Preparation method of soft-shelled turtle anti-inflammatory active peptide beverage

Abstract

The invention provides a preparation method of a soft-shelled turtle anti-inflammatory active peptide beverage, and relates to the technical field of soft-shelled turtle food deep processing. According to the preparation method of the soft-shelled turtle anti-inflammatory active peptide beverage, the soft-shelled turtle anti-inflammatory active peptide beverage comprises the following components in percentage by mass: 2.0 to 3.0 percent of peptide powder, 5.5 to 7 percent of erythritol, 4.0 to 5.0 percent of concentrated fruit juice, 0.2 to 0.3 percent of anhydrous citric acid, 0.1 to 0.15 percent of CMC (Carboxy Methylated Cellulose), 0.1 to 0.2 percent of vitamin C, 0.01 to 0.02 percent of nicotinamide and the balance of hydrogen-rich water. According to the preparation method of the soft-shelled turtle anti-inflammatory active peptide beverage, bioactive polypeptide is obtained by enzymolysis of soft-shelled turtle meat, and in-vitro verification shows that the polypeptide has the advantages of good anti-inflammatory activity, low extraction cost, simple operation, environmental protection and the like. Compared with protein, the peptide is smaller in molecular weight, easier to absorb, capable of directly entering small intestines in a complete form, fast in energy supply and high in nutritional value, has the functions of resisting fatigue and enhancing human immunity, and has a good market prospect.

Description

Technical Field

[0001] The invention relates to the technical field of deep processing of soft-shelled turtle food, and in particular to a method for preparing a soft-shelled turtle anti-inflammatory active peptide beverage. Background Art

[0002] Turtle is an aquatic economic animal. It has always been used as a nutritious food in my country. It has always been believed to have the effects of strengthening the body, beautifying the skin, and prolonging life. "Compendium of Materia Medica" mentioned: "Turtle meat nourishes yin and blood, nourishes the heart and kidneys, clears away heat and removes stasis, and strengthens the spleen and stomach. It can treat symptoms such as fatigue and night sweats, yin deficiency and yang hyperactivity, back pain and leg pain, chronic diarrhea, children's shock and pain, women's amenorrhea, and dystocia." At present, domestic manufacturers have made health products in the form of oral liquid, freeze-dried powder, and capsules using turtle as raw materials. There are also processed products such as Chinese turtle essence and turtle paste that are all the rage. There is still great potential for turtle processing to be tapped, and the prospects are broad. So far, there is no public record of using turtle anti-inflammatory active peptides as raw materials to prepare beverages.

[0003] Inflammation is a defensive response of the body to stimuli, protecting the body from harmful stimuli. However, when inflammation is not controlled, it may lead to autoimmune diseases, autoinflammatory diseases, neurodegenerative diseases or cancer. At present, commonly used anti-inflammatory drugs are gradually restricted in practical applications due to their various side effects. With the advancement of enzymatic hydrolysis technology, many food-derived peptides have been shown to have multiple physiological functions. There are potential anti-inflammatory bioactive peptides in turtle protein hydrolysate. However, in the process of preparing active peptides by protease hydrolysis, there are often some unhydrolyzed and incompletely hydrolyzed macromolecular proteins and enzymes in the hydrolysate. The use of separation and purification methods can improve its biological activity.

[0004] To this end, we have developed a new method for preparing a soft-shell turtle anti-inflammatory active peptide drink. Summary of the invention

[0005] The purpose of the present invention is to solve the above problems and provide a method for preparing a soft-shell turtle anti-inflammatory active peptide beverage. The preparation method uses the soft-shell turtle anti-inflammatory active peptide as a raw material, improves its taste and flavor, and increases consumer acceptance.

[0006] The present invention is achieved through the following technical scheme: a method for preparing a turtle anti-inflammatory active peptide beverage, which comprises the following components by mass percentage: 2.0-3.0% peptide powder, 5.5-7% erythritol, 4.0-5.0% concentrated fruit juice, 0.2-0.3% anhydrous citric acid, 0.1-0.15% CMC, 0.1-0.2% vitamin C, 0.01-0.02% niacinamide, and the remainder hydrogen-rich water.

