TWI551294B - A method to improve osteoarthritis by collagen hydrolysates derived from porcine skin. - Google Patents

Description

Method for improving degenerative arthritis products by using collagen hydrolysate of pig skin

The present invention relates to a method for extracting a collagen hydrolysate using pig skin as a raw material for producing a product for improving degenerative arthritis.

Degenerative arthritis (Osteoarthritis, OA) is the highest incidence of arthritis, occurring in older people over the age of 60, according to the World Health Organization (WHO) 2004 Global Burden of Disease report, degenerative joints Inflammation is the top 10 factors in the world with years of disability (YLDs). It can be seen that the disease burden caused by it has caused serious impact on the economic, social and psychological consumption and burden of patients. Therefore, if early prevention, diagnosis and treatment of degenerative arthritis can find more effective methods, it can save a lot of manpower and social resources. It is also an urgent task to develop related prevention and treatment methods.

The slaughtering of pigs in Taiwan's livestock slaughter is the highest. In 2011, the number of pig slaughter was about 8.79 million (Administry's Agricultural Committee, 2012). The average live weight is about 110-120 kg. If pig skin is 4% of live weight. In calculation, Taiwan's annual pig skin production can reach at least 38,000 metric tons, and according to the 2011 report on the production of livestock and poultry by-products in Taiwan, the production of fresh pork skin is only 5,402 metric tons; this is because of Taiwan pigs. In the slaughter process, except for a small number of slaughterhouses In addition to the leather material or raw materials for biomedical engineering (animal glue or collagen), most of the meat market and private slaughterhouses are treated with scalding and hair removal. Pork skins are sold. To all parts of the country. Due to the traditional consumption habits, the pig skin is not easily peeled off into a separate by-product, and the irregular pig skin and dander accompanying the raw meat cutting and trimming will eventually be discarded or processed, resulting in waste of resources. Livestock environmental issues and increased processing costs for operators. There are about 25-27% crude protein in fresh pig skin. Among nitrogenous compounds, collagen accounts for 74-76% of the total protein of pig skin, while in the dermis it is 91-92%, in addition to a small amount. Calcium, phosphorus, iron, magnesium and trace minerals are good sources of collagen. Therefore, if these pigskins and dandruffs can be developed and utilized, the large amount of waste generated by the processing of livestock products can be effectively reduced, and the added value and the profits of related industries can be enhanced.

The present inventors have in view of the problem that the irregular pigskin and dandruff generated during the slitting process of the skinned pork are not effectively utilized, resulting in waste pollution, and in the past, my research and literature pointed out that collagen hydrolyzate ( Collagen peptide) helps to improve the symptoms of degenerative arthritis. It is the idea of Nai Si and inventions. In the spirit of perseverance, it actively and continuously researches and improves, and has been tested and tested by many parties, and many times. The improvement is the present invention.

The invention provides a method for preparing a product for improving degenerative arthritis by using a collagen hydrolyzate of pig skin, which is to take pig skin of a pig and wash and scrape the residual subcutaneous fat, and steam cooking at 100 ° C to facilitate mincing, After mincing, heat to 60 ° C and then cool, remove the upper fat, store at -20 ° C for use, take the minced pork skin, extract the pig skin collagen with 0.5 M acetic acid (Acetic acid), and then take the pig skin After the collagen is cut, add 30 times of deionized water to stir Mix well and heat to 40 ° C (to make it easier to dissolve), in the following three enzymes of enzyme substrate ratio: Alcalase, Protease N, Pepsin Any one of the hydrolysis, followed by heating in a boiling water bath to 100 ° C for 10 minutes to stop the enzyme reaction (inactivation), and then cooled with 4 ° C cold water and then freeze-dried for 48 hours, the collected dry powder product was stored in -20 ° C environment for use.

The main object of the present invention is to use a pig skin extracting collagen hydrolysate to produce a method for improving the product of degenerative arthritis, thereby not only improving the added value of pig skin, increasing the income of the industry and reducing the environmental pollution of pig skin waste. In addition to the problem, a smaller molecular collagen hydrolysate can be obtained, and a product which contributes to the improvement of the condition of patients with degenerative arthritis can be produced for the benefit of the world.

