A sea cucumber intestinal peptide and its application in preparing anti-osteoporosis products

Abstract

The present invention belongs to the technical field of sea cucumber intestines and sea cucumber intestinal peptide applications, and specifically relates to a sea cucumber intestinal peptide and its application in the preparation of anti-osteoporosis products. The present invention obtains sea cucumber intestinal peptides by enzymatic hydrolysis with commercial proteases, and then achieves the purpose of removing fishy smell by microbial fermentation. The dual treatment of enzymatic hydrolysis and fermentation makes the sea cucumber intestinal peptides have higher biological activity, and obtains 10 peptide segments with the best effect, which can be used in the preparation of anti-osteoporosis products.

Description

Technical Field

[0001] The invention belongs to the technical field of application of sea cucumber intestines and sea cucumber intestinal peptides, and specifically relates to a sea cucumber intestinal peptide and application thereof in the preparation of anti-osteoporosis products. Background Art

[0002] Osteoporosis, as the most common metabolic bone disease, is characterized by bone microstructure degradation and increased brittleness. With the increasing severity of population aging, a variety of chronic metabolic diseases, including osteoporosis, have a severe impact on the elderly population. Current osteoporosis treatment drugs usually alleviate the progression of the disease by inhibiting bone resorption. It is worth noting that nutritional support or functional foods have been proven to improve osteoporosis and other related diseases by increasing bone density.

[0003] Sea cucumber is a kind of marine delicacy that can be used as both medicine and food. It is rich in protein, amino acids, fat, polypeptides, polysaccharides, saponins and other active substances, and therefore has high nutritional and medicinal value. In the process of pre-processing of sea cucumbers, a large amount of sea cucumber intestine by-products are produced, accounting for about 30% to 40% of the total weight of fresh sea cucumbers (that is, the production of sea cucumber intestines can reach 60,000 to 80,000 tons / year). "Chinese Materia Medica" also pointed out that sea cucumber intestines have the effects of calming the nerves, harmonizing the stomach, detoxifying and clearing rashes, and promoting tissue regeneration and hemostasis. Modern nutritional research has found that the nutritional value of sea cucumber intestines even exceeds that of sea cucumber body walls in some aspects. Therefore, the technology of using protease enzymatic hydrolysis is a method to efficiently obtain the nutrients in sea cucumber intestines, obtain protein polypeptide hydrolysates that are easy to absorb and have health and medicinal functions, and create significant economic and social benefits. However, after enzymatic hydrolysis of sea cucumber intestines, some aldehydes, alcohols, ketones and hydrocarbons will be produced, which makes the hydrolyzate have a heavy fishy smell and is difficult to further process and utilize. Therefore, in the further application of sea cucumber intestinal peptides, removing the fishy smell is the technical key to achieving high-value utilization and industrial development of sea cucumber intestinal hydrolysate. Summary of the invention

[0004] The technical problem to be solved by the present invention is that sea cucumber intestines, as the most important byproduct of the sea cucumber processing process, have not been fully utilized. Nutritional functional ingredients such as small molecule protein polypeptide hydrolysates that are easy to absorb and have health and medicinal functions can be obtained through protease hydrolysis. In addition, in the processing of sea cucumber and other related products, the removal of fishy smell is an important problem, so there is still a need to develop sea cucumber intestine products that are both nutritious and delicious.

[0005] In order to solve the above problems, the present invention provides a sea cucumber intestinal peptide, which is obtained by enzymatic hydrolysis with commercial protease and then fermented by microorganisms to achieve the purpose of removing fishy smell. The dual treatment of enzymatic hydrolysis and fermentation makes the sea cucumber intestinal peptide have higher biological activity and can be used in the preparation of anti-osteoporosis products.