[0007] Preferably, the concentrated fruit juice is at least one of orange juice, peach juice, grape juice and grapefruit juice.

[0008] A method for preparing a soft-shell turtle anti-inflammatory active peptide beverage comprises the following steps:

[0009] (1) Kill the fresh turtle, remove the internal organs, rinse with clean water, boil in boiling water for 30-40 minutes, remove the bones and take out the meat, and grind it with a meat grinder;

[0010] (2) taking the turtle meat obtained in step (1), adding 1% pepsin in a mass ratio of meat to water of 1:2, heating in a water bath at 37° C., pH 2-3, stirring for enzymolysis for 7 h, inactivating the enzyme, rapidly cooling, centrifuging, and obtaining a supernatant;

[0011] (3) ultrafiltration of the supernatant obtained in step (2) to obtain a turtle peptide with a molecular weight of ≤1 kDa;

[0012] (4) freeze-drying the filtrate in step (3) under vacuum;

[0013] (5) subjecting the turtle peptide powder obtained in step (4) to LC-MS / MS peptide identification;

[0014] (6) Conduct cell experiments on the anti-inflammatory activity of the peptides;

[0015] (7) Take 2.0-3.0% of the peptide powder from step (4), add 5.5-7% of erythritol, 4.0-6.0% of concentrated fruit juice, 0.2-0.3% of anhydrous citric acid, 0.1-0.15% of CMC, 0.1-0.2% of vitamin C, 0.01-0.02% of niacinamide, and the balance of hydrogen-rich water, stir to dissolve, homogenize, can, sterilize, and package into a finished beverage.

[0016] Preferably, in step (2), the enzymatic hydrolysis time is 5-6 hours, the enzyme is inactivated at 100° C. for 10-15 minutes, the centrifugal force is 10,000 r / min, and the centrifugal time is 15-20 minutes.

[0017] Preferably, the molecular weight of the ultrafiltration membrane in step (3) is 1 kDa.

[0018] Preferably, in step (4), the condenser of the vacuum freeze dryer is -82.0°C, the sample is -40°C, the vacuum degree is -1000Pa, and the drying is performed for 35-48 hours.

[0019] Preferably, the homogenization in step (5) is carried out in a homogenizer, and the homogenization time is 3-5 minutes.

[0020] Preferably, the canning in step (5) is carried out under aseptic conditions.

[0021] Preferably, in step (5), the sterilization temperature is 85-90° C. and the sterilization time is 10-15 min.

[0022] (I) Beneficial effects

[0023] The present invention provides a method for preparing a soft-shell turtle anti-inflammatory active peptide beverage, which has the following beneficial effects:

[0024] 1. This method for preparing a soft-shell turtle anti-inflammatory peptide beverage uses enzymatic hydrolysis of soft-shell turtle meat to obtain biologically active peptides. The peptides have good anti-inflammatory activity through in vitro verification, and have the advantages of low extraction cost, simple operation, and environmental protection. Peptides have smaller molecular weights than proteins and are easier to absorb. They can directly enter the small intestine in a complete form, and have fast energy supply and high nutritional value.

[0025] 2. The method for preparing a soft-shell turtle anti-inflammatory active peptide beverage adopts separation and purification means to improve its biological activity and remove some unhydrolyzed and incompletely hydrolyzed macromolecular proteins and enzymes in the enzymatic hydrolyzate;

[0026] 3. The method for preparing a turtle anti-inflammatory active peptide beverage, by adding ingredients such as erythritol, peach concentrate juice, anhydrous citric acid, etc., makes the prepared turtle anti-inflammatory active peptide beverage not only enhance the body's anti-fatigue effect, but also improve the taste and flavor of the beverage, thereby increasing consumer acceptance. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a schematic diagram of molecular docking of the pentapeptide LRFLL-MD / 2 of the present invention;

