A preparation method and application of sea cucumber peptide

Abstract

The present invention provides a preparation method and application of sea cucumber peptides, belonging to the field of marine biotechnology. The present invention improves the extraction process of sea cucumber peptides, and adopts a method of biological enzymatic hydrolysis plus low-temperature vacuum cooking, which can not only effectively reduce the loss of heat-sensitive vitamins, polysaccharides, and trace elements, and keep the nutritional components of sea cucumbers as much as possible, but more importantly, it can make the sea cucumber peptides have a better flavor. As the cooking temperature decreases, the protein and fat denaturation in the peptide powder is also reduced to a certain extent, which can reduce the flocculation precipitation caused by protein denaturation and the poor sensory perception caused by lipid denaturation of the peptide powder during the product utilization period; finally, it can also improve the extraction rate of sea cucumber peptides, reduce heat energy consumption, and reduce processing costs. It has been proven that the marine active peptide energy drink based on sea cucumber peptides has good flavor, stable shelf life and no precipitation, and is suitable for wide application in beverage products.

Description

Technical Field

[0001] The invention belongs to the technical field of marine biological active substances, and in particular, relates to a preparation method of sea cucumber peptide and application thereof. Background Art

[0002] At present, energy drinks in China are mainly caffeinated. Data show that about 80% of the world's population drinks caffeinated products every day, the most common of which are coffee (71%), soft drinks (16%) and tea (12%). Relevant surveys show that long-term drinking of caffeinated drinks can lead to addiction, and people who drink caffeinated functional drinks are more likely to have symptoms of cardiovascular rupture. Children and adolescents have weak caffeine metabolism ability, and a small amount of caffeine is likely to reach a toxic dose and cause harm to them. Therefore, if there is a product without caffeine that can achieve or even exceed the effect of caffeinated drinks, it will be a beneficial choice for both office workers with high work intensity and teenagers taking exams to enter school.

[0003] Currently, protein peptide beverage products have initially entered the market and are favored by consumers as a healthy drink among energy drinks. However, protein peptide beverage products currently also have many problems, such as easy precipitation during the shelf life, and the addition of protein peptides has a negative impact on the flavor of the product. Summary of the invention

[0004] The purpose of the present invention is to provide a preparation method of sea cucumber peptide and application thereof, so as to make up for the deficiencies of the prior art.

[0005] To achieve the above object, the present invention is achieved through the following technical solutions:

[0006] A method for preparing sea cucumber peptide comprises the following steps:

[0007] (1) pre-treating sea cucumbers and preparing sea cucumber homogenate;

[0008] (2) adding neutral protease and flavor protease for enzymolysis, and killing the enzyme after the enzymolysis is completed to obtain a crude extract of sea cucumber peptides;

[0009] (3) the crude sea cucumber peptide extract is subjected to low-temperature vacuum cooking;

[0010] (4) decolorizing the sea cucumber peptide;

[0011] (5) Collecting the components with a molecular weight of ≤10 KDa, vacuum concentrating, and spray drying to obtain sea cucumber peptides with a molecular weight range of 200-1800 Da.

[0012] Furthermore, in step (1), the pretreatment includes: removing the internal organs and inner wall muscle layer of the sea cucumber, washing it, and crushing it;

[0013] Soak in pure water below 10°C for 3-7 days (observe the salinity during this period and soak until the salinity is undetectable); mince the sea cucumber body wall and then use a colloid mill to prepare a homogenate.

[0014] Furthermore, in step (2), water, 0.01-0.2 wt% neutral protease (on a dry basis), and 0.05-0.3 wt% flavor protease (on a dry basis) are added, and enzymolysis is performed for 1-7 hours at a pH of 7-8, and the enzyme is inactivated at 95°C to obtain a crude extract of sea cucumber peptides.

[0015] Furthermore, in step (3), the low-temperature vacuum cooking conditions are: temperature range is 50°C to 80°C, and vacuum degree is 0.00MPa to -0.09MPa.

[0016] Furthermore, in step (4), the decolorization treatment may be performed by adding activated carbon and diatomaceous earth, stirring for 15-20 minutes, and then filtering by plate and frame.

[0017] Application of the sea cucumber peptide in the preparation of beverage products.

[0018] A marine active peptide energy drink comprises sea cucumber peptides.

[0019] Furthermore, the energy drink also includes oyster peptides and collagen peptides.