[0006] To achieve the above object, the present invention is implemented by the following technical scheme: a method for preparing sea cucumber intestinal peptide comprises 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 a sterile sea cucumber intestine homogenate; add papain and neutral protease in a ratio of 1:1 to the enzymatic hydrolyzate, and the added amount is 3% of the dry weight of the substrate. After enzymatic hydrolysis, the enzymes are inactivated, centrifuged, and the supernatant is taken to obtain an enzymatic hydrolyzate or the supernatant is freeze-dried and dissolved to obtain an enzymatic hydrolyzate; dietary supplementation of papain and neutral protease (1:1) 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: After adding lactic acid bacteria to the enzymatic hydrolysate for fermentation, yeast fermentation is continued. The amount of lactic acid bacteria and yeast added is 5% of the dry weight of the sea cucumber intestines. After the fermentation is completed, sterilization is carried out to obtain sea cucumber intestinal peptides. Fermentation can remove the fishy smell. At the same time, the sea cucumber intestinal peptides that are first enzymatically hydrolyzed and then fermented have higher biological activity.

[0009] First, sea cucumber intestines will produce bitter amino acids and peptides after enzymatic hydrolysis. If yeast is used for fermentation after enzymatic hydrolysis with sufficient carbon and nitrogen sources, a large amount of alcohol will be produced, which is not suitable for most people. However, using lactic acid bacteria for fermentation first will maintain the taste and mellowness of sea cucumbers and reduce the content of bitter peptides in the enzymatic hydrolysate. At the same time, it effectively prevents yeast from producing alcohol.

[0010] Sea cucumber intestines are hydrolyzed into amino acids and soluble polypeptides during the enzymatic hydrolysis process, which also causes changes in the content of free amino acids, inorganic ions, etc. Subsequently, in the production process of enzymatic hydrolysis followed by fermentation, soluble polypeptides are decomposed into amino acids and peptides with smaller molecular weights due to the combined action of microbial metabolism and microbial enzymes. Compared with peptides produced by other processes, these small molecule peptides not only remove the fishy smell of the hydrolyzate, but are also easier to digest and absorb. According to the mass spectrometry results, a higher proportion of Gly, Pro, Leu and Ile were found at the N-terminus and C-terminus of the peptides. These Gly-Pro, Ile-Gly and Leu-Gly sequences are potential osteogenic sequences.

[0011] Furthermore, the enzymatic hydrolysis condition in step (1) is 55° C. for 5 h.

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

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

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

[0015] .

[0016] The above-mentioned sea cucumber intestinal peptide can also be synthesized artificially.

[0017] An application of the 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 of rice, 10-20 parts of red beans, 1-3 parts of lotus seeds, 2-5 parts of longan, 4-6 parts of oatmeal rice, 30-40 parts of sea cucumber intestinal peptide, and 30-40 parts of wolfberry.

[0020] Lycium barbarum is a traditional Chinese medicine that is both a medicine and a food. As early as in the "Shennong Bencao Jing", its effects of nourishing the liver and kidneys, strengthening tendons and bones were recorded. In recent years, studies have found that Lycium barbarum has a variety of pharmacological activities such as anti-inflammatory, antioxidant, and anti-cancer because it is rich in polysaccharides. Animal experiments also show that Lycium barbarum polysaccharides can improve rat femoral bone density and trabecular bone structure; promote the proliferation of preosteoblasts 3T3-E1, and increase intracellular calcium ion concentration and type I collagen content. Based on the potential bone-promoting effect of Lycium barbarum, the present invention prepares a freeze-dried porridge with anti-osteoporosis effect by combining sea cucumber intestinal peptide and Lycium barbarum.

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

[0022] (1) Prepare the ingredients: prepare rice, red beans, lotus seeds, longan, and oatmeal, wash them, pour them into a container, and soak them in warm water;

[0023] (2) Boiling: Add water to the soaked materials and boil them in a container over high heat. Add wolfberry and sea cucumber intestinal peptide and continue boiling. Add seasonings after boiling and stir, and finally simmer.

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

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

[0026] Furthermore, the seasoning in step (2) is edible salt, white sugar, water, vegetable blending oil, vitamin E, and sodium D-isoascorbate.

[0027] With the improvement of residents' living standards and the development of a fast-paced lifestyle, more and more consumers are increasingly favoring foods that are convenient, fast and nutritious. In recent years, vacuum freeze drying technology has been widely used in the field of instant food processing, by giving instant products a porous structure to achieve the goal of rapid rehydration. The drying process can greatly reduce the moisture content of the product and retain its nutrients to the greatest extent. Therefore, the present invention develops a sea cucumber freeze-dried porridge suitable for the elderly, aiming to provide a technical reference for the industrial production of sea cucumber freeze-dried porridge products.