[0028] Figure 2 This is a schematic diagram of molecular docking of the pentapeptide LRFLL of the present invention with TLR4;

[0029] Figure 3 This is a schematic diagram showing the effect of different concentrations of LPS on cell viability according to the present invention;

[0030] Figure 4 This is a schematic diagram showing the effect of different concentrations of LPS on NO release in the present invention;

[0031] Figure 5 Schematic diagram showing the effect of different concentrations of the turtle anti-inflammatory peptide on NO release;

[0032] Figure 6 Schematic diagram showing the effect of different concentrations of the turtle anti-inflammatory peptide of the present invention on cell viability;

[0033] Figure 7 It is a schematic diagram of the preparation process of the present invention. DETAILED DESCRIPTION

[0034] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only 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.

[0035] Example:

[0036] A method for preparing a soft-shell turtle anti-inflammatory active peptide beverage, comprising the following components by mass percentage: 2.0-3.0% peptide powder, 5.5-7% erythritol, 4.0-5.0% concentrated fruit juice, 0.2-0.3% anhydrous citric acid, 0.1-0.15% CMC, 0.1-0.2% vitamin C, 0.01-0.02% niacinamide, and the balance hydrogen-rich water;

[0037] Wherein, the concentrated fruit juice is at least one of orange juice, peach juice, grape juice and grapefruit juice;

[0038] Specifically, a method for preparing a soft-shell turtle anti-inflammatory active peptide beverage comprises the following steps:

[0039] (1) Kill the fresh turtle, remove the internal organs, rinse with clean water, boil in boiling water for 30-40 minutes, remove the bones and take out the meat, and grind it with a meat grinder;

[0040] (2) taking the turtle meat obtained in step (1), adding 1% pepsin in a mass ratio of meat to water of 1:2, heating in a water bath at 37° C., pH 2-3, stirring for enzymolysis for 7 h, inactivating the enzyme, rapidly cooling, centrifuging, and obtaining a supernatant;

[0041] Specifically, the enzymatic hydrolysis time is 5-6 hours, the enzyme is inactivated at 100°C for 10-15 minutes, the centrifugal force is 10000 r / min, and the centrifugal time is 15-20 minutes;

[0042] (3) ultrafiltration of the supernatant obtained in step (2) to obtain a turtle peptide with a molecular weight of ≤1 kDa;

[0043] Wherein, the molecular weight of the ultrafiltration membrane is 1kDa;

[0044] (4) freeze-drying the filtrate in step (3) under vacuum;

[0045] Among them, the vacuum freeze dryer condenser is -82.0℃, the sample is -40℃, the vacuum degree is -1000Pa, and the drying time is 35-48h;

[0046] (5) The turtle peptide powder obtained in step (4) is subjected to LC-MS / MS peptide identification, and the identification result is as follows: Figure 1 As shown;

[0047] The homogenization is carried out in a homogenizer for 3-5 minutes, the canning is carried out under aseptic conditions, the sterilization temperature is 85-90°C, and the sterilization time is 10-15 minutes;

[0048] (6) Cell experiments were conducted on the anti-inflammatory activity of the peptides. Figure 2 As shown;

[0049] (7) Take 2.0-3.0% of the peptide powder from step (4), add 5.5-7% of erythritol, 4.0-6.0% of concentrated fruit juice, 0.2-0.3% of anhydrous citric acid, 0.1-0.15% of CMC, 0.1-0.2% of vitamin C, 0.01-0.02% of niacinamide, and the balance of hydrogen-rich water, stir to dissolve, homogenize, can, sterilize, and package into a finished beverage.

