 3 SV, and collect the effluent between 15-30 minutes.

[0084] Step S43: The liquid after purification is nanofiltered to remove inorganic salts and water-soluble free amino acids, and then spray-dried to obtain a polypeptide product.

[0085] Papain and trypsin were purchased from Nanning Pangbo Bioengineering Co., Ltd.

[0086] The polypeptide products obtained in the examples and comparative examples were tested for protein content and osteogenic growth peptide content. Protein content was determined according to GB5009.5 National Food Safety Standard for the Determination of Protein in Foods, and osteogenic growth peptide content was determined using the standard reference method. The test results are as follows:

[0087] Group Protein content (%) Osteogenic growth peptide content (‰) Example 1 85.3 0.22 Example 2 87.0 0.41 Example 3 84.1 0.24 Example 4 86.4 0.35 Comparative Example 1 83.6 Not detected Comparative Example 2 82.9 Not detected

[0088] As can be seen, the osteogenic growth peptide content in Examples 1, 2, 3, and 4 ranged from 0.22 to 0.41‰, while no content was detected in Comparative Examples 1 and 2. A comparison of Examples 1 and 2 shows that using bovine blood powder as a raw material resulted in a higher content of osteogenic growth peptide than using bovine bone marrow powder as a raw material. A comparison of Examples 2 with Examples 3 and 4 shows that the yield of osteogenic growth peptide was higher when prepared using the process parameters of Example 2.

[0089] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0090] Although some specific embodiments of the present application have been described in detail through examples, those skilled in the art will understand that the above examples are for illustration only and are not intended to limit the scope of the present application. Those skilled in the art will understand that the above embodiments may be modified or some technical features may be replaced with equivalents without departing from the scope and spirit of the present application. The scope of the present application is defined by the appended claims.

Claims

1. A method for preparing a polypeptide product containing osteogenic growth peptide, characterized in that: include: Step S1: using animal bone marrow or blood as raw material and sterilizing the raw material; Step S2: After sterilization, adjust the pH to 6-8, inoculate 1-3% by volume, and the initial concentration is 1-8×10 9 cfu / mL of Micrococcus luteus, inoculated with 2-5% by volume, with an initial concentration of 1-8×10 10 cfu / mL of Lactobacillus curvatus, inoculated with 0.5-4% by weight at an initial concentration of 1-8×10 8 cfu / g of Bacillus coagulans, 30-42°C, fermentation for 2-6h; adjust the temperature to 20-30°C, pH 6-8, inoculate 1-8% by volume, and the initial concentration is 1-8×10 6 cfu / mL of Geotrichum candidum, ferment for 1-5 days to obtain the fermentation broth; Step S3, adjusting the pH value of the fermentation liquid to 5-9, adding 0.5-5% of kiwifruit protease, bromelain, and alkaline protease in a ratio of 1:1-4:2-5, enzymolysis temperature 45-65° C., enzymolysis for 2-8 hours, heating to 85-95° C., and keeping warm for 20-40 minutes to obtain an enzymolysis solution; Step S4: Separate and obtain a polypeptide product containing osteogenic growth peptide from the enzymatic hydrolyzate.

2. The preparation method according to claim 1, characterized in that In step S1, the raw material is one of ox bone marrow, ox blood, sheep bone marrow, sheep blood or duck blood.

3. The preparation method according to claim 1, characterized in that In step S1, the raw materials are sterilized, including: weighing the raw materials, adding 8-20 times of water, adjusting the pH value to 6-8, heating to 75-95° C., and keeping warm for 20-40 minutes.

4. The preparation method according to claim 1, characterized in that In step S2, the pH value is adjusted to 6-7 after sterilization, and the initial concentration of 2-3 volume % is inoculated to 4-8×10 9 cfu / mL of Micrococcus luteus, inoculated with 3-5% by volume, with an initial concentration of 4-8×10 10 cfu / mL of Lactobacillus curvatus, inoculated with 2-4% by weight, with an initial concentration of 4-8×10 8 cfu / g of Bacillus coagulans, 30-37°C, fermentation for 3-6h; adjust the temperature to 25-30°C, pH 6.5-7.5, inoculate 4-8% by volume, and the initial concentration is 3-8×10 6 cfu / mL of Geotrichum candidum and ferment for 2-4 days to obtain the fermentation broth.

5. The preparation method according to claim 1, characterized in that In step S3, the pH value of the fermentation liquid is adjusted to 6-8, 1-3% of kiwifruit protease, bromelain, and alkaline protease are added in a ratio of 1:1-4:2-5, the enzymatic hydrolysis temperature is 50-60°C, the enzymatic hydrolysis is carried out for 4-6 hours, the temperature is raised to 85-95°C, and the temperature is kept for 25-35 minutes; Preferably, in step S3, the ratio of kiwi protease, bromelain and alkaline protease is 1:2-3:3-4.

6. The preparation method according to claim 1, characterized in that The polypeptide products containing osteogenic growth peptide are separated from the enzymatic hydrolysate, including: Step S41, decolorizing the enzymatic hydrolyzate to obtain a decolorized solution; Step S42: adding a decolorizing solution to the adsorptive macroporous resin at a flow rate of 1-5 SV, and collecting the effluent between 10-40 min as the refined solution; Step S43: nanofiltration is performed on the refined liquid to remove inorganic salts and water-soluble free amino acids, and spray drying is performed to obtain a polypeptide product containing osteogenic growth peptide.

7. The preparation method according to claim 6, characterized in that In step S41, the enzymatic hydrolyzate is decolorized to obtain a decolorized solution, comprising: adding 0.5-5% hydrogen peroxide to the enzymatic hydrolyzate for 20-40 minutes, then adding 0.5-5% of a mixed decolorizing agent of coconut shell activated carbon, activated clay, and diatomaceous earth in a ratio of 10-2:1:1-5, at a temperature of 55-65° C., decolorizing for 30-90 minutes, and filtering on a plate and frame; Preferably, in step S41, 2-5% hydrogen peroxide is added to the enzymatic hydrolyzate for decolorization for 20-40 minutes, and then 1-3% of a mixed decolorizing agent of coconut shell activated carbon, activated clay and diatomaceous earth is added in a ratio of 6-3:1:2-4, the temperature is 55-65°C, the decolorization is carried out for 40-60 minutes, and plate and frame filtration is performed.

8. The preparation method according to claim 6, characterized in that In step S42, a decolorizing solution is added to the adsorptive macroporous resin at a flow rate of 1-3 SV, and the effluent is collected for 15-30 min.

9. A polypeptide product containing osteogenic growth peptide prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the polypeptide product containing osteogenic growth peptide according to claim 9 in products for promoting bone growth, preventing and improving osteoporosis.

Images