The first figure is a block flow diagram of the present invention.

The second picture is a chart analysis of pig skin components.

The third panel is a graph showing the results of the test for the proliferation of rabbit synovial cells (HIG-82) by the pig skin collagen hydrolysate of the present invention.

The fourth panel is a graph showing the results of the test for the rabbit articular chondrocyte proliferation of the pig skin collagen hydrolysate of the present invention.

The fifth graph is a graph showing the results of the test for the survival rate of rabbit synovial cells (HIG-82) induced by hydrogen peroxide in the pig skin collagen hydrolysate of the present invention.

Fig. 6 is a graph showing the results of a test for catalase activity in rabbit synovial cells (HIG-82) induced by hydrogen peroxide in the pig skin collagen hydrolysate of the present invention.

The seventh figure is a rabbit pig collagen hydrolysate induced by hydrogen peroxide in rabbit synovial cells (HIG-82) A graph of the results of the superoxide dismutase activity test.

The eighth graph is a graph showing the results of the test for the content of glutathione in rabbit synovial cells (HIG-82) induced by hydrogen peroxide in the pig skin collagen hydrolysate of the present invention.

The ninth graph is a graph showing the results of the test for the activity of glutathione peroxidase in rabbit synovial cells (HIG-82) induced by hydrogen peroxide in the pig skin collagen hydrolysate of the present invention.

The tenth graph is a graph showing the results of the test for producing IL-1β in rabbit synovial cells (HIG-82) induced by lipopolysaccharide by the pig skin collagen hydrolysate of the present invention.

The eleventh graph is a graph showing the results of the test for the production of TNF-α by rabbit lipopolysaccharide-induced rabbit synoviocytes (HIG-82) by the collagen hydrolysate of the present invention.

Fig. 12 is a graph showing the results of a test for collagen secretion of rabbit synovial cells (HIG-82) after hydrogen peroxide damage by the pig skin collagen hydrolysate of the present invention.

The thirteenth graph is a graph showing the results of the test for proteoglycan secretion of rabbit synovial cells (HIG-82) after hydrogen peroxide injury by the pig skin collagen hydrolysate of the present invention.

Referring to the first figure, the present invention comprises a method for preparing a product for improving degenerative arthritis by using a collagen hydrolysate of pig skin, comprising the following steps: First, the pig skin is minced and spared: the pig skin of the pig is taken and the residue is scraped off. Subcutaneous fat, using 100 ° C steam cooking, to facilitate mincing, after mincing, heated to 60 ° C and then cooled, remove the upper fat, stored at -20 ° C for use, as shown in the second figure, pig skin composition analysis, Contains 46.1% moisture, 36.8% crude protein, 16.7% crude fat and 0.5% ash. 2. Extraction of collagen: (1) Add 10 times (w/v) 20% ethanol to the pig skin and stir at 4 °C. After 24 hours, the supernatant liquid was removed by centrifugation at 10,000 × g for 15 minutes in a refrigerated centrifuge. This step was repeated once again to achieve the effect of removing fat and impurities; (2) recovering the precipitate and Add 10 times (w/v 1:10) 0.2N sodium hydroxide solution, homogenize for 2 minutes, stir at 4 ° C for 24 hours, then centrifuge at 10,000 × g for 15 minutes, recover The precipitate was rinsed with deionized water; (3) The precipitate was dissolved back in 10 times (w/v) 0.5 M acetic acid (Acetic acid) solution, and 5 mL/mg pH 2.0 pepsin was added to the solution. Decompose at °C for 24 hours; (4) The above collagen solution was centrifuged at 10,000 × g for 1 hour, and the supernatant was collected and ice-cooled, followed by slowly adding sodium chloride to a final concentration of 2.5 M. Stir at 4 ° C for 24 hours to salt out and precipitate collagen; (5) Centrifuge the solution at 10,000 × g for 1 hour, recover the precipitate and dissolve it back into 0.5 M acetic acid (Acetic acid) to 0.05 M acetic acid (Acetic acid Dialysis for 72 hours, during which the dialysate is changed every 24 hours; (6) Finally, the dialyzed collagen solution is freeze-dried in a freeze dryer to obtain a finished collagen product, which is stored at -20 ° C for use; Preparation of collagen hydrolysate: After the collagen is cut, add 30 times of deionized water and mix evenly. After 40 ° C (to make it easier to dissolve), according to the enzyme substrate ratio of 1:50 of the following three enzymes: alkaline protease (Alcalase), Protease N (Protease N), pepsin (Pepsin), according to each Hydrolysis is carried out under specific conditions of enzyme (Alcalase: pH 8.0, 50 ° C; Protease N: pH 7.0, 50 ° C; Pepsin: pH 3.0, 37 ° C), hydrolysis time is 0-8 hours, preferably 4-6 hours, hydrolysis Thereafter, the enzyme reaction (inactivation) was stopped by heating in a boiling water bath at 100 ° C for 10 minutes, and then cooled by running water at 4 ° C in cold water, followed by freeze drying for 48 hours, and the dried powder product was collected and stored in an environment of -20 ° C for use.