[0050] Case 1:

[0051] A method for preparing a soft-shell turtle anti-inflammatory active peptide beverage comprises the following steps:

[0052] (1) Kill the fresh turtle, remove the internal organs, rinse with clean water, boil in boiling water for 30-40 minutes, remove the bones and take out the meat, and grind it with a meat grinder;

[0053] (2) taking the turtle meat obtained in step (1), adding 0.04% pepsin in a mass ratio of 1:2, heating in a water bath at 60°C, stirring for enzymolysis for 5 h, inactivating the enzyme at 100°C for 15 min, rapidly cooling, and centrifuging at 10,000 r / min for 15 min to obtain a supernatant;

[0054] (3) ultrafiltration of the supernatant obtained in step (2) using a 1 kDa nanofiltration membrane to obtain a turtle peptide with a molecular weight of ≤1 kDa;

[0055] (4) The filtrate in step (3) was freeze-dried under vacuum, with the condenser at -82°C, the sample at -40°C, the vacuum degree at 1000 Pa, and dried for 35 hours to obtain turtle anti-inflammatory active peptide powder;

[0056] (5) Take 2.0% of the peptide powder prepared in step (4), add 5.5% of erythritol, 5.0% of peach concentrate juice, 0.2% of anhydrous citric acid, 0.1% of CMC, 0.1% of vitamin C, 0.01% of niacinamide, and the balance of hydrogen-rich water, stir to dissolve, homogenize for 3 minutes, can, sterilize at 85°C for 15 minutes, and package into a finished beverage.

[0057] Case 2:

[0058] A method for preparing a soft-shell turtle anti-inflammatory active peptide beverage comprises the following steps:

[0059] (1) Kill the fresh turtle, remove the internal organs, rinse with clean water, boil in boiling water for 30-40 minutes, remove the bones and take out the meat, and grind it with a meat grinder;

[0060] (2) taking the turtle meat obtained in step (1), adding 0.04% pepsin in a mass ratio of 1:2, heating in a water bath at 60°C, stirring for enzymolysis for 6 hours, inactivating the enzyme at 100°C for 15 minutes, rapidly cooling, and centrifuging at 10,000 r / min for 20 minutes to obtain a supernatant;

[0061] (3) ultrafiltration of the supernatant obtained in step (2) using a 1 kDa nanofiltration membrane to obtain a turtle peptide with a molecular weight of ≤1 kDa;

[0062] (4) The filtrate in step (3) was freeze-dried under vacuum, with the condenser at -82°C, the sample at -40°C, the vacuum degree at 1000 Pa, and dried for 40 hours to obtain turtle anti-inflammatory active peptide powder;

[0063] (5) Take 2.0% of the peptide powder prepared in step (4), add 6% of erythritol, 4.0% of peach concentrate, 0.25% of anhydrous citric acid, 0.1% of CMC, 0.1% of vitamin C, 0.02% of niacinamide, and the balance of hydrogen-rich water, stir to dissolve, homogenize for 3 minutes, can, sterilize at 90° C. for 10 minutes, and package into a finished beverage.

[0064] Case 3:

[0065] A method for preparing a soft-shell turtle anti-inflammatory active peptide beverage comprises the following steps:

[0066] (1) Kill the fresh turtle, remove the internal organs, rinse with clean water, boil in boiling water for 30-40 minutes, remove the bones and take out the meat, and grind it with a meat grinder;

[0067] (2) taking the turtle meat obtained in step (1), adding 0.04% pepsin in a mass ratio of 1:2, heating in a water bath at 60°C, stirring for enzymolysis for 6 hours, inactivating the enzyme at 100°C for 15 minutes, rapidly cooling, and centrifuging at 10,000 r / min for 20 minutes to obtain a supernatant;

[0068] (3) ultrafiltration of the supernatant obtained in step (2) using a 1 kDa nanofiltration membrane to obtain a turtle peptide with a molecular weight of ≤1 kDa;

[0069] (4) The filtrate in step (3) was freeze-dried under vacuum, with the condenser at -82°C, the sample at -40°C, the vacuum degree at 1000 Pa, and dried for 40 hours to obtain turtle anti-inflammatory active peptide powder;

[0070] (5) Take 2.0% of the peptide powder in step (4), add 7% erythritol, 4.0% grape juice concentrate, 0.25% anhydrous citric acid, 0.15% CMC, 0.1% vitamin C, 0.02% niacinamide, and the balance hydrogen-rich water, stir to dissolve, homogenize for 4 minutes, can, sterilize at 90°C for 10 minutes, and package into a finished beverage.

