TW202337893A - Jellyfish collagen and Bacillus subtillis natto ferment that can relieve symptoms of degenerative osteoarthritis and its preparation method wherein the rough cartilage surface in knee joint tissue sections is smoothed, and the cartilage layer is thickened - Google Patents

Abstract

The present invention is a jellyfish collagen Bacillus subtillis natto ferment that can relieve the symptoms of degenerative osteoarthritis and its preparation method. Jellyfish collagen which has been proven not to have collagen induced arthritis is used as a raw material, and physical ultrasound is used to assist in extracting jellyfish collagen, and it is fermented with Bacillus subtillis natto for at least 1 day under the environmental conditions of 37 degrees of Celsius and a rotation speed of 150 rpm, to replace the traditional hydrolysis process using porcine pepsin on the market to produce the fermented jellyfish collagen of the present invention. The animal Osteoarthritis (OA) model clearly shows that the fermented product has effects of effectively reducing the difference in supporting force between the two legs of rats, smoothing the rough cartilage surface in knee joint tissue sections, and thickening the cartilage layer.

Description

Jellyfish skin collagen and Natto ferment that can relieve symptoms of degenerative osteoarthritis and its preparation method

The present invention relates to an ingredient and a preparation method used in the treatment of OA (Osteoarthritis, hereinafter referred to as "OA"), in particular, a method of using collagen that has been previously proven not to induce rheumatic osteoarthritis ( Collagen induced arthritis (CIA) doubtful jellyfish skin (Rhopilema esculentum) collagen is used as raw material; and physical ultrasound is used to assist in the extraction of jellyfish skin collagen (Jellyfish Collagen (JC)), and the process is carried out at 37 degrees Celsius and the rotation speed is 150 rpm. Under environmental conditions, it is fermented with Bacillus subtillis natto for at least 1 day to replace the traditional hydrolysis process of porcine pepsin used on the market to produce the fermented jellyfish collagen of the present invention. , referred to as FJC); the results of animal experiments have confirmed that this fermentation product can not only reduce the weight of obese OA rats, and the levels of triglycerides and total cholesterol (TC) in plasma, but also effectively inhibit the synthesis of inducible nitric oxide in plasma. enzyme (iNOS), cyclooxygenase-2 (COX-2), its products nitric oxide (NO) and prostaglandin (PGE2)... and other pro-inflammatory cytokines; it can even reduce the matrix metalloproteinase (MMP) -1 and MMP -3) content; in addition, animal OA models have also clearly shown that the ferment can reduce the difference in foot support in rats, and has the ability to smooth the rough cartilage surface in knee joint tissue sections and thicken the cartilage layer. Thick effect. Based on this, it is clearly proven that oral administration of FJC of the present invention can indeed achieve a protective effect on articular cartilage by effectively inhibiting the production of inflammation-related factors, and can have a certain degree of effect on the inflammation, pain and articular cartilage degradation caused by osteoarthritis. Its soothing effect makes it highly valuable in bone and joint health care and medical industries.

According to statistics, osteoarthritis (hereinafter referred to as "OA") is a degenerative disease of joint instability. According to research data (Appleton, 2018), more than 240 million people worldwide suffer from this disease, and Epidemiological studies have also determined that risk factors for the progression of OA include: aging, overuse or non-physiological load, obesity, trauma, hormonal disorders, or a combination of the above factors; although, so far, the cause of OA has not been clearly known. The exact cause of the disease; however, many research data (such as: Roach et al ., 2005) have clearly shown that inflammation is closely related to the process of OA, and the inflammatory cytokines produced by human metabolism will also increase over time. It is deduced that it will cause damage to articular cartilage and impair the recovery ability of its cartilage matrix. In addition, some research data (Feng et al ., 2017) clearly pointed out that OA includes: progressive erosion of articular cartilage, subchondral bone sclerosis and synovitis... and other main symptoms, but the current treatment options are still only limited. The focus is on reducing joint degeneration, improving joint flexibility and relieving joint pain... and there are always many challenges. Here we only take the current treatment for mild to moderate OA as an example. The treatment includes the use of acetaminophen and Non-steroidal anti-inflammatory drugs (NSAIDs)... and other drugs, however, research data (McAlindon et al ., 2014) have also clearly reflected that these drugs will more or less affect the heart, liver, kidneys and gastrointestinal tract... Other organs cause some adverse side effects, and these adverse side effects will become more serious as the dosage and treatment time increase. In view of this, over the years, the OA treatment industry has been relentlessly conducting research and development on this issue, hoping to eventually develop an innovative alternative treatment plan and ingredients that has no adverse side effects and can effectively alleviate the aforementioned OA symptoms, so that it can benefit numerous OA patients. Patients, this innovative alternative treatment solution and ingredient can become a biomaterial with great potential in the field of biomedical applications, thus enabling it to bring great economic value to the medical industry.

According to the investigation, since collagen is the most common component in the solid phase of articular cartilage, collagen supplementation has always been considered an important measure to prevent articular cartilage damage over time, and is the basis of the medical community after the onset of OA. It is one of the key therapeutic ingredients often chosen to support the healing process. However, traditionally, due to the insufficient supply of raw materials such as bone and leather (mainly from pigs and cattle) that provide this ingredient, the news of rising raw material prices will undoubtedly have an impact on global collagen. posing a huge threat to the supply (Khong et al ., 2018), religious factors will further restrict the use of pig and cattle derivatives among Muslim, Indian or Jewish ethnic groups, whose population not only accounts for 38.4% of the global population (Hackett et al ., 2012). The outbreaks of Bovine spongiform encephalopathy and swine fever in the 1980s and 1990s also prompted people to start to have religious and religious beliefs about collagen derived from pigs and cattle. Safety concerns, in summary, the search for raw materials that can replace pig- and bovine-source collagen has become a major issue that the industry has focused on. Subsequently, through continuous exploration and research by the industry, by-products from marine animals and marine processing industries Collagen is gradually becoming a raw material with great potential and can be used to replace porcine and bovine collagen.

Press, in the past ten years, many research materials (Barzideh et al ., 2014; Nagai et al ., 2000) have clearly revealed that jellyfish is a promising source of collagen, and its application is not restricted by any religion except In addition, and so far, no connection has been found with fatal infectious diseases; however, according to previous literature, when extracting collagen from jellyfish, it is still necessary to use various enzymes and dangerous chemicals in the extraction process and radiation... to improve the extraction rate of jellyfish collagen; among them, the traditionally used enzymes, especially pepsin, are usually pig-derived pepsin. This method not only makes the extracted jellyfish collagen be Halal/Jewish people regard it as religiously unclean food, which completely defeats the original purpose of using aquatic collagen. Currently, jellyfish are only used as a low-priced salted food in the food market. According to research literature (Brotz et al ., 2012), more and more jellyfish are multiplying in large numbers around the world, which will bring great changes to the oceans. The ecology and food market have brought serious negative impacts. Therefore, if the extraction process of jellyfish collagen can be improved, the extraction cost can be reduced or the output can be increased, it will not only turn previously neglected marine organisms (such as jellyfish) into a deep tool industry It is also expected that through the enhanced utilization of jellyfish resources, the number of jellyfish can be significantly reduced, thereby effectively reducing the damage caused to fishery ecology and environmental pollution (Khong et al ., 2018). In view of this, the inventor is thinking of using jellyfish skin to replace collagen raw materials derived from pigs, cows or chickens, which are more expensive and may have religious taboos, and fermented them with Natto bacteria. It is expected that the protease produced along with the growth of Natto bacteria can further It replaces the porcine pepsin commonly used in traditional extraction procedures on the market, thereby completely solving the religious taboo issues that may arise during the traditional extraction process of collagen.

As mentioned above, osteoarthritis is indeed a common disease among the elderly around the world, and it is by far the most common joint disease. Chang Hao occurs in the knee, hip and hand joints; among them, the knee joint has the highest incidence rate (Weber et al . al. , 2016), and more than 240 million people worldwide suffer from this disease (Appleton, 2018). In addition, according to a report from Taiwan's Ministry of Health and Welfare, the prevalence of OA in Taiwan is about 15%, and more than 3.5 million people suffer from OA disease every year. It is characterized by intra-articular inflammation, cartilage degeneration and subchondral bone reconstruction. It may eventually It causes severe joint pain or disability, and 20,000 of them require knee replacement surgery. According to reports, although there are many causes of OA, including innate factors such as age, gender, genetics and race, aging not only causes an increase in oxidative stress in articular cartilage, but also affects the synovium, subchondral bone and muscles. Increase the load on joints (Felson et al. , 1998; Loeser, 2011). Regarding the differences between patients of different genders, some studies have pointed out that the total cartilage volume of men’s tibia and patella is significantly higher than that of women, and over time, women Compared with men, the patellar cartilage defect rate is significantly higher, and there is more knee cartilage volume loss (Elbaradie et al. , 2013). The risks of other acquired changes include: obesity, activity, exercise, muscle weakness and joint Damage... and other factors (Felson et al. , 1998). Check, articular cartilage is a highly specialized joint connective tissue. Its main function is to provide a smooth and lubricated surface for the junction of bones and to provide load transmission with a low friction coefficient. Generally speaking, articular cartilage lacks blood vessels, lymphatic vessels and nerves, and is affected by the harsh biomechanical environment, making it It has only limited inherent healing and repair capabilities. Accordingly, the preservation and health of articular cartilage is of vital importance to joint health and cannot be ignored.

According to reports, cartilage is mainly composed of chondrocytes and extracellular matrix (ECM). Among them, ECM is composed of water, proteoglycan (proteoglycan), collagen and noncollagen glycoprotein (noncollagen glycoprtein)... etc. It is composed of ingredients (Aigner and Stöve, 2003), among which Type-II collagen provides a network structure and is composed of ingredients such as Proteoglycan and Non-collagenous Protein. Stabilizing its structure, necessary properties such as elasticity and high tensile strength of cartilage are provided by proteoglycans and collagen (see Sophia Fox et al. , 2009). According to relevant research data (Thomas et al. , 2007), during the progression of the initial stage of OA, the metabolic activity of chondrocytes will be up-regulated, subsequently leading to chondrocyte apoptosis, and oxidative stress, endoplasmic reticulum stress and inappropriate Mechanical stress can also induce chondrocyte apoptosis. This not only confirms that there is an obvious positive correlation between the severity of OA and cell apoptosis, but also indicates that this process plays an important role in the pathogenesis of OA. In addition, factors that affect the OA process include the following factors:

(1) Pro-inflammatory factors: At present, although the exact cause of OA is not yet clear, many research data show that inflammation is closely related to the process of OA, and pro-inflammatory cytokines, such as interleukin-1β ( Interleukin-1β (IL-1β for short), tumor necrosis factor-α (TNF-α for short) and interleukin-6 (IL-6 for short)... are all involved in the progression of OA and cartilage matrix degradation. Playing a key role (Kobayashi et al ., 2005), excessive production of pro-inflammatory cytokines will cause matrix metalloproteinases (MMPs) and disintegrin metalloproteinases with thrombospondin motifs (A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS for short) increases, thereby inducing the degradation of collagen and proteoglycans; at the same time, research data (Goldring et al ., 2000) also found that in the synovial fluid, synovial membrane, subchondral bone and cartilage of OA patients Among them, higher levels of IL-1β can promote the production of MMPs, inflammation mediators prostaglandin E2 (PGE2) and nitric oxide (NO) in chondrocytes. Under normal physiological conditions, The dynamics of ECM synthesis and degradation are maintained in the joint through the regulation of chondrocytes, but this balance will be destroyed during the process of OA, and the composition of ECM (such as collagen and proteoglycans) will be degraded by MMPs, proteolysis Excessive secretion and loss of enzymes lead to continued degradation of cartilage tissue. MMPs play a vital role in the process of OA because they are responsible for the organization, remodeling and degradation of ECM (Xue et al ., 2014). In addition, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (INOS) are stimulated by inflammatory cytokines and are expressed (Daheshia and Yao, 2008);

(2) ADAMTS: It is a kind of proteoglycanase, of which at least 19 species have been found so far. It is mainly used to break down four kinds of proteoglycans, including Aggrecan, Brevican, Neurocan and Versican, to promote cartilage matrix remodeling; if according to its receptor The types can be divided into seven types, among which ADAMTS, which breaks down Aggrecan, the main proteoglycan in joints, is used as an indicator to judge the severity of OA, such as: ADAMTS-1, ADAMTS-4, ADAMTS-5, ADAMTS-8 and ADAMTS-15 (Porter et al ., 2005); and

(3) Obesity: Press, "obesity" is actually a chronic metabolic disorder. Together with "overweight", it has been defined by the international medical community as abnormal or excessive fat accumulation that may harm health. Currently, although "obesity" It can be evaluated through the body mass index (hereinafter referred to as BMI). The conversion formula is human body weight (kg) divided by the square of height (m ² ). According to the definition of the World Health Organization (WHO), those with a BMI greater than 25 can Classified as "overweight", those with a BMI greater than or equal to 30 can be called "obese", and "obesity" has already acquired epidemic characteristics. According to epidemiological research and statistics (see WHO, 2018), in 2016 In 2016, more than 1.9 billion adults aged 18 and above in the world were "overweight"; among them, more than 650 million adults were "obese"; overall, about 13% of the world's adult "obese" population in 2016 (of which , accounting for 11% of men and 15% of women). Compared with the global population from 1975 to 2016, the global prevalence of obesity has almost tripled. In contrast, relevant research and statistics in Taiwan (National Health Service, Ministry of Health and Welfare, Taiwan , 2018 reference), Taiwan's Ministry of Health and Welfare defines "obesity" based on the physical constitution of Taiwanese people as a BMI greater than or equal to 27, and a waist circumference greater than 90 cm for men and greater than 80 cm for women. In addition, according to the Republic of China between 102 and 105 Survey results on changes in national nutrition and health status show that the prevalence of overweight and obesity among adults in Taiwan is 45.4%. Compared with 32.7% in 1982-85 and 43.4% in 1994-97, the prevalence of obesity in Taiwan has also increased. 10.7%. Check that the complications of morbid obesity basically affect all organ systems of the human body, including: cardiovascular system diseases (easily causing hypertension, atherosclerosis, peripheral vascular disease accompanied by myocardial infarction and cerebrovascular accidents...etc.) ; Respiratory system diseases (easily causing asthma, obstructive sleep apnea and obesity hypoventilation syndrome, etc.); Metabolic system diseases (easily causing type 2 diabetes, abnormal glucose tolerance, dyslipidemia, etc.); Musculoskeletal system diseases ( Easily cause intervertebral disc disease and weight-bearing osteoarthritis of hips, knees, ankles, feet, etc.); gastrointestinal system diseases (easily cause cholelithiasis, gastroesophageal reflux disease, steatohepatitis, cirrhosis, liver cancer, colorectal cancer… etc.); stress urinary incontinence, endocrine and reproductive system diseases (easily caused by polycystic ovary syndrome, increased risk of pregnancy and fetal abnormalities, male hypogonadism, endometrial cancer, breast cancer, ovarian cancer, prostate cancer and Pancreatic cancer...etc.); skin, neurological diseases (easily caused by pseudotumor cerebri, carpal tunnel syndrome...etc.) and psychological diseases (depression...etc.) (Buchwald et al ., 2007). In addition, according to Taiwan's Ministry of Health and Welfare Statistical analysis of causes of death in 2018 (National Health Administration of the Ministry of Health and Welfare, 2018 reference) shows that the top ten causes of death among Taiwanese people are (1) 206.9 cancer deaths (2) 91.5 heart disease deaths (3) per 100,000 population ) Pneumonia 56.9 people (4) Cerebrovascular disease 48.9 people (5) Diabetes 39.8 people (6) Accident injuries 29.0 people (7) Chronic lower respiratory tract disease 26.1 people (8) Hypertensive disease 25.4 people (9) Nephritis and nephropathy syndrome and renal disease, 23.4 people (10); chronic liver disease and cirrhosis, 18.3 people; the number of deaths was 133,489, accounting for 77.2% of the total number of deaths, and the majority were chronic diseases, such as: heart disease, cerebrovascular disease, diabetes, hypertension Chronic diseases such as blood pressure, nephritis, nephrotic syndrome, nephropathy, chronic liver disease and cirrhosis... among them, a total of six are related to "obesity". In addition, a report released by the U.S. Centers for Disease Control in 2016 (Murphy et al ., 2017) also clearly pointed out that 54 million obese adults in the United States suffer from OA, which may increase to 78 million in 2040, and according to statistics, More than 30% of obese patients suffer from OA. In addition, in a research report (Amin et al ., 2008), it was also confirmed that losing 5 kilograms of body weight can reduce the risk of OA by more than 50%, and it was also proven that a high-fat diet will accelerate the progression of post-traumatic OA. In summary, based on biomechanical theory, the increased risk of knee OA in obese patients is entirely due to the increased force exerted on the knee joint due to extra weight; at the same time, obese patients tend to have more adipose tissue. These adipose tissues will release inflammatory cytokines, thereby causing systemic low-grade inflammation. Among them, overexpression of IL-6 through MMP-3 will not only induce cartilage destruction, but also drive MMP-3 to participate in the decomposition of extracellular matrix. It causes collagen degradation; TNF-α inhibits the synthesis of extracellular matrix components such as collagen and proteoglycans; vascular endothelial growth factor is also secreted by adipose tissue and increases the release of IL-6 and TNF-α. Will promote the formation of osteophytes (Hashimoto et al ., 2012). In addition, another important risk factor for OA in obese patients is dyslipidemia. These patients usually have low levels of high-density lipoprotein, which may lead to cartilage fibrillation and a reduction in extracellular matrix components. In addition, obese patients often have high levels of low-density lipoprotein, which stimulates the release of vascular endothelial growth factors and free fatty acids, and activates macrophages to secrete TNF-α (King et al ., 2013). Previous literature (Ernest et al ., 2018) also proved that weight overload and meniscal instability can lead to the occurrence and development of OA.

