Oyster peptide compound yin-tonifying anti-fatigue preparation and preparation method thereof

Abstract

This invention relates to the field of functional anti-fatigue preparations, and more particularly to an oyster peptide compound yin-nourishing and anti-fatigue preparation and its preparation method. The above-mentioned oyster peptide compound yin-nourishing and anti-fatigue preparation is characterized by the following raw materials by weight: 30-60 parts oyster meat, 1-3 parts wolfberry powder, 1-5 parts yam powder, 1-2 parts astragalus polysaccharide, 2-6 parts gelatin, 1-3 parts compound protease a, 1-3 parts compound protease b, 0.1-0.5 parts transglutaminase, 0.1-1 part chitosan, 0.01-0.1 parts zinc citrate dihydrate, 10-30 parts glycerol, 1-2 parts magnesium stearate, 0.5-1.5 parts vitamin C, 1-5 parts fructooligosaccharide, and 5-10 parts lactose. This invention scientifically combines oyster peptide powder, after chelation, with wolfberry and yam, which are both medicinal and edible substances. By nourishing yin and replenishing qi to regulate the body's homeostasis, it achieves a synergistic effect in improving anti-fatigue efficacy, with significant results.

Description

Technical Field

[0001] This invention relates to the field of functional anti-fatigue preparations, and in particular to an oyster peptide compound anti-fatigue preparation and its preparation method. Background Technology

[0002] Fatigue is a physiological functional decline response that occurs when the body is under high-intensity physical exertion or long-term mental stress. It involves multiple physiological aspects such as energy metabolism disorders, increased oxidative stress levels, and neuroendocrine system imbalances.

[0003] Modern medical research shows that persistent fatigue not only leads to decreased attention and reduced exercise endurance, but may also induce weakened immunity and organ dysfunction, seriously affecting people's quality of life and health. Currently, methods for relieving fatigue mainly include rest, physical rehabilitation, and drug intervention. However, while some chemical drugs are fast-acting, they often come with side effects or the risk of dependence. In contrast, developing anti-fatigue functional foods using food-derived bioactive ingredients that combine safety and efficacy has become a research hotspot in the field of nutrition and health.

[0004] Oysters, a traditional seafood used in both medicine and food, are rich in glycogen, polypeptides, amino acids, and trace elements, and have a long history of use in combating fatigue and enhancing physical fitness. However, the large-molecule proteins in natural oysters have low bioavailability and are difficult for the human body to absorb directly and efficiently. Although current technologies often use enzymatic hydrolysis to convert them into small-molecule oyster peptides to improve absorption, conventional enzymatic hydrolysis methods often suffer from insufficient hydrolysis, resulting in insignificant or unstable anti-fatigue effects in the final product. Furthermore, the mechanism of action of simple oyster peptides in improving fatigue is singular, making it difficult to comprehensively address the complex causes of fatigue, which is a pressing technical challenge in this field. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by proposing an oyster peptide compound for tonifying Yin and combating fatigue, and its preparation method.

[0006] An oyster peptide compound preparation for nourishing yin and relieving fatigue, comprising the following raw materials by weight: 30-60 parts oyster meat, 1-3 parts wolfberry powder, 1-5 parts yam powder, 1-2 parts astragalus polysaccharide, 2-6 parts gelatin, 1-3 parts compound protease a, 1-3 parts compound protease b, 0.1-0.5 parts transglutaminase, 0.1-1 parts chitosan, 0.01-0.1 parts zinc citrate dihydrate, 10-30 parts glycerol, 1-2 parts magnesium stearate, 0.5-1.5 parts vitamin C, 1-5 parts fructooligosaccharide, and 5-10 parts lactose.

[0007] Preferably, the enzyme activity of complex protein a is 50,000-200,000 U / g, and the enzyme activity of complex protein b is 80,000-120,000 U / g.

[0008] Preferably, the complex protease a includes papain and subtilisin; the mass ratio of papain to subtilisin is 1:0.5-1.

[0009] Preferably, the complex protease b includes: flavor protease and trypsin; the mass ratio of flavor protease to trypsin is 1:1-2.

[0010] The preparation method of the above-mentioned oyster peptide compound yin-nourishing and anti-fatigue preparation includes the following steps:

[0011] S1. Homogenize oyster meat and water for 5-15 minutes, add phosphate buffer solution, add compound protease a, keep warm at 40-50℃ for 3-5 hours, raise the temperature to 90-95℃ and keep warm for 5-10 minutes to obtain pre-enzymatically hydrolyzed oysters.

[0012] S2. Centrifuge the pre-enzymatically hydrolyzed oyster liquid for 20-30 min at a temperature of 0-4℃, and retain the supernatant. Add complex proteinase b to the supernatant, incubate at 45-55℃ for 30-50 min, raise the temperature to 90-95℃ and incubate for 5-10 min, centrifuge again for 20-30 min at a temperature of 0-4℃, take the supernatant, and freeze-dry to obtain oyster peptide powder.