[0028] The beneficial effects of the present invention are:

[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 using microbial fermentation. The optimal flavor conditions of sea cucumber intestinal peptides were determined through electronic tongue and electronic nose, that is, enzymatic hydrolysis followed by fermentation. Its effect on improving osteoporosis was verified through animal experiments. Based on the mass spectrometry results, the active potential osteogenic peptides in sea cucumber intestines were given, which increased the utilization value of sea cucumber intestines and expanded the source of dietary supplements for increasing bone density and delaying bone loss.

[0030] (2) The freeze-dried sea cucumber porridge prepared by the present invention provides a freeze-dried porridge formula with anti-osteoporosis effect for the first time and combines sea cucumber intestinal peptide with wolfberry, which can completely preserve the flavor and nutritional value of the freeze-dried sea cucumber porridge and improve the utilization rate of sea cucumber intestines. The prepared freeze-dried sea cucumber porridge is delicious and has a novel taste. It is easier for the elderly to digest and absorb, and is also more attractive to young consumers.

[0031] (3) The method for preparing sea cucumber intestinal peptide provided by the present invention has good repeatability and can be preferably used in industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[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 by electronic tongue of sea cucumber intestinal hydrolysates with different deodorization treatment combinations.

[0037] Figure 6 : Electronic nose radar chart of sea cucumber intestinal hydrolysate with 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] Fig. 9 : Effects of enzymatic fermentation products on bone mineral density in osteoporotic mice.

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

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

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

[0044] Fig.13 : Sensory radar charts of MRPs of freeze-dried porridge with different formulas.

[0045] Fig.14 :Effects of freeze-dried porridge with different formulas on serum osteocalcin in osteoporotic mice.

[0046] Fig.15 :Effects of freeze-dried porridge with different formulas on serum tartrate-resistant alkaline phosphatase in osteoporotic mice.

[0047] Fig.16 :Effects of freeze-dried porridge with different formulas on bone mineral density in osteoporotic mice. DETAILED DESCRIPTION

[0048] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] Unless otherwise specified, the materials and reagents used in the following examples were purchased from the market.

[0050] Embodiment 1:

[0051] A method for preparing an anti-osteoporosis sea cucumber intestinal hydrolysate comprises the following steps:

[0052] Preparation of sea cucumber intestine hydrolysate: Take an appropriate amount of sea cucumber intestine and cut it into pieces of 1 cm in size. Add ultrapure water at a material-liquid ratio of 1:5 (m / v), add protease and stir evenly, adjust the pH to the optimal pH range of each protease with HCL solution (1 mol / L) and NaOH solution (1 mol / L) respectively (Table 2), and perform enzymolysis at the optimal temperature of 55℃ for 5 h. After the enzymolysis is completed, the sample is filtered and the supernatant is inactivated in a 90℃-100℃ water bath for 15 min to obtain sea cucumber intestine hydrolysate. After filtering the sea cucumber intestine hydrolysate through a 0.45μm microporous filter membrane, use an ultrafiltration tube with a molecular weight cutoff of 3 kDa and centrifuge at 3500 r / min for 30 min to obtain a small molecule hydrolysate. The commercial enzymes commonly used in the market for enzymatic hydrolysis of seafood are papain (100,000 u / g), neutral protease (50,000 u / g), and flavor protease (30,000 u / g) (all purchased from Solai Technology Co., Ltd.). This patent intends to fix the basic enzymatic hydrolysis parameters as 3% enzyme addition (relative to substrate dry weight), 1:5 (m / v) material-liquid ratio, 5 h enzymatic hydrolysis time, and 55°C enzymatic hydrolysis temperature. According to different enzyme ratios (single enzyme, double enzyme, triple enzyme, etc.), 13 groups of enzymes were used to enzymatically hydrolyze sea cucumber intestines, and the optimal enzymatic hydrolysis combination was determined through animal experiments (Table 3).

[0053] Table 2 Optimal conditions and enzyme activity of protease:

[0054] .

[0055] Table 3 Protease combinations and ratios:

[0056] .

[0057] Animal experiment: Healthy female C57BL6 / J mice (SPF grade), 8 weeks old, weighing 19.0±1.0 g, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. The animal breeding environment was as follows: the temperature was maintained at 22°C~24°C, the humidity was maintained at 45%~55%, and the light / dark cycle was 12 h. During the experiment, the mice were free to eat (AIN-93G type feed) and drink water.

