A sea cucumber gut peptide and application thereof in preparation of anti-osteoporosis products

Abstract

The application belongs to the technical field of sea cucumber intestine and sea cucumber intestine peptide application, and particularly relates to a sea cucumber intestine peptide and application thereof in preparation of anti-osteoporosis products. The sea cucumber intestine peptide is obtained by commercial protease enzymolysis, and then microbial fermentation is carried out to achieve the purpose of removing fishy smell. Through the double treatments of enzymolysis and fermentation, the sea cucumber intestine peptide has higher biological activity, and 10 optimal peptide segments are obtained, which can be applied to preparation of anti-osteoporosis products.

Description

Technical fields:

[0001] This invention belongs to the field of sea cucumber intestine and sea cucumber intestine peptide application technology, specifically relating to a sea cucumber intestine peptide and its application in the preparation of anti-osteoporosis products. Background technology:

[0002] Osteoporosis, the most common metabolic skeletal disease, is characterized by bone microarchitectural deterioration and increased fragility. With the increasingly severe aging population, various chronic metabolic diseases, including osteoporosis, have a detrimental impact on the elderly. Current treatments for osteoporosis typically work by inhibiting bone resorption to slow disease progression. Notably, nutritional support or functional foods have been shown to improve osteoporosis and related conditions by increasing bone density.

[0003] Sea cucumbers are a type of seafood delicacy used both as food and medicine. They are rich in proteins, amino acids, fats, polypeptides, polysaccharides, and saponins, among other active substances, thus possessing high nutritional and medicinal value. During the pre-processing of sea cucumbers, a large amount of sea cucumber intestines are generated as a byproduct, accounting for approximately 30%–40% of the total weight of fresh sea cucumbers (i.e., sea cucumber intestine production can reach 60,000–80,000 tons / year). The *Chinese Materia Medica* also indicates that sea cucumber intestines have calming, stomach-soothing, detoxifying, rash-relieving, tissue-regenerating, and hemostatic effects. Modern nutritional research has found that the nutritional value of sea cucumber intestines even surpasses that of the sea cucumber body wall in some aspects. Therefore, enzymatic hydrolysis using proteases is an efficient method to obtain the nutrients from sea cucumber intestines, yielding easily absorbed protein and polypeptide hydrolysates with health-promoting and medicinal functions, creating significant economic and social benefits. However, enzymatic hydrolysis of sea cucumber intestines produces aldehydes, alcohols, ketones, and hydrocarbons, giving the hydrolysate a strong fishy odor, making further processing and utilization difficult. Therefore, in the further application of sea cucumber intestinal peptides, removing the fishy smell is the key technology for realizing the high-value utilization and industrial development of sea cucumber intestinal hydrolysate. Summary of the Invention:

[0004] The technical problem this invention aims to solve is that sea cucumber intestines, the most significant byproduct of sea cucumber processing, are not fully utilized. Through protease hydrolysis, easily absorbed small-molecule protein and polypeptide hydrolysates with health and medicinal functions can be obtained. Furthermore, removing the fishy smell is a significant challenge in the processing of sea cucumbers and related products; therefore, there is still a need to develop sea cucumber intestine products that are both nutritious and delicious.

[0005] To address the aforementioned issues, this invention provides a sea cucumber intestinal peptide, which is obtained through enzymatic hydrolysis with commercial proteases and then subjected to microbial fermentation to remove fishy odor. Furthermore, the dual treatment of enzymatic hydrolysis and fermentation enhances the bioactivity of the sea cucumber intestinal peptide, making it suitable for use in the preparation of anti-osteoporosis products.

[0006] To achieve the above objectives, the present invention provides a method for preparing sea cucumber intestinal peptides, comprising the following steps:

[0007] (1) Enzymatic hydrolysis: Take an appropriate amount of sea cucumber intestines, cut them into pieces, add water, and sterilize the sea cucumber intestine homogenate to obtain sterile sea cucumber intestine homogenate; add papain and neutral protease in a ratio of 1:1 to the enzymatic hydrolysate, the amount of which is 3% of the dry weight of the substrate, inactivate the enzyme after enzymatic hydrolysis, centrifuge, and take the supernatant to obtain the enzymatic hydrolysate or freeze-dry the supernatant and then dissolve it to obtain the enzymatic hydrolysate; the dietary supplement of papain and neutral protease compound (1:1) enzymatic hydrolysate can promote bone formation in osteoporotic mice, so papain and neutral protease in a ratio of 1:1 are selected for enzymatic hydrolysis.