As mentioned above, research and survey literature show that the human body will suffer from OA during the life process of "birth, growth, strength, old age, and death"? It is indeed closely related to the content and quality of collagen in the human body. According to reports, collagen is a protein commonly found in animals, accounting for about 30% of the total protein content of mammals. It is mainly distributed in connective tissues in animals, such as bones, cartilage, tendons, cornea, blood vessels and skin, etc. Extracellular matrix (ECM for short), and collagen plays an important role in supporting, protecting, ion adsorbing and transporting nutrients in connective tissue and muscles, so it has extremely high water retention properties. Generally speaking, collagen is a type of glycoprotein (Glycoprotein). Collagen from vertebrates contains about 0.5-1.3% of six-carbon sugar (Hexose), while collagen from non-vertebrates contains about 3-14% sugar. In addition, the amino acid composition of collagen is also unique, allowing it to be easily distinguished from other proteins; among them, glycine (Gly) accounts for approximately 15 to 30% of the overall amino acids. Proline (Pro for short) accounts for about 12%, and hydroxyproline (Hydroxyproline for short) accounts for about 12%. General animal protein only contains a small amount of hydroxyproline. In addition, alanine (Alanine for short) Ala) accounts for about 11%, but lacks tryptophan (Try) (Krane et al ., 2008; Liu et al ., 2012). In addition, as shown in Figure 1, the main structural feature of collagen is its triple helix structure, which is composed of three polypeptide chains C1, C2 and C3 interwoven to form a collagen molecule and stabilized by hydrogen bonds (Silva et al ., 2014). The stability of the triple helix structure of collagen is related to the content of imino acids present in the molecule (Prockop and Kivirikko, 1995). Proline and hydroxyproline can penetrate through the pyrrolidine ring and hydroxyproline of imino acids. The triple helix structure is strengthened by interchain hydrogen bonds formed by the hydroxyl groups of amino acids. This special triple helix structure ensures its mechanical strength and is called procollagen, the triple helix of the collagen peptide chain. The structure is different from the ordinary α-helical structure. Although its pitch is larger, the number of amino acid residues contained in each helix is very small, only 3.3. Therefore, the triple helix of collagen appears thin and long. Moreover, the space in the middle of the helix is very small and can only accommodate one hydrogen atom. Only glycine can occupy this position. In addition, the angle between the peptide planes unique to proline is also a necessary factor for the formation of this special helical structure. This is also the main reason why the -glycine-proline-hydroxyproline-triple sequence appears alternately in the collagen peptide chain (Berisio et al ., 2004). Generally speaking, collagen can be divided into 21 types according to its structural peptide chain composition or gene locus position; among them, the five types that were first discovered, such as type I, II, III, IV and V, have been studied the most. Among them, type 1 collagen has the highest content, accounting for approximately 90% of all collagen. It is also the most widely used collagen (Prockop and Kivirikko, 1995). In addition, depending on its structure, it can be It can be roughly divided into the following categories: (1) Fibrillarcollagens, including types Ⅰ, Ⅱ, Ⅲ, and Ⅴ; (2) Reticular structure collagen, including types Ⅳ, Ⅶ, and Ⅹ; (3) Those located on the surface of fibrillar collagen (Fiber associated collagens with interrupted triple helices-FACIT), including type Ⅸ, XII, XIV, XVI, etc.; (4) Bead-shaped fibrillar collagens, including type Ⅵ; ( 5) Collagen connected to the basement membrane, including type VII; (6) Transmembrane domain collagen, including types XIII and XVII; the rest are collagens whose characteristics have not been clearly defined (Deyl and Miksik, 2000); As for collagen in various parts of the human body, its content and type also vary depending on the specificity of the tissues in each part, such as: Type I collagen; Type II collagen. Exists in cartilage, vitreous body of the eyeball, and intervertebral discs; Type III collagen mainly exists in the smooth muscle layers of skin, blood vessels, internal organs, and intestines; Type IV collagen mainly exists in the basement membrane; Type V collagen is less abundant, accounting for only 10% of all collagen, and is often The system is found along with collagen type I and type III, such as: existing in hard bone, tendon (Tendon), cornea (Cornea), skin, blood vessels, and the basement membrane of the developing fetus (Bornstein et al ., 1980; Deyl and Miksik, 2000).

Although collagen is the most ubiquitous and important component of articular cartilage, and collagen supplementation has been proven to be an important measure to effectively prevent articular cartilage damage over time, as mentioned above, due to the current global collagen Collagen supplied in the market is traditionally pig- or bovine-derived collagen. Each type of pig- or bovine-derived collagen has religious taboos due to its source of raw materials or process technology, and cannot be used by Muslims, Jews, or Hindus. It is accepted that swine or rinderpest often causes people to doubt the food safety risks of collagen derived from pigs or cattle. In view of this, in the past decade or so, the relevant medical and food industries have been working hard to target possible safety risks. On the topic of replacing pig or bovine collagen raw materials, many developments, researches and experiments have been carried out, in the hope that marine animals can be used as raw materials to extract collagen that is completely free of religious taboos and food safety risks, and can completely replace traditional pigs. source and bovine source collagen.

According to reports, edible jellyfish, commonly known as "jellyfish", has not only been an indispensable ingredient in Chinese cuisine since ancient times, but has also been well-known in traditional Chinese medicine for its curative effect on diseases such as hypertension, bronchitis, and arthritis to a certain extent. boundary (see Hsieh et al ., 2001). Check, the natural history of China's Jin Dynasty (232-300 AD) records: "There is something in the East Sea, which looks like clotted blood, several feet wide in length and radius, and is called bream fish. It has no head and no intestines in its abdomen. Where it is located , many shrimps attached to it, and the Yue people cooked it with whatever they had." This is the earliest record of eating jellyfish in history; however, only certain types of jellyfish are considered suitable for human consumption, and most of these jellyfish belong to the Rhizostomeae and Scyphozoa, and most species are distributed in subtropical and tropical waters. Please refer to Figure 2. The characteristics of these jellyfish are their tough body texture and A high proportion of protein (see Hsieh et al ., 2001), among which the most economically valuable varieties are crispy and white in texture (see Purcell et al ., 2013). Traditionally, since the quality of jellyfish J will deteriorate rapidly at room temperature, people must immediately carry out subsequent processing procedures on the jellyfish J after capturing the jellyfish J; please refer to Figure 2. First, the jellyfish J The bell jar J1 of J and the oral arm J2 hanging below are separated and washed respectively with sea water, and the gonads and mucus on them are scraped off; then, coarse salt (Sodium chloride, NaCl) and alum are usually used (Potassium alum, referred to as KAl (SO ₄ ) _{2.12H 2} O) and other ingredients, undergo multiple dehydration treatments; after repeating the dehydration treatment 2 or 3 times, process it as a salted product Among them, in addition to lowering the PH value of jellyfish products, alum can also act as a disinfectant and astringent (hardening) agent for jellyfish products, allowing the proteins in the jellyfish to be deposited and maintained in a tight structure, while table salt It can help reduce moisture and reduce the water activity inside it, thereby making the product more shelf-stable (see Purcell et al ., 2013); generally speaking, please refer to Figure 2, which is represented by the J1 part of the jellyfish bell jar. The made jellyfish product is commonly known as "jellyfish skin", and the jellyfish product made from the J2 part of the oral arm of the jellyfish is called "jellyfish head". In recent years, according to many studies in the biochemical and food fields, jellyfish ( Rhopilema esculentum) tentacle protein has shown extremely strong scavenging activity against superoxide anion free radicals, and its free radical scavenging activity remains stable at high temperatures, even at When incubated at a high temperature of 80 degrees for 30 minutes, its free radical scavenging rate was as high as 82.81%. In addition, in terms of hydroxyl free radical scavenging activity, it also showed higher scavenging power than vitamin C (Yu et al ., 2005) ; In addition, there is an animal experiment that confirmed that after feeding mice with jellyfish collagen hydrolyzate for 6 weeks, it was found that the climbing time of the mice increased in the climbing endurance test, and in the fatigue resistance test, it was found that it could be significantly improved. Reduce the levels of blood lactic acid and Blood urea nitrogen in mice, and increase liver glycogen and muscle glycogen; in aging model tests, it was also found that the Glutathione peroxidase activity in the serum of mice and the SOD activity in the liver homogenate were both reduced. , this experimental result clearly shows that jellyfish collagen hydrolyzate can significantly relieve fatigue in mice and has antioxidant effects in aging mice (see Ding et al ., 2011). Regarding the antioxidant and anti-fatigue activities of jellyfish collagen, some studies (see Hwang et al ., 2018) have shown that Nomura jellyfish ( Nemopilema nomurai ) extract can down-regulate iNOS and COX in LPS-stimulated Raw 264.7 macrophages. -2 mRNA content, and has no obvious cytotoxic effect, but has no effect on the expression of other inflammation-related genes such as IL-1β, TNF-α and IL-6; press, nerve injury induced protein 1 (abbreviated as Ninj1) is an important adhesion molecule that can reduce cell adhesion to ECM in vitro. Nomura jellyfish extract reduces the expression of Ninj1, and it reduces the inflammation at inflammatory sites in zebrafish larvae microinjected with LPS. The cumulative number of infiltrations showed an inhibitory effect on leukocytes; in addition, the study found that Nomura jellyfish extract not only inhibited the expression of MMP-2 and MMP-9 in LPS-stimulated Raw 264.7 cells, but also inhibited it in a dose-dependent manner. , blocks the nuclear translocation of NF-κB in Raw 264.7 cells. In addition, regarding the wound healing potential of jellyfish collagen, some studies (see Felician et al ., 2019) found that for in vitro wound healing activities, cells treated with jellyfish ( Rhopilema esculentum ) collagen peptides (CP) Cell migration in the wound area was significantly enhanced, and the repair rate of cell scratches treated with 6.25 mg/ml CP reached 75.49%. In terms of animal experiments, it was found that using CP ₁ extracted with collagenase II enzyme (collagenase II enzyme) and alkaline protease (Alkaline protease) and CP ₂ extracted with papain (Papain) had no negative effects on the appetite and health of mice. Effect, and it was observed after 4 days of tube feeding of 0.9g/kg CP ₁ and CP _2. Both had a significant effect on the wound healing of injured mice, and had a smaller wound area compared with the vehicle treatment group. ; In addition, between CP ₁ and CP ₂ , it was observed that CP ₁ had a greater impact on wound healing than CP ₂ on day 6; compared with the vehicle treatment group, the histology of the injured skin in the CP treatment group was Analysis showed significant signs of increased re-epithelialization, new tissue regeneration, and collagen deposition.

In view of the fact that many of the aforementioned studies have confirmed the great potential of jellyfish collagen in antioxidants and wound healing, how to make jellyfish collagen completely effective in the entire extraction process by using an easily available, cheap and high-quality marine biological and plant raw material? Without the use of bovine or porcine pepsin, jellyfish collagen can be produced in large quantities with a simple, fast and highly controllable preparation process that is completely free of religious taboos and food safety risks, and can be easily It is used to make materials for treating OA diseases, so that the jellyfish collagen not only has excellent biodegradability and biocompatibility, but also can effectively avoid the above-mentioned problems of ordinary traditional jellyfish collagen, so that it can be made based on The fermented jellyfish collagen material is more conducive to promoting the regeneration of normal joint tissue cells, thereby greatly improving the efficacy of joint regeneration. It has become a related biodegradable material in recent years. The industry has been working hard, constantly exploring, and striving for research and development, hoping to achieve breakthroughs. This is a common issue, and it is also an important issue that the present invention is eager to explore and overcome in the subsequent content.

In view of the aforementioned problems and doubts such as religious taboos and food safety risks in the selection, processing and use of traditional jellyfish collagen, the inventor has finally developed and designed a "can "Jellyfish Skin Collagen Natto Fermentation Product for Relieving Symptoms of Degenerative Osteoarthritis and its Preparation Method", with the hope that through the proposal of the present invention, the low-cost, high-quality and high-quality collagen from the jellyfish skin left over from Taiwan's food processing industry and natto bacteria are effectively reused to produce high-quality collagen materials with excellent quality, affordable price and antibacterial activity against arthritis progenitors, thereby benefiting the urgent needs of the majority of OA patients and effectively alleviating the urgent needs of these OA patients. Symptoms of degenerative osteoarthritis.

The main purpose of the present invention is to provide a jellyfish skin collagen natto ferment that can alleviate the symptoms of degenerative osteoarthritis. The fermentation product (FJC) uses commercially available salted jellyfish skin as raw material and uses physical ultrasound. After the jellyfish skin collagen (JC) is extracted with auxiliary acid, the jellyfish skin collagen (JC) is fermented with Natto bacteria for at least 1 day under the environmental conditions of 37 degrees Celsius and a rotation speed of 150 rpm, thus forming the present invention. The fermentation substance (FJC), the fermentation substance (FJC) has been confirmed by animal experiment results, not only can effectively reduce the weight of obese OA rats, plasma triglyceride and total cholesterol (TC) content, but also can effectively inhibit plasma It is still effective in the production of pro-inflammatory cytokines such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), its products nitric oxide (NO) and prostaglandin (PGE2)... Reduces the content of matrix metalloproteinases (MMP-1 and MMP-3); in addition, animal OA models also clearly show that it can effectively reduce the difference in foot support in rats, and can improve the stability of rat knee joint tissue sections. The effect of smoothing the rough cartilage surface and thickening the cartilage layer clearly proves that oral administration of the fermented substance (FJC) can indeed achieve the protective effect on articular cartilage by inhibiting the production of related inflammatory factors, and on osteoarthritis. The inflammation, pain and articular cartilage degradation caused by it produce a certain degree of soothing effect, thus having extremely high utilization value in the fields of bone and joint health care and medical industry.

Another object of the present invention is to provide a method for producing jellyfish skin collagen natto ferment that can alleviate the symptoms of degenerative osteoarthritis. It is expected that this preparation will enable the bone and joint health care and medical industry to use cheap, high-quality and easily obtained jellyfish. Using skin and natto bacteria as raw materials, through a simple, fast and highly controllable preparation process, jellyfish skin collagen and its natto bacteria fermentation product are produced in large quantities and are completely free of religious taboos and food safety risks, and the fermentation product Based on this, biochemical materials can be made that are more conducive to promoting normal joint tissue cell regeneration and are affordable, thereby greatly improving the effects of joint regeneration health care and treatment.

In order to facilitate the review committee to have a further understanding of the purpose, technical features and effects of the present invention, the detailed description is as follows:

As mentioned above, although many research data have clearly revealed that jellyfish is a promising source of collagen, its application is not subject to any religious restrictions, and so far, no relationship with any fatal infectious diseases has been found. related; however, when traditionally extracting collagen from jellyfish, it is still often necessary to use various pig or cattle-derived enzymes pepsin or other radioactive and toxic chemicals to increase the extraction rate of jellyfish collagen, especially The enzyme used to hydrolyze jellyfish to extract collagen has traditionally been pepsin from pigs or cattle. This traditional hydrolysis method not only makes the extracted jellyfish collagen considered religious by the Halal, Jewish or Hindu groups. It is not a clean food ingredient and completely defeats the original purpose of using aquatic collagen.

In view of the above problems and the hope that the industry can extract high-quality collagen that is cheap and has no religious taboos or food safety risks from the abundant jellyfish resources in oceans around the world, the inventor of this case considered In the hydrolysis process of collagen extracted from jellyfish, no animal-derived proteases (such as porcine or bovine-derived pepsin) or any other radioactive and toxic chemicals are used, and pure plant-derived enzymes are used instead. Hydrolyzable materials used. In this way, it will not only benefit the majority of OA patients and effectively alleviate the symptoms of degenerative osteoarthritis, but also significantly reduce the number of jellyfish through enhanced utilization of jellyfish resources, thus effectively reducing the damage caused by jellyfish flooding to fishery ecology and environmental pollution.