After comprehensive evaluation by anti-oxidation and collagenase inhibitory activity test, two groups of enzymes (Alcalase), Protease N (Protease N) and Pepsin (Pepsin) were selected and each group was selected. Treatment group with antioxidant capacity and collagenase inhibitory activity - A4 (alkaline protease hydrolysis for 4 hours), A8 (alkaline protease hydrolysis for 8 hours), N2 (protease N Hydrolysis for 2 hours), N8 (protease N hydrolysis for 8 hours, P2 (pepsin hydrolysis for 2 hours) and P6 (pepsin hydrolysis for 6 hours), and selected cell line HIG-82 isolated from rabbit knee joint synovium and The primary chondrocytes isolated from the knee joint cartilage of young rabbits were subjected to a cell test.

1. Pig skin collagen hydrolysate promotes cell proliferation test

Each treatment group was co-cultured with cells at 10 μg/mL, 100 μg/mL and 1 mg/mL for 7 days, and cell proliferation was measured by MTT method on days 1, 3, 5 and 7 without any hydrolysis. The culture solution of the substance was used as a control (Control). For example, the third and fourth figures are the results of the pig skin collagen hydrolysate test for rabbit synovial cells (HIG-82) and rabbit articular chondrocyte proliferation. The results showed that after 1 day of co-culture with the hydrolysate, the cell proliferation of each treatment group with a hydrolyzate concentration of 10 μg/mL was significantly higher than that of the control group (Control) (p<0.05), and the cells of the A4 and A8 treatment groups were treated. The proliferation was the most; the same results were obtained on the third day of the experiment; on the fifth day of the experiment, the cells in the A4 and P6 treatment groups proliferated most, and the other treatment groups were lower or no significant difference compared with the control group (Control); In the middle, the cell proliferation was the highest in the A4 treatment group, and the A8 treatment group was the second. Except for the P6 treatment group and the control group (Control), the cell proliferation was significantly higher in the other treatment groups than in the control group (Control) (p<0.05). ). The results of promoting cell proliferation were also observed at a hydrolyzate concentration of 100 μg/mL and 1 mg/mL, but did not show any significant difference as the hydrolyzate concentration increased.

In rabbit chondrocytes, cell proliferation was significantly higher in the N2, N8 and P2 treatment groups than in the control group (p<0.05) after co-culture with the hydrolysate at a concentration of 10 μg/mL for 1 day. Although it was higher than the control group, it did not reach significant difference. On the third day of the experiment, the cell proliferation of the N2, N8 and P6 treatment groups was significantly higher than that of the control group (Control) (p<0.05), and the cell proliferation of other groups. There was no significant difference from the control group (Control); on the 5th day of the experiment, each group and the control There was no significant difference between the groups. Among them, the cells in the A4 and N2 treatment groups had the most proliferation. On the 7th day, the cell proliferation in the A4, A8, N2 and N8 treatment groups was significantly higher than that in the control group (Control). <0.05). When the pig skin collagen hydrolysate concentration was 100 μg/mL and 1 mg/mL co-cultured with rabbit chondrocytes, it was found that the hydrolysate inhibited cell proliferation, so the hydrolyzate concentration in the subsequent cell test was 10 μg/mL. get on. The results of this experiment showed that low concentration (10 μg/mL) of pig skin collagen hydrolysate also promoted the proliferation of rabbit synovial cells (HIG-82) and rabbit articular chondrocytes.