[0020] A marine active peptide energy drink: 10-20g / L crystalline fructose, 20-40g / L maltitol, 1-5g / L pectin, 2-15g / L oyster peptide, 2-10g / L sea cucumber peptide, 30-50g / L collagen peptide, 0.4-0.9g / L natural taurine, 0.1-0.5g / L sodium hyaluronate.

[0021] Furthermore, the preparation method of the oyster peptide is specifically as follows: the edible part of fresh oysters is frozen and then homogenized, 0.05-0.3wt% animal composite protease (on a dry basis) and 0.01-0.2wt% neutral protease (on a dry basis) are added, and enzymolysis is performed for 1-7h to obtain a crude oyster peptide extract; the crude peptide extract is decolorized and then filtered, the filtrate after filtration is desalted, and the desalted solution is spray-dried to obtain oyster peptide.

[0022] The advantages and beneficial effects of the present invention are:

[0023] The present invention improves the extraction process of sea cucumber peptides, and adopts a method of biological enzymatic hydrolysis plus low-temperature vacuum cooking, which can not only effectively reduce the loss of heat-sensitive vitamins, polysaccharides and trace elements and keep the nutritional components of sea cucumbers as much as possible, but more importantly, can make the sea cucumber peptides have better flavor. As the cooking temperature decreases, the protein and fat denaturation in the peptide powder is also reduced to a certain extent, which can reduce the flocculation precipitation caused by protein denaturation and the poor sensory perception caused by lipid denaturation of the peptide powder during the product utilization period; finally, the extraction rate of sea cucumber peptides can be improved, heat energy consumption can be reduced, and processing costs can be reduced.

[0024] The marine active peptide energy drink based on sea cucumber peptides is compounded with marine active peptides such as oyster peptides and collagen peptides. The drink has good flavor, without the special flavors of sea cucumber and oyster, and is stable during the shelf life without precipitation. It has been verified in practice that the anti-fatigue effect of this energy drink is better than the "caffeine" energy drinks on the market. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further described below in conjunction with embodiments.

[0026] Embodiment 1:

[0027] This example studies the enzyme preparation and conditions for enzymolysis by experimental analysis.

[0028] 1. Enzyme preparation screening: By screening five kinds of enzymes, neutral protease, papain, flavor protease, aquatic protease, and animal compound enzymes, different combinations were made, the end point of enzymolysis was determined by the residue content, and the enzyme was inactivated to obtain the crude extract of sea cucumber peptide for sensory evaluation. Referring to the national standard GB / T 16291.1-2012, the sensory evaluation team of this study selected 16 men and women without taste disorders, including 8 women and 8 men (aged between 25 and 32), and the temperature of the tasting room was controlled at room temperature (25±2℃). According to the characteristics of the experiment, the reference solution was used to train the team members. The reference solutions are as follows: sweetness (sucrose solution 16g / L); sourness (citric acid solution 1g / L); astringency (quercetin solution 0.5g / L); bitterness (caffeine 0.5g / L).

[0029] The taste of characteristic flavor is relatively complex. This evaluation uses sea cucumber soup made from sea cucumber water boiled in plain water as the reference solution (10g / L boiled for 60min). The flavor score is 10 points based on the six reference solutions, and the relative flavor of the enzymatic hydrolysate is evaluated. And the score is judged from 0-10, and the higher the score, the heavier the flavor. The evaluation is divided into two steps: smelling and tasting. The sea cucumber cooking liquid is cooled to 35℃ and stored in a 20mL disposable plastic cup. The subjects need to smell its flavor first, and then taste it back and forth on the front, middle, back and sides of the tongue for about 15S. They can spit it out or swallow it, rinse their mouths with distilled water, and repeat the previous operation after 5min. The final result is the average of the scores of each group member.

[0030] Table 1 Sensory evaluation of different sea cucumber enzymatic hydrolysates

[0031] variety Sweetness sour bitterness Astringency Characteristic flavor Neutral protease 2 1 7 none 1 Papain 3 5 4 2 3 Flavor protease 2 3 5 1 2 Aquatic protease 5 3 3 1 3 Animal compound protease 3 1 4 3 4

[0032] Note: All ratings remove the highest and lowest scores and take the average value

[0033] According to Table 1, the characteristic flavors are ranked from high to low as follows: animal compound protease > aquatic protease = papain > flavor protease > neutral protease.

[0034] The order of bitterness from high to low is: neutral protease > flavor protease > papain = animal complex protease > aquatic protease.

[0035] The purpose of this preparation is to obtain a sea cucumber hydrolysate with a bitter main taste and a lighter characteristic flavor, so the neutral protease and flavor protease in Table 1 are comprehensively selected.