[0008] (2) Fermentation: Lactic acid bacteria are added to the enzymatic hydrolysate for fermentation, followed by yeast fermentation. The amount of lactic acid bacteria and yeast added is 5% of the dry weight of the sea cucumber intestine. After fermentation, the sea cucumber intestine peptides are sterilized. Fermentation can remove the fishy smell, and the sea cucumber intestine peptides that are first enzymatically hydrolyzed and then fermented have higher biological activity.

[0009] Firstly, enzymatic hydrolysis of sea cucumber intestines produces bitter amino acids and peptides. If yeast fermentation is used first after enzymatic hydrolysis, assuming sufficient carbon and nitrogen sources, it will produce a large amount of alcohol, making it unsuitable for most people. However, fermentation with lactic acid bacteria first preserves the flavor and richness of the sea cucumber, reducing the content of bitter peptides in the hydrolysate. Simultaneously, it effectively prevents the production of alcohol by yeast.

[0010] Sea cucumber intestines are hydrolyzed into amino acids and soluble polypeptides during enzymatic hydrolysis, which also alters the content of free amino acids and inorganic ions. Subsequently, the production process of enzymatic hydrolysis followed by fermentation, due to the combined action of microbial metabolism and enzymes, further breaks down the soluble polypeptides into amino acids and smaller peptides. Compared to peptides produced by other processes, these smaller peptides not only remove the fishy odor from the hydrolysate but are also easier to digest and absorb. Furthermore, mass spectrometry results revealed a high proportion of Gly, Pro, Leu, and Ile sequences at the N- and C-termini of the peptides; these Gly-Pro, Ile-Gly, and Leu-Gly sequences are potential osteogenic sequences.

[0011] Furthermore, the enzymatic hydrolysis conditions in step (1) are 55°C for 5 hours.

[0012] Furthermore, in step (2), the fermentation conditions for lactic acid bacteria are 37℃ for 8 hours; and the fermentation conditions for yeast are 42℃ for 8 hours.

[0013] A sea cucumber intestinal peptide prepared using the above method comprises the following sequence:

[0014] Table 1. Amino acid sequence of sea cucumber intestinal peptides:

[0015]

[0016] The aforementioned sea cucumber intestinal peptides can also be synthesized artificially.

[0017] The application of the above-mentioned sea cucumber intestinal peptide in the preparation of anti-osteoporosis products.

[0018] Furthermore, the product is freeze-dried sea cucumber intestine porridge.

[0019] Furthermore, the freeze-dried sea cucumber intestine porridge includes the following ingredients: 5-10 parts rice, 10-20 parts red beans, 1-3 parts lotus seeds, 2-5 parts longan, 4-6 parts oat rice, 30-40 parts sea cucumber intestine peptides, and 30-40 parts goji berries.

[0020] Goji berries are a traditional Chinese medicine used in both food and medicine. As early as the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), their effects of nourishing the liver and kidneys and strengthening muscles and bones were recorded. Recent studies have found that goji berries, due to their rich polysaccharide content, possess various pharmacological activities such as anti-inflammatory, antioxidant, and anti-cancer effects. Animal experiments have also shown that goji berry polysaccharides can improve femoral bone density and trabecular bone structure in rats; promote the proliferation of pre-osteoblast 3T3-E1 cells; and increase intracellular calcium ion concentration and type I collagen content. Based on the potential bone-promoting effects of goji berries, this invention prepares a freeze-dried porridge with anti-osteoporosis effects by combining sea cucumber intestinal peptides and goji berries.

[0021] Furthermore, the preparation method of the above-mentioned freeze-dried sea cucumber intestine porridge includes the following steps:

[0022] (1) Preparation: Prepare rice, red beans, lotus seeds, longan and oat rice. After washing, pour them into a container and add warm water to soak.

[0023] (2) Boiling: Add water to the soaked materials and boil them in a container over high heat. After boiling, add wolfberries and sea cucumber intestinal peptides and continue to boil. After boiling, add seasonings and stir. Finally, simmer.

[0024] (3) Pre-freezing in molds: Divide the cooked seafood porridge into molds, cool to room temperature, and then put it in a cold storage at -20℃ for pre-freezing;

[0025] (4) Freeze-drying: Place the pre-frozen sea cucumber porridge into a vacuum freeze dryer and dry it for 8 to 20 hours at a vacuum degree of 40 to 250 Pa and a temperature of -75°C.

