Method for extracting antifreeze peptide from sheepskin bone byproduct and application of antifreeze peptide

Abstract

The invention discloses a method for extracting antifreeze peptide from skin and bone byproducts of livestock and poultry animals and application of the antifreeze peptide, and relates to a method for extracting antifreeze peptide with high thermal hysteresis activity from skin and bone byproducts of a local variety sheepskin and bone of Xinjiang and application of the antifreeze peptide. Comprising an application of the sheep bone antifreeze peptide extracted by a method for extracting the antifreeze peptide from the sheepskin bone by-product to protect the cell integrity of fresh meat of livestock and poultry under low-temperature stress and an application of the sheep bone antifreeze peptide extracted by the method for extracting the antifreeze peptide from the sheepskin bone by-product to influence the survival rate of lactobacillus under low-temperature stress. Meanwhile, the heat stagnation activity of the extracted antifreeze peptide is gt; 5 DEG C; the sheep bone antifreeze peptide has the advantages that the sheep bone antifreeze peptide accounts for 24.8% when the molecular weight range is 1000 Daltons or above, the sheep bone antifreeze peptide accounts for 65.0% when the molecular weight range is 100-1000 Daltons, and the sheep bone antifreeze peptide accounts for 10.2% when the molecular weight range is lower than 180 Daltons; according to the method for hydrolyzing the sheepskin bone byproducts through the alkaline protease, the additional value and the utilization rate of the sheep bones and the sheepskin are improved to a great extent, and effective utilization of the sheep byproducts and maximization of the economic value are facilitated.

Description

Technical Field

[0001] The invention relates to a method and application of extracting antifreeze peptides with high thermal hysteresis activity from skin and bone byproducts of livestock and poultry, in particular to a method and application of extracting antifreeze peptides from sheep skin and bone byproducts. Background Art

[0002] Natural antifreeze peptides (AFPs) are a new type of green food antifreeze agent derived from natural animals, plants, insects, bacteria and fungi. They are a type of peptide that can non-colligatively reduce the freezing point of a solution and has the function of reducing the growth rate of ice crystals and inhibiting recrystallization in a low-temperature environment. Since the mid-20th century, scientists have successively isolated antifreeze proteins from polar marine fish, which can inhibit the growth of ice crystals and reduce the freezing point of solutions. With the continuous deepening of research, researchers have found that the carriers of antifreeze activity exist in certain special fragments on the polypeptide chain of antifreeze proteins. For example, it was found in Arctic and Antarctic cod that antifreeze proteins are a family of polymers composed of the tripeptide repeating unit glycine-glycine-threonine. In the American yellow cap flounder, it was found that antifreeze peptides composed of 8 amino acids, and glycine alone accounted for 60% of all amino acids. Studies have shown that the molecular weight of antifreeze peptides is concentrated in the range of 150 to 2000 Da, accounting for about 80% of the total antifreeze peptides, indicating that most antifreeze peptide molecules are short peptides of 2 to 18 amino acid residues.

[0003] Antifreeze peptides can be used as cryoprotectants in the food industry to extend the shelf life of food, in the biomedical field to protect cells and tissues from freezing damage, and in agriculture to improve the cold resistance of plants and expand the range of crops. Researchers have successfully isolated and purified antifreeze peptides from collagenase complexes such as fish, chicken skin, pig skin, and donkey bones, and conducted in-depth research on their properties.

[0004] Although different types of antifreeze peptides have been found in plants, marine fish, insects and fungi, high cost and low yield are still the main factors limiting the industrial preparation of antifreeze peptides. Therefore, finding suitable antifreeze peptide raw materials and extracting antifreeze peptides with high thermal hysteresis activity are issues that need to be urgently addressed.

[0005] At the same time, sheep bones and sheepskin are the main carriers of collagen. From the analysis of the specific amino acid sequence and spatial structure of collagen, collagen contains highly active antifreeze peptides. Therefore, it is of great significance to design a method and application for extracting antifreeze peptides from sheepskin bone by-products. Summary of the invention

[0006] The object of the present invention is to provide a method and application of extracting antifreeze peptides from sheepskin bone byproducts to solve the problems raised in the above background technology.