[0071] Case 4:

[0072] A method for preparing a soft-shell turtle anti-inflammatory active peptide beverage comprises the following steps:

[0073] (1) Kill the fresh turtle, remove the internal organs, rinse with clean water, boil in boiling water for 30-40 minutes, remove the bones and take out the meat, and grind it with a meat grinder;

[0074] (2) taking the turtle meat obtained in step (1), adding 0.04% pepsin in a mass ratio of 1:2, heating in a water bath at 60°C, stirring for enzymolysis for 6 hours, inactivating the enzyme at 100°C for 15 minutes, rapidly cooling, and centrifuging at 10,000 r / min for 20 minutes to obtain a supernatant;

[0075] (3) ultrafiltration of the supernatant obtained in step (2) using a 1 kDa nanofiltration membrane to obtain a turtle peptide with a molecular weight of ≤1 kDa;

[0076] (4) The filtrate in step (3) was freeze-dried under vacuum, with the condenser at -82°C, the sample at -40°C, the vacuum degree at 1000 Pa, and dried for 40 hours to obtain turtle anti-inflammatory active peptide powder;

[0077] (5) Take 3.0% of the peptide powder in step (4), add 6% of erythritol, 6.0% of grape juice concentrate, 0.25% of anhydrous citric acid, 0.15% of CMC, 0.1% of vitamin C, 0.02% of niacinamide, and the balance of hydrogen-rich water, stir to dissolve, homogenize for 3 minutes, can, sterilize at 90°C for 10 minutes, and package into a finished beverage.

[0078] Case 5:

[0079] A method for preparing a soft-shell turtle anti-inflammatory active peptide beverage comprises the following steps:

[0080] (1) Kill the fresh turtle, remove the internal organs, rinse with clean water, boil in boiling water for 30-40 minutes, remove the bones and take out the meat, and grind it with a meat grinder;

[0081] (2) taking the turtle meat obtained in step (1), adding 0.04% pepsin in a mass ratio of 1:2, heating in a water bath at 60°C, stirring for enzymolysis for 6 hours, inactivating the enzyme at 100°C for 15 minutes, rapidly cooling, and centrifuging at 10,000 r / min for 20 minutes to obtain a supernatant;

[0082] (3) ultrafiltration of the supernatant obtained in step (2) using a 1 kDa nanofiltration membrane to obtain a turtle peptide with a molecular weight of ≤1 kDa;

[0083] (4) The filtrate in step (3) was freeze-dried under vacuum, with the condenser at -82°C, the sample at -40°C, the vacuum degree at 1000 Pa, and dried for 40 hours to obtain turtle anti-inflammatory active peptide powder;

[0084] (5) Take 2.0% of the peptide powder prepared in step (4), add 7% of erythritol, 5.0% of orange juice concentrate, 0.25% of anhydrous citric acid, 0.15% of CMC, 0.1% of vitamin C, 0.02% of niacinamide, and the balance of hydrogen-rich water, stir to dissolve, homogenize for 3 minutes, can, sterilize at 90°C for 10 minutes, and package into a finished beverage.