First of all, according to previous research reports (see Priest, 1993), Bacillus subtilis is a Gram-positive rod-shaped bacterium that is an aerobic or facultative anaerobic bacterium. Although it can perform aerobic respiration in an aerobic environment, it can also use glucose and nitrate to grow anaerobically under facultative anaerobic conditions. Its size is quite small, only 0.8 µm × 1.5~1.8 µm, and is similar to ordinary Saprophytes also have the property of producing heat-resistant spores. In addition, other research reports (see Burdett, 1988, Wier et al., 1983 and Dring et al., 1976) also pointed out that Bacillus subtilis reproduces by binary fission and mostly grows in soil, hay, sewage and other environments. To cope with environmental changes and lack of nutrient sources, it will produce endospores to resist adverse environments. The spores themselves are heat-resistant and contain a layer of calcium salt in the spore cortex, which has heat-resistant properties. When cells transition from exponential phase to stationary phase and enter the sporulation phase, many enzymes and antibiotics are also synthesized and secreted out of the cells to seek new nutritional sources or to resist adverse external environments. (Errington et al., 1993); Bacillus subtilis is an excellent host cell that can directly secrete a large number of expressed proteins into the culture medium (Doi, 1986); in addition, because it can produce high amounts of soluble and stable enzyme, therefore, Bacillus subtilis is the strain most commonly used in industry to produce target metabolites (see Liebs, 1988); so far, as many as 65 strains of Bacillus subtilis have been isolated, many of which have been Used as a host for fermentation industry because most Bacillus subtilis are non-pathogenic bacteria and are recognized as GRAS (generally regarded as safe) microorganisms by the U.S. Food and Drug Administration (FDA). In addition, it is an excellent secretor and can secrete a large amount of proteins and metabolites, and can provide many useful substances to humans and produce a variety of hydrolytic enzymes (see Priest et al., 1977).

Secondly, natto is one of the subspecies of Bacillus subtilis, and was first purified and isolated from natto by Professor Shin Sawamura of the University of Tokyo in 1905, and the strain was named Bacillus natto ( Sawamura, 1906 (cf. Sawamura, 1906). The earliest documented record of natto in Japan appears in the book "New Sarugakuki" in 1286 AD, which means "beans of Nasho (i.e., temple kitchen)". At that time, Japanese beans were ingested by monks who were particular about observing the precepts. The main source of protein, so natto is also called "temple natto" (see Xu, 2005); the traditional method of making natto is to wash the soybeans and soak them overnight and then steam them; then wrap them in straw and put them at 100 degrees Celsius Sterilize it in water and keep it at 42 degrees Celsius for one day; because the rice straw contains Bacillus subtilis, which will produce spores and is highly heat-resistant, it will not be damaged during the sterilization process and can also be cultured at high temperatures by accelerating growth. It inhibits other bacterial species and causes the fermentation of soybeans to produce sticky filaments (see Tanimoto, 2001). This sticky filament is actually the main source of the flavor of natto. Some people do not like to eat natto because it has a very strong smell. and taste, mainly caused by fermentation by-products such as NH4+ produced during the fermentation of natto (Tsuji, 1982). The natto bacteria in natto are nattokinase contained in the sticky filaments produced during soybean fermentation. ), the main components of the viscous filaments are composed of poly-γ-glutamic acid (γPGA for short) and fructan. Its function is to prevent nattokinase from losing its function (see Tsuji, 1982), and poly After purification, glutamic acid has many uses in food processing, such as: as a thickening agent for fruit juice drinks, anti-aging health food and antifreeze...etc.

In view of this, the inventor thought of effectively utilizing the natto bacteria in the large amount of high-quality natto by-products left after food processing in Taiwan's food processing industry, in the hydrolysis process of extracting jellyfish collagen (JC). , ferment it with Bacillus subtillis natto under environmental conditions of 37 degrees Celsius and a rotation speed of 150 rpm for at least 1 day to completely replace the traditional hydrolysis process using porcine pepsin on the market to produce the invention. Jellyfish skin collagen fermented product (Fermented Jellyfish Collagen, referred to as FJC); the present invention provides a "jellyfish skin collagen natto fermented product that can alleviate the symptoms of degenerative osteoarthritis and its preparation method", please refer to Figure 3 , the manufacturing method includes the following steps (note, the following process steps are the best steps, data and conditions summarized by the inventor after many process implementations, repeated analysis and adjustment of the details and conditions, therefore, For the purpose of simplifying the description, the present invention only takes the best steps, data and conditions as examples to illustrate the preparation method in the subsequent description; however, when the preparation method of the present invention is actually implemented, it is not limited to these, and its steps, data and conditions are not limited to this. and conditions can also be fine-tuned according to actual needs. As long as the fine-tuning range of the data and conditions is within ±5% of the data and conditions listed below, the main purpose of the invention should still be achieved and the required results can be achieved. The fermentation material is also the scope of rights that the present invention intends to claim for protection here; at the same time, the raw materials used in the preparation method of the present invention are not limited to natto from the high-quality natto by-products left after processing by Taiwan’s soy food processing industry. Bacteria, other plant-derived strains can also be used and replaced according to actual needs. This is also within the scope of the rights claimed by the present invention, and shall be stated in advance.

Please refer to Figure 3 again. In a preferred embodiment of the present invention, the method for making the FJC of the present invention includes the following steps:

(100) Desalting: The jellyfish used in the present invention is the salted jellyfish skin commonly sold and used in the market. Before using the salted jellyfish skin in the present invention, it must be desalted first. , the salted jellyfish skin used in the preferred embodiment of the present invention is provided by "Zelin Food Co., Ltd." in Fuzhou, China, and the desalination process is based on and improved from previous research data Kimura -Suda et al . (1995) disclosed that in the preferred embodiment of the present invention, the desalination treatment is to thoroughly clean the salt on the surface of the salted jellyfish skin with clean water;

(101) Then, cut the washed jellyfish skin into jellfish strips one by one with a thickness of about 1 to 1.5 millimeters (mm) and a width of about 2 to 5 centimeters (cm);

(102) Then, immerse the jellyfish skin filaments in clean water 10 times its total weight, and wash the jellyfish skin filaments repeatedly for 3 hours while slowly stirring with a magnet, and replace them every hour Clean water to thoroughly wash away the salt remaining in the salted jellyfish skin;

(103) Ultrasound-assisted acid extraction of collagen: The extraction process used in this preferred embodiment of the present invention is based on and improved from the method disclosed in previous research data (Khong et al. (2018)). 4 degrees, complete the following extraction steps in order:

(103-1) The desalted jellyfish skin filaments were subjected to alkaline hydrolysis pretreatment for 2 hours with a sodium hydroxide (NaOH) solvent (w/v=1/2) with a concentration of 0.1 mol (M) ( alkaline pretreatment) to form a hydrolyzed mixture of the jellyfish skin filaments;

(103-2) Then, wash the insoluble matter in the hydrolysis mixture with water several times until the washing water becomes neutral;

(103-3) Then, add acetic acid (CH ₃ COOH, also known as acetic acid) solvent (w/v=1/2) with a concentration of 0.5 mol (M) to the hydrolysis mixture, and use a homogenizer to The hydrolysis mixture was subjected to ultrasonic vibration for 2 hours to make the hydrolysis mixture become a completely homogeneous first mixed solution, and then the first mixed solution was rapidly stirred with a magnet for 2 hours, and a high-speed centrifuge was used to 10000×g The first mixed solution is centrifuged for one hour at a rotating speed, and after obtaining the precipitate, the precipitate is back-dissolved in acetic acid with a concentration of 0.5 molar (M) to form a second mixed solution; And adding sodium chloride (NaCl) to the second mixed solution to salt it out, so that the final concentration of the second mixed solution reaches 4.5 moles (M);

(103-4) Finally, use a high-speed centrifuge at a speed of 10,000 × g to centrifuge the second mixed solution for one hour at 4 degrees Celsius to obtain the precipitate, and then dissolve the precipitate back In 0.5 (M) acetic acid, a third mixed solution was formed; and at 4 degrees Celsius, the third mixed solution was dialyzed against deionized water with a concentration of (v/v=1/10). Finally, After freeze-drying the obtained third mixed solution, the freeze-dried powder of jellyfish collagen (JC for short) of the present invention can be obtained.

In order to truly understand the basic components of these jellyfish skin collagen (JC) freeze-dried powders, the inventor specifically conducted the following analysis: First, the inventor used the analysis method disclosed in previous research data (AOAC, 1997). The basic components of these jellyfish skin collagens (including: moisture, ash, crude fat, crude protein and carbohydrates, etc.) are subject to subsequent detection and analysis; among them, the "moisture analysis" involves washing the crucible (including the crucible lid) After drying at 105 degrees Celsius, place 0.3 grams of dry jellyfish skin collagen (JC) freeze-dried powder in a crucible, and place it in a constant temperature drying oven to dry at 105 degrees Celsius. Take it out every 2 hours and place it in a crucible. After cooling in the dryer for 30 minutes, weigh; in this way, the procedures of drying at 105 degrees Celsius, cooling for 30 minutes, and weighing are repeated until the weighing result reaches a constant weight. The calculation formula is moisture content (%) = [1- (Weight after drying to constant weight/original weight of sample) ] It is placed in an ashing furnace and ashed at 550 degrees Celsius for more than 6 hours. Then it is taken out and placed in a desiccator to cool for 30 minutes before weighing. In this way, drying at 105 degrees Celsius, cooling for 30 minutes and weighing are repeated every 3 hours. program until the weighing result reaches a constant weight. The calculation formula is ash content (%) = (weight of constant weight after ashing/original weight of sample) x 100%; "Crude fat analysis" is based on drying 3.0 grams (g) Wrap the jellyfish skin collagen (JC) powder in a cylindrical filter paper, put it into a Soxhlet extractor, and connect it to a fat collection bottle. Add 200 milliliters (ml) of ether (Ether) to the upper end of the Soxhlet extractor, and connect a condenser tube and reflux in a constant temperature water bath at 50 to 60 degrees Celsius for 16 hours; when the extraction is completed, quickly remove the fat Collect the bottle, recover the ether from the Soxhlet extractor, dry the fat collection bottle in an oven at 105 degrees Celsius, take it out, place it in a desiccator to cool and then weigh it; in this way, repeat the process at 105 degrees Celsius. Drying, cooling and weighing procedures are carried out until the weighing result reaches a constant weight. The calculation formula is crude fat content (%) = (weight of constant weight of fat collection bottle after fat extraction/weight of fat collection bottle) x sample weight x 100% "Crude protein analysis" uses the "Kjeldahl nitrogen method" to determine the total nitrogen. The method is to take 0.5 grams (g) of dried jellyfish skin collagen (JC) powder, place it in a decomposition tube, and place it in a decomposition tube. Add 5 grams (g) of catalyst (the weight ratio of potassium sulfate (K ₂ SO ₄ ) to copper sulfate pentahydrate (CuSO ₄ .5H ₂ O) is 9:1) and 15 ml ( ml) of concentrated sulfuric acid (Sulfuric acid) with a concentration of 18N. Then, place the decomposition tube into a protein decomposition furnace and heat it to 380~400 degrees Celsius until the liquid in the decomposition tube is decomposed into a light blue clear state. Then, take out the decomposition tube and cool it, add 70 milliliters (ml) of ultrapure water (ddH2O) and 60 milliliters (ml) of 35% sodium hydroxide (NaOH) solvent, and distill it out with a total nitrogen distiller. Nitrogen; then, use 20 ml of 4% boric acid as the absorbent, add 2 drops of phenol red indicator, collect for 5 minutes, and titrate with 0.1N sulfuric acid until it turns pink. So far, the calculation formula is crude protein content (%) = [(sample titer) - (blank group titer)] x 0.1 x 14.007 x [6/(sample weight x 1000)] x 100%; "Carbohydrate Analysis" Based on the calculation formula [100% - (moisture content) - (ash content) - (crude fat content) - (crude protein content)], after calculation, it can be known that the jellyfish skin collagen (JC) powder contains Carbohydrate content, and obtain the basic ingredients of jellyfish skin collagen (JC) powder shown in Table 1 below. Table 1. Basic ingredients of jellyfish skin collagen (JC) powder basic ingredients Net weight(%) Moisture 2.92 ± 0.05 Ash 81.53 ± 0.29 Crude lipid 0.15 ± 0.13 Crude protein 10.27 ± 0.09 Carbohydrates 5.13

(104) The jellyfish skin collagen (JC) freeze-dried powder was fermented according to the following steps:

(104-1) First, secondary activation of the lactic acid bacteria for fermentation is performed: in the preferred embodiment of the present invention, the lactic acid bacteria for fermentation are selected from Bacillus subtillis natto for fermentation. The strain of Bacillus subtillis natto is It was purchased from the "Bioresource Collection and Research Center (BCRC)" of the Institute of Food Industry. Before fermenting the jellyfish skin collagen freeze-dried powder in the present invention, the purchased product must be stored in a freezer. The lactic acid bacteria are inoculated into fresh culture medium for secondary activation. The fresh culture medium is based on and improved from the BCRC formula. The method is to prepare the culture medium solution with distilled water so that each liter of the culture medium solution contains 100 grams (g) of glucose. (Glucose), 10.0 grams (g) of yeast extract (Yeast Extract), 1.5 grams (g) of dipotassium hydrogen phosphate (K ₂ HPO ₄ ), 0.5 grams (g) of sodium acetate (CH ₃ COONa), 0.2 Grams (g) of magnesium sulfate heptahydrate (MgSO ₄ .7H ₂ O) and 0.038 grams (g) manganese sulfate monohydrate (MnSO4.H2O) and other ingredients, and at 37 degrees Celsius, place the aqueous culture medium solution in a After 24 hours of cultivation in a constant-temperature incubator, the fresh culture medium will be formed for use in secondary activation of lactic acid bacteria;

(104-2) Use an Erlenmeyer flask for small-scale culture of lactic acid bacteria: The method is based on what was disclosed in the previous literature (see Yin et al ., 2002). First prepare a sterilized Erlenmeyer flask and place it in a sterile operation on the table; then, inoculate 100 milliliters (ml) of the fresh culture medium with 1% of the activated lactobacilli; then, culture it at 37 degrees Celsius, and every three hours, take out and measure the pH value and light Density value (OD, 600nm), and adjust its pH with sodium hydroxide (NaOH) titration to make the pH value reach about 6, and observe the growth curve for dozens of hours; after about 12 hours, the growth reaches After the end of the logarithmic growth phase (i.e., OD 600 nm ＞ 0.8, bacterial load is about 10 ⁷ - 10 ⁸ CFU/ml), use a high-speed centrifuge at a speed of 8000 × g for 20 minutes at 4 degrees Celsius. Centrifuge, collect the bacterial cells, add the bacterial cells to 200 milliliters (ml) of sterilized physiological saline with a concentration of 0.85%, suspend the bacterial cells on the physiological saline, and then centrifuge them under the above conditions. , after removing the supernatant liquid once, add 100 milliliters (ml) of sterilized physiological saline with a concentration of 0.85%, and suspend the bacteria on the physiological saline and place it in an environment of 4 degrees Celsius for later use.

(104-3) Large-scale fermentation of jellyfish skin collagen freeze-dried powder: The method is based on what was disclosed in previous literature (see Pan and Wang, 1998). The collagen freeze-dried powder was added before extraction from jellyfish skin. The weight percentage is 50% distilled water (Distilled water), 4% sucrose (Sucrose), 2% sodium chloride (NaCl), 1% glucose (Glucose) and 1% of the aforementioned bacteria and other ingredients, and the Put the other ingredients together into a beaker with a capacity of one liter (L) to form the fermented jellyfish skin collagen solution required by the present invention, open the top of the beaker, seal it with aluminum foil, and move it to 37 degrees Celsius. Fermentation is carried out in a fermentation tank (or incubator) at a certain temperature; during the fermentation process, first, the pH meter (pH meter) must be calibrated, and the required utensils must be sterilized in a high-temperature sterilization kettle (at a high temperature of 121 degrees Celsius) , sterilized for 15 minutes); then, adjust the rotation speed of the fermentation tank to 150 rpm/min, the temperature to 35 degrees Celsius, and the pH value range to be controlled between 5.9 and 6.1 (titrated with 3 molar sodium hydroxide (NaOH) measured), let the jellyfish skin collagen solution to be fermented be fermented, and observe and record the consumption of sodium hydroxide every three hours until the sodium hydroxide is no longer consumed (representing the termination of lactic acid fermentation). Once the fermentation is terminated, Take out the jellyfish skin collagen fermentation liquid and freeze-dry it to obtain the jellyfish skin collagen fermentation product (FJC) of the present invention.