[0026] Furthermore, the seasonings mentioned in step (2) are edible salt, white sugar, water, vegetable blended oil, vitamin E, and sodium D-isoascorbate.

[0027] With the improvement of living standards and the development of a fast-paced lifestyle, more and more consumers are favoring convenient, quick, and nutritious foods. In recent years, vacuum freeze-drying technology has been widely used in the processing of ready-to-eat foods, achieving rapid rehydration by giving ready-to-eat products a porous structure. The drying process can significantly reduce the moisture content of the product while maximizing the retention of its nutrients. Therefore, this invention develops a freeze-dried sea cucumber porridge suitable for the elderly, aiming to provide technical reference for the industrial production of freeze-dried sea cucumber porridge products.

[0028] The beneficial effects of this invention are as follows:

[0029] (1) The application of sea cucumber intestinal peptides in anti-osteoporosis products was given. The optimal conditions for enzymatic hydrolysis of sea cucumber intestines were determined through animal experiments, and further deodorization was carried out by microbial fermentation. The optimal flavor conditions for sea cucumber intestinal peptides were determined by electronic tongue and electronic nose, i.e. enzymatic hydrolysis followed by fermentation. The effect of improving osteoporosis was verified through animal experiments. Based on the mass spectrometry results, potential active osteogenic peptides in sea cucumber intestines were given, which improved the utilization value of sea cucumber intestines and expanded the dietary supplement source for improving bone density and delaying bone loss.

[0030] (2) The freeze-dried sea cucumber porridge prepared by this invention is the first to provide a freeze-dried porridge formula with anti-osteoporosis and combines sea cucumber intestinal peptide with wolfberry. It can completely preserve the flavor and nutritional value of the freeze-dried sea cucumber porridge, improve the utilization rate of sea cucumber intestine, and make the freeze-dried sea cucumber porridge taste delicious and have a novel taste. It is easier for the elderly to digest and absorb, and it can also attract younger consumers.

[0031] (3) The sea cucumber intestinal peptide preparation method provided by the present invention has good reproducibility and can be better used in industrial production. Attached Figure Description

[0032] Figure 1 Effects of different combinations of protease hydrolysates on serum osteocalcin in osteoporotic mice.

[0033] Figure 2 Effects of different combinations of protease hydrolysates on serum osteoprotegerin in osteoporotic mice.

[0034] Figure 3 Effects of different combinations of protease hydrolysates on serum tartrate-resistant alkaline phosphatase in osteoporotic mice.

[0035] Figure 4 Effects of different combinations of protease hydrolysates on bone mineral density in osteoporotic mice.

[0036] Figure 5 PCA detection of sea cucumber intestinal enzymatic hydrolysates using electronic tongue assays with different deodorization treatment combinations.

[0037] Figure 6 Electronic nose radar image of sea cucumber intestinal hydrolysates from different deodorization treatment combinations.

[0038] Figure 7 Effects of enzymatic fermentation products on serum osteocalcin in osteoporotic mice.

[0039] Figure 8 Effects of enzymatic fermentation products on serum tartrate-resistant alkaline phosphatase in osteoporotic mice.

[0040] Figure 9 Effects of enzymatic fermentation products on bone density in osteoporotic mice.

[0041] Figure 10 Effects of different synthetic peptides on serum tartrate-resistant alkaline phosphatase in osteoporotic mice.

[0042] Figure 11 Effects of different synthetic peptides on serum osteocalcin in osteoporotic mice.

[0043] Figure 12 Effects of different synthetic peptides on bone mineral density in osteoporotic mice.

[0044] Figure 13 Sensory radar charts of MRPs for freeze-dried porridge with different formulations.

[0045] Figure 14 Effects of different freeze-dried porridge formulations on serum osteocalcin in osteoporotic mice.

[0046] Figure 15 Effects of different freeze-dried porridge formulations on serum tartrate-resistant alkaline phosphatase in osteoporotic mice.

[0047] Figure 16 Effects of different freeze-dried porridge formulations on bone density in osteoporotic mice. Detailed implementation method:

[0048] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0049] Unless otherwise specified, all materials and reagents used in the following examples were commercially available.