[0036] Example 1 (enzymatic hydrolysis + low-temperature vacuum cooking vacuum degree -0.06MPa to -0.1MPa)

[0037] Remove the internal organs and inner wall muscle layer of the sea cucumber, wash it, and crush it; soak it in RO below 10°C for 3-7 days (observe the salinity during the period and soak it until the salinity is undetectable), mince the sea cucumber body wall with a meat grinder, and then prepare it into a homogenate with a colloid mill, add water and 0.01-0.2% neutral protease and flavor protease, enzymolysis for 1-7 hours, pH 7-8, and obtain a crude sea cucumber peptide extract; seal the crude sea cucumber peptide extract and start low-temperature vacuum cooking, the temperature range during the cooking process is 50°C to 80°C, and the vacuum degree is -0.06MPa to -0.1MPa. Release the pressure, add the sea cucumber peptide to activated carbon and diatomaceous earth, stir for 15-20min, then filter by plate and frame, collect the components with a molecular weight of ≤10KDa, vacuum concentrate, spray dry, and obtain a sea cucumber peptide with a molecular weight range of 200-1800Da with a yield of 6.2%.

[0038] Example 2 (hot water extraction + low temperature vacuum cooking vacuum degree -0.06MPa ~ -0.1MPa)

[0039] Remove the internal organs and inner wall muscle layer of the sea cucumber, wash it, and crush it; soak it in RO below 10°C for 3-7 days (observe the salinity during the period and soak it until the salinity is undetectable), mince the sea cucumber body wall with a meat grinder, and then prepare it into a homogenate with a colloid mill, add water and cook it at a temperature of 95°C for 1-7 hours, with a pH of 7-8, to obtain a crude sea cucumber peptide extract; seal the crude sea cucumber peptide extract and start low-temperature vacuum cooking, the temperature range during the cooking process is 80°C to 100°C, and the vacuum degree is -0.06MPa to -0.1MPa. Release the pressure, add the sea cucumber peptide to activated carbon and diatomaceous earth, stir for 15-20min, then filter it with a plate and frame, collect the components with a molecular weight of ≤10KDa, vacuum concentrate them, and spray dry them to obtain a sea cucumber peptide with a molecular weight range of 200-1800Da with a yield of 3.3%.

[0040] Example 3 (enzymatic hydrolysis + low temperature and low pressure cooking, vacuum degree -0.03MPa ~ 0.06MPa)

[0041] Remove the internal organs and inner wall muscle layer of the sea cucumber, wash it, and crush it; soak it in RO below 10°C for 3-7 days (observe the salinity during the period and soak it until the salinity is undetectable), mince the sea cucumber body wall with a meat grinder, and then prepare it into a homogenate with a colloid mill, add water and 0.01-0.2% neutral protease and flavor protease, enzymolysis for 1-7 hours, pH 7-8, and inactivate the enzyme at 95°C to obtain a crude sea cucumber peptide extract; seal the crude sea cucumber peptide extract and start low-temperature vacuum cooking, the temperature range during the cooking process is 50°C to 80°C, and the vacuum degree is -0.03MPa to -0.06MPa. Release the pressure, add the sea cucumber peptide to activated carbon and diatomaceous earth, stir for 15-20min, then filter by plate and frame, collect the components with a molecular weight of ≤10KDa, vacuum concentrate, spray dry, and obtain a sea cucumber peptide with a molecular weight range of 200-1800Da with a yield of 4.6%.

[0042] Example 4 (enzymolysis + low-temperature normal pressure cooking, vacuum degree 0.00MPa~0.03MPa)

[0043] Remove the internal organs and inner wall muscle layer of the sea cucumber, wash it, and crush it; soak it in RO below 10°C for 3-7 days (observe the salinity during the period and soak it until the salinity is undetectable), mince the sea cucumber body wall with a meat grinder, and then prepare it into a homogenate with a colloid mill, add water and 0.01-0.2% neutral protease and flavor protease, enzymolysis for 1-7 hours, pH 7-8, and inactivate the enzyme at 95°C to obtain oyster peptide crude extract; seal the sea cucumber peptide crude extract and start low-temperature vacuum cooking, the temperature range during the cooking process is 50°C to 80°C, and the vacuum degree is 0.00MPa to -0.03MPa. Release the pressure, add the sea cucumber peptide to activated carbon and diatomaceous earth, stir for 15-20min, then filter by plate and frame, collect the components with a molecular weight of ≤10KDa, vacuum concentrate, spray dry, and obtain a sea cucumber peptide with a molecular weight range of 200-1800Da with a yield of 3.7%.

