Chicken cartilage extract, preparation method and application thereof

Abstract

The invention relates to the technical field of cartilage extraction, and in particular to a chicken breast cartilage extract, a preparation method and an application thereof. The chicken breast cartilage extract uses chicken breast cartilage raised for 40 to 60 days as a raw material, and the obtained cartilage extract has a higher chondroitin sulfate A / C value.

Description

Technical Field

[0001] The invention relates to the technical field of cartilage extraction, and in particular to a chicken breast cartilage extract, a preparation method and application thereof. Background Art

[0002] Cartilage is a tough and elastic connective tissue found primarily in joints and supporting bones.

[0003] Cartilage extract is made from high-quality animal cartilage using a special enzymatic hydrolysis process. It usually contains many bioactive ingredients, such as collagen, sodium chondroitin sulfate and other natural bioactive substances. Therefore, cartilage extract is widely used in the health food or functional food industry.

[0004] In order to improve the utilization value of cartilage extracts, there are also many studies related to cartilage extracts. For example, the patent application with patent publication number CN114045322A discloses a method for extracting chicken cartilage extracts from chicken breast cartilage and its products. When extracting type II collagen, chicken breast cartilage is used as a raw material, and type II collagen is obtained through defatting, grinding, acid hydrolysis, enzymolysis, electrophoresis dialysis, secondary grinding, sterilization, and then type II collagen is prepared through carrier membrane preparation, secondary electrophoresis, and vacuum sealing to obtain products. The chicken cartilage extract obtained by the invention is a small molecule polypeptide with a hydrolysis molecular weight of about several thousand, which is easily absorbed. The prepared dressing film product can provide cells with the required collagen polypeptide, promote cell proliferation and differentiation, and achieve the effect of wound repair. Another example is the patent application with patent publication number CN112544778A which discloses a method for preparing a high-quality cartilage extract with high purity and good taste, and the application of the prepared cartilage extract. By using only one enzyme, the effect of using multiple enzymes in combination can be achieved, which not only improves the product quality, but also simplifies the production process, thereby improving both the product quality and the production efficiency. It can control impurities and effectively reduce the sodium content in the product, and significantly improves the color, taste and application areas of the cartilage extract.

[0005] At present, the relevant research on cartilage extracts mainly focuses on the extraction, separation and purification methods, and there is little research on the characteristics and quality of the raw materials themselves. Summary of the invention

[0006] The present invention starts from the raw materials, conducts in-depth research on the characteristics of the raw materials, and obtains a cartilage extract with a higher chondroitin sulfate A / C value.

[0007] The present invention focuses on the A / C value of cartilage extract. According to the research of the article "The Molecular Evolution of Cartilage", the content of chondroitin sulfate A in human body decreases year by year after the age of 1, and remains at a relatively low level after the age of 45; the content of chondroitin sulfate C in human body hardly changes with age. It can be seen that with the increase of age, it is necessary to select chicken breast cartilage of specific quality as raw material to obtain cartilage extract with higher chondroitin sulfate A content to make up for the problem of decreased chondroitin sulfate A content in human body.

[0008] Therefore, the present invention aims to provide a cartilage extract with a higher A / C value, so as to meet the human body's demand for chondroitin sulfate A.

[0009] To this end, the first aspect of the present invention provides a chicken breast cartilage extract, which uses chicken breast cartilage raised for 40 to 60 days as raw material, especially 45 to 60 days.

[0010] More preferably, the raw material is chicken breast cartilage of 40 to 60 days, especially 45 to 60 days, in captivity. In the chicken breast cartilage of this growth period, the chondroitin sulfate A / C ratio is up to 4.1.

[0011] The second aspect of the present invention provides a chicken sternum cartilage extract, using the chicken sternum cartilage of caged chickens as a raw material.

[0012] More preferably, the raw material is chicken breast cartilage of 40 to 60 days, especially 45 to 60 days, in captivity. In the chicken breast cartilage of this growth period, the chondroitin sulfate A / C ratio is up to 4.1.