2. Effect of hydrolysate on the survival rate of hydrogen peroxide-induced glioma cells (HIG-82)

Hydrogen peroxide in the body can be SOD (‧O ₂ ^- +‧O ₂ ^- +2H ⁺ H ₂ O ₂ +O ₂ ) conversion, macrophage phagocytosis, sulfhydryl oxidation and other redox reactions, which are highly susceptible to diffusion through the cell membrane to a Fenton reaction with trace metals such as copper or iron ( Fenton reaction), which in turn produces more unstable hydroxyl radicals. Free radicals can participate in intracellular and extracellular reactions to degrade articular cartilage and accelerate the course of degenerative arthritis. Under the cycle of hypoxia and reperfusion oxygen, rabbit synovial cells-HIG-82 (non-chondrocytes) will release freely. Therefore, in this study, rabbit anti-oxidation was carried out with rabbit synovial cells (HIG-82) - cell damage was induced with 1 mM hydrogen peroxide and different groups of pig skin collagen hydrolysate were added (10 μg/mL). ) as a treatment group, while cells that were not added with any hydrolyzate and not treated with hydrogen peroxide were used as a control group; no hydrolyzate was added, only hydrogen peroxide was added as a negative control group; Hydrogen peroxide damage and adding 2500Unit/mL catalase (Positive control) as positive control group, and then treated by MTT method, the effect of hydrolysate on the survival rate of rabbit synovial cells (HIG-82) was determined. The result is as shown in the fifth figure. It was found that the survival rate of rabbit synovial cells (HIG-82) decreased to 46.9% after 3 hours of induction with 1 mM hydrogen peroxide. However, the addition of pig skin collagen hydrolysate significantly increased rabbit synovial cells (HIG- 82) Survival rate (p<0.05), showing that each group of hydrolysate has the ability to scavenge hydrogen peroxide active oxygen molecules and protect cells from oxidative damage induced by hydrogen peroxide. The six groups of hydrolysate are A8 and N8. The cell survival rate was the highest, 73.5 and 74.9%, respectively. It is speculated that the effect may be due to aromatic or lysine and arginine such as Phenylalanine and Tyrosine. Provided by a branched base amino acid such as Arginine, and the contents of these amino acids in pig skin collagen are 821.2, 121.6, 1255.9 and 2966.0 mg/100 g, respectively.

The effect of hydrolysate on the antioxidant enzyme system in rabbit synovial cells (HIG-82)

In this experiment, 1 mM hydrogen peroxide was used to oxidize the cells and different groups of pig skin collagen hydrolysate (10 μg/mL) were added as the treatment group, while cells without any hydrolyzate and not treated with hydrogen peroxide were used as a control. Control (Control); no hydrolyzate added, only hydrogen peroxide was added as a negative control group; hydrogen peroxide was added and 2500 units/mL catalase was added as a positive control group (Positive control) ), and measured the content of catalase, superoxide dismutase, glutathione and glutathione peroxidase in a commercial analysis kit.

(1) Catalase (Catalase)

Catalase is mostly located in the primary peroxisomes of aerobic cells in the body, such as liver cells, kidney cells, and red blood cells. It consists of four identical units, each containing the original heme and Fe ^{3 . + The} tightly bound active region allows only small molecules of protein to enter, so it can only reduce small molecules such as H ₂ O ₂ , methyl or ethyl peroxide, and cannot reduce macromolecules such as lipid peroxides. From the results of the hydrolyzate of the sixth figure hydrolysate induced by hydrogen peroxide in rabbit synovial cells (HIG-82), it can be found that the negative control group is induced by 1 mM H ₂ O ₂ . Intracellular catalase activity was significantly reduced (p<0.05), and its activity was 5.3 nmol/min/mL; except for the P2 treatment group, other hydrolysate treatment groups were more negative than the negative control group. The activity of catalase in cells was significantly increased (p<0.05), and the improvement effect of A8 (10.8nmol/min/mL) and N2 (10.6nmol/min/mL) was better. There was no significant difference in the positive control group (11.4 nmol/min/mL).