[0013] S3. Add gelatin, astragalus polysaccharide, and chitosan to citric acid solution and stir evenly. Add transglutaminase to the solution, shake in a water bath at 50-60℃ for 5-10 hours, and inactivate at 90-95℃ for 5-15 minutes. Separate the reaction solution using an ultrafiltration membrane with a molecular weight cutoff of 10kDa, collect the retentate, and freeze-dry to obtain activated gelatin.

[0014] S4. Mix oyster peptide powder, zinc citrate dihydrate, glycerin, and water. Adjust the pH of the system to 4-5. Stir at 60-70℃ for 1-2 hours. Add activated gelatin and continue stirring for 10-30 minutes to form wet granules. Dry and granulate. Add wolfberry powder, yam powder, magnesium stearate, vitamin C, fructooligosaccharides, and lactose and mix evenly. Compress into tablets.

[0015] Preferably, in S2, the centrifugal speed is 5000-10000 r / min.

[0016] Preferably, in S3, the concentration of the citric acid solution is 0.5-1.2 mol / L.

[0017] Preferably, in step S4, a citric acid solution with a concentration of 0.1-0.5 mol / L is used to adjust the pH of the system to 4-5.

[0018] Preferably, in step S4, after adding activated gelatin and stirring, wet granules are prepared using a 5-10 mesh sieve.

[0019] Preferably, in step S4, the wet particles are dried by blowing air at a temperature of 50-60°C.

[0020] Compared with existing technologies, the present invention has the following advantages:

[0021] This invention features high utilization of natural ingredients and employs a dual-complex protease hydrolysis technology to obtain small-molecule oyster peptides. These peptides are not only highly pure but also have a very high proportion of small-molecule peptides, making them easier to absorb. Furthermore, the combination with wolfberry and yam enhances the yin-nourishing effect, solving the problems of poor absorption and limited efficacy in existing food products.

[0022] This invention uses a combination of gelatin, chitosan, and astragalus polysaccharide to enhance antioxidant capacity. Furthermore, when combined with oyster peptide powder, it effectively protects and improves the stability of the oyster peptide powder in vivo, promoting the release of active peptides in the intestines. The oyster peptide powder, when combined and chelated with zinc citrate dihydrate, effectively promotes the absorption and utilization of nutrients by the human body, resulting in excellent antioxidant effects while ensuring anti-fatigue efficacy.

[0023] This invention scientifically combines oyster peptide powder with medicinal and edible substances from wolfberry and yam after chelation. By nourishing yin and replenishing qi, it regulates the body's homeostasis and achieves a synergistic effect in improving anti-fatigue efficacy. The effect is significant, and the preparation process is simple, making it suitable for large-scale promotion and application. Attached Figure Description

[0024] Figure 1 This is a comparison chart of the DHMBA content and protein content of the oyster peptide powders obtained in Example 5 and Comparative Example 1.

[0025] Figure 2 This is a comparison chart showing the mass ratio of peptides with a relative molecular weight <1000 in the total protein of the oyster peptide powders obtained in Example 5 and Comparative Example 1.

[0026] Figure 3 The graph shows a comparison of the duration of pole climbing and running at exhaustion for mice in the blank control group, Example 5 group, Comparative Example 1 group, Comparative Example 2 group, and Comparative Example 3 group. Detailed Implementation

[0027] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0029] The oyster meat used below was sourced from oyster farming areas in Dalian. The goji berry powder used below was purchased from the Bozhou Traditional Chinese Medicine Market in Anhui Province; its origin is Zhongning County, Ningxia. The yam powder used below was purchased from the Bozhou Traditional Chinese Medicine Market in Anhui Province; its origin is Wenxian County, Henan Province. The astragalus polysaccharide used below was purchased from Qingdao Yuekang Biotechnology Co., Ltd. The gelatin (food grade) used below was purchased from Hebei Pengyu Biotechnology Co., Ltd. The chitosan (food grade) used below was purchased from Hebei Tuohai Biotechnology Co., Ltd. The zinc citrate dihydrate used below was purchased from Nantong Feiyu Food Technology Co., Ltd. The fructooligosaccharides used below were purchased from Hebei Hongtao Bioengineering Co., Ltd. The lactose used below was purchased from Shandong Jiju Biotechnology Co., Ltd.

[0030] The papain, subtilisin, flavor protease, and trypsin used below were purchased from Shandong Jiju Biotechnology Co., Ltd. The transglutaminase (food grade) used below was purchased from Hebei Jiuxing Chemical Products Co., Ltd.