[0044] By comparing the above Example 1 (enzymatic hydrolysis + low-temperature vacuum cooking with a vacuum degree of -0.06MPa to -0.1MPa) and Example 2 (hot water extraction + low-temperature vacuum cooking with a vacuum degree of -0.06MPa to -0.1MPa), it can be seen that under the same extraction conditions (same vacuum degree), the enzymatic hydrolysis yield is nearly twice the water extraction yield. By comparing Examples 1 (enzymatic hydrolysis + low-temperature vacuum cooking with a vacuum degree of -0.06MPa to -0.1MPa), 3 (enzymatic hydrolysis + low-temperature low-pressure cooking, vacuum degree of -0.03MPa to 0.06MPa), and 4 (enzymatic hydrolysis + low-temperature normal pressure cooking, vacuum degree of 0.00MPa to 0.03MPa), it can be seen that under the same enzymatic hydrolysis conditions, the yield of sea cucumber peptides is different with different vacuum degrees, and the yield of sea cucumber peptides increases with increasing vacuum degree.

[0045] In order to determine the effect of different processes on the stability of sea cucumber peptide powder in beverages, the raw materials in Example 1 (enzymatic hydrolysis + low-temperature vacuum cooking), Example 2 (hot water extraction + vacuum extraction) and Example 4 (enzymatic hydrolysis + atmospheric pressure extraction) were prepared into beverages and placed at 4°C and 37°C for 3 months to observe the product stability.

[0046] Table 2 Effects of different enzyme preparations and enzymolysis time on the system

[0047]

[0048] As can be seen from the above table, the sea cucumber peptide prepared in Example 1 did not generate precipitation during the storage period of 3 months at 4°C and 37°C, which is more advantageous for the stability of the later product system; Example 2 flocculated and precipitated in the second week of storage at 4°C and the third week of storage at 37°C, which is not conducive to the stability of the product system; Example 4 flocculated and precipitated in the fourth week of storage at 4°C and the fifth week of storage at 37°C, which is not conducive to the stability of the product system. Therefore, considering the stability of the system, the method of Example 1 was selected to prepare the sea cucumber peptide.

[0049] In order to verify the effect of vacuum extraction technology on the flavor of sea cucumber peptides, the sea cucumber peptide powders prepared in Example 1 (enzymatic hydrolysis + low-temperature vacuum cooking) and Example 4 (enzymatic hydrolysis + low-temperature normal pressure cooking) were selected for beverage compounding.

[0050] Example 5

[0051] Use Example 1 Sea Cucumber Peptide Low GI Sweetener: crystalline fructose 10-20g / L, maltitol 20-40g / L, 1-5g / L pectin, 2-15g / L oyster peptide, 2-10g / L sea cucumber peptide, 30-50g / L collagen peptide, 0.4-0.9g / L taurine, 0.1-0.5g / L sodium hyaluronate, 0.05-0.2g / L flavor.

[0052] Filter through a filter with a filtration level of 5 to 200 μm. After filtration, the liquid is transparent, odorless, and has no visible matter to the naked eye.

[0053] After filtration, the material is sterilized at UHT108-115℃ for 15-45S and then filled.

[0054] Example 6: Use the low GI version of the sea cucumber peptide sweetener in Example 4: crystalline fructose 10-20g / L, maltitol 20-40g / L, 1-5g / L pectin, 2-15g / L oyster peptide, 2-10g / L sea cucumber peptide, 30-50g / L collagen peptide, 0.4-0.9g / L taurine, 0.1-0.5g / L sodium hyaluronate, 0.05-0.2g / L flavor.

[0055] Filter through a filter with a filtration level of 10-50μm. After filtration, the liquid is transparent, odorless, and has no visible matter to the naked eye;

[0056] After filtration, the material is sterilized at UHT108-115℃ for 15-45S and then filled.

[0057] Example 7: Use the sea cucumber peptide version of the sugar alcohol version of the sweetener in Example 1: erythritol 20-30g / L, maltitol 10-20g / L, 1-5g / L pectin, 2-15g / L oyster peptide, 2-10g / L sea cucumber peptide, 30-50g / L collagen peptide, 0.4-0.9g / L taurine, 0.1-0.5g / L sodium hyaluronate, 0.05-0.2g / L flavor.

[0058] Filter through a filter with a filtration level of 10 to 50 μm. After filtration, the liquid is transparent, odorless, and has no visible matter to the naked eye;

[0059] After filtration, the material is sterilized at UHT108-115℃ for 15-45S and then filled.