(2) Superoxide dismutase (SOD)

SOD is an antioxidant enzyme containing metal ions, which can rapidly disproportionate the most toxic superoxide anion in ROS into hydrogen peroxide and oxygen by continuous oxidation-reduction reaction of metal ions in the active site. Hydrogen peroxide is then passed through hydrogen peroxide. The reduction and reoxidation of the enzyme (Catalase) into oxygen and water, to achieve the role of scavenging superoxide anion. SOD is divided into the SOD type of Cu/Zn-SOD, Mn-SOD and extracellular SOD (Extracellul ar SOD, EC-SOD) mammalian extracellular and intracytoplasm according to the position of the transition metal ion contained in the active center of the enzyme. Mainly Cu/Zn-SOD, while Mn-SOD exists on the mitochondria. As shown in the seventh figure, the results of SOD activity in rabbit synovial cells (HIG-82) induced by hydrogen peroxide showed that the negative control group was induced by 1 mM H ₂ O ₂ (Negative control). The intracellular SOD activity was significantly reduced to 2.3 U/mL (p<0.05); except for the N8 treatment group, the SOD activity of the other treatment groups was higher than that of the negative control group, but only the P6 treatment group. SOD activity was significantly higher than Negative control (p<0.05) and activity was 3.4 U/mL.

(3) Glutathione content and glutathione peroxidase (Glutathi One peroxide, GPx)

GSH is a natural antioxidant substance containing sulfur in living organisms. It is a three-peptide consisting of glutamic acid, cysteine (Cysteine) and glycine (Glycine). It is divided into reduced glutathione. (GSH) and oxidized glutathione (Glutathione disulphide, GSSG). GSH can be catalyzed by GPx to oxidized GSSG, and then reduced to GSH by glutathione reductase, and GSH is abundantly present in the cytoplasm, so the GSH/GSSG ratio is generally higher in normal cells, and appropriate GSH concentration can maintain the activity of GPx; GSH can be used as cofactor in the presence of GPx, and decomposes peroxidation such as H ₂ O ₂ , lipid hydroperoxides (LOOH) and peroxynitrite (ONOO ^- ). To reduce oxidative damage to cells. Its reaction formula is as follows:

For example, Figure 8 and Figure 9 show the content of glutathione (GSH) and glutathione peroxidase (GPx) in rabbit synovial cells (HIG-82) induced by hydrogen peroxide in pig skin collagen hydrolysate. The result of activity. In terms of GSH content, the results showed that the intracellular GSH content was negatively controlled by hydrogen peroxide, and the negative control content (14.2 μM) was significantly lower than that of the control group (Control), while in the hydrolysate treatment group. The A8 and P6 treatment groups significantly increased the GSH content (p<0.05), and the contents were 16.6 μM and 18.5 μM, respectively. Similar results were obtained in the determination of GPx activity. Under the induction of hydrogen peroxide, the GPx activity of the negative control group was significantly lower than that of the control group (Control), which was 18.9 nmol/min/rmL (p<0.05). However, in each hydrolysate-treated group, except for P6, the GPx activity was significantly increased to 24.1 nmol/min/mL (p<0.05), and the GPx activities of the other treatment groups were not significantly different from the Negative control group (Control).

(4) Effect of hydrolysate on the secretion of cytokines in rabbit synovial cells (HIG-82)

Synovial cells secrete regulatory factors that promote inflammation, including cytokines (TNF-α, IL-1β, IL-6), prostaglandins, chemokines, matrix metalloproteinases (MMPs), and their inhibitors and loops. Oxidase, etc., thereby regulating the function of chondrocytes and the conversion of cartilage matrix. While IL-1β and TNF-α play a role in promoting the decomposition of cartilage matrix in cartilage metabolism, in this experiment, lipopolysaccharide (LPS) induces cells to produce an inflammatory response and explores pig skin collagen hydrolysate on rabbits. Membrane cells (HIG-82) secrete the effects of pro-inflammatory cytokines. For example, the tenth and eleventh images are the results of the production of IL-1β and TNF-α by rabbit collagen hydrolysate induced by lipopolysaccharide (LPS) in rabbit synovial cells (HIG-82). The results showed that the treatment group of pig skin collagen hydrolysate significantly inhibited the production of IL-1β (p<0.05), and the secretion decreased by 28-38%. There was no significant difference between the treatment groups; In the effect of α-generation, the secretion amount of each treatment group was significantly inhibited (p<0.05), and the treatment group with N8, P2 and P6 had the best effect, and the inhibition amount was 72-77%.