[0058] Experimental grouping and design: After 3 days of adaptive feeding, the mice underwent bilateral ovariectomy to establish an osteoporosis model. All mice were randomly divided into 13 groups, including normal group (Normal), model group (Model), positive control group (alendronate sodium, ALF, 0.15 μg / kg·bw), and sea cucumber intestinal enzymatic hydrolysis group (a, b, c, d, e, f, g, h, i, j, k, l, m), with 6 mice in each group. During the experiment, the mice were gavaged with the corresponding test substance (200 mg / kg·bw) at a volume of 10 mL / kg·bw, once a day, for 90 consecutive days.

[0059] Serum biochemical index determination: The levels of OCN, OPG and tartrate-resistant acid phosphatase (TRAP) in serum were determined using ELISA kits, and the specific operations were carried out according to the kit instructions.

[0060] Determination of bone mineral content: After grinding the femoral tissue, a mixed solution of nitric acid and perchloric acid (5:2, v / v) was added for digestion. After the volume was fixed, the calcium and phosphorus contents were determined using an AA-6880 atomic absorption spectrophotometer.

[0061] Bone density measurement: After the femoral tissue was fixed in 10% neutral formaldehyde for 48 h, its bone density was measured by GK99 dual-energy X-ray absorptiometry. The scanning conditions were: small animal mode, voltage 80 kV, current 165 mA.

[0062] OCN and OPG are matrix proteins secreted by osteoblasts, and TRAP is a protein degrading enzyme secreted by osteoclasts. Figure 1-Figure 3 As shown, the results of serum biochemical levels showed that compared with the sham operation group (Sham), the levels of serum bone formation markers OCN and OPG in the model group (OVX) mice were significantly reduced, and the bone resorption marker TRAP was significantly increased, entering a "low bone formation" state. The supplementation of different complex enzyme combinations significantly increased the serum bone formation markers OCN and OPG. It is worth noting that the d group (papaya: neutral = 1:1) had the highest effect, increasing the levels by 44.9% and 17.3%, respectively (p < 0.05), and inhibited the abnormal increase of the bone resorption marker TRAP (27.0%, p < 0.05), indicating that dietary supplementation of papaya and neutral protease compound (1:1) enzymatic hydrolysate can promote bone formation in osteoporotic mice.

[0063] The results of mouse bone density were as follows Figure 4 As shown, compared with the sham operation group (Sham), the bone density of the model group (OVX) mice was significantly reduced. Dietary supplementation with a combination of protease and enzymatic hydrolysates could significantly increase the density of mice, among which the papaya and neutral protease combination group (1:1) had the most significant effect.

[0064] The rate and total amount of mineral deposition in bones can reflect the overall state of bones. As shown in Table 4, the bone mineral content (bone calcium, bone phosphorus) of the model group (OVX) was significantly reduced compared with the sham group, and the enzymatic hydrolysates of different combinations of proteases had an improvement effect. It is worth noting that the peptide in group d (papaya: neutral = 1:1) significantly increased the calcium-phosphorus ratio and inhibited the loss of minerals, indicating that dietary supplementation of papaya and neutral protease combination group (1:1) enzymatic hydrolysates can delay bone loss in osteoporotic mice.

[0065] Table 4 Effects of different combinations of protease hydrolysates on bone mineral content in osteoporotic mice:

[0066] .

[0067] Note: ** indicates P < 0.01 significant level compared with the Sham group; ## indicates P < 0.01 significant level compared with the OVX group.

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

[0069] Embodiment 2:

[0070] This example provides a method for removing the fishy smell of sea cucumber intestinal peptides and the application of the product thereof, which specifically comprises the following steps:

[0071] Considering that some aldehydes, alcohols, ketones and hydrocarbons are produced during the enzymatic hydrolysis of sea cucumber intestines, which makes the hydrolyzate produce a strong fishy smell, and will 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 pieces of 1 cm in size. Add ultrapure water at a solid-liquid ratio of 1:5 (m / v), sterilize the sea cucumber intestine homogenate at 121℃ for 10 min, and obtain 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 it at 37℃ for 8 h, and then continue to add a certain amount of yeast (Angel Yeast Co., Ltd.) and ferment it at 42℃ for 8 h. After fermentation, sterilize the sterile sea cucumber intestine homogenate at 100℃, and the amount of lactic acid bacteria and yeast added is 5% of the dry weight of the sea cucumber intestine.