[0060] Example 8: Use the sea cucumber peptide version of the sugar alcohol version of the sweetener in Example 4: erythritol 20-30g / L, maltitol 10-20g / L, 1-5g / L pectin, 2-15g / L oyster peptide, 2-10g / L sea cucumber peptide, 30-50g / L collagen peptide, 0.4-0.9g / L taurine, 0.1-0.5g / L sodium hyaluronate, 0.05-0.2g / L flavor.

[0061] Filter through a filter with a filtration level of 10 to 50 μm. After filtration, the liquid is transparent, odorless, and has no visible matter to the naked eye;

[0062] After filtration, the material is sterilized at UHT108-115℃ for 15-45S and then filled.

[0063] The sea cucumber peptide powder prepared in Example 1 and Example 4 was used to verify the effects of its application in products on the sensory organs and system.

[0064] Referring to the national standard GB / T 16291.1-2012, the sensory evaluation panel of this study selected 15 males and females without taste disorders, including 9 females and 6 males (aged between 25 and 32), and the temperature of the evaluation room was controlled at room temperature (25±2°C). The beverages prepared in Example 1 and Example 2 were placed in a group, and in Example 7 and Example 4 were placed in a group at 4°C, 25°C and 37°C for 3 months. The appearance and quantity of the samples were consistent, and each taster was given a group of samples. The aroma, sweetness, sourness, fishy smell (whether it was anti-fishy) and freshness of the samples were evaluated differently, and the results of the outstanding flavor were filled in the evaluation table in Table 3.

[0065] Table 3 Directed pairwise comparison evaluation table

[0066]

[0067]

[0068] The statistical results of the differences between the 2 oyster peptide drinks by 15 tasters are as follows:

[0069] Table 4 Statistical table of difference pair evaluation results

[0070]

[0071]

[0072] It can be seen from the above table that in terms of aroma index, 12 people in Group 1 felt that the aroma of Example 5 was better than that of Example 6, and 11 people in Group 2 felt that the aroma of Example 7 was better than that of Example 8; in terms of sweet and sour feeling, 10 people said that the difference was not obvious; in terms of anti-fishy smell, 13 people in Group 1 felt that the anti-fishy smell of Example 6 was more serious than that of Example 5, and 12 people in Group 2 felt that the anti-fishy smell of Example 8 was more serious than that of Example 7; in terms of freshness, Example 5 and Comparison 3 had better freshness. In summary, the sea cucumber peptide powder prepared by bio-enzymatic hydrolysis combined with low-temperature cooking technology has a good effect on flavor in beverage applications. In beverage preparation applications, the taste is more delicate and coordinated, and the flavor is more stable and excellent. Compared with sea cucumber peptide products prepared by a single bio-enzymatic hydrolysis method, beverages prepared by this method have a better taste, a more stable system, no anti-fishy smell during the shelf life, and better physical sensation for consumers.

[0073] In order to determine the effect of sea cucumber peptide powder on the stability of beverages, the beverages of Group 1 Examples 5 and 6, and Group 2 Examples 7 and 8 were placed at 4°C, 25°C and 37°C for 3 months, respectively, and the product stability was observed. If there was no precipitation in each group, it was marked with "N", and if there was precipitation in all groups, it was marked with "O". If one of the beverages in each group had precipitation, the corresponding example was written. The results are shown in Table 3.

[0074] Table 5 Experimental records

[0075]

[0076]

[0077] As can be seen from Table 5, Examples 6 and 8 are more likely to precipitate, which shows that the peptide powder prepared in Example 1 not only has a stable flavor and no fishy smell, but is also more stable under high temperature and high pressure (sterilization) or pH < 4 (acidity regulator) conditions than the peptide powder extracted by a simple enzymatic hydrolysis process.

[0078] The marine active peptide energy drink made with this formula has a sweet and sour and refreshing taste, a light fruity aroma, and no special flavors such as sea cucumber and oyster. It has a stable shelf life and its anti-fatigue effect is better than that of commercially available "caffeine" energy drinks.