[0031] Example 1

[0032] An oyster peptide compound preparation for nourishing yin and relieving fatigue, the raw materials of which include: 30g oyster meat, 1g wolfberry powder, 1g yam powder, 1g astragalus polysaccharide, 2g gelatin, 1g compound protease a with an enzyme activity of 50000U / g, 1g compound protease b with an enzyme activity of 80000U / g, 0.1g transglutaminase, 0.1g chitosan, 0.01g zinc citrate dihydrate, 10g glycerol, 1g magnesium stearate, 0.5g vitamin C, 1g fructooligosaccharide, and 5g lactose.

[0033] Complex protease a is composed of papain and subtilisin in a mass ratio of 1:0.5. Complex protease b is composed of flavor protease and trypsin in a mass ratio of 1:1.

[0034] The preparation method of the above-mentioned oyster peptide compound yin-nourishing and anti-fatigue preparation includes the following steps:

[0035] S1. Add oyster meat and water to a high-pressure homogenizer and homogenize for 5 minutes. Add to 150g of phosphate buffer solution with pH 6.5, add complex proteinase a, keep warm at 40℃ for 3 hours, and then heat to 90℃ and keep warm for 5 minutes to obtain pre-enzymatically hydrolyzed oysters.

[0036] S2. Centrifuge the pre-enzymatically hydrolyzed oyster liquid at 0℃ for 20 min at a speed of 5000 r / min, retain the supernatant, add complex proteinase b, incubate at 50℃ for 30 min, raise the temperature to 90-95℃ and incubate for 5 min, centrifuge at 0℃ for 20 min at a speed of 5000 r / min, take the supernatant, and freeze-dry to obtain oyster peptide powder;

[0037] S3. Add gelatin, astragalus polysaccharide and chitosan to 20g of 0.5mol / L citric acid solution and stir well. Add transglutaminase to the solution, shake in a water bath at 55℃ for 8h, and inactivate at 90℃ for 5min. Separate the reaction solution with an ultrafiltration membrane with a molecular weight cutoff of 10kDa, collect the retentate, and freeze-dry to obtain activated gelatin.

[0038] S4. Mix oyster peptide powder, zinc citrate dihydrate, glycerin, and deionized water. Adjust the pH of the system to 4 using a 0.1 mol / L citric acid solution. Stir at 60℃ for 1 hour. Add activated gelatin and continue stirring for 10 minutes at a stirring speed of 200 r / min to obtain a soft mass. Pass the mass through a 5-mesh sieve to obtain wet granules. Dry the granules at 50℃ using forced air drying. Add wolfberry powder, yam powder, magnesium stearate, vitamin C, fructooligosaccharides, and lactose and mix well. Compress the mixture into tablets and sterilize with ultraviolet light.

[0039] Example 2

[0040] An oyster peptide compound preparation for nourishing yin and relieving fatigue, the raw materials of which include: 60g oyster meat, 3g wolfberry powder, 5g yam powder, 2g astragalus polysaccharide, 6g gelatin, 3g compound protease a with an enzyme activity of 200,000U / g, 3g compound protease b with an enzyme activity of 120,000U / g, 0.5g transglutaminase, 1g chitosan, 0.1g zinc citrate dihydrate, 30g glycerol, 2g magnesium stearate, 1.5g vitamin C, 5g fructooligosaccharide, and 10g lactose.

[0041] Complex protease a is composed of papain and subtilisin in a 1:1 mass ratio. Complex protease b is composed of flavor protease and trypsin in a 1:2 mass ratio.

[0042] The preparation method of the above-mentioned oyster peptide compound yin-nourishing and anti-fatigue preparation includes the following steps:

[0043] S1. Add oyster meat and water to a high-pressure homogenizer and homogenize for 15 minutes. Add to 300g of pH 7 phosphate buffer solution and add complex protein a. Keep at 50℃ for 5 hours and then heat to 95℃ for 10 minutes to obtain pre-enzymatically hydrolyzed oysters.

[0044] S2. Centrifuge the pre-enzymatically hydrolyzed oyster liquid at 4℃ for 30 min at a speed of 10000 r / min, retain the supernatant, add complex proteinase b, incubate at 55℃ for 50 min, raise the temperature to 95℃ and incubate for 10 min, centrifuge at 4℃ for 30 min at a speed of 10000 r / min, take the supernatant, and freeze-dry to obtain oyster peptide powder.

[0045] S3. Add gelatin, astragalus polysaccharide and chitosan to 40g of 1.2mol / L citric acid solution and stir well. Add transglutaminase to the solution, shake in a water bath at 55℃ for 8h, and inactivate at 95℃ for 15min. Separate the reaction solution with an ultrafiltration membrane with a molecular weight cutoff of 10kDa, collect the retentate, and freeze-dry to obtain activated gelatin.