[0050] Example 1:

[0051] A method for preparing sea cucumber intestinal enzymatic hydrolysate with osteoporosis-preventing properties, specifically comprising the following steps:

[0052] Preparation of sea cucumber intestine enzymatic hydrolysate: Take an appropriate amount of sea cucumber intestine and cut it into 1cm pieces. Add ultrapure water at a material-to-liquid ratio of 1:5 (m / v). Add protease and stir evenly. Adjust the pH to the optimal pH range of each protease using HCl solution (1mol / L) and NaOH solution (1mol / L) respectively (Table 2). Enzymatic hydrolysis is carried out at the optimal temperature of 55℃ for 5 hours. After enzymatic hydrolysis, filter the sample and inactivate the enzyme in a water bath at 90℃-100℃ for 15 minutes to obtain sea cucumber intestine enzymatic hydrolysate. Filter the sea cucumber intestine enzymatic hydrolysate through a 0.45μm microporous membrane and centrifuge at 3500r / min for 30 minutes using an ultrafiltration tube with a molecular weight cutoff of 3kDa to obtain a small molecule enzymatic hydrolysate. Commonly available commercial enzymes for enzymatic hydrolysis of seafood include papain (100,000 u / g), neutral protease (50,000 u / g), and flavor protease (30,000 u / g) (all purchased from Solex Technology Co., Ltd.). This patent proposes to fix the basic enzymatic hydrolysis parameters as follows: enzyme addition of 3% (relative to substrate dry weight), material-to-liquid ratio of 1:5 (m / v), hydrolysis time of 5 hours, and hydrolysis temperature of 55℃. Thirteen groups of enzymes with different ratios (single enzyme, double enzyme, triple enzyme, etc.) were used to hydrolyze sea cucumber intestines, and the optimal hydrolysis combination was determined through animal experiments (Table 3).

[0053] Table 2. Optimal conditions and enzyme activity for proteases:

[0054] Types of proteases Optimal stability (°C) Optimal pH Enzyme activity (U / g) Papain 55-65 6.0-7.0 6.0×104 neutral protease 50-60 6.0-7.0 1.0×105 Flavor proteases 50-55 6.0-7.0 3×105 .

[0055] Table 3. Protease combinations and ratios:

[0056] Protease Combination Enzyme ratio [a]papain 3％ [b]Neutral protease 3％ [c]flavor protease 3％ [d] Papaya: Neutral 1：1 [e] Papaya: Neutral 1：2 [f] Papaya: Neutral 2：1 [g] Papaya: Flavor 1：1 [h] Papaya: Flavor 1：2 [i] Papaya: Flavor 2：1 [j] Neutral: Flavor 1：1 [k] Neutral: Flavor 1：2 [l] Neutral: Flavor 2：1 [m] Papaya: Neutral: Flavor 1：1：1 .

[0057] Animal experiments: Healthy female C57BL6 / J mice (SPF grade), 8 weeks old, weighing 19.0±1.0g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The animal housing environment was maintained at 22℃~24℃, humidity at 45%~55%, with a 12h light / 12h dark cycle. Mice had free access to food (AIN-93G type feed) and water during the experiment. Experimental grouping and design: After 3 days of acclimatization, the mice underwent bilateral ovariectomy to establish an osteoporosis model. All mice were randomly divided into 13 groups (n=6 per group): Normal group, Model group, Positive control group (alendronate sodium, ALF, 0.15μg / kg·bw), and Sea Cucumber Intestinal Enzymatic Digestion group (a, b, c, d, e, f, g, h, i, j, k, l, m). During the experiment, mice were administered the corresponding test substance (200 mg / kg bw) by gavage at a volume of 10 mL / kg bw once a day for 90 consecutive days.

[0058] Serum biochemical markers were measured: the levels of OCN, OPG, and tartrate-resistant acid phosphatase (TRAP) in serum were determined using an ELISA kit. The specific procedures were performed in accordance with the kit instructions.

[0059] Bone mineral content determination: After grinding the femoral tissue, a nitric acid-perchloric acid mixed solution (5:2, v / v) was added for digestion. After adjusting the volume, the calcium and phosphorus contents were determined using an AA-6880 atomic absorption spectrophotometer.

[0060] Bone mineral density measurement: Femoral tissue was fixed in 10% neutral formaldehyde for 48 hours, and its bone mineral density was measured using a GK99 dual-energy X-ray absorptiometry system. Scanning conditions were: small animal mode, voltage 80 kV, current 165 mA.