[0007] To achieve the above object, the present invention provides the following technical solutions: A method for extracting antifreeze peptides from sheep skin bone byproducts, the steps of extracting antifreeze peptides from sheep bones are as follows: Step 1: Put the sheep bones into liquid nitrogen for rapid freezing, remove the bone marrow and crush them; Step 2: Degreasing the sheep bones with n-hexane, washing, drying the bones, grinding them to 125 mesh, grinding them into bone powder, and obtaining preliminarily processed sheep bone powder; Step 3: Remove impurities with sodium hydroxide, filter and decalcify twice with citric acid, rinse with distilled water and soak with citric acid for >4 hours, enzymatically hydrolyze with pepsin, discard the precipitate after centrifugation, and adjust the pH of the supernatant to neutral to inactivate the enzyme; Step 4: Collect the precipitate after salting out with sodium chloride, dissolve the precipitate in acetic acid solution, and dialyze with sodium dihydrogen phosphate, acetic acid and distilled water in sequence to obtain collagen solution, and freeze-dry the collagen solution in a vacuum freeze dryer for later use; Step 5: Weigh the sheep bone freeze-dried powder and dissolve it in acetic acid to prepare a collagen solution, add alkaline protease, heat it in a water bath, and keep the pH of the collagen solution unchanged during the process; after the enzymatic hydrolysis is completed, boil it to inactivate the enzyme, cool it to room temperature, adjust the pH value to neutral, and centrifuge to obtain the supernatant to obtain sheep bone collagen polypeptide; Step 6: Using ice affinity adsorption method to separate polypeptides with antifreeze activity from sheep collagen polypeptides, which are recorded as antifreeze peptides; The method steps for extracting antifreeze peptides from sheepskin are as follows: S1: shave the raw material and cut it into pieces, soak the pieces in n-hexane to remove fat, and then wash them with distilled water; S2: The sheepskin weight and liquid volume are added with sodium hydroxide in a certain proportion to remove impurities and proteins, rinsed with distilled water, soaked with citric acid for >4 hours, enzymatically hydrolyzed with pepsin, centrifuged and discarded, and the pH of the supernatant is adjusted to neutral to inactivate the enzyme; S3: Add NaCl powder to the supernatant for salting out to make the final concentration of NaCl 0.5M, let it stand and centrifuge to collect the precipitate, dissolve it in acetic acid, and dialyze it with sodium dihydrogen phosphate, acetic acid and distilled water as the dialysis external solution in sequence. Repeat each step once, and freeze-dry the sample solution in the dialysis bag for later use; S4: Weigh sheepskin collagen and dissolve it in acetic acid to prepare a collagen solution, and add alkaline protease; S5: After the enzymatic hydrolysis is completed, the enzyme is boiled to inactivate the enzyme, cooled to room temperature, the pH value is adjusted to neutral, and the supernatant is collected by centrifugation to obtain sheepskin collagen enzymatic hydrolysis polypeptides; S6: Add sheepskin collagen peptides to the snake-shaped ice affinity adsorption device to collect antifreeze peptides, which are recorded as antifreeze peptides.

[0008] As a further solution of the present invention: in the step 2, the solid-liquid ratio of the sheep bone to n-hexane is 1:4 (W / V), the degreasing time is 5 hours, and the n-hexane temperature is 65-75° C.; In the step 3, the mass of bone powder and the volume of liquid are 1:10, the concentration of sodium hydroxide is 0.1 mol / L, the concentration of citric acid decalcification is 0.1 mol / L, the concentration of citric acid foaming is 0.5 mol / L, the mass of pepsin and the volume of the foamed solution is 0.25% (w / v), and the enzymatic hydrolysis time is 48 hours; In the step 4, the sodium chloride concentration is 0.5 mol / L, the salting-out time is 4 h, the precipitate after salting-out is dissolved with 0.5 mol / L acetic acid, dialyzed with sodium dihydrogen phosphate with a pH of 8.6 and a concentration of 0.02 mol / L, then with acetic acid with a concentration of 0.01 mol / L, and finally dialyzed with distilled water, the dialysis time for each step is 4 h, and each step is repeated once.