[0046] S4. Mix oyster peptide powder, zinc citrate dihydrate, glycerin, and deionized water. Adjust the pH of the system to 5 using a 0.5 mol / L citric acid solution. Stir at 70℃ for 2 hours. Add activated gelatin and continue stirring for 30 minutes at a stirring speed of 400 r / min to obtain a soft mass. Pass the mass through a 10-mesh sieve to obtain wet granules. Dry the granules at 60℃ using forced air drying. Add wolfberry powder, yam powder, magnesium stearate, vitamin C, fructooligosaccharides, and lactose and mix well. Compress the mixture into tablets and sterilize with ultraviolet light.

[0047] Example 3

[0048] An oyster peptide compound preparation for nourishing yin and relieving fatigue, the raw materials of which include: 40g oyster meat, 2.5g wolfberry powder, 2g yam powder, 1.8g astragalus polysaccharide, 3g gelatin, 1.5g compound protease a with an enzyme activity of 150,000U / g, 1.5g compound protease b with an enzyme activity of 110,000U / g, 0.4g transglutaminase, 0.3g chitosan, 0.08g zinc citrate dihydrate, 15g glycerol, 1.8g magnesium stearate, 0.8g vitamin C, 4g fructooligosaccharide, and 7g lactose.

[0049] Complex protease a is composed of papain and subtilisin in a mass ratio of 1:0.9. Complex protease b is composed of flavor protease and trypsin in a mass ratio of 1:1.2.

[0050] The preparation method of the above-mentioned oyster peptide compound yin-nourishing and anti-fatigue preparation includes the following steps:

[0051] S1. Add oyster meat and water to a high-pressure homogenizer and homogenize for 12 minutes. Add to 200g of phosphate buffer solution with pH 6.8, add complex proteinase a, keep warm at 42℃ for 4.5 hours, raise the temperature to 92℃ and keep warm for 9 minutes to obtain pre-enzymatically hydrolyzed oysters.

[0052] S2. Centrifuge the pre-enzymatically hydrolyzed oyster liquid at 1℃ for 28 min at a speed of 7000 r / min, retain the supernatant, add complex proteinase b, incubate at 52℃ for 35 min, raise the temperature to 93℃ and incubate for 7 min, centrifuge at 3℃ for 22 min at a speed of 9000 r / min, take the supernatant, and freeze-dry to obtain oyster peptide powder.

[0053] S3. Add gelatin, astragalus polysaccharide and chitosan to 25g of 1mol / L citric acid solution and stir well. Add transglutaminase to the solution, shake in a water bath at 60℃ for 5h, and inactivate at 91℃ for 12min. Separate the reaction solution with an ultrafiltration membrane with a molecular weight cutoff of 10kDa, collect the retentate, and freeze-dry to obtain activated gelatin.

[0054] S4. Mix oyster peptide powder, zinc citrate dihydrate, glycerol, and deionized water. Adjust the pH of the system to 4.5 using a 0.2 mol / L citric acid solution. Stir at 62℃ for 100 min. Add activated gelatin and continue stirring for 15 min at a stirring speed of 350 r / min to obtain a soft mass. Pass the mass through a 10-mesh sieve to obtain wet granules. Dry the granules at 52℃ using forced air drying. Add wolfberry powder, yam powder, magnesium stearate, vitamin C, fructooligosaccharides, and lactose and mix well. Compress the mixture into tablets and sterilize with ultraviolet light.

[0055] Example 4

[0056] An oyster peptide compound preparation for nourishing yin and relieving fatigue, the raw materials of which include: 50g oyster meat, 1.5g wolfberry powder, 4g yam powder, 1.2g astragalus polysaccharide, 5g gelatin, 2.5g compound protease a with an enzyme activity of 100,000U / g, 2.5g compound protease b with an enzyme activity of 90,000U / g, 0.2g transglutaminase, 0.7g chitosan, 0.02g zinc citrate dihydrate, 25g glycerol, 1.2g magnesium stearate, 1.2g vitamin C, 2g fructooligosaccharide, and 9g lactose.

[0057] Complex protease a is composed of papain and subtilisin in a mass ratio of 1:0.7. Complex protease b is composed of flavor protease and trypsin in a mass ratio of 1:1.8.

[0058] The preparation method of the above-mentioned oyster peptide compound yin-nourishing and anti-fatigue preparation includes the following steps:

[0059] S1. Add oyster meat and water to a high-pressure homogenizer and homogenize for 8 minutes. Add to 240g of phosphate buffer solution with pH 6.8, add complex proteinase a, keep warm at 48℃ for 3.5 hours, raise the temperature to 94℃ and keep warm for 7 minutes to obtain pre-enzymatically hydrolyzed oysters.

[0060] S2. Centrifuge the pre-enzymatically hydrolyzed oyster liquid at 3℃ for 22 min at a speed of 9000 r / min, retain the supernatant, add complex proteinase b, incubate at 45℃ for 45 min, raise the temperature to 91℃ and incubate for 9 min, centrifuge at 1℃ for 28 min at a speed of 7000 r / min, take the supernatant, freeze dry to obtain oyster peptide powder.