During the aforementioned preparation process, the inventor specifically measured the bacterial count and pH value of the jellyfish skin collagen fermentation broth, and obtained 0, 1, 2, and 3 of the jellyfish skin collagen fermentation broth shown in Figure 4. In addition to the pH changes of the fermentation broth over 4 days, the following antioxidant capabilities of the jellyfish skin collagen fermentation broth were specifically analyzed, which are briefly described below:

(A1) Analysis of the ability to scavenge DPPH free radicals: This analysis is based on what was disclosed in the previous literature (Om P. Sharma et al ., 2009) and was adjusted; the method is to take 20 microliters (μL) of JC ( FJC), add 100 microliters (μL) of DPPH (1,1-diphenyl-2-picrylhydrazyl, 1,1-diphenyl-2-trinitophenylhydrazyl) at a concentration of 0.5 millimol per liter (mM) (dissolved in Methanol), let it stand for about 30 minutes at room temperature in a dark environment, then measure its absorbance value under the irradiation of colored light with a wavelength of 517nm, and then calculate its clearance rate according to the following formula, and use trolox as the control group: DPPH Clearance rate (%) = [1- (sample absorbance value) / (control group absorbance value)] × 100 %; that is, the jellyfish skin collagen (JC) and its fermentation 1, 2, 3 and DPPH free radical scavenging ability of 4-day fermentation broth (FJC). Table 2. DPPH free radical scavenging ability of jellyfish skin collagen (JC) and its fermentation broth (FJC) fermented for 1, 2, 3 and 4 days The DPPH radical scavenging (%) ¹ JC 8.49 ± 0.03 ^a2 FJC-day 1 16.32 ± ^0.07b FJC-day 2 40.81 ± ^1.01c FJC-day 3 47.16 ± 2.05 ^days FJC-day 4 47.35 ± 0.31 ^days

(A2) Analysis of the ability to scavenge ABTS free radicals (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)): This analysis is based on the previous literature (Miller et al ., 1993). And make adjustments; the method is to prepare 20 microliters (μL) of peroxidase with a concentration of 44U/ml and 20 microliters (μL) of phosphate buffered saline (PBS buffer) with a concentration of 750 μmol. 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS ^{․ +} ) per liter (μM) and 140 μL microliter (μL) of 75 μM concentration H ₂ O ₂ and other ingredients; mix evenly by shaking 6 Minutes later, stable blue-green ABTS cationic radicals are generated; then, add 40 microliters (μL) of appropriately diluted JC (FJC); after shaking evenly, measure the absorbance value at a wavelength of 734 nm. In addition, prepare trolox solutions with concentrations of 0.05, 0.1, 0.15, 0.2, 0.25, and 0.3 millimoles per liter (mM) to make a standard curve, and use this standard curve to convert jellyfish skin collagen (JC) and its fermentation broth (FJC) The equivalent concentration of trolox (mM) is often expressed as TEAC, and its clearance rate is calculated according to the following formula: ABTS ^{․ +} Clearance rate (%) = [(Absorbance value of control group - Absorbance value of sample) / Absorbance value of control group] × 100%; that is, the jellyfish skin collagen (JC) and its fermentation 1, 2, shown in Table 3 below can be obtained ABTS cationic radical scavenging ability of fermentation broth (FJC) at 3 and 4 days. Table 3. ABTS cationic radical scavenging ability of jellyfish skin collagen (JC) and its fermentation broth (FJC) fermented for 1, 2, 3 and 4 days The ABTS ^{․ +} scavenging (%) ¹ JC 11.33 ± 0.08 ^{a 2} FJC-day 1 19.61± ^0.88b FJC-day 2 25.86 ± ^0.49c FJC-day 3 27.12 ± 0.63 ^days FJC-day 4 27.08 ± 0.09 ^days

(A3) Analysis of the ability to chelate ferrous ions: This analysis is based on the previous literature (Kuda et al . 2005) and was adjusted; the method is to add 100 microliters (μL) of JC (FJC) , 100 microliters (μL) of water and 25 microliters (μL) of ferrous chloride (FeCl ₂ ) with a concentration of 0.5 mmol per liter (mM) are uniformly mixed into a mixed solution, and the mixed solution is detected to be colored at a wavelength of 550nm. The absorbance value (A ₁ ) under light irradiation, then add 25 microliters (μL) of 2.5 millimoles per liter concentration (mM) of iron detection reagent (Ferrozine) in water to the mixture, and let it stand for 20 minutes. , detect its absorbance value (A ₂ ) under the irradiation of colored light with a wavelength of 550nm, and then use deionized water as a control group to calculate its ability to chelate ferrous ions according to the following formula: Chelating ability (chelatind effect %) = [1 - (A ₂ - A ₁ ) / (Control group A ₂ - Control group A ₁ )] × 100%; that is, the jellyfish skin collagen (JC) shown in Table 4 below and its fermentation 1, 2, 3 and 4 can be obtained Ferrous ion chelating ability of fermented broth (FJC). Table 4. Ferrous ion chelating capacity of jellyfish skin collagen (JC) and fermentation broth (FJC) fermented for 1, 2, 3 and 4 days Chelating Fe ²⁺ effect (%) JC 0.11 ± 0.01 ^{a 1} FJC-day 1 0.13± ^0.01ab FJC-day 2 0.16 ± ^0.01b FJC-day 3 0.23 ± ^0.03c FJC-day 4 0.24 ± ^0.03c

(A4) Analysis of reducing ability: This analysis is based on the previous literature (Senevirathne et al . 2006) and adjusted; the method is to take 100 microliters (μL) of JC (FJC) and remove the Ionized water was used as the control group. Add 100 microliters (μL) of phosphate buffer solution and 1% potassium hexacyanoferrate (K ₃ Fe(CN) ₆ ), mix evenly, and then incubate in a water bath at 50 degrees Celsius for 20 minutes. After cooling in an ice bath, add 100 microliters (μL) of 10% trichloroethanoic acid (TCA), and centrifuge it for 10 minutes at 3000 rpm. Take 100 Add 100 microliters (μL) of deionized water and 20 microliters (μL) of ferrous chloride (FeCl ₃ ) to the supernatant and react for 10 minutes, then detect it under 700nm wavelength colored light irradiation. The absorbance value can be used to obtain the reducing power of jellyfish skin collagen (JC) and its fermentation broth (FJC) after fermentation for 1, 2, 3 and 4 days as shown in Table 5 below, and then analyze its reducing power. Table 5. Reducing power of jellyfish skin collagen (JC) and fermentation broth (FJC) after 1, 2, 3 and 4 days of fermentation Reducing power (Absorbance at 700 nm) JC 0.09 ± 0.00 ^{a 1} FJC-day 1 0.23 ± ^0.00b FJC-day 2 0.27± ^0.00c FJC-day 3 0.27 ± ^0.01c FJC-day 4 0.27 ± ^0.01c

In addition, in order to further understand the following biochemical properties of jellyfish skin collagen (JC) and its fermentation product (FJC), the inventor has carried out the following measurements and analyzes on each of the biochemical properties one by one, which are briefly described as follows:

(B1) Determination and analysis of protein content: This analysis is based on the previous literature (Bradford (1976)) and was adjusted; the method is to first mix Bradford reagent with ultrapure water (ddH ₂ O) After diluting 5 times and filtering with a 0.22 micron (μm) mesh filter, dilute the jellyfish skin collagen fermentation broth (FJC) and take 20 microliters (μL) into a 96-well culture plate, and then place it in the Add 200 microliters (μL) of Bradford reagent to the culture plate, mix the two, and let the two interact for 15 minutes in a dark environment, and then detect the absorbance value under the irradiation of colored light with a wavelength of 595nm. In addition, use Bovine serum albumin (BSA) with a concentration of 0~200 μg/ml is used as a standard solution. A standard curve is drawn based on the standard solution, and then a linear regression equation is calculated based on it to calculate the protein in JC (FJC). concentration.

(B2) Determination and analysis of collagen amino acids: The method is to take 1 milligram (mg) of JC (FJC) and place it into a special test tube for amino acid analysis, and add 1 ml ( ml) of L-norleucine solvent (where L-norleucine is dissolved in 6N grade hydrochloric acid (HCl) with a concentration of 25g/ml as a heterodyne standard) and 1 ml (ml) of 6 N grade hydrochloric acid (HCl), and after vacuuming, place it in an oven at 150 degrees Celsius for 10 hours of hydrolysis reaction. Finally, after repeatedly removing the hydrochloric acid (HCl) with a vacuum concentrator, the buffer solution can be diluted to a volume, and an amino acid analyzer can be used to measure and analyze the amino acids, and obtain the results shown in Table 6 below. Amino acid composition of jellyfish skin collagen (JC) and its fermented product (FJC). Table 6. Amino acid composition of jellyfish skin collagen (JC) and its fermented product (FJC) Amino acid JC (residue/1000 residue) FJC (residue/1000 residue) Glycine 333.08 324.84 Glutamic acid 115.65 103.21 Proline 102.16 95.63 Alanine 95.86 100.74 Aspartic acid 76.84 76.86 Arginine 51.93 55.87 Hydroxyproline 38.00 46.86 Serine 31.59 29.85 Lysine 29.71 30.22 Threonine 28.83 27.33 Leucine 24.90 30.68 Valine 19.06 22.21 Isoleucine 15.37 11.78 Phenylalanine 14.26 14.86 Methodine 10.32 8.57 Tyrosine 9.58 7.23 Cysteine 2.86 2.40 Histidine 0.00 0 Total 1000 1000

(B3) Measurement and analysis of UV absorption spectrum: The method is to dissolve freeze-dried JC (FJC) in acetic acid with a concentration of 0.5 mole (M) to obtain a concentration of 1mg/ml, and Use a centrifuge to perform centrifugation at 5000 rpm for 15 minutes, and detect the absorbance of the supernatant under the irradiation of colored light with a wavelength of 200 to 400 nm. Then its characteristic spectrum and maximum value can be recorded in the 0.1 nm wavelength range. The absorbance values were obtained to obtain the ultraviolet absorption spectra of the jellyfish skin collagen (JC) and its fermentation product (FJC) of the present invention shown in Figures 5 and 6 respectively.

(B4) Determination and analysis of protein by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE): This analysis is based on the disclosure of the previous literature (Weber and Osbron, 1969) and is adjusted; its method The method of discontinuous SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis) is used for protein identification and analysis. First, Separating Gel and Stacking Gel are prepared according to the components shown in Table 7 below, and the Separating Gel is slowly and evenly mixed. Pour it into a glass plate (slab), and add a water layer until it polymerizes into a gel. When the water layer and the colloid form an interface, the gel formation is completed. Remove the water layer with filter paper, then pour the Stacking Gel and put it in A one-tooth comb. After the gel is formed, the comb is pulled out and placed in an electrophoresis tank. Table 7. Composition of SDS-PAGE film 8% Separating Gel 4% Stacking Gel H ₂ O 7.15ml 32.22ml 1.5M Tris pH8.8 3.8ml ---- 0.5M Tris pH6.8 ---- 0.38ml 40% Acrylamide/Bis 3.75ml 0.38ml 10% SDS 0.15ml 0.03ml TEMED 0.006ml 0.003ml 10% APS 0.15ml 0.03ml Total 15.00ml 3.00ml

Next, the jellyfish skin collagen (JC) and its fermentation product (FJC) of the present invention are uniformly mixed with the buffer solution in a ratio of 5:1, and then heated at 100 degrees Celsius for 5 minutes. After cooling, 15 microliters ( μL) was injected into a sample tank, and Tris-glycine SDS buffer solution was used as running buffer; electrophoresis was performed at 70 Volt voltage for 30 minutes, and then the voltage was adjusted to 150 Volt for 2 hours; electrophoresis film Coomassie brilliant blue R-250 dye was used to stain for 1 hour, and then destained with destaining solution (10% Acetic acid and 20% Methanol) until the background was clear, and mixed with 5 μL (μL) of protein standard and collagen. Protein standard comparison can be used to evaluate the molecular weight and collagen composition.

(B5) Measurement and analysis of Fourier transform infrared spectroscopy (FT-IR): This analysis is based on what was disclosed in the previous literature (Camacho et al ., 2001) and was adjusted. The method is to divide 1 milligram (mg) into After the jellyfish skin collagen (JC) and its fermented product (FJC) of the present invention are evenly mixed with potassium bromide (KBr) in a ratio of 1:100, they are put into a molding machine and placed in a tableting machine. In this tableting machine, a vacuum pump was used to evacuate and compress the sample tablets, and potassium bromide (KBr) tablets were used as the background value. An FT-IR (Fourier Transform Infrared Spectroscopy) spectrometer was used to detect the infrared spectrum of the sample ingot. The number of scans was 16 times, the resolution was 8cm-1, and the scanning range was 4000~400 cm ^-1 , that is, The Fourier transform infrared spectra of the jellyfish skin collagen (JC) and its fermentation product (FJC) of the present invention shown in Figures 7 and 8 were obtained respectively.

(B6) Determination and analysis of peptide concentration: The method is to use O-phthaldialdehyde (OPA) mixed solution to determine the peptide content in the jellyfish skin collagen (JC) and its fermentation product (FJC) of the present invention. The method is to take 25 ml (ml) of 100 mmol (mM) sodium tetraborate, 2.5 ml (ml) of 20% sodium dodecyl sulphate (SDS), 1 ml (ml) of OPA (mixed by dissolving 40 milligrams (mg) of OPA in 1 milliliter (ml) of methanol) and 100 microliters (μL) of β-mercaptoethanol and other drugs, and these drugs were removed Ionized water was quantified to 50 milliliters (ml) in a brown bottle. Take 0.2 grams (g) of the fermented jellyfish skin collagen (FJC) powder of the present invention and quantify it to 10 milliliters (ml) with deionized water. The concentration is 20 mg/ml. Filter it through a 0.2 μm filter membrane and then dilute it appropriately. Take 2 milliliters (ml) of the OPA mixed solution and 50 μL of FJC and mix it evenly at room temperature and let it stand for 2 minutes. Use a spectrophotometer to measure the absorbance value under the irradiation of colored light with a wavelength of 340 nm. It can be determined by the Leu-Gly standard material. The obtained standard calibration curve is converted into the peptide content in mg/g (Nakamura et al ., 1995).

In addition, in order to further understand the following immune properties of the jellyfish skin collagen (JC) and its fermentation product (FJC) of the present invention, the inventors have carried out the following cell culture, experiments and measurements on each of the immune properties. Here is a brief summary: The instructions are as follows:

(C1) Culture of macrophages RAW264.7: The method is to culture macrophages RAW264.7 in a solution containing L-glutamine, penicillin, streptomycin, and hydrochloric acid. Dulbecco's Modified Eagle Medium-low glucose (DMEM-LG) medium containing ingredients such as Pyridoxine hydrochloride, Sodium pyruvate and Sodium bicarbonate, and adding 10% fetus After adding Fetal bovine serum (FBS for short), place it in a constant temperature incubator at 37 degrees Celsius for culture; after two to three days, replace the medium, and when the cells are full, remove the old medium and replace it with Use Phosphate buffered saline (PBS) to clean the attached cells in the culture dish; after removing the PBS, add 3 ml (ml) of culture medium, scrape the cells with a cell scraper, and transfer them to a 15 ml volume. (ml) centrifuge tube, and use a centrifuge to centrifuge the tube at a speed of 800 rpm for 3 minutes (min); then, remove the supernatant in the centrifuge tube and collect the obtained cell pellet. After dispersing with an appropriate amount of culture medium, add 1 ml (ml) of the cell solution into a culture dish containing 1 ml (ml) FBS and 8 ml (ml) DMEM-LG. Shake the cells evenly with a figure-eight shake method, and use Observe the cells under an inverted microscope, and place the culture dish in a constant-temperature incubator for culture.

(C2) Culture of human chondrosarcoma cell SW-1353: According to reports, human chondrosarcoma cell SW-1353 is a type of fibroblast, a cell that synthesizes extracellular matrix and collagen, and is one of the biological connective tissues. Basic structure, which plays an important role in animal wound healing, this kind of fibroblasts must be cultured in a solution containing 3.65 millimol (mM) concentration of L-glutamine (glutamine), 90 units/ml ( ml) concentration of penicillin (Penicillin), 90 microliter (μl)/ml (ml) concentration of streptomycin (Streptomycin), 18 millimol (mM) concentration of zwitterionic sulfonic acid buffer (HEPES) and 23.57 milliseconds In a medium containing a 1:1 mixture of F-12 and DMEM (Dulbecco's modified eagles medium) with molar (mM) concentrations of sodium bicarbonate and other ingredients, and adding 10% FBS to the medium, culture it at Celsius Place in a constant temperature incubator containing 5% carbon dioxide (CO ₂ ) at 37 degrees; after two to three days, replace the culture medium. When the cells have grown to fill the plate, remove the old culture medium and clean the attached cells in the culture plate with PBS. , after removing the PBS, add 1 ml (ml) of 1x trypisn-EDTA to the cell culture plate and allow it to incubate for about 3 to 5 minutes. After confirming with a microscope that the cells have detached from the bottom of the culture plate, add 1 ml (ml) of DMEM F-12. Culture medium and 3 ml (ml) of PBS, rinse all the cells, and transfer them to a 15 ml (ml) sterile centrifuge tube. Use a centrifuge at 800 rpm to run the sterile centrifuge tube for 3 minutes. ); then, remove the supernatant in the centrifuge tube. Dissolve the obtained cell pellet with an appropriate amount of culture medium. Then add 1 milliliter (ml) of the cell solution to a petri dish and shake it in a figure-eight manner. Disperse the cells evenly and observe the cells using an inverted microscope. Then, place the culture dish in a constant-temperature incubator for culture. After four hours, observe the cell attachment and growth.