[0009] In step 5, the concentration of the collagen solution is 10 mg / mL, alkaline protease is added to the collagen solution, the enzyme substrate ratio is 26,000 u / g collagen, the solution pH is adjusted to 9, the heating temperature is 59° C., and the heating time is 5.6 h; In S6, the sheepskin collagen polypeptide is added to an ice affinity adsorption device precooled to -10°C, and the antifreeze peptide is adsorbed on ice crystals in the condenser tube, and the temperature of the condenser tube is raised to 5°C, and the antifreeze peptide is collected and recorded as the antifreeze peptide.

[0010] As a further solution of the present invention: in the step 2, the solid-liquid ratio of the sheep bone to n-hexane is 1:4 (W / V), the degreasing time is 5 hours, and the n-hexane temperature is 65-75° C.; In the step 3, the mass of bone powder and the volume of liquid are 1:10, the concentration of sodium hydroxide is 0.1 mol / L, the concentration of citric acid decalcification is 0.1 mol / L, the concentration of citric acid foaming is 0.5 mol / L, the mass of pepsin and the volume of the foamed solution is 0.25% (w / v), and the enzymatic hydrolysis time is 48 hours; In the step 4, the sodium chloride concentration is 0.5 mol / L, the salting-out time is 4 h, the precipitate after salting-out is dissolved with 0.5 mol / L acetic acid, dialyzed with sodium dihydrogen phosphate with a pH of 8.6 and a concentration of 0.02 mol / L, then with acetic acid with a concentration of 0.01 mol / L, and finally dialyzed with distilled water, the dialysis time for each step is 4 h, and each step is repeated once.

[0011] In step 5, the concentration of the collagen solution is 10 mg / mL, the pH of the collagen solution is adjusted to 9 after alkaline protease is added, the heating temperature is 50° C., and the heating time is 6 h; In step six, the sheep bone collagen polypeptide is added to the snake-shaped ice affinity adsorption device, and the antifreeze peptide is adsorbed on the ice crystals in the condenser tube, and the temperature of the condenser tube is raised to 5° C. to collect the antifreeze peptide.

[0012] Compared with the prior art, the present invention has the following beneficial effects: The thermal hysteresis activity of the antifreeze peptide extracted by the present invention is >5°C; The sheep bone antifreeze peptide extracted by the present invention significantly improves the survival rate of lactobacilli under low temperature stress; The invention discloses a method for hydrolyzing sheepskin and bone byproducts by using alkaline protease, aiming to greatly improve the added value and utilization rate of sheep bones and sheepskins, and is conducive to realizing the effective utilization of sheep byproducts and maximizing the economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a picture of sheep collagen.

[0014] Figure 2 The thermal hysteresis activity and ice crystal content of the collagen solution and antifreeze peptide obtained in the present invention are shown in FIG.

[0015] Figure 3 Schematic diagram of the effect of antifreeze peptides on the freezing survival rate of Lactobacillus acidophilus.

[0016] Figure 4 This is a comparison of untreated tissue cells under -4°C freezing temperature stress and cell tissue under the action of antifreeze peptides. DETAILED DESCRIPTION

[0017] 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.

[0018] See also Figures 1 to 4 In an embodiment of the present invention, a method and application of extracting antifreeze peptides from sheep skin bone byproducts is provided. The steps of extracting antifreeze peptides from sheep bones are as follows: Step 1: Put the sheep bones into liquid nitrogen for rapid freezing, remove the bone marrow and crush them; Step 2: Degreasing the sheep bones with n-hexane, washing, drying the bones, grinding them to 125 mesh, grinding them into bone powder, and obtaining preliminarily processed sheep bone powder; Step 3: Remove impurities with sodium hydroxide, filter and decalcify twice with citric acid, rinse with distilled water and soak with citric acid for >4 hours, enzymatically hydrolyze with pepsin, discard the precipitate after centrifugation, and adjust the pH of the supernatant to neutral to inactivate the enzyme; Step 4: Collect the precipitate after salting out with sodium chloride, dissolve the precipitate in acetic acid solution, and dialyze with sodium dihydrogen phosphate, acetic acid and distilled water in sequence to obtain collagen solution, and freeze-dry the collagen solution in a vacuum freeze dryer for later use; Step 5: Weigh the sheep bone freeze-dried powder and dissolve it in acetic acid to prepare a collagen solution, add alkaline protease, heat it in a water bath, and keep the pH of the collagen solution unchanged during the process; after the enzymatic hydrolysis is completed, boil it to inactivate the enzyme, cool it to room temperature, adjust the pH value to neutral, and centrifuge to obtain the supernatant to obtain sheep bone collagen polypeptide; Step 6: Using ice affinity adsorption method to separate polypeptides with antifreeze activity from sheep collagen polypeptides, which are recorded as antifreeze peptides; In the step 2, the solid-liquid ratio of the sheep bone to n-hexane is 1:4 (W / V), the degreasing time is 5 hours, and the n-hexane temperature is 65-75° C.; In the step 3, the mass of bone powder and the volume of liquid are 1:10, the concentration of sodium hydroxide is 0.1 mol / L, the concentration of citric acid decalcification is 0.1 mol / L, the concentration of citric acid foaming is 0.5 mol / L, the mass of pepsin and the volume of the foamed solution is 0.25% (w / v), and the enzymatic hydrolysis time is 48 hours; In the step 4, the sodium chloride concentration is 0.5 mol / L, the salting-out time is 4 h, the precipitate after salting-out is dissolved with 0.5 mol / L acetic acid, dialyzed with sodium dihydrogen phosphate with a pH of 8.6 and a concentration of 0.02 mol / L, then with acetic acid with a concentration of 0.01 mol / L, and finally dialyzed with distilled water, the dialysis time for each step is 4 h, and each step is repeated once.