[0061] OCN and OPG are matrix proteins secreted by osteoblasts, while TRAP is a protein-degrading enzyme secreted by osteoclasts. Figures 1-3 As shown, serum biochemical results indicated that, compared with the sham-operated group (Sham), the model group (OVX) mice had significantly lower levels of serum bone formation markers OCN and OPG, and significantly higher levels of bone resorption marker TRAP, entering a state of "low bone formation." Supplementation with different complex enzyme combinations significantly increased serum bone formation markers OCN and OPG. Notably, group d (papaya:neutral = 1:1) showed the highest effect, increasing levels by 44.9% and 17.3%, respectively (p<0.05), and inhibiting the abnormal increase in bone resorption marker TRAP (27.0%, p<0.05). This suggests that dietary supplementation with a 1:1 mixture of papaya and neutral protease hydrolysates can promote bone formation in osteoporotic mice.

[0062] Mouse bone mineral density results as follows Figure 4 As shown, compared with the sham group, the model group (OVX) mice had significantly lower bone mineral density. Dietary supplementation with protease hydrolysate significantly increased bone density in mice, with the papaya and neutral protease combination (1:1) showing the most significant effect.

[0063] The rate and total amount of mineral deposition in bones can reflect the overall state of the bones. As shown in Table 4, the bone mineral content (bone calcium and bone phosphorus) in the model group (OVX) was significantly reduced compared with the sham-operated group. Different combinations of protease hydrolysates had an ameliorative effect on this. Notably, the d group (papaya:neutral = 1:1) significantly increased the calcium-to-phosphorus ratio and inhibited bone mineral loss, indicating that dietary supplementation with a combination of papaya and neutral protease hydrolysates (1:1) can delay bone loss in osteoporotic mice.

[0064] Table 4. Effects of different combinations of protease hydrolysates on bone mineral content in osteoporotic mice:

[0065]

[0066]

[0067] Note: ** indicates a significance level of P < 0.01 compared to the Sham group; ## indicates a significance level of P < 0.01 compared to the OVX group.

[0068] In summary, among different sea cucumber intestine complex enzyme hydrolysis combinations, the 1:1 complex hydrolysate of papain and neutral protease effectively inhibited bone loss in osteoporotic mice and promoted the formation of osteogenic metabolites, exhibiting the best effect. Therefore, the 1:1 complex hydrolysate of papain and neutral protease, with an enzyme addition of 3% (relative to substrate dry weight), a hydrolysis time of 5 hours, and a hydrolysis temperature of 55℃ were selected as the subsequent hydrolysis conditions.

[0069] Example 2:

[0070] This example provides a method for deodorizing sea cucumber intestine peptides and the application of the product, specifically including the following steps:

[0071] Considering that the enzymatic hydrolysis of sea cucumber intestines produces aldehydes, alcohols, ketones, and hydrocarbons, resulting in a strong fishy smell in the hydrolysate, which can affect the taste of the product in further product development, we use microbial fermentation to remove the fishy smell.

[0072] Take an appropriate amount of sea cucumber intestines and cut them into 1cm pieces. Add ultrapure water at a material-to-liquid ratio of 1:5 (m / v). Sterilize the sea cucumber intestine homogenate at 121℃ for 10 minutes to obtain a sterile sea cucumber intestine homogenate. Fermentation conditions: Add a certain amount of lactic acid bacteria (strain: Lactobacillus plantarum Q7) to the sterile sea cucumber intestine homogenate and ferment at 37℃ for 8 hours. Then, add a certain amount of yeast (Angel Yeast Co., Ltd.) and ferment at 42℃ for 8 hours. After fermentation, sterilize the sterile sea cucumber intestine homogenate at 100℃. The amount of lactic acid bacteria and yeast added is 5% of the dry weight of the sea cucumber intestines.

[0073] Experimental grouping and design: Four combinations were set up: fermentation followed by enzymatic hydrolysis (A), enzymatic hydrolysis followed by fermentation (B), simple fermentation (C), and simple enzymatic hydrolysis (D). In groups A and B, according to the enzymatic hydrolysis combination and enzyme dosage determined in Example 1, a compound enzyme was added to the sterile sea cucumber intestine homogenate at 3% of the sea cucumber intestine weight before and after fermentation. After enzyme inactivation, the mixture was centrifuged at 3500 r / min for 30 min at room temperature, and the supernatant was collected and freeze-dried.