(C3) Cell viability assay (MTT assay): This assay is based on the previous literature (Mosmann T., 1983) and adjusted; the method is to use PBS to wash the cultured cells and then use a centrifuge at 800 rpm Centrifuge it for 3 minutes (min) at a rotating speed, and wash the resulting cell pellet with 3 ml (ml) of DMEM containing 2% FBS. Use a cell counter to count the cells and adjust the concentration to 2x10 ⁵ cells /ml. Add the cell solution to a 96 well culture plate to stick it to the plate, then add the sample serially diluted with culture medium, culture it in an incubator at 37 degrees Celsius and containing 5% CO ₂ for 18 to 24 hours, and then add After 4 hours of reaction with 100μl/well MTT (1mg/ml), use a oscillator to shake for 5 minutes, and then detect the absorbance value under the irradiation of colored light with a wavelength of 570nm, and calculate the relative cell survival rate according to the following formula: Relative cell viability (%) = [(A _sample - A _blank ) / (A _control - A _blank )] x 100, and the macrophage RAW 264.7 shown in Figure 9 is obtained using the jellyfish skin collagen ferment (FJC) of the present invention 24-hour cell viability.

(C4) Measurement of nitric oxide (NO) release: This measurement is based on the previous literature (Guevara et al ., 1998) and has been adjusted. The method is to count the cells with a cell counter and adjust the number of cells to 2x10 ⁵ cells/ml, take 1 ml (ml) and place it in a 12-well culture plate. Incubate for 4 hours to allow the cells to adhere to the plate. Then aspirate the cell culture medium and add JC, FJC or MIA of different concentrations that have been diluted in the test tube in advance. (2.5 μM), incubate in an incubator for 24 hours at 37 degrees Celsius and containing 5% CO ₂ , then take 100 μl each of the cell supernatant and the serially diluted standard (0.1M nitrine) ( μl) into a 96well culture plate, add 50 μl (μl) of SUL solution (0.1% sulfanilamide solution in 2.5% phosphoric acid), mix, and then add 50 μl (μl) of NED solution (0.1% N-( 1-naphthyl) ethylenediamine dihydTaiwanhloride in ddH2O) is mixed and left to react for 10 minutes. Use an ELISA reader to measure the absorbance value under the irradiation of colored light with a wavelength of 540 nm. A standard curve can be made based on this and calculated by the interpolation method. The release amount of NO was obtained by using the jellyfish skin collagen fermentation product (FJC) and LPS of the present invention for 24 hours to obtain the NO release amount of the macrophage RAW 264.7 shown in Figure 10.

(C5) NBT reduction analysis of reactive oxygen species (ROS): This analysis is based on the previous literature (Spitz and Oberley, 1989) and was modified. The method is to count the cells with a cell counter. Adjust the number of cells to 2x10 ⁵ cells/ml, take 1 ml (ml) and place it in a 12well culture plate. Incubate for 4 hours to allow the cells to adhere to the plate. Then aspirate the cell culture medium and add different concentrations of JC that have been diluted in the test tube. , FJC or IL-1β (10 μg/well), culture it in an incubator for 24 hours at 37 degrees Celsius and containing 5% CO _2. Then, with Trypsin action, use PBS to pour the cells into a centrifuge tube, and then Use a centrifuge at 800 rpm for 15 minutes (min), remove the supernatant, and add 0.3 ml (ml) of NBT (Nitroblue tetrazolium) solution (0.1 mg/ml NBT, 5% FBS and 3% DMSO dissolved in 10ml DMEM/F12), incubate for 1 hour, then use a centrifuge to centrifuge at a speed of (1500 xg for 15 minutes (min), remove the supernatant and add 200 μl ( μl) of DMSO to dissolve the purple crystals in the cells, place it in an ultrasonic shaker for 5 minutes, take out 200 μl (μl), place it in a 96 well culture plate, and use an ELISA reader to measure the absorbance value under the irradiation of colored light with a wavelength of 570 nm. , that is, the reactive oxygen species (ROS) content can be calculated accordingly.

In addition, the inventors wanted to further and gain a deeper understanding of whether jellyfish skin collagen and its fermentation products are indeed effective in relieving symptoms related to osteoarthritis (OA) in non-surgical treatments for cartilage repair in osteoarthritis. Relevant animal surgeries and experiments were also carried out one by one for the following items. We would like to briefly explain and analyze them as follows:

(D1) Surgery to induce osteoarthritis in rats: This surgery is based on what was disclosed in previous literature (Hayami et al. , 2006), using anterior cruciate ligament transection/meniscal removal surgery (ACLT/MMx surgery). It is hoped that the knee joints of rats will be damaged and become unstable first, thereby accelerating the induction of early symptoms of osteoarthritis (OA). The method is to first inject Zoletil ^® 50 (1 ml/g) subcutaneously into the rats. The mouse was anesthetized, and after removing the hair near the right knee joint, the skin was disinfected with iodine. Then, the skin was incised longitudinally on the knee joint to expose the ligaments, the anterior cruciate ligament was cut, and the medial meniscus was removed; after the operation Flush the surgical site with sterile saline and stop bleeding; then, suture the joint cavity with catgut (such as 4-0 chromic catgut manufactured by Taiwan Jiahe Medical Materials), and then suture the skin with 3-0 nylon suture (Braided silk) ; Finally, the rats were injected with 10 mg/kg of cephalosporin antibiotic subcutaneously to prevent bacterial infection of the wounds after surgery.

(D2) For the breeding and grouping of experimental rats: the feeding method was in accordance with the "Rule of the Terrestrial Animal Experiment Center, College of Life Sciences, National Taiwan Ocean University", 56 rats purchased from "Lesco Biotechnology Co., Ltd." for 5 weeks Age-old male Sprague-Dawley rats were individually housed in an environment of 23 ± 1 degrees Celsius, a humidity of 40 to 60%, and a 12-hour light-dark cycle (i.e., 7:00 AM to 6:59 PM for light, 7 :00 PM~6:59 AM in a dark state) in a stainless steel rat cage; the feeders are powdered and tablet feed (Laboratory Rodent Diet 5001, PMI®Lab Diet®, Saint Louis, Missouri, USA) and distilled water. The supply of feed and distilled water adopts the ad libitum feeding method with unlimited supply. After one week of pre-breeding, except for the control group (control group), which continues to be fed with tablet feed, the other groups are fed with a high-fat diet (High Fat Diet, (referred to as HFD) for 6 weeks to induce obesity. Among them, rats that were successfully induced to suffer from osteoarthritis through the aforementioned surgery of semilunar cartilage and anterior cruciate ligament damage were fed by tube feeding. The jellyfish skin collagen (JC) and its fermented product (FJC) of the present invention were consumed every week. Measure your weight. The grouping was based on what was revealed in previous literature (Long, 2016). After 1 week of domestication, they were randomly divided into 7 groups at 6 weeks of age; then 2 groups were randomly selected as the control group (control) and the osteoarthritis group ( OA); and after the other 5 groups were fed a high-fat diet for 6 weeks to induce obesity, the osteoarthritis group and each of the 5 groups with high-fat diet-induced obesity were respectively subjected to the aforementioned surgery for semilunar cartilage and anterior cruciate ligament damage. to successfully induce osteoarthritis; finally, feed glucosamine sulfate or different doses (such as: 1 times the dose, 20 mg/kg b.w.; 2 times the dose, 40 mg/kg b.w. or 5 times the dose, 100 mg/kg b.w.) using jellyfish skin collagen ferment (FJC), the experimental rats were divided into 7 groups, with 8 rats in each group, forming the following grouping status: 1. Sham operation control group (Control): fed general feed; 2. Osteoarthritis group (OA): fed general feed; 3. Osteoarthritis group (OBOA): fed HFD; 4. FJC1 group: fed HFD + osteoarthritis + fed 1 times the dose (20 mg/kg b.w.) of FJC of the present invention; 5. FJC2 group: fed HFD + osteoarthritis + fed 2 times the dose (40 mg/kg b.w.) FJC of the present invention; 6. FJC5 group: Feed HFD + Osteoarthritis + Feed 5 times the dose (100 mg/kg b.w.) FJC of the present invention; and 7. GS group: HFD + osteoarthritis + glucosamine sulfate (100g/kg b.w.) group.

(D3) Collection and processing of experimental animal samples: After feeding the rats with jellyfish skin collagen ferment (FJC) for 6 weeks, the rats were fasted for at least 12 hours, and then the rats were weighed individually, and each This body weight was used as the final body weight of each rat; then, each rat was euthanized with carbon dioxide; then, each rat was euthanized using a syringe pre-moistened with heparin (Heparin, 500 IU/ml) , draw blood from the peritoneal hepatic portal vein, and temporarily store the blood in a low-temperature environment; then, each rat is dissected to remove its organs, including: heart, liver, spleen, kidney, abdominal fat and accessory fat...etc. , and weigh the organs, and use a part of the blood obtained as whole blood, and put the rest into a centrifuge tube, and use a high-speed centrifuge at a speed of 1570 xg at 4 degrees Celsius. After the centrifuge tube is centrifuged for 15 minutes, the supernatant (i.e., plasma) is collected, and the plasma is moved into a new centrifuge tube and stored in a refrigerator at -80 degrees Celsius for subsequent related work. Used during evaluation and analysis.

(D4) Assessment of plasma functional indicators: This assessment mainly tests, analyzes and evaluates the following indicators one by one. Here is a brief description as follows:

(D4-1) Triglyceride (TG): The detection and analysis of this indicator is based on the triglyceride fluorescence detector of the commercially available Randox brand TG detection kit to analyze the TG content in plasma. The principle is as follows After TG is hydrolyzed by lipase and catalyzed by a series of peroxidases, the H ₂ O ₂ produced will react with 4-aminophenazone and 4-chlorophenol. (4-chlorophenol) undergoes a biochemical reaction to produce a purple-red quinoneimine compound, which serves as an indicator; the detection and analysis steps are to uniformly mix plasma and reagents in a ratio of 1:100, and at room temperature After letting it stand for 10 minutes to fully react, measure its absorbance value under the irradiation of colored light with a wavelength of 500nm. This can then be compared with the absorbance value of the standard. According to the following formula, the TG in the plasma of each rat can be converted. Concentration: TG concentration (mg/ dl) = (A _simple - A _block )/(A _standard - A _blank ) × Standard concentration

(D4-2) Total cholesterol (TC): The detection and analysis of this indicator still uses the aforementioned triglyceride fluorescence detector to analyze the total cholesterol content in plasma. The principle is that TC will be hydrolyzed by enzymes. With the oxidation products H ₂ O ₂ , 4-aminophenazone and phenol (Phenol), it is catalyzed by catalase (peroxidase) to produce quioneimine, which serves as an indicator; its detection and analysis steps are based on plasma Mix evenly with the reagent in a ratio of 1:100, and let it stand at room temperature for 10 minutes to fully react. Then, measure the absorbance value under the irradiation of colored light with a wavelength of 500 nm, and then it can be compared with the standard. The absorbance value is converted to the TC concentration in the plasma of each rat according to the following formula: TC concentration (mg/ dl) = (A _simple -A _block )/(A _standard -A _blank ) × Standard concentration

(D4-3) Low-density lipoprotein cholesterol (LDL-C for short): The detection and analysis of this indicator uses the Fortress LDL-C detection kit to analyze the low-density lipoprotein cholesterol content in plasma. The principle is to add heparin to the plasma. (Heparin) will cause the LDL-C in it to precipitate at the isoelectric point, while high-density lipoprotein cholesterol (HDL-C) and very low-density lipoprotein cholesterol (VLDL-C) ) will still stay in the supernatant; the detection and analysis steps are to take 100 microliters (μl) of each rat plasma and mix it evenly with 500 microliters (μl) of precipitating agent, and let it stand at room temperature. After allowing it to fully react for 15 minutes, use a centrifuge to perform centrifugation at 3000 rpm for 20 minutes. Take 100 microliters (μl) of the supernatant and measure the total cholesterol concentration with a cholesterol biochemical reagent kit. By subtracting the cholesterol content of the supernatant from the total cholesterol content in the plasma, the LDL-C content in the plasma of each rat can be obtained.

(D4-4) High-density lipoprotein cholesterol (HDL-C): The detection and analysis of this indicator still uses the aforementioned Fortress HDL-C detection kit to analyze the high-density lipoprotein cholesterol content in the plasma of each rat. The principle is to In the presence of magnesium ions, adding phosphotungstic acid to plasma can precipitate LDL-C, VLDL-C and chylomicrons, and after centrifugation, the HDL -C can be detected in the supernatant; the detection and analysis steps are to mix 50 microliters (μl) of plasma with 500 microliters (μl) of precipitant, react at room temperature for 15 minutes, and use centrifugation After centrifuging it for 20 minutes at a speed of 800 High-density lipoprotein concentration in rat plasma.

(D4-5) Leptin: The detection and analysis of this indicator was carried out using the Rat Leptin Enzyme Immunometric Assay detection kit (Assay Designs, Ins., Ann Arbor, MI, USA) to analyze the leptin in the plasma of each rat. Leptin content, one of the 96-well plates provided in the test kit already contains Leptin capture antibody; the detection and analysis steps are to first dilute the rat plasma 5 times with Reagent diluent, and then add 100 microliters (μl) of the serially diluted standard and each rat plasma was added to the 96-well plate, and allowed to stand at room temperature for 2 hours to fully react; then, use Wash buffer to wash the 96-well plate at least 3 times to remove excess matrix; then, Add 100 microliters (μl) of Leptin detection antibody to the 96-well plate, and let it stand at room temperature for 1 hour to allow it to fully react; then, wash the 96-well plate with Wash buffer at least 3 times, and then add 100 μl (μl) of Streptavidin-HRP reagent was added to the 96-well plate, and allowed to stand at room temperature for 30 minutes to fully react; then, the 96-well plate was washed with Wash buffer at least 3 times, and then 50 μl ( μl) of Substrate solution, and let it stand at room temperature for 20 minutes to allow it to fully react; finally, after adding 50 μl (μl) of Stop solution, immediately detect its absorbance value under 450 nm wavelength colored light irradiation. And based on calibration with the standard, the leptin concentration in the plasma of each rat can be obtained.

(D4-6) Adiponectin: The detection and analysis of this indicator uses the Taiclone Rat diponectin ELISA detection kit (Assay pro LLC) test method to detect and analyze the adiponectin content in the plasma of each rat. The detection and analysis steps are to add 50 microliters (μl)/well of the standard or each rat plasma to a 96-well culture plate, and place it in an incubator for 2 hours at 37 degrees Celsius to allow it to fully react. Then, wash 5 times with wash buffer; then, add 50 μl/well of Biotinylated Mouse Adiponectin Antibody, and place it in an incubator at 37 degrees Celsius for 1 hour to allow it to fully react, and then use wash buffer After washing 5 times, add 50 microliters (μl)/well of Streptavidase-peroxidase Conjugate, and place it in the incubator for 30 minutes at 37 degrees Celsius to allow it to fully react. Then wash 5 times with wash buffer and add 50 Microliter (μl)/well of Chromogen Substrate, and place it in an incubator at 37 degrees Celsius for 10 minutes to fully react; finally, add 50 microliter (μl)/well of Stop solution until the color changes from After the blue changes to yellow, immediately use an ELISA reader to detect the absorbance value under colored light irradiation with wavelengths of 450 nm and 570 nm, and calibrate it with the standard to obtain the adiponectin in the rat plasma. concentration.

(D4-7) Lipid peroxidation (Malondialdehyde, referred to as MDA) test: This test is based on what was disclosed in previous literature (Placer et al ., 1966). The method is to take 0.1 milliliter (ml) of each rat plasma. Homogenize the solution and put it into a test tube and use PBS as a blank group. Add 0.2 milliliters (ml) of reaction reagents (containing 15%, w/v trichloroacetic acid in 0.25N HCL and 0.375%, w/v thiobarbituric). acid in 0.25N HCL and other ingredients) were shaken and evenly mixed, and then treated in a water bath at 100 degrees Celsius for 15 minutes. Then, use running water to cool to room temperature c.4, and then add 0.3 milliliters (ml) of normal Butanol (n-butanol), and shake vigorously to mix evenly and fully react; then, use a centrifuge to perform centrifugation at 500 xg for 10 minutes, take the supernatant and measure its wavelength with an ELISA reader The absorbance value under 532 nm colored light irradiation, and compared with the standard substance (5 nM 1,1,3,3-tetramethoxypropane), the MDA concentration in the plasma of each rat can be obtained according to the following formula: Plasma Medium MDA concentration (nM/ ml) = [(Asample - Ablank)/ (Astandard - Ablank)] x5

(D5) Glutathione peroxidase (GPx) detection: The detection and analysis of this indicator are based on those disclosed in previous literature (Bohnenkamp and Weser, 1975), using the Randox Superoxide Dismutase (SOD) detection kit. To detect and analyze the superoxide dismutase activity in the plasma of each rat; the principle is to use the superoxide radicals generated by xanthine (Xanthine) and xanthine oxidase (XOD), which will interact with 2, 3-Bis(4-nitrophenyl)-5-phenyltetrazolium chloride hydrate (2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride, referred to as I.N.T) reacts to form red color Formazan, and SOD activity is the inhibitory ability to inhibit this reaction; the experimental method is to take 5 microliters (μl) of each rat plasma and add 170 microliters (μl) of the reaction reagent (containing 0.05 mmol ( 0.025 mmol of Xanthine and 0.025 mmol of 2,3-bis(4-nitrophenyl)-5-phenyltetrazolium chloride hydrate). 25 microliters (μl) of Xanthine oxidase (80 U/L) was allowed to react at room temperature for 30 seconds, and the absorbance under colored light irradiation with a wavelength of 505 nm was detected initially and after 3 minutes. value, and use the standard substance to make a standard curve. Substitute the absorbance value measured in each rat plasma to calculate its SOD activity.