[0019] In step 5, the concentration of the collagen solution is 10 mg / mL, alkaline protease is added to the collagen solution, the enzyme substrate ratio is 26,000 u / g collagen, the solution pH is adjusted to 9, the heating temperature is 59° C., and the heating time is 5.6 h; In step 6, the sheep bone collagen polypeptide is added to an ice affinity adsorption device precooled to -10°C, and the antifreeze peptide is adsorbed on ice crystals in the condenser tube, and the temperature of the condenser tube is raised to 5°C to collect the antifreeze peptide; The method steps for extracting antifreeze peptides from sheepskin are as follows: S1: shave the raw material and cut it into pieces, soak the pieces in n-hexane to remove fat, and then wash them with distilled water; S2: The sheepskin weight and liquid volume are added with sodium hydroxide in a certain proportion to remove impurities and proteins, rinsed with distilled water, soaked with citric acid for >4 hours, enzymatically hydrolyzed with pepsin, centrifuged and discarded, and the pH of the supernatant is adjusted to neutral to inactivate the enzyme; S3: Add NaCl powder to the supernatant for salting out to make the final concentration of NaCl 0.5M, let it stand and centrifuge to collect the precipitate, dissolve it in acetic acid, and dialyze it with sodium dihydrogen phosphate, acetic acid and distilled water as the dialysis external solution in sequence. Repeat each step once, and freeze-dry the sample solution in the dialysis bag for later use; S4: Weigh sheepskin collagen and dissolve it in acetic acid to prepare a collagen solution, and add alkaline protease; S5: After the enzymatic hydrolysis is completed, the enzyme is boiled to inactivate the enzyme, cooled to room temperature, the pH value is adjusted to neutral, and the supernatant is collected by centrifugation to obtain sheepskin collagen enzymatic hydrolysis polypeptides; S6: Add sheepskin collagen peptides to the snake-shaped ice affinity adsorption device to collect antifreeze peptides, which are recorded as antifreeze peptides.

[0020] In S1, the raw material is shaved and cut into pieces of 0.2 cm x 0.2 cm, and the pieces are soaked in n-hexane for 6 hours, the material-liquid ratio of sheepskin to n-hexane is 1:2 (W / V), the temperature of n-hexane is 65-75° C., and then washed with distilled water; In the S2, 0.1M sodium hydroxide is added to the sheepskin at a ratio of 1:10 in mass to liquid volume to remove impurities and proteins overnight, and then the sheepskin is rinsed with distilled water and soaked with 0.5M citric acid for >4 hours, and then enzymatically hydrolyzed with pepsin for 48 hours, with the mass of pepsin and the volume of the solution after soaking being 0.25% (w / v), and the precipitate is discarded after centrifugation, and the pH of the supernatant is adjusted to neutral to inactivate the enzyme; In S3, NaCl powder is added to the supernatant for salting out to a final concentration of 0.5 M NaCl, and the precipitate is collected by centrifugation after standing for 4 hours, dissolved in 0.5 M acetic acid, and dialyzed with sodium dihydrogen phosphate having a pH of 8.6 and a concentration of 0.02 M, 0.01 M acetic acid and distilled water as the dialysis external solution in sequence. The dialysis time for each step is 4 hours, and each step is repeated once; In the S4, sheepskin freeze-dried powder is weighed and dissolved in 0.1 M acetic acid to prepare a collagen solution with a concentration of 10 M, alkaline protease is added, the enzyme substrate ratio is 26,000 u / g collagen, the pH is adjusted to 9, and heated at 59° C. for 5.6 h, and the pH of the collagen solution is kept unchanged during the process; In S6, sheepskin collagen polypeptide is added to the snake-shaped ice affinity adsorption device, and the antifreeze peptide is adsorbed on the ice crystals in the condenser tube, and the temperature of the condenser tube is raised to 5° C., and the antifreeze peptide is collected and recorded as the antifreeze peptide.