[0074] Electronic tongue and electronic nose analysis: Accurately transfer 30 mL of the test solution into the electronic tongue sample cup. Before measurement, perform self-testing, activation, calibration, and diagnostic steps on the electronic tongue to ensure the reliability and stability of the collected data. Prepare a RefSol reference solution using 30 mmol / L KCl solution and 0.3 mmol / L tartaric acid solution. Place the sensor in the reference solution and zero it for 30 s, then begin the measurement. The test time is 30 s. After the test, rinse with the reference solution for 3 s, and then perform a second taste test for 30 s. Each sample is repeated 4 times, and the last 3 results are taken as the test results.

[0075] Accurately transfer 30 mL of sample into an empty vial with the electronic nose tip, seal the vial, and allow it to stand at 26°C for 30 min before testing. Measurement parameters: cleaning time 100 s, zeroing time 5 s, pre-injection time 8 s, measurement time 60 s, carrier gas flow rate 400 mL / min. Each sample is measured 4 times, and the last 3 measurements are taken as the test results.

[0076] Table 5: Representative types of electronic nose sensors:

[0077]

[0078] Animal experiments: Healthy female C57BL6 / J mice (SPF grade), 8 weeks old, weighing 19.0±1.0g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Animals had free access to water, and all conditions except diet were the same as in Example 1. After 3 days of acclimatization, the mice underwent bilateral ovariectomy to establish an osteoporosis model. Based on the experimental groupings described above, the following 5 groups were established: model group (OVX), groups A, B, C, and D (n=6). During the experiment, mice were administered the corresponding test substance (200mg / kg / bw) by gavage at a volume of 10mL / kg·bw once daily for 90 consecutive days.

[0079] Reference Example 1 for serum biochemical indicators and bone mineral density measurement.

[0080] Principal component analysis was performed on the characteristic parameters of the electronic tongue, and the results are as follows: Figure 5 As shown, the contribution rate of the first principal component (PCA) was 75.86% for different treatment groups, the contribution rate of the second principal component (PCA) was 12.66%, and the sum of PC1 and PC2 was 88.52%. In the PCA results, the greater the distance between samples on the PC1 axis, the greater the difference between samples. The figure shows that compared with group D, groups A, B, and C do not overlap on the PC1 axis, indicating that the samples have good differentiation after deodorization and their flavors are independent. Group B, after enzymatic hydrolysis followed by fermentation, is significantly different from group A, indicating that the deodorization method of enzymatic hydrolysis followed by fermentation has a significant impact on the flavor of sea cucumber sausage.

[0081] An electronic nose can detect various taste indicators of food using artificial lipid membrane sensor technology. Radar images of sea cucumber intestinal hydrolysate before and after deodorization using an electronic nose are shown below. Figure 6 As shown, compared with the untreated sample, the lactic acid bacteria compound treatment significantly reduced volatile substances. The results are consistent with the sensory evaluation results.

[0082] like Figure 7-8 As shown, serum biochemical results indicated that, compared to the model group (OVX), the enzymatically hydrolyzed and fermented sea cucumber intestinal peptides effectively reduced the level of the bone resorption marker TRAP and increased the level of the bone formation marker OCN in mice. Among these, the effect of group B (enzymatic hydrolysis followed by fermentation) was more significant compared to group A (i.e., fermentation followed by enzymatic hydrolysis) (P < 0.01). Similar to the serum bone metabolism results, group B (enzymatic hydrolysis followed by fermentation) significantly increased osteoporotic bone mineral density, indicating that the sea cucumber intestinal peptides obtained through enzymatic hydrolysis followed by fermentation have higher biological activity. Figure 9 ).

[0083] Based on the above results, the samples in group B were desalted, with three copies of each sample. Peptide sequences were determined using a Q Exactive mass spectrometer, and the raw data files were searched in the sea cucumber collagen database downloaded from NCBI using Proteome Discoverer 2.4. The search parameters were set as follows: potential dynamic modification: oxidation (M, P, K); enzyme specificity: non-specific; fragment mass tolerance: 0.02 Da; precursor mass tolerance: 10 ppm; false discovery rate (FDR) of peptide identification results ≤ 1%. The potential for bioactivity of the identified peptide sequences was predicted using Peptide Ranker.