(D6) Superoxide Dismutase (SOD): The detection and analysis of this indicator are also based on those disclosed in previous literature (Bohnenkamp and Weser, 1975), and the Randox Superoxide Dismutase (SOD) detection kit is used to analyze each Superoxide dismutase activity in rat plasma; its principle is to use superoxide free radicals generated by xanthine and xanthine oxidase (XOD) to react with 2,3-bis(4-nitro) Phenyl)-5-phenyltetrazolium chloride hydrate (I.N.T) reacts to form red Formazan, and SOD activity is the inhibitory ability to inhibit this reaction; the experimental method is to take 5 microliters (μl) of each rat plasma ), and add 170 microliters (μl) of the reaction reagent (which contains 0.05 millimoles (mM) of xanthine (Xanthine) and 0.025 millimoles (mM) of I.N.T), mix evenly, and then add 25 microliters (μl) of value, and use the standard substance to make a standard curve. Substitute the absorbance value measured in each rat plasma to calculate its SOD activity.

(D7) Aspartate Aminotransferase (AST): The detection and analysis of this indicator uses the Randox Aspartate Aminotransferase (AST) detection kit to detect and analyze the aspartate aminotransferase in the rat plasma. Aminase activity.

(D8) Glutamic Pyruvic Transaminase (ALT): The detection and analysis of this indicator is based on the Randox Glutamic Pyruvic Transaminase (ALT) detection kit to detect and analyze the alanine transamination in the plasma of each rat. Enzyme activity.

(D9) Blood urea nitrogen (BUN): The detection and analysis of this indicator are based on those disclosed in previous literature (Lyman et al ., 1986). Randox Urea detection kit is used to detect and analyze the respective indicators. Urea content in rat plasma; the principle is that urea will be hydrolyzed to produce ammonia and carbon dioxide in the presence of water and urease, and ammonia will combine with α-oxoglutarate in water; at the same time, nicotine amide adenine dinuclear Nicotinamide adenine dinucleotide (Nicotinamide adenine dinucleotide, abbreviated as coenzyme or NAD ⁺ ) will produce L-glutamate (L-glutamate) and coenzyme (NAD ⁺ ) under the action of glutamate dehydrogenase (Glutamate dehydrogenase); The method is to take 2 microliters (μl) of the standard or each rat plasma and add it to a 96-well plate, and add 200 microliters (μl) of the reaction reagent (containing 10 U/ml urease) to each well. (urease), 2U/ml glutamate dehydrogenase (glutamatedehydrogenase), 0.26mmol/ml, 3mmol/ml adenosine-2-diphosphate and 14mmol/ml α-ketoglutarate ( α-oxoglutarate), after mixing thoroughly and reacting for 30 seconds, detect its absorbance value under 340 nm wavelength colored light irradiation after 1, 2 and 3 minutes, which can be used to compare the absorbance value of the standard, according to the following formula , convert to the concentration of urea nitrogen in the plasma of each rat: Blood urea nitrogen concentration (mg/dL) =ΔA _sample / ΔA _standard × Standard concentration

(D10) Creatinine: The detection and analysis of this indicator are based on the previous literature (Baxmann, 2008). Randox Creatinine detection kit is used to detect and analyze the creatinine content in the plasma of each rat; its method 2 milliliters (ml) of the standard or each rat plasma was added to a 96-well plate, and 100 microliters (μl) of sodium hydroxide (sodium hydroxide) with a concentration of 0.32 mol/ml was added to each well. ) Reaction reagents, once thoroughly mixed and allowed to react for 30 seconds, detect their absorbance values under irradiation with 492 nm wavelength colored light after 1, 2 and 3 minutes, which can then be compared with the absorbance values of the standard, according to the following formula, Convert the creatinine concentration in the plasma of each rat: Creatinine content (mg/ dL) =ΔA _sample / ΔA _standard × Standard concentration

(D11) Determination of protease concentration, including determination of the concentrations of the following three indicators:

(D11-1) Matrix metalloproteinase-1 (MMP-1 for short) concentration: The detection and analysis of this indicator uses the Taiclone Rat Matrix Metalloproteinase 1 ELISA detection kit. The 96-well plate provided in the detection kit has been pre-coated with (Coating) MMP-1 specific antibody; the method is to add 100 microliters (μl)/well of the standard or each rat plasma to an ELISA 96-well plate, and place the 96-well plate at 37 degrees Celsius After reacting for 1.5 hours in a temperature incubator, pour out the standard or rat plasma in the well, add 100 μl/well of biotinylated detection Ab., shake evenly, and Place the 96-well plate in an incubator at 37 degrees Celsius. After reacting for 1 hour, wash it three times with wash buffer and then add 100 μl/well of antibody protein labeling reagent (HRP). conjugate), and place the 96-well plate in an incubator at 37 degrees Celsius. After reacting for 30 minutes, wash it 5 times with wash buffer, and then add 90 microliters (μl)/well of base. Material chromogenic reagent (substrate reagent), and place the 96-well plate in an incubator at 37 degrees Celsius. After reacting for 15 minutes, add 50 microliters (μl)/well of stop solution (Stop solution) immediately. Use an ELISA reader to detect the absorbance value under the irradiation of colored light with a wavelength of 450 nm, which can be compared with the absorbance value of the standard. According to the relevant formula, the concentration of matrix metalloproteinase-1 in the plasma of each rat can be converted.

(D11-2) Matrix metalloproteinase-3 (MMP-3 for short): The detection and analysis of this indicator uses the Taiclone Rat Matrix Metalloproteinase 3 ELISA detection kit. The 96-well plate provided in the detection kit has been pre-coated with MMP. -3 specific antibody; the method is to add 100 microliters (μl)/well of the standard or each rat plasma to an ELISA 96-well plate, and place the 96-well plate in an incubator at 37 degrees Celsius. After the reaction for 1.5 hours, pour out the standard or rat plasma in the well, add 100 μl/well of biotinylated detection Ab., shake evenly, and mix the Place the 96-well plate in an incubator at 37 degrees Celsius. After reacting for 1 hour, wash it three times with Wash buffer. Add 100μL/well HRP conjugate. Place it in an incubator at 37 degrees Celsius and react for 30 minutes. Wash the eluent (Wash buffer) 5 times, then add 90 μl/well of substrate reagent, and place it in an incubator at 37 degrees Celsius for 15 minutes. After adding 50 μl/well of Stop solution, immediately use an ELISA reader to detect its absorbance value under colored light irradiation with a wavelength of 450 nm, which can be compared with the absorbance value of the standard. According to the relevant formula , to calculate the concentration of matrix metalloproteinase-3 in the plasma of each rat.

(D11-3) Matrix metalloproteinase-13 (MMP-13): The detection and analysis of this indicator uses the Taiclone Rat Matrix Metalloproteinase 13 ELISA detection kit; the 96-well plate provided in the detection kit has been pre-coated with MMP- The specific antibody of 13; the method is to add 100 microliters (μl)/well of the standard or each rat plasma to an ELISA 96-well plate, and place the 96-well plate in an incubator at 37 degrees Celsius. , after reacting for 1.5 hours, pour out the standard or rat plasma in the well, add 100 μl/well of biotinylated detection Ab., shake evenly, and mix the 96 Place the well plate in an incubator at 37 degrees Celsius. After reacting for 1 hour, wash it three times with Wash buffer. Add 100 microliters (μl)/well of antibody protein labeling reagent (HRP conjugate) and let it stand at 37 degrees Celsius. In an incubator at a temperature of In the box, after reacting for 15 minutes, add 50 microliters (μl)/well of Stop solution, and immediately use an ELISA reader to detect the absorbance value under colored light irradiation with a wavelength of 450 nm, which can be compared with the standard. The absorbance value was converted into the concentration of matrix metalloproteinase-13 in the plasma of each rat according to the relevant formula.

(D12) Determination of pro-inflammatory factors:

(D12-1) Inducible nitric oxide synthase (iNOS): The detection and analysis of this indicator uses the Elabscience Rat Inducible nitric oxide synthase ELISA detection kit. The 96-well plate provided by the detection kit has Pre-coated with iNOS-specific antibodies; the method is to add 100 microliters (μl)/well of the standard or each rat plasma to the ELISA 96-well plate, and place the 96-well plate at 37 degrees Celsius. After the reaction for 1.5 hours, pour out the standard or rat plasma in the well, add 100 microliters (μl)/well of biotinylated detection Ab. and shake evenly. and place the 96-well plate in an incubator at 37 degrees Celsius. After reacting for 1 hour, wash it three times with wash buffer and add 100 μl/well of antibody protein labeling reagent ( HRP conjugate), place it in an incubator at 37 degrees Celsius, react for 30 minutes, wash 5 times with wash buffer, and then add 90 μl/well of substrate reagent ), place it in an incubator at 37 degrees Celsius, react for 15 minutes, add 50 μl/well of Stop solution, and immediately use an ELISA reader to detect its absorbance under colored light irradiation with a wavelength of 450 nm. The value can be compared with the absorbance value of the standard substance, and according to the relevant formula, the concentration of inducible nitric oxide synthase in the plasma of each rat can be calculated.

(D12-2) Enzyme Cyclooxygenase-2 (COX-2): The detection and analysis of this indicator is carried out using the Elabscience Rat Cyclooxygenase ELISA detection kit. The 96-well plate provided by the detection kit has been pre-coated with Specific antibody for COX-2; the method is to add 100 microliters (μl)/well of standard or each rat plasma to the ELISA 96-well plate, and place the 96-well plate at 37 degrees Celsius. In the incubator, after 1.5 hours of reaction, pour out the standard or rat plasma in the well, add 100 μl/well of biotinylated detection Ab., shake evenly, and Place the 96-well plate in an incubator at 37 degrees Celsius. After reacting for 1 hour, wash it three times with wash buffer and add 100 μl/well of antibody protein conjugate reagent (HRP conjugate). ), place it in an incubator at 37 degrees Celsius, react for 30 minutes, wash 5 times with wash buffer, and then add 90 μl/well of substrate reagent. Place it in an incubator at 37 degrees Celsius and react for 15 minutes. After adding 50 μl/well of Stop solution, immediately use an ELISA reader to detect the absorbance value under colored light with a wavelength of 450 nm. , that is, the concentration of the enzyme cyclooxygenase-2 in the plasma of each rat can be calculated based on the absorbance value of the reference standard and the relevant formula.

(D12-3) Nitric oxide (NO): According to reports, NO generated in the animal body is generally oxidized into nitrite ions (Nitrite, NO ₂ ^- ) and nitrate ions (Nitrate, NO ₃ ^- ), so By measuring nitrite ions (NO ₂ ^- ), the production amount of nitric oxide (NO) can be indirectly calculated. The detection of this indicator uses the Griess assay. In this assay, the reaction reagent can react with NO ₂ ^- to generate a red solution, and the different accumulation amounts of NO ₂ ^- will cause different color concentrations of the solution, so the solution can be at the wavelength of Under the irradiation of colored light of 540 nm, different absorbance values are shown, which are used as the basis for quantitative analysis. The method is based on what was disclosed in the previous literature (Schmidt, et al ., 1992). First, take 50 microliters (μl) of After adding each rat plasma to a 96-well plate, add 25 microliters (μl) of 1% Sulfanilamide (SUL) solution, mix evenly, protect from light for 10 minutes, then add 25 microliters (μl) ) and a concentration of 1% N-1-Naphthylethylenediamine dihydrochloride (NED for short, which exists in a 2.5% concentration of phosphoric acid (Phosphoric acid) solution), as soon as Mix evenly and let it stand for 10 minutes. Immediately use an ELISA reader to detect the absorbance value under the irradiation of colored light with a wavelength of 540 nm. This can then be used to compare the absorbance value of the standard substance. According to the relevant formula, the concentration in each rat plasma can be calculated. Nitrogen oxide concentration; the standard strain is serially diluted with 0.1 μM sodium nitrite, and the measured absorbance value is used to prepare a standard curve, and a linear regression curve is used to predict the NO release amount.

(D12-4) Prostaglandin E2 (PGE2): The detection and analysis of this indicator uses the Taiclone Rat Prostaglandin E2 ELISA detection kit. The 96-well plate provided in the detection kit has been pre-coated with PGE2. The specific antibody was added to an ELISA 96-well plate, adding 50 μl/well of standard or rat plasma, and then immediately adding 50 μl of biotinylated reagent (Biotinylated Detection). Ab working solution), and place the 96-well plate in an incubator at 37 degrees Celsius. After reacting for 45 minutes, pour out the standard or rat plasma in the wells and wash with wash buffer. 3 times, add 100 μl/well of antibody protein labeling reagent (HRP conjugate), place it in an incubator at 37 degrees Celsius, react for 30 minutes, wash 5 times with wash buffer, and then Add 90 μl/well of substrate reagent and place it in an incubator at 37 degrees Celsius. After reacting for 15 minutes, add 50 μL/well of stop solution and mix immediately. Use an ELISA reader to detect the absorbance value under the irradiation of colored light with a wavelength of 450 nm, which can be compared with the absorbance value of the standard. According to the relevant formula, the concentration of prostaglandin E2 in the plasma of each rat can be converted.

(D12-5) Tumor Necrosis Factor-α (TNF-α for short): The detection and analysis of this indicator uses the Elabscience Rat Tumor Necrosis Factor-α ELISA detection kit. The 96-well test kit provides The plate has been pre-coated with TNF-α specific antibodies. The method is to add 100 μl/well of the standard or each rat plasma to the ELISA 96-well plate, and add the 96-well Place the plate in an incubator at 37 degrees Celsius and react for 1.5 hours. Then pour out the standard or rat plasma in the well and add 100 μl/well of biotinylated detection reagent. Ab.) After shaking evenly, place the 96-well plate in an incubator at 37 degrees Celsius. After reacting for 1 hour, wash it three times with eluent (Wash buffer), and add 100 microliters (μl)/well of Antibody protein labeling reagent (HRP conjugate), placed in an incubator at 37 degrees Celsius, reacted for 30 minutes, washed 5 times with eluent (Wash buffer), and then added 90 microliters (μl)/well of substrate for display. Place the substrate reagent in an incubator at 37 degrees Celsius. After reacting for 15 minutes, add 50 μl/well of Stop solution, and immediately use an ELISA reader to detect the wavelength of 450 nm. The absorbance value under colored light irradiation can be compared with the absorbance value of the standard, and the concentration of tumor necrosis factor α in the plasma of each rat can be calculated according to the relevant formula.

(D12-6) Type II collagen: The detection and analysis of this indicator uses the Elabscience Rat Type II collagen ELISA detection kit. The 96-well plate provided in the detection kit has been pre-coated with (Coating) The specific antibody for Type II collagen is prepared on an ELISA 96-well plate, adding 100 microliters (μl) / well of the standard or each rat plasma, and placing the 96-well plate at 37 degrees Celsius for culture In the box, react for 1.5 hours. Then, pour out the standard substance or rat plasma in the well, add 100 microliters (μl) / well of biotinylated detection Ab., and incubate at 37 degrees Celsius. In the incubator, after reacting for 30 minutes, wash with wash buffer 5 times, then add 90 μl/well of substrate reagent, and place it in an incubator at 37 degrees Celsius. , after reacting for 15 minutes, add 50 μl/well of Stop solution, and immediately use an ELISA reader to detect its absorbance value under colored light irradiation with a wavelength of 450 nm, which can be compared with the standard. The absorbance value is converted into the concentration of type II collagen in the plasma of each rat according to the relevant formula.