(5) Effect of hydrolysate on collagen secretion of rabbit synovial cells (HIG-82)

Collagen is the most important structural molecule that constitutes cartilage ECM, accounting for 95% of articular cartilage. It can provide lubrication of articular cartilage during exercise friction, and the course of degenerative arthritis is often accompanied by collagen denaturation and lysis. The stable inflation pressure in the joint is destroyed, which in turn changes the structure of the articular cartilage. Joint synovial cells and chondrocytes are the cells in the joints for the study of arthritis. Both of them can secrete collagen. Here, the easily obtained rabbit synovial cells (HIG-82) are used to investigate the hydrolysis of collagen in pig skin. The amount of collagen secretion after 12, 24, and 48 hours of co-culture, the twelfth figure is the result. The results show that the negative control group ( Negative control) After the stimulation with hydrogen peroxide, the amount of collagen secretion decreased with the culture time; after 12 hours of co-culture, the collagen secretion of each treatment group was significantly higher than that of the negative control group (p< 0.05), in which the collagen secretion in the A8 and N2 treatment groups was significantly higher than that in the positive control group (p<0.05), and the secretion levels were 2.46 μg/mL and 2.41 μg/mL, respectively. There was no significant difference in the positive control group. At the 24th hour, the collagen secretion of each treatment group continued to increase, which was significantly higher than that of the negative control group (p<0.05), A8, N2, N8. The collagen content of P2 and positive control group was significantly higher than that of positive control group (p<0.05), and the secretion amount was 2.99μg/mL, 2.69μg/mL, 2.75μg/mL, respectively. And 2.73μg/mL, the other treatment groups were not significantly different from the positive control group (Positive control); after 48 hours of culture, the A8 and N2 treatment groups had almost the same amount of collagen secretion as the control group (Control). The results of this experiment showed that the pig skin collagen hydrolysate has the ability to inhibit the activity of collagenase in MMPs. Therefore, it is speculated that in addition to protecting the rabbit synovial cells (HIG-82) from hydrogen peroxide, the A4 and N2 treatment groups may also It can inhibit cell collagen from being degraded by inhibiting MMPs.

(6) Effect of hydrolysate on the secretion of proteoglycan in rabbit chondrocytes induced by hydrogen peroxide

Proteoglycans and collagen fibers are the main structural components of articular cartilage, which are responsible for dispersing and transferring the pressure exerted by the joints during exercise and load. In this test, the primary chondrocytes isolated from rabbits were oxidatively damaged with 1 mM hydrogen peroxide, and the effect of the collagen secretion of pig skin on the amount of proteoglycan secretion was determined. Results As shown in Fig. 13, the secretion of proteoglycans in chondrocytes after co-culture with each hydrolysate-treated group was significantly higher than that in the negative control group (p<0.05), with the effects of N2, N8 and P6. Better and positive control group (Positive co Ntrol) no significant difference.

It can be seen from the above that the pig skin collagen hydrolysate has good antioxidant capacity in an in vitro test, and can enhance the activity of the antioxidant enzyme system in the cell and protect the cells from oxidative damage, stimulate the cells to secrete collagen and protein. An extra-cartilage matrix such as a polysaccharide; at the same time, it can also inhibit the secretion of inflammatory factors-cytokines (IL-1β and TNF-α) and the activity of collagenase (MMP-1), and extract collagen from pig skin. The preparation of hydrolysate improves the product of degenerative arthritis disease, thereby not only improving the added value of pig skin, increasing the profitability of the industry and reducing the environmental pollution of pig skin waste, but also obtaining a smaller molecular collagen hydrolysate, and Produce products that contribute to the improvement of the condition of patients with degenerative arthritis, benefit the world, and have both environmental and practical benefits.