[0085] Case 6:

[0086] A method for preparing a soft-shell turtle anti-inflammatory active peptide beverage comprises the following steps:

[0087] (1) Kill the fresh turtle, remove the internal organs, rinse with clean water, boil in boiling water for 30-40 minutes, remove the bones and take out the meat, and grind it with a meat grinder;

[0088] (2) taking the turtle meat obtained in step (1), adding 0.04% pepsin in a mass ratio of 1:2, heating in a water bath at 60°C, stirring for enzymolysis for 6 hours, inactivating the enzyme at 100°C for 15 minutes, rapidly cooling, and centrifuging at 10,000 r / min for 20 minutes to obtain a supernatant;

[0089] (3) ultrafiltration of the supernatant obtained in step (2) using a 1 kDa nanofiltration membrane to obtain a turtle peptide with a molecular weight of ≤1 kDa;

[0090] (4) The filtrate in step (3) was freeze-dried under vacuum, with the condenser at -82°C, the sample at -40°C, the vacuum degree at 1000 Pa, and dried for 40 hours to obtain turtle anti-inflammatory active peptide powder;

[0091] (5) Take 3.0% of the peptide powder in step (4), add 7% erythritol, 5.0% orange juice concentrate, 0.3% anhydrous citric acid, 0.15% CMC, 0.1% vitamin C, 0.02% niacinamide, and the balance hydrogen-rich water, stir to dissolve, homogenize for 4 minutes, can, sterilize at 90°C for 10 minutes, and package into a finished beverage.

[0092] Case 7:

[0093] A method for preparing a soft-shell turtle anti-inflammatory active peptide beverage comprises the following steps:

[0094] (1) Kill the fresh turtle, remove the internal organs, rinse with clean water, boil in boiling water for 30-40 minutes, remove the bones and take out the meat, and grind it with a meat grinder;

[0095] (2) taking the turtle meat obtained in step (1), adding 0.04% pepsin in a mass ratio of 1:2, heating in a water bath at 60°C, stirring for enzymolysis for 6 hours, inactivating the enzyme at 100°C for 15 minutes, rapidly cooling, and centrifuging at 10,000 r / min for 20 minutes to obtain a supernatant;

[0096] (3) ultrafiltration of the supernatant obtained in step (2) using a 1 kDa nanofiltration membrane to obtain a turtle peptide with a molecular weight of ≤1 kDa;

[0097] (4) The filtrate in step (3) was freeze-dried under vacuum, with the condenser at -82°C, the sample at -40°C, the vacuum degree at 1000 Pa, and dried for 40 hours to obtain turtle anti-inflammatory active peptide powder;

[0098] (5) Take 2.0% of the peptide powder prepared in step (4), add 7% of erythritol, 5.0% of grapefruit concentrated juice, 0.25% of anhydrous citric acid, 0.15% of CMC, 0.1% of vitamin C, 0.02% of niacinamide, and the balance of hydrogen-rich water, stir to dissolve, homogenize for 3 minutes, can, sterilize at 90° C. for 10 minutes, and package into a finished beverage.

[0099] Case 8:

[0100] A method for preparing a soft-shell turtle anti-inflammatory active peptide beverage comprises the following steps:

[0101] (1) Kill the fresh turtle, remove the internal organs, rinse with clean water, boil in boiling water for 30-40 minutes, remove the bones and take out the meat, and grind it with a meat grinder;

[0102] (2) taking the turtle meat obtained in step (1), adding 0.04% pepsin in a mass ratio of 1:2, heating in a water bath at 60°C, stirring for enzymolysis for 6 hours, inactivating the enzyme at 100°C for 15 minutes, rapidly cooling, and centrifuging at 10,000 r / min for 20 minutes to obtain a supernatant;

[0103] (3) ultrafiltration of the supernatant obtained in step (2) using a 1 kDa nanofiltration membrane to obtain a turtle peptide with a molecular weight of ≤1 kDa;

[0104] (4) The filtrate in step (3) was freeze-dried under vacuum, with the condenser at -82°C, the sample at -40°C, the vacuum degree at 1000 Pa, and dried for 40 hours to obtain turtle anti-inflammatory active peptide powder;

[0105] (5) Take 3.0% of the peptide powder in step (4), add 7% of erythritol, 6.0% of grapefruit concentrated juice, 0.25% of anhydrous citric acid, 0.15% of CMC, 0.2% of vitamin C, 0.02% of niacinamide, and the balance of hydrogen-rich water, stir to dissolve, homogenize for 4 minutes, can, sterilize at 90°C for 10 minutes, and package into a finished beverage.