[0013] The third aspect of the present invention provides a method for preparing a chicken sternum cartilage extract, using chicken sternum cartilage raised for 40 to 60 days as raw material, preferably, using chicken sternum cartilage raised for 45 to 60 days as raw material.

[0014] More preferably, chicken breast cartilage aged 40 to 60 days, especially 45 to 60 days, is used as the raw material.

[0015] The above preparation method specifically comprises the following steps:

[0016] S1. Take fresh chicken breast bones, pre-treat them, and rinse them with clean water;

[0017] S2. Boil the rinsed fresh chicken breast bones in water for more than 4 hours, then cool to 50℃ to 55℃ and adjust the pH to 6-7;

[0018] S3. Add protease to the chicken breast bone in step S2, perform sufficient enzymolysis, and after the enzymolysis is completed, heat up to inactivate the enzyme, filter to obtain a clear filtrate, remove ash by nanofiltration, concentrate, and perform vacuum freeze-drying to obtain a chicken breast cartilage extract.

[0019] More preferably, the protease is one or more of papain, bromelain, alkaline protease, keratinase and neutral protease.

[0020] More preferably, the added amount of the protease is 1% (w / v) 200,000 enzyme activity units, and the enzymolysis time is 4-7 hours.

[0021] Further preferably, the pretreatment includes the operation of removing residual muscle, fat and bloody red bone.

[0022] The fourth aspect of the present invention provides the use of the chicken sternal cartilage extract for the treatment and prevention of bone-related diseases such as arthritis and osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The trend curve of the content of sodium chondroitin sulfate in cartilage of caged and free-range chickens in different growth cycles;

[0024] Figure 2 The curve diagram of the change trend of the chondroitin sulfate A / C ratio in the cartilage of caged and free-range chickens in different growth cycles;

[0025] Figure 3 This is a curve chart showing the change trend of collagen in cartilage of caged and free-range chickens at different growth cycles;

[0026] Figure 4 It is a bar graph of mechanical pain values ​​of rats in different treatment groups;

[0027] Figure 5 This is a bar graph showing the results of measuring blood inflammatory factors in rats in different treatment groups;

[0028] Figure 6 HE staining images of joint tissues of mice in each group. DETAILED DESCRIPTION

[0029] The technical scheme of the present invention is further described in detail below through specific examples. It should be understood that the implementation of the present invention is not limited to the following examples, and any formal modifications and / or changes made to the present invention will fall within the scope of protection of the present invention. The parameter ranges involved include endpoint values ​​unless otherwise specified.

[0030] In the present invention, unless otherwise specified, all parts and percentages are by weight, and the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are all conventional methods in the art unless otherwise specified.

[0031] Example 1 Preparation of Chicken Sternum Cartilage Extract

[0032] 1.1 Raw material screening for preparation of chicken sternal cartilage extract

[0033] Free-range and caged white-feathered chickens of 30, 45, 60, 90, 120 and 150 days were respectively selected, fresh chicken breast bones were taken after slaughter, residual muscle, fat and blood-red bones were removed, the chickens were rinsed with clean water, crushed and then freeze-dried.

[0034] Confinement refers to raising chickens in cages or on flat ground in a factory, where the chickens have a small range of activity and are automatically fed at regular intervals.

[0035] Free-range refers to outdoor (mostly under forest) farming, where chickens have a large range of activities and can catch insects, fruits, etc. on their own in addition to feed.

[0036] 1.2 Preparation of chicken sternal cartilage extract

[0037] Place the chicken breast bone obtained in 1.1 in a beaker, add water to cover the cartilage, boil in a water bath for more than 4 hours, then cool to 55°C, adjust the pH to 6-7, add 1% (w / v) 200,000 enzyme activity units of papain, and fully enzymolyze for 6 hours. After the enzymolysis is completed, heat to 80°C to inactivate the enzyme, filter to obtain a clear filtrate, remove ash by nanofiltration, concentrate and vacuum freeze-dry to obtain a chicken breast cartilage extract.