[0021] The invention discloses an application of sheep bone antifreeze peptides extracted by the method of extracting antifreeze peptides from sheep skin and bone byproducts to protect the integrity of fresh meat cells of livestock and poultry under low temperature stress.

[0022] The invention discloses an application of sheep bone antifreeze peptides extracted from sheep skin bone byproducts to the storage of food-grade beneficial strains under low temperature stress.

[0023] The molecular weight of the antifreeze peptide is further determined by ultra-high performance liquid chromatography, and the specific determination method is to determine the molecular weight of the antifreeze peptide by ultra-high performance liquid chromatography; The thermal hysteresis activity of antifreeze peptides was further detected by differential scanning calorimetry. The thermal hysteresis activity of antifreeze peptides was detected by cooling the sample to complete freezing, then heating it to 10°C at a rate of 1°C / min and holding it for 5 minutes to obtain the melting heat (ΔHm) and melting point (Tm) of the collagen solution. The sample was then cooled to -20°C and held for 5 minutes, then heated at a rate of 1°C / min until the sample was partially melted, i.e., reached its retention temperature (Th), and held for 3 minutes, then the temperature was lowered from Th to -20°C, and the above process was repeated under different Th conditions for 3 minutes. The initial crystallization temperature (T0) and crystallization heat (ΔHr) of the sample at different Th were recorded, and the thermal hysteresis activity (thermal hysteresis activity = Th-T0) and ice crystal content [ice crystal content = (1-ΔHr / ΔHm) × 100%] were calculated; like Figure 2 It is shown that the thermal hysteresis activity of the collagen obtained by the present invention is 1.8°C, and the ice crystal content is 27%. The thermal hysteresis activity of the antifreeze peptide after separation and purification is 5.2°C, and the ice crystal content is reduced to 11%. Embodiment 1

[0024] The specific steps of extracting antifreeze peptides from sheep bones are as follows: The sheep bones were placed in liquid nitrogen for rapid freezing, and the bone marrow was removed and crushed. The sheep bones were defatted with n-hexane. The solid-liquid ratio of sheep bones to n-hexane was 1:4 (W / V), the defatting time was 5 hours, and the n-hexane temperature was 65-75°C. The defatted bones were washed, and after the bones were dried, they were crushed to 125 mesh and ground into bone powder to obtain the preliminarily processed sheep bone powder; 0.1M sodium hydroxide was added at a ratio of 1:10 to the mass of the bone powder and the volume of the liquid to remove the impurities, and after filtering, 0.1M citric acid was used for decalcification twice in a row, and after rinsing with distilled water, 0.5M citric acid was used for soaking for >4 hours, and pepsin was used for enzymatic hydrolysis for 48 hours. The mass of pepsin and the volume of the solution after soaking were 0.25% (w / v). After centrifugation, the precipitate was discarded, and the pH of the supernatant was adjusted to neutral to inactivate the enzyme. After salting out with 0.5M sodium chloride for 4 hours, the precipitate is collected, and the precipitate is dissolved in 0.5M acetic acid solution, and dialyzed with sodium dihydrogen phosphate with a pH of 8.6 and a concentration of 0.02M, 0.01M acetic acid and distilled water in sequence, the dialysis time for each step is 4 hours, and each step is repeated once to obtain a collagen solution, and the collagen solution is placed in a vacuum freeze dryer for freeze drying for standby use; weigh the sheep bone freeze-dried powder, dissolve it in 0.1M acetic acid to prepare a collagen solution with a concentration of 10M, add alkaline protease, adjust the pH to 9, and heat at 50°C for 6 hours, and keep the pH of the collagen solution unchanged during the process; after the enzymatic hydrolysis is completed, boil and inactivate the enzyme, cool to room temperature, adjust the pH value to neutral, centrifuge and take the supernatant to obtain sheep bone collagen polypeptide; add the sheep bone collagen polypeptide to a snake-shaped ice affinity adsorption device, wait for the antifreeze peptide to be adsorbed on the ice crystals in the condenser, raise the temperature of the condenser to 5°C, collect the antifreeze peptide, and record it as antifreeze peptide; Embodiment 2