The determination and analysis of osteoarthritis-related factors are as follows:

(E-1) Bipedal balance test (Incapacitance test): The measurement and analysis of this indicator are based on those disclosed in previous literature (Mapp et al ., 2010). The method is based on the bipedal balance test and is suitable for measuring hind limb standing. The force distribution of the weight at that time can be used to evaluate the degree of discomfort and condition of the animal's joints. Press, under normal circumstances, the weight distribution of the rat's hind limbs will be more even, and the pressure difference between the two feet will be close to 0; conversely, if the rat's unilateral knee joint is injured, the uninjured side's hind limbs will bear more weight. , causing the pressure difference between the feet to increase. In the aforementioned measurements, the weight distribution of the rat's hind limbs was measured using a bipedal balance pain meter. The method is to put the tested rat into an acrylic box equipped with a 65∘ inclined plate to train the tested rat to stand on its hind limbs in advance; when the test is officially carried out, the acrylic box will be placed on a bipedal balance pain tester, so that when each tested rat stands on its hind limbs, the bipedal balance pain tester can measure and record the pressure on both hind limbs. , after repeating the above measurement process 5 times, and taking the average value, the bipedal balance test value of each tested rat can be obtained.

(E-2) Histological analysis of the knee joint: The measurement and analysis of this indicator were performed by sacrificing the rat at the end of the experiment, taking out the knee joint and shaving the excess muscle tissue, and then immersing the knee joint in formaldehyde content of 10%. formalin, and then sent to Lipei Co., Ltd. in Taichung, Taiwan at room temperature for anatomy and histopathological diagnosis, and outsourcing the production of Hematoxylin and Eosin (H&E) and Safranin of knee joint tissue. -O stained sections.

According to the above, in the aforementioned experiments of the present invention, the experimental results are expressed as mean ± standard deviation (Mean ± S.D.), and the statistical analysis of the experimental data is based on Graph Pad Prism 5 and Statistical Product & Service Solution (SPSS) 22.0 software is used for statistical processing. The single-factor score difference and statistical differences analysis of specific group comparisons are analyzed by One-way analysis of variance (One-way ANOVA). The differences between groups are then analyzed by multiple comparisons. Test method (Multipe comparison method), that is, Dunnett's Test is used for post hoc comparison. When P<0.05, it means there is a statistically significant difference.

In view of the aforementioned experimental results, the following biochemical properties of the jellyfish skin collagen (JC) freeze-dried powder and its fermentation product (FJC) of the present invention are explained and analyzed one by one as follows:

(F-1) Amino acid composition analysis: According to previous literature (Paul Szpak, 2001), collagen is a protein with a very special amino acid composition. Generally, glycine accounts for about 1/1 of all amino acid residues. 3 Collagen, the two main sequences are Gly-Pro-X and Gly-X-Hyp, where X is an amino acid other than Gly, Pro and Hyp. After conversion, Gly will fall around 330 residue/1000 residue. It also has high levels of alanine but lacks tryptophan. The aforementioned experimental results of the present invention show that the Gly of JC and FJC are 333.08 and 324.84 residue/1000 residue respectively, and the remaining amino acids with higher proportions are glutamic acid, proline and alanine respectively, which means that the Gly of the present invention The amino acid composition of JC and FJC is consistent with that of collagen.

(F-2) Fourier transform infrared spectroscopy (FTIR for short): According to reports, Fourier transform infrared spectroscopy is mainly used to analyze the functional groups and secondary structure of collagen. The aforementioned experimental results of the present invention show that the JC and FJC of the present invention have five absorption bands, namely Amide A; Amide B; Amide I; Amide II; Amide III; and the values of the main absorption bands of the two are Amide A 3405.51 respectively. cm ^-1 and 3334.36 cm ^-1 , Amide B 2924.50 cm ^-1 and 2918.81 cm ^-1 , Amide I 1659.32 cm ^-1 and 1649.39 cm ^-1 , Amide II 1552.62 cm ^-1 and 1555.46 cm ^-1 , Amide III 1239.53 cm ^-1 and 1235.26 cm ^-1 . After comparing this result with what was disclosed in the previous literature (Karima Belbachir et al. 2009), it should be inferred that the JC and FJC extracted by the present invention are consistent with the functional groups of collagen.

(F-3) UV absorption spectrum: Generally speaking, substances with conjugated double bonds have the ability to absorb ultraviolet light. Among the 20 basic amino acids, there are 4 types with conjugated double bonds. , including: tryptophan, tyrosine, phenylalanine and histidine, etc. Among them, tryptophan has the strongest absorption of ultraviolet light with a wavelength of 280 nm. Therefore, generally speaking, most proteins are exposed to colored light with a wavelength of 280 nm. There will be a significant UV absorption peak under all conditions. However, since collagen contains almost no tryptophan, this strong absorption will not appear according to previous literature (Zhang et al ., 2008). Peak, which can be used as a basis for identifying collagen, and the main absorption peak of collagen appears near the irradiation of colored light with a wavelength of 230 nm. Because the above-mentioned experimental results of the present invention show that the FJC of the present invention has a colored light with a wavelength of 230 nm. There is the strongest absorption peak at the light irradiation point, and there is no obvious absorption front at the 280 nm colored light irradiation point. This experimental result fully indicates that the JC and FJC extracted by the present invention indeed belong to collagen.

In addition, the present invention is still based on the aforementioned experimental results, and the following biochemical properties of the jellyfish skin collagen fermentation broth (FJC) of the present invention are analyzed and explained one by one as follows:

(G-1) DPPH (1,1-diphenyl-2-trinitophenylhydrazine, referred to as DPPH) free radical scavenging ability: According to reports, DPPH is a stable nitrogen-containing free radical that can be used to detect jellyfish skin collagen of the present invention The hydrogen supply capacity of the fermentation broth decreases when it is reduced, so the absorbance value can be measured as one of the antioxidant capacity indicators. Accordingly, please refer to Table 8 below. It can be observed from the above-mentioned experimental results of the present invention that the FJC of the present invention obviously has better DPPH free radical scavenging ability than JC, and this DPPH free radical scavenging ability will increase with fermentation. As the number of days increased, the clearance rate increased rapidly from the initial 8.49% to 47.16% on the third day. This increase slowed down on the fourth day of fermentation. Table 8. DPPH free radical scavenging ability of jellyfish skin collagen (JC) and its fermentation broth (FJC) fermented for 1, 2, 3 and 4 days The DPPH radical scavenging (%) ¹ JC 8.49 ± 0.03 ^{a 2} FJC-day 1 16.32 ± ^0.07b FJC-day 2 40.81 ± ^1.01c FJC-day 3 47.16 ± 2.05 ^days FJC-day 4 47.35 ± 0.31 ^days

(G-2) ABTS ^{․ ＋} Clearing ability: ABTS ^{․ +} is a long-lived cationic free radical, ABTS ^{․ ＋} Scavenging ability is provided by antioxidants to remove ABTS ^{․ +} The ability of free radicals. Accordingly, please refer to Table 9 below. It can be observed from the above-mentioned experimental results of the present invention that the FJC of the present invention obviously has better ABTS than JC ^{․ +} clearing ability, and this one ABTS ^{․ +} The scavenging ability will increase as the number of fermentation days increases. The scavenging rate rapidly increases from the initial 11.33% to 27.12% on the third day. This increase slows down on the fourth day of fermentation. Table 9. The scavenging ability of ABTS cationic free radicals of jellyfish skin collagen (JC) and its fermentation broth (FJC) fermented for 1, 2, 3 and 4 days. The ABTS ^{․ +} scavenging (%) ¹ JC 11.33 ± 0.08 ^{a 2} FJC-day 1 19.61± ^0.88b FJC-day 2 25.86 ± ^0.49c FJC-day 3 27.12 ± 0.63 ^days FJC-day 4 27.08 ± 0.09 ^days

(G-3) Ability to chelate ferrous ions: Generally speaking, divalent metal ions such as Fe ²⁺ or Cu ²⁺ can catalyze the Fenton reaction and the Harber-Weiss reaction. As it progresses, more hydroxyl free radicals are generated, which promotes lipid oxidation and causes oxidative damage. However, according to previous literature (Kitazawa and Iwasaki, 1999), since the chelating agent structure has two pairs of shared electrons, it cannot react with free radicals. Instead, it can chelate divalent metal ions to effectively inhibit the movement of metal ions. The effect of catalyzing the generation of hydroxyl radicals.

In addition, the present invention also confirmed the relevant reducing ability of FJC of the present invention through the following cell experiments:

(G-4) The effect of FJC of the present invention on the survival rate of RAW 264.7 and SW1353 cells;

(G-5) Determination of the survival rate of RAW 264.7 and SW1353 cells by FJC of the present invention under the condition of inducing inflammation;

(G-6) Effect of FJC of the present invention on NO release when inducing inflammation.

Finally, the present invention has confirmed the effect of the FJC of the present invention on symptoms related to osteoarthritis through the following animal experiments:

(H-1) The effect of FJC of the present invention on the body weight and organ weight percentage of obese osteoarthritis rats: From the aforementioned animal experiment results, it can be clearly observed that among the body weights of these rats, the Control group and OA that did not induce obesity The body weight of the group was significantly lower than that of other groups. Among the obesity-induced groups, the OBOA group had the heaviest weight, followed by the group fed FJC. Based on this experimental result, please refer to Figure 11. It should be It can be inferred that the higher pain in the hind limbs of the rats in the OBOA group led to the reduced willingness to exercise in the rats in the OBOA group, making their weight higher than that of the other high-fat diet groups. Although there was no significant difference between the groups in the percentage of organ weight shown in the liver and spleen parts; however, there was a significant increase in the percentage of organ weight shown in the kidney part in the OBOA group. Based on this, it should be inferred that It is the result of the burden on the kidneys caused by obesity, and this result can be significantly improved after feeding more than twice the dose of FJC; in addition, the results of accessory testicular fat and abdominal fat can also be clearly found , the weight of the OBOA group increased significantly compared with the weight of the Control group; among them, regarding the accessory fat, a significant weight reduction effect was seen after feeding five times the dose of FJC, while for abdominal fat, The same effect was observed by feeding only twice the dose of FJC. Based on this, it should be confirmed that the FJC of the present invention can indeed improve the problem of increased kidney, accessory testicular fat and abdominal fat weight. But is this result caused by the FJC of the present invention having an improvement effect on obesity or osteoarthritis? It is still unknown.

(H-2) The effect of FJC of the present invention on obesity-related indicators in obese osteoarthritis rats: With reference to previous literature (Boudewijn Klop et al ., 2013), typical obesity dyslipidemia is generally caused by TG and Elevated free fatty acids, reduced HDL-C accompanied by HDL dysfunction and normal or mildly elevated LDL-C with small and dense LDL composition ultimately lead to increased risk factors such as blood sugar, insulin and blood pressure, thereby increasing the risk of cardiovascular disease. Risk of disease. In addition, please refer to Figures 12A~D. It can be clearly seen from the aforementioned animal experiment results that the TG and TC concentrations in the blood of the Control group and the OA group that have not induced obesity are both lower, while the TG in the blood of the OBOA group Compared with it, there is a significant increase in the concentration of TG and TC. This phenomenon can slightly reduce the concentration of TG and TC in the blood in the GS group after feeding FJC of the present invention, but there is a difference between feeding one, two and five times the dose. There is no significant difference. In addition, it can be clearly seen from the aforementioned animal experiment results that there is no significant difference in HDL concentration between the general group and the obese group. However, significant differences in HDL concentration can be found in the GS group fed with two or five times the dose of FJC. Increase, and it can be observed that the LDL concentration in the blood of the obesity-induced group has a significant increase, but this situation cannot be suppressed in the GS group after administration of FJC of the present invention. Based on this, it should be confirmed that twice the dose of FJC can improve dyslipidemia in rats, but it does not inhibit the production of LDL.

(H-3) Leptin and adiponectin content in plasma: As disclosed in previous literature (Diwan et al ., 2018), Leptin and Adiponectin are adipocyte hormones, and their expression levels will increase in obesity. , among which leptin is a key hormone that regulates energy intake and consumption by controlling appetite and glucose metabolism. The concentration of leptin in the circulation is directly proportional to the total body fat. When it is lacking, it will lead to out-of-control food intake, obesity and diabetes. . The experimental results showed that compared with the Control group, the concentrations of Leptin and Adiponectin in the other groups increased significantly, and slight inhibition was observed after administration of higher doses of FJC. It has been confirmed that five times the dose of FJC can slightly improve leptin and adiponectin abnormalities in rats, but the effect is not obvious, so FJC may not have a significant improvement effect on obesity.

(H-4) The effect of FJC of the present invention on oxidative stress in obese osteoarthritis rats: With reference to the previous literature (Ramalho-Santos et al ., 2008), it can be seen that under oxidative stress, free radicals will attack Polyunsaturated fatty acids are eventually peroxidized to cause plasma lipid peroxidation (MDA). Regarding this problem, the present invention analyzes the degree of lipid peroxidation. Please refer to Figures 13A to C. The results show that the OBOA group The MDA content of the control group was significantly higher than that of the Control group, and the MDA content could be significantly reduced after six weeks of tube feeding of each dose of FJC and GS, and the effects of the two and five times dose and GS groups were better. Based on this, it should be confirmed One dose of the FJC of the present invention can slow down the degree of lipid peroxidation in obese osteoarthritis rats, but the improvement effect is better when fed with two or five times the dose. In addition, given that obesity is an overall chronic inflammatory response, it will secrete inflammatory cytokines, and osteoarthritis will also promote the secretion of inflammatory cytokines, and obesity will increase free fatty acids in the blood, leading to lipid excess. Oxidation causes oxidative stress in the body. Regarding this issue, the present invention measures the activity of the antioxidant enzymes glutathione peroxidase (GPx) and superoxide dismutase (SOD). Please refer to Chapter 13 As shown in the figure, it can be clearly seen from the measurement results that the antioxidant enzyme activity of the group induced into OA was significantly lower than that of the Control group, while the GPx activity was significantly lower after administration of two or five times the doses of FJC and GS. This situation was improved after that, and the improvement in SOD activity was clearly shown by feeding the FJC group with double the dose. Based on this, it should be confirmed that feeding twice the dose of FJC can alleviate the lower antioxidant enzyme activity in obese osteoarthritis rats.

(H-5) Effect of FJC of the present invention on liver and kidney function in obese osteoarthritis rats: Refer to the previous literature (Ida M. Washington and Gerald Van Hoosier, 2012). When liver or muscle bruises, trauma, necrosis, After infection or tumor formation, the AST concentration in the blood will increase, while the ALT content will only increase when the cells are obviously necrotic. In addition, in the case of hepatitis, congestive heart failure, liver or bile duct injury, or myopathy ALT levels may also increase, so ALT levels are often used as an indicator to measure liver function. Regarding this, it can be found from the aforementioned animal experiment results. As shown in Table 10 below, after feeding different doses of FJC of the present invention for 6 weeks The results of the analysis of aspartate transaminase, alanine transaminase, urea nitrogen and creatinine contents in the plasma of obese osteoarthritis rats: Table 10. After feeding different doses of FJC of the present invention for 6 weeks Results of analysis of aspartate transaminase, alanine transaminase, urea nitrogen and creatinine contents in plasma of obese osteoarthritis rats: AST ALT BUN Creatinine (U/L) (U/L) (mmol/L) (mg/dL) Control 23.15 ± ^2.13b 18.44 ± ^1.37c 5.80± ^0.09c 0.58 ± ^0.04c OA 25.23 ± ^3.47b 18.14 ± ^1.46c 5.91 ± ^0.07b 0.71± ^0.04b OBOA 31.47 ± 1.89 ^a 21.85 ± 1.30 ^a 6.07 ± 0.09 ^a 0.79 ± 0.04 ^a OBOA+FJC1 31.34 ± 2.61 ^a 20.04 ± 3.15 ^abc 6.05 ± 0.06 ^a 0.70± ^0.08b OBOA+FJC2 31.43 ± 2.20 ^a 19.88 ± ^1.57ab 6.06 ± 0.05 ^a 0.67 ± ^0.08b OBOA+FJC5 30.64 ± 1.86 ^a 20.47± ^1.46ab 6.06± ^0.07a 0.68 ± ^0.07b OBOA+GS 29.90± ^1.25a 21.48± ^0.99ab 6.05± ^0.14a 0.74 ± 0.03 ^a

The experimental results showed that the levels of liver function indicators AST and ALT were comparable to those of the Control and OA groups. There was a significant increase in AST and ALT in the blood of the induced obesity group, but no improvement was found in the GS group after each dose of FJC. situation. Based on this, it should be inferred that FJC of the present invention should have nothing to do with the improvement of liver function. In addition, according to the previous literature (Mazze et al ., 2000), it can be clearly found from the organ weight percentage that the kidney weight percentage is heavier in the OBOA group. When further observing the renal function index in plasma, given that BUN is Metabolites of protein, and creatinine is a metabolite of muscle. When the kidneys are damaged, their filtration function will decrease, causing the concentration of BUN and creatinine in the blood to increase. This can be found from the above-mentioned animal experiment results of the present invention. BUN in the OBOA group and creatinine were significantly higher than those in the Control group, and there was no improvement in BUN in each dosage group and the positive control group; as for creatinine, it can be observed from the aforementioned animal experiment results of the present invention, and its concentration was significant in each dosage group. decreased, but there was no effect in the GS group. Accordingly, it should be confirmed that a double dose of FJC of the present invention should be able to improve renal damage to a certain extent.