[0038] 1.3 Determination of index components in chicken sternal cartilage extract

[0039] The content of chondroitin sulfate sodium and chondroitin sulfate A and C was determined using the content determination method under chondroitin sulfate sodium in the Chinese Pharmacopoeia (2020 edition); the collagen content was determined using the GB / T 9695.23 hydroxyproline determination method, with a conversion factor of 8, that is, collagen content (%) = hydroxyproline content (%) × 8. The results are shown in Table 1.

[0040]

[0041] The results in Table 1 show that in the process of preparing chicken sternal cartilage extract using chicken sternal cartilage as raw material, the content of sodium chondroitin sulfate and the ratio of its isomers in the prepared cartilage extract are different due to the different growth time and feeding methods of the chickens: (1) The content of sodium chondroitin sulfate in chicken sternal cartilage of free-range chickens and caged chickens is similar during the same growth period, but the collagen content of caged chicken sternal cartilage is higher and the chondroitin sulfate A / C ratio is higher; (2) Under the same feeding method, as the chickens grow, the content of chondroitin sulfate A, the isomer of sodium chondroitin sulfate in chicken sternal cartilage, increases first and then decreases, while the content of chondroitin sulfate C remains almost unchanged. At about 45 days of feeding, the chondroitin sulfate A / C values ​​in chickens of different feeding methods all reach the highest, with the chondroitin sulfate A / C value of caged chickens reaching the highest 4.1 and the chondroitin sulfate A / C value of free-range chickens reaching the highest 3.6.

[0042] Example 2 Study on the efficacy of chicken sternal cartilage extract

[0043] 2.1 Experimental modeling and grouping

[0044] Male SPF Wistar rats, weighing 180-220 g, 8 rats in each group, were divided into normal control group (A), OA model group (B), low-dose chicken sternal cartilage extract group (C), and high-dose chicken sternal cartilage extract group (D). Rats in group A were injected with 50 μL of normal saline in the knee joint cavity. After modeling, groups C and D were gavaged with chicken sternal cartilage extract (low dose 45 mg / kg / day, high dose 180 mg / kg / day), and groups A and B were gavaged with the same volume of normal saline once a day for 5 consecutive weeks. Among them, chicken sternal cartilage extract was prepared from white-feathered chicken sternal cartilage from chickens raised in a cage and grown for 45 days. Plantar mechanical pain was measured every week during the experiment. After the experiment, blood was collected from the heart to analyze the levels of IL-6 and TNF-α in serum. At the same time, the knee joints of the right hind limbs of rats in each group were fixed with 4% paraformaldehyde and decalcified for pathological examination (HE staining).

[0045] 2.2 Experimental testing indicators

[0046] 2.2.1 Mechanical pain index detection

[0047] The French bioseb Bio-EVF5 electronic mechanical analgesia instrument was used to measure the mechanical pain threshold before modeling (0th day) and on the 7th, 14th, 21st, 28th and 35th days after modeling, and the differences in mechanical pain among the rats in each group were observed.

[0048] 2.2.2 Blood index examination

[0049] After the experiment, blood was collected from the heart, and the levels of IL-6 and TNF-α in serum were detected using ELISA kits to observe the changes in blood inflammatory factors in rats in each group.

[0050] 2.2.3 Pathological examination

[0051]

[0052] 2.3 Statistical analysis

[0053] All data were analyzed by ANOVA using SPSS software (version number). The mechanical pain values ​​and blood-related factors of the four groups of rats were expressed as mean ± standard deviation (x ± s). The overall comparison between the groups was performed by one-way analysis of variance, and the pairwise comparison between the groups was performed by LSD-t test. P < 0.05 was considered statistically significant.

[0054] 2.4 Results of the study on the efficacy of chicken sternal cartilage extract

[0055] 2.4.1 Mechanical pain results of rats in each group

[0056] See also Figure 4 And as shown in Table 3 below.