[0061] S3. Add gelatin, astragalus polysaccharide and chitosan to 35g of 0.8mol / L citric acid solution and stir well. Add transglutaminase to the solution, shake in a 50℃ water bath for 10h, and inactivate at 93℃ for 8min. Separate the reaction solution with an ultrafiltration membrane with a molecular weight cutoff of 10kDa, collect the retentate, and freeze-dry to obtain activated gelatin.

[0062] S4. Mix oyster peptide powder, zinc citrate dihydrate, glycerin, and deionized water. Adjust the pH of the system to 4.5 using a 0.4 mol / L citric acid solution. Stir at 68℃ for 80 min. Add activated gelatin and continue stirring for 25 min at a stirring speed of 250 r / min to obtain a soft mass. Pass the mass through a 10-mesh sieve to obtain wet granules. Dry the granules at 58℃ using forced air drying. Add wolfberry powder, yam powder, magnesium stearate, vitamin C, fructooligosaccharides, and lactose and mix well. Compress the mixture into tablets and sterilize with ultraviolet light.

[0063] Example 5

[0064] An oyster peptide compound preparation for nourishing yin and relieving fatigue, the raw materials of which include: 45g oyster meat, 2g wolfberry powder, 3g yam powder, 1.5g astragalus polysaccharide, 4g gelatin, 2g compound protease a with an enzyme activity of 120000U / g, 2g compound protease b with an enzyme activity of 100000U / g, 0.3g transglutaminase, 0.5g chitosan, 0.05g zinc citrate dihydrate, 20g glycerol, 1.5g magnesium stearate, 1g vitamin C, 3g fructooligosaccharide, and 8g lactose.

[0065] Complex protease a is composed of papain and subtilisin in a mass ratio of 1:0.8. Complex protease b is composed of flavor protease and trypsin in a mass ratio of 1:1.5.

[0066] The preparation method of the above-mentioned oyster peptide compound yin-nourishing and anti-fatigue preparation includes the following steps:

[0067] S1. Add oyster meat and water to a high-pressure homogenizer and homogenize for 10 min. Add to 220 g of phosphate buffer solution with pH 6.8, add complex proteinase a, keep warm at 45℃ for 4 h, raise the temperature to 93℃ and keep warm for 8 min to obtain pre-enzymatically hydrolyzed oysters.

[0068] S2. Centrifuge the pre-enzymatically hydrolyzed oyster liquid at 2℃ for 25 min at a speed of 8000 r / min, retain the supernatant, add complex proteinase b, incubate at 45℃ for 40 min, raise the temperature to 92℃ and incubate for 8 min, centrifuge at 2℃ for 25 min at a speed of 8000 r / min, take the supernatant, freeze dry to obtain oyster peptide powder.

[0069] S3. Add gelatin, astragalus polysaccharide and chitosan to 30g of 0.9mol / L citric acid solution and stir well. Add transglutaminase to the solution, shake in a 60℃ water bath for 10h, and inactivate at 92℃ for 10min. Separate the reaction solution with an ultrafiltration membrane with a molecular weight cutoff of 10kDa, collect the retentate, and freeze-dry to obtain activated gelatin.

[0070] S4. Mix oyster peptide powder, zinc citrate dihydrate, glycerol, and deionized water. Adjust the pH of the system to 4.5 using a 0.3 mol / L citric acid solution. Stir at 65℃ for 90 min. Add activated gelatin and continue stirring for 20 min at a stirring speed of 300 r / min to obtain a soft mass. Pass the mass through a 10-mesh sieve to obtain wet granules. Dry the granules in a forced-air drying process at 52-58℃. Add wolfberry powder, yam powder, magnesium stearate, vitamin C, fructooligosaccharides, and lactose and mix well. Compress the mixture into tablets and sterilize with ultraviolet light.

[0071] Comparative Example 1

[0072] An oyster peptide compound preparation for nourishing yin and relieving fatigue, the raw materials of which include: 45g oyster meat, 2g wolfberry powder, 3g yam powder, 1.5g astragalus polysaccharide, 4g gelatin, 4g compound protease with an enzyme activity of 100,000 U / g, 0.3g transglutaminase, 0.5g chitosan, 0.05g zinc citrate dihydrate, 20g glycerol, 1.5g magnesium stearate, 1g vitamin C, 3g fructooligosaccharide, and 8g lactose.

[0073] The complex protease is composed of flavor protease and trypsin in a mass ratio of 1:1.5.

[0074] The preparation method of the above-mentioned oyster peptide compound yin-nourishing and anti-fatigue preparation includes the following steps:

[0075] S1. Add oyster meat and water to a high-pressure homogenizer and homogenize for 10 minutes. Add to 220g of phosphate buffer solution with pH 6.8, add half of the complex protease, keep warm at 45℃ for 4 hours, and then heat to 93℃ and keep warm for 8 minutes to obtain pre-enzymatically hydrolyzed oysters.