[0073] Experimental grouping and design: Four combinations were set up, namely, fermentation followed by enzymolysis group (A), enzymolysis followed by fermentation group (B), simple fermentation group (C) and simple enzymolysis group (D). For group A and group B, according to the enzymolysis combination and enzyme addition amount determined in Example 1, complex enzymes were added to the sterile sea cucumber intestine homogenate before and after fermentation at 3% of the weight of the sea cucumber intestine. After inactivating the enzyme, the mixture was centrifuged at 3500 r / min for 30 min at room temperature, and the supernatant was taken and freeze-dried.

[0074] Electronic tongue and electronic nose analysis: Accurately pipette 30 mL of the test solution into the electronic tongue sample cup. Before measurement, perform self-test, activation, calibration and diagnosis on the electronic tongue to ensure the reliability and stability of the collected data. Prepare the Ref Sol reference solution with 30 mmol / L KCl solution and 0.3 mmol / L tartaric acid solution. Place the sensor in the reference solution and return to zero for 30 seconds before starting the measurement. The test time is 30 seconds. After the test, wash with the reference solution for 3 seconds and perform the aftertaste measurement again for 30 seconds. Repeat each sample 4 times, and take the last 3 times as the test result.

[0075] Accurately transfer 30 mL of sample into the electronic nose headspace bottle, seal it with a cap, and let it stand at 26°C for 30 min before testing. Determination parameters: cleaning time 100 s, zeroing time 5 s, pre-injection time 8 s, determination time 60 s, carrier gas flow rate 400 mL / min, each sample is measured 4 times, and the last 3 times are taken as the test results.

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

[0077] .

[0078] Animal experiment: Healthy female C57BL6 / J mice (SPF grade), 8 weeks old, weighing 19.0±1.0 g, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. The animals were given free access to water, and the conditions other than diet were the same as those in Example 1. After 3 days of adaptive feeding, the mice underwent bilateral ovariectomy to establish an osteoporosis model. According to the above experimental grouping, the following 5 groups were set up: model group (OVX), A, B, C, and D groups (n=6). During the experiment, the mice were gavaged with the corresponding test substance (200 mg / kg·bw) at a volume of 10 mL / kg·bw, once a day, for 90 consecutive days.

[0079] Serum biochemical indexes and bone density determination reference example 1.

[0080] The characteristic parameters of the electronic tongue were subjected to principal component analysis, and the results were as follows: Figure 5As shown in the figure, the contribution rate of the first principal component of different treatment groups is 75.86%, the contribution rate of the second principal component is 12.66%, and the sum of PC1 and PC2 is 88.52%. In the PCA results, the greater the distance between the samples on the PC1 axis, the greater the difference between the samples. As can be seen from the figure, compared with group D, groups A, B and C do not overlap on the PC1 axis, indicating that the samples have better differentiation after deodorization and the flavors are independent of each other. Group B is farther away from group A after enzymatic hydrolysis followed by fermentation, indicating that the deodorization method of enzymatic hydrolysis followed by fermentation has a significant effect on the flavor of sea cucumber sausage.

[0081] Electronic noses can use artificial lipid membrane sensor technology to detect various taste indicators of food. The electronic nose radar image of sea cucumber intestine hydrolysate before and after deodorization is shown in the figure below. Figure 6 As shown in the figure, compared with the undeodorized samples, the lactic acid bacteria complex treatment significantly reduced the volatile substances. The results were consistent with the sensory evaluation results.

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

[0083] Based on the above results, the samples of group B were desalted, and each sample was performed in triplicate. The peptide sequences were determined using a Q Exactive mass spectrometer, and the raw data files were searched against 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: nonspecific; fragment mass tolerance: 0.02 Da; precursor mass tolerance: 10 ppm; the false discovery rate (FDR) of peptide identification results was ≤1%. Peptide Ranker was used to predict the possibility that the peptide sequence had biological activity for the identified peptides.