[0106] Experimental results: Based on the above cases 1 to 8, the following research was conducted:

[0107] (1) Use the Pepti deRanker program (URL link:

[0108] https: / / bioware.ucd.i ecompass / biowareweb / Serverpages / peptideranker.php) predicted the biological activity of the oligopeptides (dipeptides to pentapeptides) in the above collected peptides, and used 0.5 as the threshold for activity prediction. When the peptide prediction score was greater than this value, it was considered to have potential biological activity;

[0109] (2) Molecular docking of pepsin-hydrolyzed oligopeptides: Molecular docking studies were performed using AutoDokTOO ls software. The protein crystal structures of MD-2 and TLR4 were obtained from the RCSB protein database (PDB), and water molecules in the crystal structures were removed and the structures were optimized before docking;

[0110] (3) LPS-induced RAW264.7 cells to establish an inflammatory model: Take cells in the logarithmic growth phase, discard the original culture medium, wash three times with PBS, add 3 mL of complete culture medium, scrape the bottom of the culture flask with a sterile cell scraper, blow the cells to make them evenly distributed, and obtain a single cell suspension. Use a cell counting plate to count the cells under a microscope to make the cell density in the suspension 5×105 / mL, and inoculate the suspension in a 96-well plate at 100 μL per well. After culturing for 24 hours, aspirate the original culture medium and add 100 μL of LPS solution of the corresponding concentration (0.1, 0.5, 1.0, 1.5 μg / mL) to each well. Determine its NO content and cell viability;

[0111] (4) Determination of nitric oxide (NO) content: The NO content was detected by the Griess method. Cells in the logarithmic phase were inoculated in a 96-well plate, with 100 μL per well. The culture plate was placed in an incubator for overnight culture. After the cells adhered to the wall (usually 12 to 24 hours), the old culture medium of the culture plate was discarded, and then 100 μL of LPS or peptide samples of different concentrations were added to each well and incubated for a specified time. After the culture was completed, the supernatant of the cell culture fluid was collected and the NO content was determined using a NO detection kit;

[0112] (5) Determination of cell viability: Cell viability was determined by the CCK-8 method. Cells in the logarithmic phase were inoculated in a 96-well plate with 100 μL per well. The culture plate was placed in an incubator for overnight culture. After the cells adhered to the wall (usually 12 to 24 hours), the old culture medium of the culture plate was discarded, and then 100 μL of different concentrations of LPS or the peptide sample to be tested was added to each well and incubated for 24 hours. After the culture was completed, the cell morphology was observed and recorded using an inverted microscope. Then 10 μL of CCK-8 solution was placed in each well. After incubation in the incubator for 1 hour, the cell viability was calculated by measuring the absorbance at 450 nm using an enzyme reader and calculated according to the following formula:

[0113] Cell viability % = (OD of drug-treated cells / OD of control cells)*100;

[0114] (6) Sensory evaluation of soft-shell turtle anti-inflammatory peptide beverage: The soft-shell turtle anti-inflammatory peptide beverage prepared in the example was evaluated by taste, smell and color. Ten sensory assessors (5 males and 5 females, aged 20-28) who had undergone sensory evaluation training formed a sensory evaluation team.

[0115]

[0116]

[0117] Table 1 Sensory scoring criteria for soft-shell turtle anti-inflammatory peptide beverages

[0118]

[0119] Table 2 Sensory evaluation results of soft-shell turtle anti-inflammatory peptide beverage

[0120]

[0121] Table 3 Molecular docking scores of pepsin-hydrolyzed oligopeptides

[0122] In summary, the present invention uses enzymatic hydrolysis of turtle meat to obtain biologically active peptides, and the peptides have good anti-inflammatory activity through in vitro verification, and have the advantages of low extraction cost, simple operation, and environmental protection. The molecular weight of peptides is smaller than that of proteins, and they are easier to absorb. They can directly enter the small intestine in a complete form, and have fast energy supply and high nutritional value. At the same time, by adding ingredients such as erythritol, peach concentrate juice, and anhydrous citric acid, the prepared turtle anti-inflammatory active peptide beverage can enhance the body's anti-fatigue while also improving the taste and flavor of the beverage, thereby increasing consumer acceptance.