[0084] Table 6. Identification and Activity Prediction of Peptide Sequences from Sea Cucumber Intestine Enzymatic Hydrolysis and Fermentation

[0085]

[0086] aFrom PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ).

[0087] Example 3:

[0088] This example provides an application of sea cucumber intestinal peptides, specifically including the following steps:

[0089] The sea cucumber intestinal bioactive peptides predicted in Example 2 were biosynthesized by Shanghai Sangon Biotech Co., Ltd. using the Fmoc-peptide solid-phase synthesis method. HPLC and MS sequence analysis showed that the purity of all ten peptides—GPAGPTGPTGPA(①), DGGDVFR(②), GPAGTPTGPTGPAG(③), MLLILLL(④), IGPIGPGTG(⑤), IGPIGPT(⑥), PGPGPF(⑦), PGGPFP(⑧), MLLLPVM(⑨), and LGPLGP(⑩)—was greater than 98.89%. Chemically synthesized peptides, due to the controllable raw materials and processes, exhibit high purity and good safety for clinical applications. However, the numerous steps and various solvents involved in the synthesis process inevitably affect the bioactivity of the peptides; therefore, further verification of the bioactivity of the synthesized peptides is necessary.

[0090] Experimental grouping and design: Healthy female C57BL6 / J mice (SPF grade), 8 weeks old, weighing 19.0±1.0g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Animals had free access to water, and all conditions except diet were the same as in Example 1. After 3 days of acclimatization, the mice underwent bilateral ovariectomy to establish an osteoporosis model. Twelve groups were set up as follows: model group (OVX), enzymatic fermentation group (B), and synthetic peptide intervention group (①-⑩) (n=5). During the experiment, mice were administered the corresponding enzymatic fermentation group test substance (200mg / kg·bw) by gavage at a volume of 10mL / kg·bw, and the synthetic peptide (100mg / kg·bw) once daily for 90 consecutive days.

[0091] Reference Example 1 for serum biochemical indicators and bone mineral density measurement.

[0092] To verify the bioactivity of the above peptides, osteoporotic mice were further treated with chemically synthesized peptides. For example... Figure 10-11 As shown, compared with the model (OVX) group, the synthetic peptides significantly increased serum osteocalcin levels, decreased the level of the bone resorption marker TRAP, and increased bone mineral density in osteoporotic mice. Figure 12 It is noteworthy that the promoting effect of several synthetic peptides on osteoporosis mice at a dose of 100 mg / kg·bw was comparable to that of group B at a dose of 200 mg / kg·bw, with the effective concentration being reduced by about half, suggesting that these synthetic peptides may be potential active factors in SCIP.

[0093] Example 4:

[0094] This example provides a method for preparing freeze-dried sea cucumber porridge for osteoporosis and its specific application, specifically for delaying bone loss and increasing bone density in patients with osteoporosis. The method includes the following steps:

[0095] (1) Preparation: Prepare rice, red beans, lotus seeds, longan and oat rice, and wash them. The weight ratio is 5:20:1:1:2:4. Pour the rice, red beans, lotus seeds, longan and oat rice into a container and add warm water to soak. The mass of the warm water should be twice the total weight of the raw materials. Keep the water temperature at 40-50℃ and soak for about 30 minutes.

[0096] (2) Boiling: Boil the soaked material in a container over high heat for about 30 minutes at a material-to-liquid ratio of 1:5g / mL. After boiling, add 30g of wolfberry and 30g of sea cucumber intestinal peptide prepared in Example 2, and continue boiling for another 10 minutes. After boiling, add edible salt, white sugar, water, vegetable blended oil, vitamin E, and sodium D-isoascorbate for seasoning, stir, and finally simmer for 5 minutes.

[0097] (3) Pre-freezing in molds: Divide the cooked seafood porridge into molds, cool to room temperature and then put it in a cold storage at -20℃ for pre-freezing.

[0098] (4) Freeze-drying: Place the pre-frozen sea cucumber porridge into a vacuum freeze dryer and dry it for 8 to 20 hours at a vacuum degree of 40 to 250 Pa and a temperature of -75°C.

[0099] The sea cucumber freeze-dried porridge product produced by the above method has a soft, sweet taste, uniform color per tube, unique flavor, and high nutritional and therapeutic value.