[0025] The specific steps for extracting antifreeze peptides from sheepskin are as follows: The raw material was shaved and cut into pieces (0.2cmx0.2cm), and the pieces were soaked in n-hexane for 6 hours, with the ratio of sheepskin to n-hexane being 1:2 (W / V), and the temperature of n-hexane being 65-75℃, and then washed with distilled water. 0.1M sodium hydroxide was added to the sheepskin at a ratio of 1:10 in terms of mass and liquid volume to remove impurities and proteins overnight, and then rinsed with distilled water and soaked in 0.5M citric acid for >4 hours, and hydrolyzed with pepsin for 48 hours, with the mass of pepsin and the volume of the solution after soaking being 0.25% (w / v), and the precipitate was discarded after centrifugation, and the pH of the supernatant was adjusted to neutral to inactivate the enzyme. NaCl powder was added to the supernatant for salting out, so that the final concentration of NaCl was 0.5M. After standing for 4 hours, the precipitate was collected by centrifugation and dissolved in 0.5M acetic acid, and dialyzed with sodium dihydrogen phosphate with a pH of 8.6 and a concentration of 0.02M, 0.01M acetic acid and distilled water as the dialysis external solution in turn. The dialysis time for each step was 4 hours, and each step was repeated once. The sample solution in the dialysis bag was freeze-dried for use. The sheepskin freeze-dried powder was weighed and dissolved in 0.1M acetic acid to prepare a collagen solution with a concentration of 10M, alkaline protease was added, the pH was adjusted to 9, and heated at 50℃ for 6 hours. The pH of the collagen solution was kept unchanged during the process; after the enzymatic hydrolysis was completed, the enzyme was boiled to kill the enzyme, cooled to room temperature, the pH value was adjusted to neutral, and the supernatant was centrifuged to obtain sheepskin collagen polypeptide; the sheepskin collagen polypeptide was added to the snake-shaped ice affinity adsorption device, and the antifreeze peptide was adsorbed on the ice crystals in the condenser tube. The temperature of the condenser tube was raised to 5℃, and the antifreeze peptide was collected and recorded as antifreeze peptide. Embodiment 3

[0026] The specific steps for verifying the ability of antifreeze peptides to protect strains are as follows: Lactobacillus acidophilus was activated at 37°C for 18 h. During the growth stability period, the bacterial culture was collected by centrifugation, washed with sterile water, and diluted with physiological saline to OD 600 The value is about 0.8. Antifreeze peptides were added to the above bacterial suspension to prepare equal volumes of bacterial suspensions with final concentrations of 1%, 5%, and 10% (v / v) as the experimental group, and bacterial suspensions added with sterile water were used as the negative control group, and bacterial suspensions added with cell freezing solution were used as the positive control group. Take 50 μL of each of the above groups of samples and add them to 10 mL of MRS liquid culture medium, culture at 37°C for 16 hours, measure the absorbance at 600 nm, and obtain the concentration of the bacterial solution before freezing. Then freeze it at -4°C for 3 days, take it out and thaw it, and culture it in the same way, and measure the absorbance. The formula for bacterial survival rate is: Bacterial survival rate = [OD after freezing 600 nm / OD after freezing 600 nm) × 100%]; like Figure 3As shown, after freezing at -4°C, the survival rate of the bacteria in the negative control group was only 53.1%, and that in the positive control group was 61.5%, while the survival rate of the bacteria in the group with antifreeze peptides was significantly higher than that in the control group, and the survival rate of the group with 10% antifreeze peptides was as high as 95.76%. This indicates that the antifreeze peptides obtained by the present invention can effectively inhibit the size of ice crystals at low temperatures, reduce the mechanical damage to the cell membrane during the freezing process, and thus maintain the metabolic activity of the cells. Embodiment 4