[0123] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a soft-shell turtle anti-inflammatory active peptide beverage, characterized in that: Calculated by mass percentage, it contains the following components: 2.0-3.0% peptide powder, 5.5-7% erythritol, 4.0-5.0% concentrated fruit juice, 0.2-0.3% anhydrous citric acid, 0.1-0.15% CMC, 0.1-0.2% vitamin C, 0.01-0.02% niacinamide, and the balance hydrogen-rich water.

2. The method for preparing a soft-shell turtle anti-inflammatory active peptide beverage according to claim 1, characterized in that: The concentrated fruit juice is at least one of orange juice, peach juice, grape juice and grapefruit juice.

3. The method for preparing a soft-shell turtle anti-inflammatory active peptide beverage according to claim 1, characterized in that: The following steps are involved: (1) Kill the fresh turtle, remove the internal organs, rinse with clean water, boil in boiling water for 30-40 minutes, remove the bones and take out the meat, and grind it with a meat grinder; (2) taking the turtle meat obtained in step (1), adding 1% pepsin in a mass ratio of meat to water of 1:2, heating in a water bath at 37° C., pH 2-3, stirring for enzymolysis for 7 h, inactivating the enzyme, rapidly cooling, centrifuging, and obtaining a supernatant; (3) ultrafiltration of the supernatant obtained in step (2) to obtain a turtle peptide with a molecular weight of ≤1 kDa; (4) freeze-drying the filtrate in step (3) under vacuum; (5) subjecting the turtle peptide powder obtained in step (4) to LC-MS / MS peptide identification; (6) Conduct cell experiments on the anti-inflammatory activity of the peptides; (7) Take 2.0-3.0% of the peptide powder from step (4), add 5.5-7% of erythritol, 4.0-6.0% of concentrated fruit juice, 0.2-0.3% of anhydrous citric acid, 0.1-0.15% of CMC, 0.1-0.2% of vitamin C, 0.01-0.02% of niacinamide, and the balance of hydrogen-rich water, stir to dissolve, homogenize, can, sterilize, and package into a finished beverage.

4. The method for preparing a soft-shell turtle anti-inflammatory active peptide beverage according to claim 3, characterized in that: In the step (2), the enzymatic hydrolysis time is 5-6 hours, the enzyme is inactivated at 100° C. for 10-15 minutes, the centrifugal force is 10000 r / min, and the centrifugal time is 15-20 minutes.

5. The method for preparing a soft-shell turtle anti-inflammatory active peptide beverage according to claim 3, characterized in that: The molecular weight of the ultrafiltration membrane in step (3) is 1 kDa.

6. The method for preparing a soft-shell turtle anti-inflammatory active peptide beverage according to claim 3, characterized in that: In the step (4), the condenser of the vacuum freeze dryer is -82.0°C, the sample is -40°C, the vacuum degree is -1000Pa, and the drying is performed for 35-48 hours.

7. The method for preparing a soft-shell turtle anti-inflammatory active peptide beverage according to claim 3, characterized in that: In the step (5), the homogenization is carried out in a homogenizer, and the homogenization time is 3-5 minutes.

8. The method for preparing a soft-shell turtle anti-inflammatory active peptide beverage according to claim 3, characterized in that: The filling in step (5) is carried out under aseptic conditions.

9. The method for preparing a soft-shell turtle anti-inflammatory active peptide beverage according to claim 3, characterized in that: In step (5), the sterilization temperature is 85-90° C. and the sterilization time is 10-15 min.

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