[0084] Table 6 Sequence identification and activity prediction of sea cucumber intestinal enzymatic fermentation peptides:

[0085] .

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

[0087] Embodiment 3:

[0088] This example provides an application of sea cucumber intestinal peptide, which specifically includes the following steps:

[0089] The sea cucumber intestinal bioactive peptides predicted in Example 2 were biosynthesized by Shanghai Shenggong Co., Ltd. through Fmoc-peptide solid phase synthesis. According to HPLC and MS sequence analysis, the purity of the ten peptides GPAGPTGPTGPA (①), DGGDVFR (②), GPAGPTGPTGPAG (③), MLLILLL (④), IGPIGPTG (⑤), IGPIGPT (⑥), PGPGPF (⑦), PGGPFP (⑧), MLPLPVM (⑨), and LGPLGP (⑩) was greater than 98.89%. Chemically synthesized polypeptides have high purity and good safety in clinical application because of the controllable raw materials and processes. However, in the process of synthesis, due to the large number of steps involved and the use of multiple solvents. It is inevitable that the biological activity of the polypeptide will be affected to a certain extent, so it is necessary to further verify the biological activity of the synthesized polypeptide.

[0090] Experimental grouping and design: Healthy female C57BL6 / J mice (SPF grade), 8 weeks old, weighing 19.0±1.0 g, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. The animals were given free access to water, and the other conditions except for the diet were the same as those in Example 1. After 3 days of adaptive feeding, the mice underwent bilateral ovariectomy to establish an osteoporosis model. The following 12 groups were set up: model group (OVX), enzymatic fermentation group (B) and synthetic peptide intervention group (①-⑩) (n=5). During the experiment, the mice were gavaged with the corresponding enzymatic fermentation group test substance (200 mg / kg·bw) and synthetic peptide (100 mg / kg·bw) at a volume of 10 mL / kg·bw once a day for 90 consecutive days.

[0091] Serum biochemical indexes and bone density determination reference example 1.

[0092] In order to verify the biological activity of the above peptides, chemically synthesized peptides were further used to intervene in osteoporotic mice. Figure 10-11 As shown in the results, compared with the model (OVX) group, the synthetic peptides could significantly increase serum osteocalcin levels, reduce the level of bone resorption marker TRAP, and improve the bone density of osteoporotic mice ( Fig.12 It is worth noting that the promoting effect of several synthetic peptides on osteoporotic mice at a dose of 100 mg / kg·bw was comparable to that of group B at a dose of 200 mg / kg·bw, and their effective concentrations were reduced by about 1 times, suggesting that these synthetic peptides may be potential active factors in SCIP.

[0093] Embodiment 4:

[0094] This example provides a method for preparing freeze-dried sea cucumber porridge for preventing osteoporosis and its specific application, which is specifically used to delay bone loss and improve bone density in patients with osteoporosis, and specifically includes the following steps:

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

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

[0097] (3) Pre-freezing in molds: Divide the cooked seafood porridge into molds, cool to room temperature, and then place 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 at a vacuum degree of 40 to 250 Pa and a temperature of -75°C for 8 to 20 h.

[0099] The anti-osteoporosis sea cucumber freeze-dried porridge product produced by the above method has a soft, glutinous and sweet taste, a uniform color per tube, a 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 rich sensory evaluation experience. The samples were evaluated 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 according to the actual situation, and take the average value after scoring for data analysis.

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

[0102] .

[0103] Functional evaluation experimental grouping: According to the methods in Examples 1 and 2, deodorized sea cucumber intestinal peptides were prepared, and according to 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±1.0 g, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. The animals had free access to water, and the other conditions except for the diet were the same as in Example 1. After 3 days of adaptive feeding, the mice underwent bilateral ovariectomy to establish an osteoporosis model. All mice were randomly divided into four groups, including a 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), with 6 mice in each group. During the experiment, the mice were given the corresponding freeze-dried porridge at a food intake of 5 g, once a day, for 90 consecutive days.

[0104] Serum biochemical indexes and bone density measurement reference example 1.