[0076] S2. Centrifuge the pre-enzymatically hydrolyzed oyster liquid at 2℃ for 25 min at a speed of 8000 r / min, retain the supernatant, add the remaining complex protease, incubate at 45℃ for 40 min, raise the temperature to 92℃ and incubate for 8 min, centrifuge at 2℃ for 25 min at a speed of 8000 r / min, take the supernatant, and freeze-dry to obtain oyster peptide powder.

[0077] S3. Add gelatin, astragalus polysaccharide and chitosan to 30g of 0.9mol / L citric acid solution and stir well. Add transglutaminase to the solution, shake in a 60℃ water bath for 10h, and inactivate at 92℃ for 10min. Separate the reaction solution with an ultrafiltration membrane with a molecular weight cutoff of 10kDa, collect the retentate, and freeze-dry to obtain activated gelatin.

[0078] S4. Mix oyster peptide powder, zinc citrate dihydrate, glycerol, and deionized water. Adjust the pH of the system to 4.5 using a 0.3 mol / L citric acid solution. Stir at 65℃ for 90 min. Add activated gelatin and continue stirring for 20 min at a stirring speed of 300 r / min to obtain a soft mass. Pass the mass through a 10-mesh sieve to obtain wet granules. Dry the granules in a forced-air drying process at 52-58℃. Add wolfberry powder, yam powder, magnesium stearate, vitamin C, fructooligosaccharides, and lactose and mix well. Compress the mixture into tablets and sterilize with ultraviolet light.

[0079] Comparative Example 2

[0080] An oyster peptide compound preparation for nourishing yin and relieving fatigue, the raw materials of which include: 45g oyster meat, 2g wolfberry powder, 3g yam powder, 1.5g astragalus polysaccharide, 4g gelatin, 2g compound protease a with an enzyme activity of 120000U / g, 2g compound protease b with an enzyme activity of 100000U / g, 0.3g transglutaminase, 0.5g chitosan, 20.05g glycerol, 1.5g magnesium stearate, 1g vitamin C, 3g fructooligosaccharide, and 8g lactose.

[0081] Complex protease a is composed of papain and subtilisin in a mass ratio of 1:0.8. Complex protease b is composed of flavor protease and trypsin in a mass ratio of 1:1.5.

[0082] The preparation method of the above-mentioned oyster peptide compound yin-nourishing and anti-fatigue preparation includes the following steps:

[0083] S1. Add oyster meat and water to a high-pressure homogenizer and homogenize for 10 min. Add to 220 g of phosphate buffer solution with pH 6.8, add complex proteinase a, keep warm at 45℃ for 4 h, raise the temperature to 93℃ and keep warm for 8 min to obtain pre-enzymatically hydrolyzed oysters.

[0084] S2. Centrifuge the pre-enzymatically hydrolyzed oyster liquid at 2℃ for 25 min at a speed of 8000 r / min, retain the supernatant, add complex proteinase b, incubate at 45℃ for 40 min, raise the temperature to 92℃ and incubate for 8 min, centrifuge at 2℃ for 25 min at a speed of 8000 r / min, take the supernatant, freeze dry to obtain oyster peptide powder.

[0085] S3. Add gelatin, astragalus polysaccharide and chitosan to 30g of 0.9mol / L citric acid solution and stir well. Add transglutaminase to the solution, shake in a 60℃ water bath for 10h, and inactivate at 92℃ for 10min. Separate the reaction solution with an ultrafiltration membrane with a molecular weight cutoff of 10kDa, collect the retentate, and freeze-dry to obtain activated gelatin.

[0086] S4. Mix oyster peptide powder, glycerin, and deionized water. Adjust the pH of the system to 4.5 using a 0.3 mol / L citric acid solution. Stir at 65℃ for 90 min. Add activated gelatin and continue stirring for 20 min at a stirring speed of 300 r / min to obtain a soft mass. Pass the mass through a 10-mesh sieve to obtain wet granules. Dry the granules at 52-58℃ using forced air drying. Add wolfberry powder, yam powder, magnesium stearate, vitamin C, fructooligosaccharides, and lactose and mix well. Compress the mixture into tablets and sterilize with ultraviolet light.

[0087] Comparative Example 3

[0088] An oyster peptide compound preparation for nourishing yin and relieving fatigue, the raw materials of which include: 45g oyster meat, 2g wolfberry powder, 3g yam powder, 4g gelatin, 2g compound protease a with an enzyme activity of 120000U / g, 2g compound protease b with an enzyme activity of 100000U / g, 0.3g transglutaminase, 2g chitosan, 0.05g zinc citrate dihydrate, 20g glycerol, 1.5g magnesium stearate, 1g vitamin C, 3g fructooligosaccharides, and 8g lactose.