[0079] Example 9 Activity Test

[0080] 1 Materials and Methods

[0081] 1.1 Sample: Marine active peptide beverage (concentrated solid powder of Example 5) is provided by Qingdao Marine Food Nutrition and Health Innovation Research Institute. The sample submitted for inspection is a milky white solid with a mass of 545.985g. It is prepared into 1L solution (10 times concentrated solution) with deionized water for stand-by use. 1.2 Comparative sample: Red Bull Vitamin Flavored Drink, produced by Tiansi Pharmaceutical Healthcare Co., Ltd. (Thailand). The sample is a yellow liquid and the recommended human intake is 250ml per day. Sample batch number: 265HN08, shelf life: 18 months. Before use, evaporate 10000ml of vitamin flavored drink at 50°C into 1000ml for stand-by use. Sample batch number: 20221205-001, shelf life: 12 months. 1.3 Experimental animals: Healthy SPF-grade Kunming male mice, weighing about 18-22 g, were provided by Beijing Weitonglihua Experimental Animal Technology Co., Ltd. The experimental animals were raised in a barrier environment with an experimental environment temperature of 22℃±2℃ and a relative humidity of 50%±10%. They were used in the experiment after one week of adaptive breeding.

[0082] 1.4 Dosage setting and test substance administration method: The recommended human dose of marine active peptide drink is 330 ml per day, which is equivalent to 5.5 ml / kg·bw per day based on a body weight of 60 kg. The low, medium and high doses of experimental mice are determined according to 5 times, 10 times and 30 times the recommended human dose, that is, the daily drinking amount of the marine active peptide drink group is: 27.5 ml / kg·bw for the low dose group, 55 ml / kg·bw for the medium dose group and 165 ml / kg·bw for the high dose group, with 8 mice in each group. The comparison sample is dosed at 30 times the recommended human intake, that is, the daily drinking amount of Red Bull vitamin flavored beverage for experimental mice is 125 ml / kg·bw, 114 ml / kg·bw and 75 ml / kg·bw respectively. The test substance was administered to mice orally once a day. The negative control group was gavaged with an equal volume of deionized water instead of the test substance solution, and various indicators of physical fatigue relief function were measured after continuous administration for 30 days.

[0083] 1.5 Main instruments and reagents: animal balance, electronic balance, lead sheet, electric glass homogenizer, low-speed centrifuge, spectrophotometer, constant temperature swimming box, constant temperature water bath, glassware, disposable quantitative blood tube, test tube, centrifuge tube, micropipette, surgical instruments. Urea, glycogen and blood lactate test kits were purchased from Nanjing Jiancheng Biological Company.

[0084] 1.6 Experimental methods

[0085] 1.6.1 Weighted swimming test: 30 minutes after the last administration of the test sample, mice with a lead sheet of 5% of their body weight at the base of their tails were placed in a swimming box with a water depth of 35 cm and a water temperature of 25°C ± 1.0°C. The time from the start of swimming to the death of the mice was recorded with a stopwatch, i.e., the weighted swimming time of the mice.

[0086] 1.6.2 Serum urea determination: 30 minutes after the last administration of the test substance, the mice in each group swam in 30℃ water without weight for 90 minutes. After being fished out and dried, they rested for 60 minutes, and then 0.5ml of blood was collected from the eyeballs. The blood was placed in a 4℃ refrigerator for about 3 hours, and after the blood coagulated, it was centrifuged at 3000rpm / min for 15 minutes. The serum was taken according to the instructions of the kit, and the urea content in the serum of each mouse was calculated according to the concentration of the standard tube and the corresponding absorbance.

[0087] 1.6.3 Determination of liver glycogen: 30 minutes after the last administration of the test substance, the animal was killed, the liver was rinsed with physiological saline and dried with filter paper, and 100 mg of liver was accurately weighed. According to the instructions of the kit, 300 μl of alkaline solution was taken, added to a test tube, boiled in a boiling water bath for 20 minutes, cooled in running water, and glycogen hydrolyzate was prepared. 9.6 ml of distilled water was added to the glycogen hydrolyzate to prepare a 1% liver glycogen detection solution, and liver glycogen determination was performed according to the instructions of the kit.

[0088] 1.6.4 Blood lactate determination: 30 minutes after the last administration of the test sample, 20 μl of blood was collected from the inner canthus of the mouse eye using a capillary tube treated with anticoagulant heparin and placed in an EP tube. 120 μl of protein precipitant was added to each tube, gently inverted to mix, allowed to stand for 10 minutes, centrifuged at 3500 rpm / min for 8 minutes, and the supernatant was taken for lactate determination. After the first blood collection, the mouse was allowed to swim without weight in water at 30°C for 10 minutes. After the mouse was taken out, 20 μl of blood was collected from the inner canthus immediately. After the mouse rested quietly for 20 minutes, another 20 μl of blood was collected. The operation was the same as above. According to the instructions of the kit, the blood lactate content at different times was calculated according to the concentration and absorbance value of the standard tube.