[0027] The specific steps for verifying the protective ability of antifreeze peptides on fresh meat tissue cells of livestock and poultry are as follows (taking fresh mutton tissue as an example): After removing the fascia and large pieces of fat from the fresh lamb leg meat after slaughter, the meat was processed into 3×3 cm block samples. The control group was set as the untreated lamb sample, and the experimental group was set as the lamb sample soaked in 2% antifreeze peptide for 20 minutes. All samples were refrigerated at -4℃, and samples were taken on the 0th, 5th, 10th, 15th and 20th days for observation by paraffin section.

[0028] like Figure 4 As shown in the figure, under the freezing temperature stress of -4℃, the untreated tissue cells ruptured to a greater extent, while the cell tissues under the action of antifreeze peptides were less broken. With the increase of low temperature stress time, the growth rate of the broken proportion of treated and untreated cell tissues was also very different. Antifreeze peptides greatly inhibited the growth and recrystallization of ice crystals near the tissue, thus having a more obvious protective effect on meat tissue cells.

[0029] The molecular weight distribution of sheepskin bone antifreeze peptide is shown in the following table: Sheepskin bone antifreeze peptide molecular weight distribution table

[0030] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for extracting antifreeze peptides from sheepskin bone byproducts, characterized in that: The method steps for extracting antifreeze peptides from sheep bones are as follows: Step 1: Put the sheep bones into liquid nitrogen for rapid freezing, remove the bone marrow and crush them; Step 2: Degreasing the sheep bones with n-hexane, washing, drying the bones, grinding them to 125 mesh, grinding them into bone powder, and obtaining preliminarily processed sheep bone powder; Step 3: Remove impurities with sodium hydroxide, filter and decalcify twice with citric acid, rinse with distilled water and soak with citric acid for >4 hours, enzymatically hydrolyze with pepsin, discard the precipitate after centrifugation, and adjust the pH of the supernatant to neutral to inactivate the enzyme; Step 4: Collect the precipitate after salting out with sodium chloride, dissolve the precipitate in acetic acid solution, and dialyze with sodium dihydrogen phosphate, acetic acid and distilled water in sequence to obtain collagen solution, and freeze-dry the collagen solution in a vacuum freeze dryer for later use; Step 5: Weigh the sheep bone freeze-dried powder and dissolve it in acetic acid to prepare a collagen solution, add alkaline protease, heat it in a water bath, and keep the pH of the collagen solution unchanged during the process; after the enzymatic hydrolysis is completed, boil it to inactivate the enzyme, cool it to room temperature, adjust the pH value to neutral, and centrifuge to obtain the supernatant to obtain sheep bone collagen polypeptide; Step 6: Using ice affinity adsorption method to separate polypeptides with antifreeze activity from sheep collagen polypeptides, which are recorded as antifreeze peptides; The method steps for extracting antifreeze peptides from sheepskin are as follows: S1: shave the raw material and cut it into pieces, soak the pieces in n-hexane to remove fat, and then wash them with distilled water; S2: The sheepskin weight and liquid volume are added with sodium hydroxide in a certain proportion to remove impurities and proteins, rinsed with distilled water, soaked with citric acid for >4 hours, enzymatically hydrolyzed with pepsin, centrifuged and discarded, and the pH of the supernatant is adjusted to neutral to inactivate the enzyme; S3: Add NaCl powder to the supernatant for salting out to make the final concentration of NaCl 0.5M, let it stand and centrifuge to collect the precipitate, dissolve it in acetic acid, and dialyze it with sodium dihydrogen phosphate, acetic acid and distilled water as the dialysis external solution in sequence. Repeat each step once, and freeze-dry the sample solution in the dialysis bag for later use; S4: Weigh sheepskin collagen and dissolve it in acetic acid to prepare a collagen solution, and add alkaline protease; S5: After the enzymatic hydrolysis is completed, the enzyme is boiled to inactivate the enzyme, cooled to room temperature, the pH value is adjusted to neutral, and the supernatant is collected by centrifugation to obtain sheepskin collagen enzymatic hydrolysis polypeptides; S6: Add sheepskin collagen peptides to the snake-shaped ice affinity adsorption device to collect antifreeze peptides, which are recorded as antifreeze peptides.