[0089] Complex protease a is composed of papain and subtilisin in a mass ratio of 1:0.8. Complex protease b is composed of flavor protease and trypsin in a mass ratio of 1:1.5.

[0090] The preparation method of the above-mentioned oyster peptide compound yin-nourishing and anti-fatigue preparation includes the following steps:

[0091] S1. Add oyster meat and water to a high-pressure homogenizer and homogenize for 10 min. Add to 220 g of phosphate buffer solution with pH 6.8, add complex proteinase a, keep warm at 45℃ for 4 h, raise the temperature to 93℃ and keep warm for 8 min to obtain pre-enzymatically hydrolyzed oysters.

[0092] S2. Centrifuge the pre-enzymatically hydrolyzed oyster liquid at 2℃ for 25 min at a speed of 8000 r / min, retain the supernatant, add complex proteinase b, incubate at 45℃ for 40 min, raise the temperature to 92℃ and incubate for 8 min, centrifuge at 2℃ for 25 min at a speed of 8000 r / min, take the supernatant, freeze dry to obtain oyster peptide powder.

[0093] S3. Add gelatin and chitosan to 30g of 0.9mol / L citric acid solution and stir until homogeneous. Add transglutaminase and shake in a 60℃ water bath for 10h. Inactivate at 92℃ for 10min. Separate the reaction solution using an ultrafiltration membrane with a molecular weight cutoff of 10kDa. Collect the retentate and freeze-dry to obtain activated gelatin.

[0094] S4. Mix oyster peptide powder, zinc citrate dihydrate, glycerol, and deionized water. Adjust the pH of the system to 4.5 using a 0.3 mol / L citric acid solution. Stir at 65℃ for 90 min. Add activated gelatin and continue stirring for 20 min at a stirring speed of 300 r / min to obtain a soft mass. Pass the mass through a 10-mesh sieve to obtain wet granules. Dry the granules in a forced-air drying process at 52-58℃. Add wolfberry powder, yam powder, magnesium stearate, vitamin C, fructooligosaccharides, and lactose and mix well. Compress the mixture into tablets and sterilize with ultraviolet light.

[0095] The 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) content of the oyster peptide powders obtained in Example 5 and Comparative Example 1 was determined using liquid chromatography-mass spectrometry. The protein content (on a dry basis) and relative molecular weight of the oyster peptide powders obtained in Example 5 and Comparative Example 1 were determined using Soxhlet extraction and high performance size exclusion chromatography.

[0096] like Figure 1 and Figure 2 As shown, the DHMBA content, protein content, and the mass ratio of peptides with a relative molecular weight <1000 in the total protein of the oyster peptide powder obtained in Example 5 were significantly higher than those in Comparative Example 1, confirming that alternating enzymatic hydrolysis with two complex proteases can completely decompose the protein in oysters into small molecule peptides and effectively improve the yield of DHMBA.

[0097] Sixty healthy, qualified SPF-grade, 8-week-old male ICR mice were selected and, after 1 week of acclimatization feeding, were randomly divided into 5 groups (blank control group, Example 5 group, Comparative Example 1 group, Comparative Example 2 group, and Comparative Example 3 group), with 12 mice in each group.

[0098] The oyster peptide compound yin-nourishing and anti-fatigue preparations obtained in Example 5 and Comparative Examples 1-3 were ground separately and added to drinking water to form suspensions for later use. The Example 5 group was administered the suspension of the oyster peptide compound yin-nourishing and anti-fatigue preparation obtained in Example 5 by gavage, with a gavage dose of 100 mg / kg; the Comparative Example 1 group was administered the suspension of the oyster peptide compound yin-nourishing and anti-fatigue preparation obtained in Comparative Example 1 by gavage, with a gavage dose of 100 mg / kg; the Comparative Example 2 group was administered the suspension of the oyster peptide compound yin-nourishing and anti-fatigue preparation obtained in Comparative Example 2 by gavage, with a gavage dose of 100 mg / kg; the Comparative Example 3 group was administered the suspension of the oyster peptide compound yin-nourishing and anti-fatigue preparation obtained in Comparative Example 3 by gavage, with a gavage dose of 100 mg / kg; the blank control group was administered an equal volume of physiological saline by gavage.

[0099] Thirty minutes after gavage, mice in each group underwent pole climbing and running tests at exhaustion.

[0100] The exhaustion pole-climbing experiment was conducted as follows: A 40cm long, 8mm diameter glass rod was used as the climbing frame, with the lower end suspended vertically above the ground at a height of 25cm, and the upper end leaving a 5.5cm gap. The exhaustion time of the mice was recorded. Before the formal experiment, the mice underwent pole-climbing training once. The experiment was repeated 3 times, and the average of the three pole-climbing times was taken as the final exhaustion time. The climbing rod was cleaned after each experiment.