[0105] Food sensory evaluation is the most direct and practical method for testing food. The results of sensory evaluation show that ( Fig.13 ), the color and umami scores of the porridge with sea cucumber intestinal peptide and wolfberry increased, its sweetness was moderate, and its bitterness was significantly reduced, which shows that the enzymatic fermentation reaction increased the content of volatile substances with umami taste, which may be due to the effect of small molecule peptides. In general, the sea cucumber intestinal peptide and wolfberry compound group has a mellow taste and delicious taste.

[0106] The functional evaluation results showed that ( Figure 14-15 ), compared with the model, different formulas of freeze-dried porridge can increase serum OCN levels and inhibit TRAP levels. Compared with the group with only wolfberry (LB) and the group with only sea cucumber intestinal peptide (SCIP), the group with sea cucumber intestinal peptide and wolfberry (SCIP-LB) had the most significant effect. Further bone density measurement revealed that ( Fig.16 ), the SCIP-LB group significantly increased the bone density of mice, indicating that the combination of sea cucumber intestinal peptide and wolfberry can effectively improve osteoporosis.

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

Claims

1. An application of a sea cucumber intestinal peptide composition in the preparation of an anti-osteoporosis product, characterized in that: The sea cucumber intestinal peptide composition comprises sea cucumber intestinal peptides with amino acid sequences as shown in SEQ ID NO. 1, 2, 4-6.

2. The use according to claim 1, characterized in that: The product is freeze-dried sea cucumber intestine porridge.

3. The use according to claim 2, characterized in that: The freeze-dried sea cucumber intestine porridge comprises the following ingredients: 5-10 parts of rice, 10-20 parts of red beans, 1-3 parts of lotus seeds, 2-5 parts of longan, 4-6 parts of oatmeal, 30-40 parts of the sea cucumber intestine peptide composition according to claim 1, and 30-40 parts of wolfberry.

4. The use according to claim 2 or 3, characterized in that The preparation method of freeze-dried sea cucumber intestine porridge comprises the following steps: (1) Prepare the ingredients: prepare rice, red beans, lotus seeds, longan, and oatmeal, wash them, pour them into a container, and soak them in warm water; (2) Boiling: Add water to the soaked materials and boil them in a container over high heat. Add wolfberry and sea cucumber intestinal peptide composition after boiling, continue boiling, add seasoning after boiling, stir, and finally stew; (3) Pre-freezing in molds: Divide the cooked seafood porridge into molds, cool to room temperature, and then place in a cold storage at -20°C for pre-freezing; (4) Freeze-drying: Place the pre-frozen sea cucumber porridge into a vacuum freeze dryer and dry it at a vacuum degree of 40 to 250 Pa and a temperature of -75°C for 8 to 20 h.

5. The use according to claim 4, characterized in that: The seasoning in step (2) is edible salt, white sugar, water, vegetable blending oil, vitamin E, and D-sodium isoascorbate.

6. The method for preparing the sea cucumber intestinal peptide according to claim 1, characterized in that The following steps are involved: (1) Enzymatic hydrolysis: Take an appropriate amount of sea cucumber intestines, chop them into pieces, add water, and sterilize the sea cucumber intestine homogenate to obtain a sterile sea cucumber intestine homogenate; add papain and neutral protease in a ratio of 1:1 to the sea cucumber intestine homogenate, and the added amount is 3% of the dry weight of the substrate. After enzymatic hydrolysis, inactivate the enzymes, centrifuge, and take the supernatant to obtain a sea cucumber intestine enzymatic hydrolysate or freeze-dry the supernatant and then dissolve it to obtain a sea cucumber intestine enzymatic hydrolysate; filter the sea cucumber intestine enzymatic hydrolysate through a 0.45 μm microporous filter membrane, and then use an ultrafiltration tube with a molecular weight cutoff of 3 kDa to centrifuge at 3500 r / min for 30 min to obtain a small molecule enzymatic hydrolysate; the enzymatic hydrolysis conditions are 55°C for 5 h; (2) Fermentation: After adding lactic acid bacteria to the small molecule enzymatic hydrolysate for fermentation, yeast fermentation is continued. The amount of lactic acid bacteria and yeast added is 5% of the dry weight of the sea cucumber intestine. After the fermentation is completed, sterilization is carried out to obtain sea cucumber intestinal peptides. The fermentation conditions of lactic acid bacteria are 37°C for 8 h; the fermentation conditions of yeast are 42°C for 8 h.

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