(H-6) The effect of FJC of the present invention on the inflammatory response in obese osteoarthritis rats: According to previous literature (Gregg B. Fields, 2014), matrix metalloproteinase was first discovered in 1962 and has the ability to cleave Collagen and extracellular matrix capabilities. So far, at least 28 different types of matrix metalloproteinases have been discovered in this family. Their classification is mainly named according to their ability to cleave different extracellular matrices; among them, MMP-1 is also called "interstitial collagenase" ” or “fibroblast collagenase”, MMP-3 can degrade type II, III, IV, IX and X collagen, as well as proteoglycans, fibronectin, laminin and elastin...etc. In addition, MMP-3 can also activate other MMPs, such as MMP-1, MMP-7 and MMP-9, making MMP-3 play a vital role in connective tissue remodeling. This can be confirmed by the aforementioned animal experiments of the present invention. As a result, please refer to Figure 14A~B. The levels of MMP-1 and MMP-3 in the blood of the OA and OBOA groups were significantly higher than those of the Control group, which means that feeding the FJC of the present invention can significantly inhibit their production. For MMP-1, part of the group fed with 5 times the dose of FJC had the best inhibitory effect, while a dose-dependent decrease in MMP-3 content was observed with the FJC of the present invention. As for the contents of iNOS, COX-2 and their products NO and PGE2 in plasma, according to previous literature (P. Needleman and P. T. Manning, 1999), iNOS and COX-2 can produce NO and PGE2 by inducing enzymatic pathways. It can cause inflammation and related tissue damage. The above-mentioned substances found in the joints of patients with osteoarthritis seem to be closely related to the progressive symptoms of pain, swelling and cartilage destruction. This can also be observed from the aforementioned animal experiment results. Please refer to Figure 15A~D. The levels of iNOS, COX-2, NO and PGE2 in the plasma of the OA and OBOA groups were significantly increased compared with the Control group. , after feeding to FJC and GS, their contents were significantly inhibited. However, in addition to the dose-dependent inhibition of iNOS at each dose of FJC, there was no significant difference between each dose and COX-2, NO and PGE2. Based on this, it should be confirmed that one dose of FJC can inhibit the levels of pro-inflammatory factors such as iNOS, COX-2, NO and PGE2 in plasma and reduce them.

(H-7) The effect of the FJC of the present invention on the pain-supporting force difference of both feet of obese osteoarthritis rats: the supporting force difference of the Control group will gradually tend to 0 over time, and finally the supporting force difference of the OA and OBOA groups The difference was significantly higher. Regarding this, please refer to Figure 16. It can also be observed from the aforementioned animal experiment results that the force difference of the rats fed FJC and the positive control drug group was significantly reduced. This also fully indicates that the rats fed FJC After FJC, it can obviously effectively relieve the pain caused by OA; among them, the relief effect of feeding 2 and 5 times the dose of FJC is more obvious.

(H-8) Effect of FJC of the present invention on knee joint cartilage in obese osteoarthritis rats: Safranin O-Fast green staining method was used to evaluate cartilage tissue. Please refer to Figure 17, where the cartilage of the Control group can be observed. The surface is smooth and complete, but the OA and OBOA groups that underwent ACLT + MMx surgery have obvious cartilage destruction, thinner cartilage layers, rough surfaces, and reduced safranin O-Fast green staining, indicating that the protein content of the cartilage tissue in the OA and OBOA groups There is loss of polysaccharides. In each dose of FJC group, it was observed that cartilage degeneration was inhibited and cartilage surface roughness was improved. Among them, the recovery effect of FJC at five times the dose was the most significant. These findings clearly demonstrate that taking a double dose of FJC of the present invention can alleviate cartilage matrix degradation and articular cartilage destruction in obese osteoarthritis rats.

Based on the above conclusion, the present invention extracts jellyfish skin collagen (JC) from jellyfish with abundant marine resources through ultrasound-assisted acid extraction, and then uses cheap and high-quality natto Bacillus subtilis from by-products of the agricultural or food processing industries as raw materials. Using mature and convenient preparation procedures and equipment, the jellyfish skin collagen is fermented so that the triple helix structure of the jellyfish skin collagen remains unchanged, and the jellyfish skin collagen of the present invention can alleviate the symptoms of degenerative osteoarthritis. Protein Natto ferment (FJC), according to many of the aforementioned experiments, the FJC of the present invention not only shows a significant protective effect on RAW 264.7 cells, but can also effectively inhibit the production of nitric oxide induced by LPS; in addition, according to the aforementioned animal experiments , the FJC of the present invention can still effectively inhibit the expression of pro-inflammatory cytokines NO, COX-2, TNF-α and MMPs... etc. in OA rats, clearly showing its protective effect on articular cartilage; not only that, the aforementioned animal experiments It also clearly shows that the FJC of the present invention has the ability to effectively inhibit cartilage degradation in animal OA models, thereby effectively improving the antioxidant and biocompatibility of jellyfish skin collagen... and other material properties, thereby making the jellyfish skin collagen of the present invention Fermentation materials can be used to create innovative materials for the treatment of degenerative osteoarthritis. Since the arthritis progenitor system is a chronic inflammatory disease related to the accumulation of microorganisms, and the jellyfish skin collagen fermentation product of the present invention has been confirmed by many experiments to have antibacterial activity against arthritis progenitors, according to this, it can not only It can effectively prevent or improve arthritis symptoms, and can fully achieve the purpose of effective administration. Therefore, in the clinical treatment process of jellyfish skin collagen, it has great potential to effectively and accurately relieve the symptoms of degenerative osteoarthritis, and It can greatly improve the therapeutic effect and efficiency of jellyfish skin collagen, thus having extremely high utilization value in the bone and joint health care and medical industries, and the obtained jellyfish skin collagen fermentation material can become an important material in the field of biomedical applications. Innovative biomaterials with huge potential.

According to the above, the above is only one of the best specific embodiments of the present invention, but the technical features of the present invention are not limited thereto. Anyone familiar with the art can easily think of equivalent changes in the field of the present invention. or modifications, can be covered by the following patent scope of this case.

[customary knowledge] C1, C2, C3: polypeptide chain J:Jellyfish J1: bell jar J2: Oral arm [Invention] 100~104: steps

[Picture 1] A schematic diagram of the triple helix structure of collagen; [Picture 2] A schematic diagram of the appearance and cross-section of a jellyfish; [Figure 3] is a schematic flow chart of the manufacturing method of the present invention; [Figure 4] is a schematic diagram of the changes in pH value of the fermentation broth (FJC) of the jellyfish skin collagen (JC) fermentation for 0, 1, 2, 3 and 4 days; [Figure 5] is the ultraviolet absorption spectrum of jellyfish skin collagen (JC) of the present invention; [Figure 6] is the ultraviolet absorption spectrum of the jellyfish skin collagen fermentation product (FJC) of the present invention; [Figure 7] is the Fourier transform infrared spectrum of the jellyfish skin collagen (JC) of the present invention; [Figure 8] is the Fourier transform infrared spectrum of the jellyfish skin collagen fermentation product (FJC) of the present invention; [Figure 9] A schematic diagram of the cell survival rate of macrophages RAW 264.7 treated with the jellyfish skin collagen ferment (FJC) of the present invention for 24 hours; [Figure 10] A schematic diagram of the NO release amount of macrophage RAW 264.7 when the jellyfish skin collagen ferment (FJC) and LPS of the present invention are used together for 24 hours; [Figure 11] It is a schematic diagram of analyzing the body weight of obese osteoarthritis rats after feeding different doses of jellyfish skin collagen ferment (FJC) of the present invention for 6 weeks; [Figure 12A~D] shows the effects of (a) triglyceride and (b) total cholesterol in the plasma of obese osteoarthritis rats after feeding different doses of jellyfish skin collagen ferment (FJC) of the present invention for 6 weeks. , Schematic diagram of analysis of (c) high-density lipoprotein HDL-C and (d) low-density lipoprotein LDL-C content; [Figure 13A~C] shows the effects of (a) lipid peroxidation MDA content, (b) glutathione content in the plasma of obese osteoarthritis rats after feeding different doses of jellyfish skin collagen ferment (FJC) of the present invention for 6 weeks. Schematic diagram of the analysis of the effects of thione peroxidase GPx activity (activity) and (c) superoxide dismutase SOD activity; [Figure 14A~B] shows the effects of (a) matrix metalloproteinase MMP-1 in the plasma of obese osteoarthritis rats after feeding different doses of jellyfish skin collagen ferment (FJC) of the present invention for 6 weeks; (b) Schematic diagram of analysis of matrix metalloproteinase MMP-3 and (c) matrix metalloproteinase MMP-13 content; [Figure 15A~D] shows the effects of (a) inducible nitric oxide synthase, (b) in the plasma of obese osteoarthritis rats after feeding different doses of jellyfish skin collagen ferment (FJC) of the present invention for 6 weeks. ) Schematic diagram of analysis of cyclooxygenase-2, (c) nitric oxide NO and (d) prostaglandin E2 content; [Figure 16] It is a schematic diagram of analyzing the difference in pain and balance support of both feet of obese osteoarthritis rats after feeding different doses of jellyfish skin collagen ferment (FJC) of the present invention for 6 weeks; and [Figure 17] is a schematic diagram of the analysis of rat cartilage in different groups using the Safranin O-Fast green staining method.

100~104: steps

Claims (7)

A jellyfish skin collagen Natto fermentation product (FJC) that can alleviate the symptoms of degenerative osteoarthritis. The fermentation product (FJC) uses commercially available salted jellyfish skin as raw material, and uses physical ultrasonic auxiliary acid to extract it. After removing the jellyfish skin collagen (JC), the jellyfish skin collagen (JC) is fermented with Natto bacteria in a fermentation tank environment of 37 degrees Celsius and a rotation speed of 150 rpm for at least 1 day to form the fermentation product (FJC). ), the fermentation product (FJC) can reduce the body weight, plasma triglyceride and total cholesterol (TC) contents of obese OA rats, and can effectively inhibit inducible nitric oxide synthase (iNOS), epoxy Synthase-2 (COX-2) and its products nitric oxide (NO) and prostaglandins (PGE2) produce pro-inflammatory cytokines and can effectively reduce the levels of matrix metalloproteinases (MMP-1 and MMP-3). , and has the effect of effectively reducing the difference in foot support in rats, smoothing the rough cartilage surface in knee joint tissue sections, and thickening the cartilage layer. This fermented substance (FJC) can indeed inhibit the related The production of inflammatory factors achieves the protective effect on articular cartilage, and has a certain degree of soothing effect on the inflammation, pain and articular cartilage degradation caused by osteoarthritis. A method for preparing jellyfish skin collagen Natto ferment (FJC) that can alleviate the symptoms of degenerative osteoarthritis. The preparation method uses generally commercially available salted jellyfish skin as raw material and includes the following steps: First, the salted jellyfish skin must be desalted with clean water to clean the salt on the surface of the salted jellyfish skin; Then, cut the washed jellyfish skin into jellyfish skin filaments one by one; Next, clean the jellyfish skin filaments; Using an ultrasonic-assisted acid extraction method, jellyfish skin collagen was extracted from the washed jellyfish skin filaments; Finally, the jellyfish skin collagen (JC) is fermented for at least 1 day with Natto bacteria in a fermentation tank environment of 37 degrees Celsius and a rotation speed of 150 rpm, to obtain the jellyfish skin collagen fermentation product (JC). FJC). According to the production method described in claim 2, the jellyfish skin filaments are in a specification of about 1 to 1.5 millimeters (mm) in thickness and about 2 to 5 centimeters (cm) in width. The preparation method as described in claim 3, wherein the step of cleaning the jellyfish skin filaments is to immerse the jellyfish skin filaments in clean water 10 times its total weight, and slowly stir the jellyfish skin filaments with a magnet. Wash the jellyfish skin filaments repeatedly for at least 3 hours, and replace the water every hour to clean out the salt in them. The preparation method as described in claim 4, wherein the ultrasonic-assisted acid extraction method is performed at 4 degrees Celsius, and the following steps are completed in sequence: First, the desalted and washed jellyfish skin filaments were subjected to alkaline hydrolysis pretreatment (alkaline pretreatment) with a sodium hydroxide (NaOH) solvent (w/v=1/2) with a concentration of 0.1 mole (M) for 2 hours. ), to form a hydrolysis mixture of the jellyfish skin filaments; Then, wash the insoluble matter in the hydrolysis mixture with water several times until the washing water becomes neutral; Subsequently, an acetic acid (CH3COOH) solvent (w/v=1/2) with a concentration of 0.5 mol (M) was added to the hydrolysis mixture, and a homogenizer was used to conduct ultrasonic vibration of the hydrolysis mixture for 2 hours, so that After the hydrolysis mixture reaches a completely homogenized first mixed solution, the first mixed solution is quickly stirred with a magnet for 2 hours, and a high-speed centrifuge is used to perform a step on the first mixed solution at a speed of 10,000 × g. centrifuge for an hour, and after obtaining the precipitate, the precipitate is back-dissolved in acetic acid with a concentration of 0.5 mol (M) to form a second mixed solution; and chlorine is added to the second mixed solution. Salt out it with sodium (NaCl) so that the final concentration of the second mixed solution reaches 4.5 moles (M); and Finally, use a high-speed centrifuge to centrifuge the second mixed solution for one hour at a speed of 10,000 × g to obtain the precipitate, and then dissolve the precipitate back in 0.5 (M) acetic acid to form a The third mixed solution is dialyzed against deionized water with a concentration of (v/v=1/10). Finally, the obtained third mixed solution is freeze-dried to obtain jellyfish. Freeze-dried powder of skin collagen (JC). The preparation method as described in claim 5, wherein the fermentation process includes the following steps: First, it is necessary to inoculate the purchased Bacillus subtillis natto for fermentation into fresh culture medium for secondary activation; Use an Erlenmeyer flask to culture lactic acid bacteria in a small amount: first prepare a sterilized Erlenmeyer flask and place it on a sterile operating table; then, inoculate 100 ml of activated lactic acid bacteria with 1% of the bacterial solution (ml) of the fresh culture medium; then, culture it at 37 degrees Celsius, and every three hours, take out the pH value and optical density value, and adjust the pH value with sodium hydroxide (NaOH) titration, so that the pH value reaches About 6, and observe the growth curve for dozens of hours; after about 12 hours, after the growth reaches the end of the logarithmic proliferation phase, use a high-speed centrifuge at a speed of 8000 × g for 20 minutes at 4 degrees Celsius. Process, collect the bacterial cells, add the bacterial cells to 200 milliliters (ml) of sterilized physiological saline with a concentration of 0.85%, suspend the bacterial cells on the physiological saline, and then centrifuge them under the above conditions. After removing the supernatant once, add 100 milliliters (ml) of sterilized physiological saline with a concentration of 0.85%, and suspend the bacteria on the physiological saline and place it in an environment of 4 degrees Celsius for later use; Large-scale fermentation of jellyfish skin collagen freeze-dried powder: the method is to add 50% distilled water, 4% sucrose, 2% by weight to the jellyfish skin collagen freeze-dried powder. Sodium chloride (NaCl), 1% glucose (Glucose) and 1% of the aforementioned bacteria and other ingredients, and put these ingredients together into a beaker with a capacity of one liter (L) to form a jellyfish skin to be fermented Collagen solution, and open the top of the beaker, cover it with aluminum foil and seal it, then move it to a fermentation tank or incubator at 37 degrees Celsius for fermentation; during the fermentation process, first, the pH meter must be calibrated, and the pH meter must be calibrated. The equipment to be used is sterilized in a high-temperature sterilization kettle; then, the rotation speed of the fermentation tank is adjusted to 150rmp/min, the temperature is 35 degrees Celsius, and the pH value range is controlled between 5.9 and 6.1, so that the jellyfish skin collagen to be fermented The solution is fermented, and the consumption of sodium hydroxide is observed and recorded every three hours until the sodium hydroxide is no longer consumed, which means the lactic acid fermentation is terminated. After the fermentation is terminated, the jellyfish skin collagen fermentation liquid is taken out and freeze-dried. That is, the jellyfish skin collagen fermentation product (FJC) of the present invention is obtained. The preparation method of claim 6, wherein the fresh culture medium is prepared by using distilled water to prepare a culture medium aqueous solution such that each liter of the culture medium aqueous solution contains 100 grams (g) of glucose (Glucose), 10.0 grams (g) of Yeast Extract, 1.5 grams (g) of dipotassium hydrogen phosphate (K ₂ HPO ₄ ), 0.5 grams (g) of sodium acetate (CH ₃ COONa), 0.2 grams (g) of magnesium sulfate heptahydrate ( MgSO _{4.7H 2} O) and 0.038 grams (g) of manganese sulfate monohydrate (MnSO4.H2O), and put the culture medium solution into a constant temperature incubator for 24 hours at 37 degrees Celsius, that is, The fresh culture medium is formed for use in secondary activation of the lactic acid bacteria.

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