[0057]

[0058] The results showed that from the first week to the fifth week after modeling, the mechanical pain values ​​of rats in the OA model group were significantly decreased compared with the normal control group (P < 0.05). From the first week to the third week, there was no significant difference in the mechanical pain values ​​of rats in the high-dose and low-dose groups of chicken sternal cartilage extract compared with the OA model group. At the fourth week, the mechanical pain values ​​of rats in the high-dose and low-dose groups of chicken sternal cartilage extract were significantly increased compared with the OA model group (P < 0.05). Compared with week 0, the mechanical pain values ​​of rats in the high-dose and low-dose groups of chicken sternal cartilage extract decreased at the first week after modeling, and the mechanical pain values ​​showed an overall upward trend in the 2nd, 3rd, 4th, and 5th weeks. The mechanical pain value of the high-dose group of chicken sternal cartilage extract was close to that of the normal control group at the 5th week, but there was still a significant difference (P < 0.05).

[0059] 2.4.2 Results of blood inflammatory factors in rats of each group

[0060] Results Figure 5 And as shown in Table 4 below.

[0061]

[0062] Compared with the normal control group, the IL-6 and TNF-α levels in the blood of the OA model group increased significantly (p<0.05). Compared with the OA model group, the IL-6 and TNF-α levels in the high and low dose groups of chicken sternal cartilage extract decreased significantly (p<0.05), indicating that chicken sternal cartilage extract has a good anti-inflammatory effect.

[0063] 2.4.3 Pathological results of rats in each group

[0064] HE staining was performed on the joint tissues of mice in each treatment group. Figure 6 .

[0065] according to Figure 6 show:

[0066] Normal control group (A): The joint structure is normal, the joint surface is covered with hyaline cartilage and the surface is smooth. The joint cavity is normal, the synovial surface is smooth, and there is no abnormality in the joint capsule and surrounding fibrous tissue. The bone tissue structure under the joint surface is normal. The chondrocytes and matrix staining are normal, and the tide line is complete and clearly visible.

[0067] OA model group (B): The hyaline cartilage covering the surface of the bone joint became thinner, local chondrocytes degenerated and necrotized, and the number of chondrocytes decreased; hyperplastic fibrous tissue was seen on the surface of the articular cartilage, the joint cavity narrowed, and local joint surface adhesions occurred. The synovial tissue was congested and thickened, local synovial cells degenerated, necrotized, and disappeared, and fibroblasts proliferated around the synovial cells. The bone tissue below the local bone joint was affected, and the trabeculae were broken. The chondrocytes and matrix were lightly stained, the integrity of the tide line was destroyed, and the Mankin's score was significantly higher than that of the normal control group (p<0.05).

[0068] Low-dose group (C): The hyaline cartilage covering the surface of the bone joint became thinner, local chondrocytes proliferated, and the number of chondrocytes increased; hyperplastic fibrous tissue was visible on the surface of the articular cartilage. The synovial tissue was congested and thickened, and fibroblasts proliferated around the synovial cells. The staining of chondrocytes and cartilage matrix decreased, and the integrity of the tide line was destroyed. The Mankin's score was significantly lower than that of the OA model group (p<0.05).

[0069] High-dose group (D): The hyaline cartilage covering the local bone joint surface became thinner and the surface was not smooth. Most samples had chondrocyte hyperplasia and the number of chondrocytes increased. The local synovial tissue was congested and thickened. The bone tissue below the bone joint was normal. The staining of chondrocytes and cartilage matrix decreased, and the integrity of the tide line was destroyed. The Mankin's score was significantly lower than that of the OA model group (p<0.05).

[0070]

[0071] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a chicken breast cartilage extract, characterized in that: The specific steps include the following: S1. Take fresh chicken breast bones from 45 to 60-day-old chickens, pre-treat them, and rinse them with clean water; S2. Boil the rinsed fresh chicken breast bones in water for more than 4 hours, then cool to 50℃ to 55℃ and adjust the pH to 6-7; S3. Add protease to the chicken sternum of step S2, perform sufficient enzymolysis, heat up to inactivate the enzyme after the enzymolysis is completed, filter to obtain a clear filtrate, remove ash by nanofiltration, concentrate and perform vacuum freeze-drying to obtain a chicken sternal cartilage extract, wherein the chondroitin sulfate A / C ratio in the chicken sternal cartilage extract is as high as 4.1.

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