[0089] 1.7 Experimental data statistics: The data obtained from the experiment are all numerical variable data. All data were analyzed by variance analysis using SPSS statistical software. ANOVA was used for homogeneous variances, and rank sum test was used for unequal variances.

[0090] 1.8 Result determination: Weighted swimming time: If the weighted swimming time of the test sample group is significantly longer than that of the control group, and the difference is significant, the experimental result can be determined to be positive; Urea: If the serum urea of ​​the test sample group is lower than that of the negative control group, and the difference is significant, the experimental result can be determined to be positive; Liver glycogen: If the liver glycogen content of the test sample group is significantly higher than that of the negative control group, and the difference is significant, the experimental result can be determined to be positive; Blood lactate: The area under the blood lactate curve at three time points is used as the judgment standard. If the area of ​​any test group is smaller than that of the negative control group, and the difference is significant, the experimental result can be determined to be positive. If the weighted swimming test result is positive, and any two of the three biochemical indicators of blood lactate, serum urea, and liver glycogen are positive, it can be determined that the test sample has the function of relieving physical fatigue.

[0091] 2 Results

[0092] 2.1 Body weight changes of experimental mice

[0093] As shown in Tables 6 to 9, there was no significant difference in the initial body weight, mid-term body weight and body weight at the end of the experiment in the weight-bearing swimming test, serum urea test, liver glycogen test and blood lactate test (P>0.05). The above results suggest that the marine active peptide drink and the control sample Red Bull vitamin drink have no adverse effects on the weight gain of mice.

[0094] Table 6 Initial, mid-term and final body weights of mice in the weight-bearing swimming experiment (g, )

[0095]

[0096] Note: Comparative sample: Red Bull vitamin flavored drink.

[0097] Table 7 Initial, mid-term and final body weights of mice in serum urea experiment (g, )

[0098]

[0099] Note: Comparative sample: Red Bull vitamin flavored drink.

[0100] Table 8 Initial, mid-term and final body weights of mice in the glycogen experiment (g, )

[0101]

[0102] Table 9 Initial, mid-term and final body weights of mice in the blood lactate experiment (g, )

[0103]

[0104]

[0105] Note: Comparative sample: Red Bull vitamin flavored drink.

[0106] 2.2 Weighted swimming time of mice

[0107] As can be seen from Table 10, after oral administration of different doses of the test substance to mice for 30 days, compared with the negative control group, the weighted swimming time of mice in the medium and high dose groups of marine active peptide drinks and the high dose group of the control sample (Red Bull vitamin drink) was significantly prolonged, and the difference was significant (P<0.05, P<0.01); among them, the extended time of the marine active peptide energy drink was 7.64±1.87, significant P<0.01, while the extended time of the high dose group of the control sample (Red Bull vitamin drink) was 4.97±1.15, significant P>0.05.

[0108] Table 10 Effect of marine active peptide drinks on weight-bearing swimming time of mice

[0109]

[0110] Note: Comparative sample: Red Bull vitamin flavored drink.

[0111] 2.3 Serum urea content in mice

[0112] As can be seen from Table 11, after mice were gavaged with different doses of the test substance for 30 days, compared with the negative control group, the serum urea content of mice in the medium and high dose groups of the marine active peptide drink was significantly reduced after swimming for 90 minutes without weight and resting for 60 minutes (P<0.05, P<0.01); the serum urea nitrogen content of the high dose group of the control sample was also significantly reduced (P<0.05).

[0113] Table 11 Effects of marine active peptide drinks on serum urea in mice

[0114]

[0115]

[0116] Note: Comparative sample: Red Bull vitamin flavored drink.

[0117] 2.4 Mouse liver glycogen content

[0118] As can be seen from Table 12, after oral administration of different doses of the test substance to mice for 30 days, the liver glycogen content of the mice in the high-dose group was significantly increased compared with that in the negative control group (P<0.05); in the comparison samples, the liver glycogen content of the mice in the high-dose Red Bull Vitamin Drink group was significantly increased (P<0.05).

[0119] Table 12 Effects of marine active peptide beverage on liver glycogen content in mice

[0120]

[0121] Note: Comparative sample: Red Bull vitamin flavored drink.

[0122] 2.5 Blood lactate content in mice

[0123] As can be seen from Table 13, after oral administration of different doses of the test substance to mice for 30 days, there was no significant difference in the blood lactate content of mice in each dose group before swimming, 0 min rest after swimming, and 20 min rest, as well as the area under the blood lactate curve at these three time points compared with the negative control group (P>0.05).