[0100] Sensory evaluation: The sensory evaluation panel consisted of 10 members (5 men and 5 women) with extensive sensory evaluation experience. They evaluated the samples according to the fishy smell evaluation criteria in Table 7. The fishy smell of the enzymatic hydrolysate was used as a reference, with a score of 4. The stronger the fishy smell, the higher the score. Members could retain one decimal place as appropriate. The scores were averaged for data analysis.

[0101] Table 7 Sensory evaluation criteria for the fishy smell of the samples:

[0102]

[0103] Functional evaluation experiment grouping: Following the methods in Examples 1 and 2, deodorized sea cucumber intestinal peptides were prepared. Based on Example 3, the following products were prepared: sea cucumber intestinal peptide freeze-dried porridge, sea cucumber intestinal peptide and wolfberry compound freeze-dried porridge, and wolfberry freeze-dried porridge samples. Healthy female C57BL6 / J mice (SPF grade), 8 weeks old, weighing 19.0±1.0g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Animals had free access to water, and all conditions except diet were the same as in Example 1. After 3 days of acclimatization, the mice underwent bilateral ovariectomy to establish an osteoporosis model. All mice were randomly divided into four groups of 6 per group: model group (OVX), sea cucumber intestinal peptide freeze-dried porridge (SCIP-O), sea cucumber intestinal peptide and wolfberry compound freeze-dried porridge (SCIP-P), and wolfberry freeze-dried porridge (SCIP-Q). During the experiment, mice were given 5g of the corresponding freeze-dried porridge once daily for 90 consecutive days.

[0104] Reference Example 1 for serum biochemical indicators and bone mineral density measurement.

[0105] Sensory evaluation is the most direct and practical method for inspecting food. Sensory evaluation results indicate that (…). Figure 13 The porridge made with sea cucumber intestinal peptides and goji berries showed improved scores in terms of color and umami flavor, with a moderate sweetness and significantly reduced bitterness. This indicates that the enzymatic fermentation reaction increased the content of volatile substances with umami flavor, possibly due to the action of small molecule peptides. Overall, the porridge made with sea cucumber intestinal peptides and goji berries has a rich and delicious taste.

[0106] Functional evaluation results show that ( Figure 14-15 Compared with the model, all different freeze-dried porridge formulations increased serum OCN levels and inhibited TRAP levels. The combination of sea cucumber intestinal peptide and wolfberry (SCIP-LB) showed the most significant effect compared to the groups with only wolfberry (LB) and sea cucumber intestinal peptide (SCIP). Further analysis using bone mineral density measurements revealed… Figure 16 The SCIP-LB group significantly improved bone density in mice, indicating that the combination of sea cucumber intestinal peptide and wolfberry can effectively improve osteoporosis.

[0107] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A sea cucumber intestinal peptide, the amino acid sequence of which is shown in SEQ ID NO.

7.

2. The use of the sea cucumber intestinal peptide according to claim 1 in the preparation of an anti-osteoporosis drug.

3. The method for preparing sea cucumber intestinal peptide according to claim 1, characterized in that: Synthesized by Fmoc-peptide solid-phase synthesis method.

4. The method for preparing sea cucumber intestinal peptide according to claim 1, characterized in that... Includes the following steps: (1) Enzymatic hydrolysis: Take an appropriate amount of sea cucumber intestines, cut them into small pieces, add water, and then sterilize the sea cucumber intestine homogenate to obtain sterile sea cucumber intestine homogenate. Add papain and neutral protease in a 1:1 ratio to the enzymatic hydrolysate, at a concentration of 3% of the substrate dry weight, and perform enzymatic hydrolysis. After enzyme inactivation, centrifugation is performed, and the supernatant is collected to obtain the enzyme hydrolysate, or the supernatant is freeze-dried and then dissolved to obtain the enzyme hydrolysate. (2) Fermentation: After adding lactic acid bacteria to the enzymatic hydrolysate for fermentation, yeast fermentation continues. The amounts of lactic acid bacteria and yeast added are... It is 5% of the dry weight of sea cucumber intestines; after fermentation, it is sterilized to obtain sea cucumber intestine peptides.

5. The preparation method according to claim 4, characterized in that: In step (1), the enzymatic hydrolysis conditions are 55℃ for 5 hours.

6. The preparation method according to claim 4, characterized in that: In step (2), the fermentation conditions for lactic acid bacteria are 37℃. Fermentation time: 8 hours; the fermentation conditions for yeast are 42℃ for 8 hours.

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