[0101] The exhaustive running experiment was conducted as follows: Mice were placed in a rotating fatigue apparatus and subjected to exhaustive running at a moderate intensity (12 m / min). Exhaustion was defined as the mice being unable to continue running, stopping at the back of the track, experiencing rapid breathing and muscle tremors, and still being unable to continue running after an electric shock. The duration of exhaustive running was recorded.

[0102] like Figure 3 As shown, the duration of pole climbing and running at exhaustion in the mice of Example 5 group was significantly higher than that in the other groups, confirming that the anti-fatigue effect of the oyster peptide compound yin-nourishing and anti-fatigue preparation obtained in this invention is significant and stable.

[0103] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A compound oyster peptide preparation for nourishing yin and relieving fatigue, characterized in that, The raw materials, by weight, include: 30-60 parts oyster meat, 1-3 parts wolfberry powder, 1-5 parts yam powder, 1-2 parts astragalus polysaccharide, 2-6 parts gelatin, 1-3 parts complex protease a, 1-3 parts complex protease b, 0.1-0.5 parts transglutaminase, 0.1-1 part chitosan, 0.01-0.1 parts zinc citrate dihydrate, 10-30 parts glycerol, 1-2 parts magnesium stearate, 0.5-1.5 parts vitamin C, 1-5 parts fructooligosaccharides, and 5-10 parts lactose.

2. The oyster peptide compound yin-nourishing and anti-fatigue preparation according to claim 1, characterized in that, The enzyme activity of complex protein a is 50,000-200,000 U / g, and the enzyme activity of complex protein b is 80,000-120,000 U / g.

3. The oyster peptide compound yin-nourishing and anti-fatigue preparation according to claim 1, characterized in that, Complex protease a includes: papain and subtilisin; the mass ratio of papain to subtilisin is 1:0.5-1.

4. The oyster peptide compound yin-nourishing and anti-fatigue preparation according to claim 1, characterized in that, Complex protease b includes: flavor protease and trypsin; the mass ratio of flavor protease to trypsin is 1:1-2.

5. A method for preparing an oyster peptide compound yin-nourishing and anti-fatigue preparation as described in any one of claims 1-4, characterized in that, Includes the following steps: S1. Homogenize oyster meat and water for 5-15 minutes, add phosphate buffer solution, add compound protease a, keep warm at 40-50℃ for 3-5 hours, raise the temperature to 90-95℃ and keep warm for 5-10 minutes to obtain pre-enzymatically hydrolyzed oysters. S2. Centrifuge the pre-enzymatically hydrolyzed oyster liquid for 20-30 min at a temperature of 0-4℃, and retain the supernatant. Add complex proteinase b to the supernatant, incubate at 45-55℃ for 30-50 min, raise the temperature to 90-95℃ and incubate for 5-10 min, centrifuge again for 20-30 min at a temperature of 0-4℃, take the supernatant, and freeze-dry to obtain oyster peptide powder. S3. Add gelatin, astragalus polysaccharide, and chitosan to citric acid solution and stir evenly. Add transglutaminase to the solution, shake in a water bath at 50-60℃ for 5-10 hours, and inactivate at 90-95℃ for 5-15 minutes. Separate the reaction solution using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, collect the retentate, and freeze-dry to obtain activated gelatin. S4. Mix oyster peptide powder, zinc citrate dihydrate, glycerin, and water. Adjust the pH of the system to 4-5. Stir at 60-70℃ for 1-2 hours. Add activated gelatin and continue stirring for 10-30 minutes to form wet granules. Dry and granulate. Add wolfberry powder, yam powder, magnesium stearate, vitamin C, fructooligosaccharides, and lactose and mix evenly. Compress into tablets.

6. The preparation method of the oyster peptide compound yin-nourishing and anti-fatigue preparation according to claim 5, characterized in that, In S2, the centrifugal speed is 5000-10000 r / min.

7. The preparation method of the oyster peptide compound yin-nourishing and anti-fatigue preparation according to claim 5, characterized in that, In S3, the concentration of citric acid solution is 0.5-1.2 mol / L.

8. The preparation method of the oyster peptide compound yin-nourishing and anti-fatigue preparation according to claim 5, characterized in that, In S4, the pH of the system is adjusted to 4-5 using a citric acid solution with a concentration of 0.1-0.5 mol / L.

9. The preparation method of the oyster peptide compound yin-nourishing and anti-fatigue preparation according to claim 5, characterized in that, In S4, after adding activated gelatin and stirring, wet granules are prepared using a 5-10 mesh sieve.

10. The preparation method of the oyster peptide compound yin-nourishing and anti-fatigue preparation according to claim 5, characterized in that, In S4, wet particles are dried by blowing air at a temperature of 50-60℃.

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