[0124] Table 13 Effects of marine active peptide drinks on blood lactate content of mice at three time points

[0125]

[0126] Note: Comparative sample: Red Bull vitamin flavored drink.

[0127] Table 14 Effects of marine active peptide drinks on the area under the blood lactate curve of mice at three time points

[0128]

[0129] Note: Comparative sample: Red Bull vitamin flavored drink.

[0130] Animal experiments have shown that oral administration of marine active peptide drinks to mice at doses of 27.5ml / kg·bw, 55ml / kg·bw and 165ml / kg·bw for 30 consecutive days had no adverse effects on the weight gain of mice; it could significantly prolong the weight-bearing swimming time of mice; reduce serum urea production during exercise; and increase liver glycogen reserves. According to the criteria for relieving physical fatigue in the Ministry of Health's "Technical Specifications for Inspection and Evaluation of Health Foods" (2003 edition), it can be considered that marine active peptide drinks have the function of relieving physical fatigue.

[0131] In summary, the sea cucumber peptide powder extracted by low-temperature vacuum cooking combined with bio-enzymatic hydrolysis process not only improves the extraction rate, but also reduces energy consumption and process costs in production. In the actual application of liquid beverages, compared with the traditional single bio-enzyme preparation, it not only has good flavor sensory perception, but also the system is relatively stable, which greatly improves the scope of application of sea cucumber peptide raw materials in the food field.

[0132] The present invention uses sea cucumber peptides, oyster peptides and taurine as the main anti-fatigue substances. Through the compounding of the three, the effect of commercially available coffee and caffeine can be achieved, or even better, while avoiding the side effects of large doses or long-term use of caffeine that damage the human central nervous system, leading to heart disease and increased blood pressure. After the improved process of the present invention, the prepared sea cucumber peptides and oyster peptides have a good anti-fatigue effect, and the scientific enzymatic hydrolysis and deodorization process can make the above two marine active peptides have a better flavor, and no flocculation precipitation caused by protein will occur during the shelf life.

[0133] On the basis of the above embodiments, the present invention continues to describe in detail the technical features involved therein and the functions and roles played by the technical features in the present invention, so as to help technicians in this field fully understand the technical solution of the present invention and reproduce it.

[0134] Finally, although this specification is described according to implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A method for preparing sea cucumber peptide, characterized in that: The preparation method comprises the following steps: (1) pre-treating sea cucumbers and preparing sea cucumber homogenate; (2) adding water, 0.01-0.2 wt% neutral protease, and 0.05-0.3 wt% flavor protease, performing enzymolysis for 1-7 h, at a pH of 7-8, and inactivating the enzyme at 95° C. to obtain a crude extract of sea cucumber peptides; (3) The crude sea cucumber peptide extract is subjected to low-temperature vacuum cooking, wherein the conditions of the low-temperature vacuum cooking are: a temperature range of 50° C. to 80° C., and a vacuum degree of 0.00 MPa to -0.09 MPa; (4) Decolorizing the sea cucumber peptide; (5) Collect the components with a molecular weight of ≤10KDa, perform vacuum concentration, and spray dry to obtain sea cucumber peptides with a molecular weight range of 200-1800Da.

2. The preparation method according to claim 1, characterized in that In the step (1), the pretreatment includes: removing the internal organs and inner wall muscle layer of the sea cucumber, washing and crushing; soaking in pure water below 10°C for 3-7 days; mincing the sea cucumber body wall, and then preparing it into a homogenate using a colloid mill.

3. The preparation method according to claim 1, characterized in that: In the step (4), the decolorization treatment is as follows: adding activated carbon and diatomaceous earth, stirring for 15-20 minutes, and then filtering by plate and frame.

4. Use of the sea cucumber peptide prepared according to claim 1 in beverage products.

5. A marine active peptide energy drink, characterized in that: The energy drink comprises the sea cucumber peptide prepared according to claim 1.

6. The marine active peptide energy drink according to claim 5, characterized in that: The energy drink also includes oyster peptides and collagen peptides.

7. The marine active peptide energy drink according to claim 5, characterized in that: The marine active peptide energy drink includes 10-20g / L crystalline fructose, 20-40g / L maltitol, 1-5g / L pectin, 2-15g / L oyster peptide, 2-10g / L sea cucumber peptide, 30-50g / L collagen peptide, 0.4-0.9g / L natural taurine, and 0.1-0.5g / L sodium hyaluronate.

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