2. The method for extracting antifreeze peptides from sheepskin bone byproducts according to claim 1, characterized in that: In the step 2, the solid-liquid ratio of the sheep bone to n-hexane is 1:4 (W / V), the degreasing time is 5 hours, and the n-hexane temperature is 65-75° C.; In the step 3, the mass of bone powder and the volume of liquid are 1:10, the concentration of sodium hydroxide is 0.1 mol / L, the concentration of citric acid decalcification is 0.1 mol / L, the concentration of citric acid foaming is 0.5 mol / L, the mass of pepsin and the volume of the foamed solution is 0.25% (w / v), and the enzymatic hydrolysis time is 48 hours; In the step 4, the sodium chloride concentration is 0.5 mol / L, the salting out time is 4 hours, the precipitate after salting out is dissolved with 0.5 mol / L acetic acid, dialyzed with sodium dihydrogen phosphate with a pH of 8.6 and a concentration of 0.02 mol / L, then with acetic acid with a concentration of 0.01 mol / L, and finally dialyzed with distilled water, the dialysis time for each step is 4 hours, and each step is repeated once; In step 5, the concentration of the collagen solution is 10 mg / mL, alkaline protease is added to the collagen solution, the enzyme substrate ratio is 26,000 u / g collagen, the solution pH is adjusted to 9, the heating temperature is 59° C., and the heating time is 5.6 h; In step six, the sheep bone collagen polypeptide is added to an ice affinity adsorption device precooled to -10°C, and the antifreeze peptide is adsorbed on ice crystals in the condenser tube, and the temperature of the condenser tube is raised to 5°C to collect the antifreeze peptide.

3. The method for extracting antifreeze peptides from sheepskin bone byproducts according to claim 1, characterized in that: In S1, the raw material is shaved and cut into pieces of 0.2 cm x 0.2 cm, and the pieces are soaked in n-hexane for 6 hours, the material-liquid ratio of sheepskin to n-hexane is 1:2 (W / V), the temperature of n-hexane is 65-75° C., and then washed with distilled water; In the S2, 0.1M sodium hydroxide is added to the sheepskin at a ratio of 1:10 in mass to liquid volume to remove impurities and proteins overnight, and then the sheepskin is rinsed with distilled water and soaked with 0.5M citric acid for >4 hours, and then enzymatically hydrolyzed with pepsin for 48 hours, with the mass of pepsin and the volume of the solution after soaking being 0.25% (w / v), and the precipitate is discarded after centrifugation, and the pH of the supernatant is adjusted to neutral to inactivate the enzyme; In S3, NaCl powder is added to the supernatant for salting out to a final concentration of 0.5 M NaCl, and the precipitate is collected by centrifugation after standing for 4 hours, dissolved in 0.5 M acetic acid, and dialyzed with sodium dihydrogen phosphate having a pH of 8.6 and a concentration of 0.02 M, 0.01 M acetic acid and distilled water as the dialysis external solution in sequence. The dialysis time for each step is 4 hours, and each step is repeated once; In the S4, sheepskin freeze-dried powder is weighed and dissolved in 0.1 M acetic acid to prepare a collagen solution with a concentration of 10 M, alkaline protease is added, the enzyme substrate ratio is 26,000 u / g collagen, the pH is adjusted to 9, and heated at 59° C. for 5.6 h, and the pH of the collagen solution is kept unchanged during the process; In S6, the sheepskin collagen polypeptide is added to an ice affinity adsorption device precooled to -10°C, and the antifreeze peptide is adsorbed on ice crystals in the condenser tube, and the temperature of the condenser tube is raised to 5°C, and the antifreeze peptide is collected and recorded as the antifreeze peptide.

4. Use of the sheep bone antifreeze peptide extracted by the method for extracting antifreeze peptides from sheep skin and bone byproducts according to claim 1 in protecting the integrity of fresh meat cells of livestock and poultry under low temperature stress.

5. Use of the sheep bone antifreeze peptide extracted by the method for extracting antifreeze peptides from sheep skin and bone byproducts according to claim 1 in storing food-grade beneficial strains under low temperature stress.

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