Claims (5)

The invention relates to a method for preparing collagen hydrolysate of pig skin, comprising the following steps: a. smashing pig skin: use pig skin of pig to clean and scrape residual subcutaneous fat, steam cooking at 100 ° C, to mince and twist After crushing, heat to 60 ° C and then cool, remove the upper fat, spare; b. extract collagen: (1) add the pig skin into 10 times (w / v) 20% ethanol, stir at 4 ° C for 24 hours Then, the upper layer liquid was removed by centrifugation at 10,000 × g for 15 minutes in a refrigerated centrifuge. This step was repeated once again to achieve the effect of removing fat and impurities; (2) the precipitate was recovered and added 10 times (w/v 1:10). In a 0.2N sodium hydroxide solution, homogenize for 2 minutes, stir at 4 ° C for 24 hours, centrifuge at 10,000 × g for 15 minutes, recover the precipitate and rinse with deionized water; 3) The precipitate was dissolved in 10 times (w/v) 0.5 M acetic acid (Acetic acid) solution, and 5 mL/mg pH 2.0 pepsin (Pepsin) was added and decomposed at 4 ° C for 24 hours; (4) The collagen solution was centrifuged at 10,000 × g for 1 hour, and the supernatant was collected and ice-cooled, followed by slow addition of sodium chloride. After the final concentration reached 2.5 M, the mixture was stirred at 4 ° C for 24 hours to salt out and precipitate the collagen; (5) The solution was centrifuged at 10,000 × g for 1 hour, and the precipitate was recovered and dissolved in 0.5 M acetic acid (Acetic acid). Dialysis with 0.05M acetic acid (Acetic acid) for 72 hours, during which the dialysate is changed every 24 hours; (6) lyophilized collagen solution is freeze-dried in a freeze dryer to obtain a finished collagen product; c. Collagen hydrolysis Preparation: After the collagen is cut, add 30 times of deionized water and stir evenly, and after heating to 40 °C, the following three enzymes with enzyme substrate ratio: 1:50: Alcalase, Protease N (Alphaase) Hydrolysis is carried out by any of Protease N) and pepsin. The conditions of the alkaline protease (Alcalase) are pH 8.0, 50 ° C, and the protease The conditions of N (Protease N) are pH 7.0 and 50 ° C. The conditions of the pepsin are pH 3.0, 37 ° C, and the hydrolysis time is 0-8 hours. After hydrolysis, the enzyme reaction is stopped by heating in a boiling water bath at 100 ° C for 10 minutes. (Inactivated), and then dried in cold water at 4 ° C for 48 hours, and the dried powder was collected to improve the product of degenerative arthritis. The method for producing a collagen hydrolysate of pig skin according to claim 1, wherein the pig skin is ground and stored at -20 ° C for use. The method for producing a collagen hydrolysate of pig skin according to claim 1, wherein the extracted collagen product is stored at -20 ° C for use. The method for producing a collagen hydrolysate of pig skin according to claim 1, wherein the improved degenerative arthritis product is stored in an environment of -20 ° C for use. A use of collagen hydrolysate of pig board to improve degenerative arthritis, the pig skin of the pig is washed and the residual subcutaneous fat is scraped off, and steamed at 100 ° C for centrifugation, mincing, heating to After cooling at 60 ° C, remove the upper fat, store at -20 ° C for later use, take the minced pork skin, extract the pig skin collagen with 0.5 M acetic acid (Acetic acid), and then cut the pig skin collagen finished product. Add 30 times of deionized water to stir evenly, and after heating to 40 ° C (to make it easier to dissolve), the following three enzymes with an enzyme substrate ratio of 1:50: Alcalase, Protease N And any one of pepsin is hydrolyzed, and then heated to 100 ° C for 10 minutes in a boiling water bath to stop the enzyme reaction (inactivation), and then cooled by cold water at 4 ° C, freeze-dried for 48 hours, and the dried powder is collected to obtain Good anti-oxidation ability, and can enhance the activity of the antioxidant enzyme system in the cell and protect the cells from oxidative damage, stimulate the cells to secrete extra-cartilage matrix such as collagen and proteoglycan, and also inhibit fine A proinflammatory cytokine factor (IL-1β and TNF-α) secretion of collagenase The activity of (MMP-1) improves the degenerative arthritis product.