CN114766553A

CN114766553A - Sturgeon and dragon's tendon chewable tablet and preparation method thereof

Info

Publication number: CN114766553A
Application number: CN202210480605.XA
Authority: CN
Inventors: 赵元晖; 韩贵新; 汪金林; 薛长湖; 陈泽凡; 刘康; 徐新星; 高瑞昌; 白帆; 许贺; 黄瑜晴; 徐鹏
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2022-05-05
Filing date: 2022-05-05
Publication date: 2022-07-22

Abstract

The invention discloses sturgeon and dragon tendon chewable tablets, which belong to the technical field of aquatic product processing and comprise the following components: 10 parts of sturgeon longjin powder, 0 part or 45-60 parts of milk powder, 0 part or 30-60 parts of coconut powder and 0 part or 1-5 parts of magnesium stearate; the sturgeon longjin powder is prepared by the following method: drying sturgeon dragon bones at 105-110 ℃ for 60-110 min, adding into water, treating at 121 ℃ for 15-25 min, filtering, and spray-drying to obtain the sturgeon dragon bones. The preparation method of the sturgeon and dragon tendon chewable tablet comprises the following steps: mixing sturgeon longjin powder, milk powder and coconut powder, adding ethanol solution to obtain wet mixture, sieving, granulating, drying, adding magnesium stearate, and tabletting. The sturgeon and dragon tendon chewable tablet has the effects of relieving joint inflammation pain and promoting osteoblast differentiation, and can be eaten as a health-care product with the effects of relieving joint inflammation pain and promoting osteoblast differentiation.

Description

Sturgeon and dragon's tendon chewable tablet and preparation method thereof

Technical Field

The invention relates to sturgeon and dragon bone chewable tablets and a preparation method thereof, belonging to the technical field of aquatic product processing.

Background

Sturgeons, also known as sturgeons, have extremely high economic and scientific research values and are of various types including siberian sturgeons, dabigatran sturgeons, acipenser schrencki, chinese sturgeons and the like, and the more common edible sturgeons in the market are hybrid sturgeons. Sturgeon keel is the spinal cord of sturgeon, is glittering and translucent, has strong flexibility and enough elasticity, and is named as keel. The sturgeon longjin has low fat content, contains a large amount of colloid and protein, also contains various nutritional factors, has the effects of enriching blood, tonifying qi, strengthening kidney, stimulating appetite, clearing heat, detoxicating, nourishing yin, beautifying, improving eyesight, clearing away heart-fire, moistening dryness, regulating lung and the like, is very effective in preventing osteoporosis and enhancing organism immunity, is a superexcellent tonic, can be comparable with ginseng and pilose antler, and is a traditional delicious food in China.

At present, the sturgeon breeding industry in China is more prosperous, but the main processing direction of sturgeons is still a caviar product, the utilization rate of sturgeon processing waste is low, and most sturgeons are processed into feed with low added value or directly treated as garbage. The sturgeon longjin is generally used as a food raw material to be directly stewed in dishes, which is not beneficial to the development and utilization of sturgeon longjin products. The Chinese patent application of CN 108813427A discloses a method for making a dried sturgeon keel, which is to dry the sturgeon keel at the temperature of 40-80 ℃, the humidity of 0-40% and the wind speed of 8-25 m/s, so that the hardness, the elasticity, the chewiness and the resilience of the sturgeon keel are kept while the moisture content and the water activity of the sturgeon keel are reduced, and the prepared dried sturgeon keel has delicious taste, good mouthfeel, safety and health after being soaked in water; however, the sturgeon keel is processed in a low-temperature (lower than the boiling point of water) mode, so that the eating mode of the sturgeon keel is not changed, the flavor of the sturgeon keel is not improved, and a new product form is not formed.

Osteoporosis (OP) is one of the common chronic diseases, old people and postmenopausal women are high-incidence people with Osteoporosis, and old people with older age are more prone to Osteoporosis fracture when falling down; the growth of age and loss of bone mass also contribute to the development of degenerative joint disease Osteoarthritis (OA). With the increasing aging trend of the population, bone diseases such as osteoporosis and the like seriously affect the health and life of people.

The traditional ideas of homology of medicine and food, homology of medicine and food and use of medicine and food have a long history in China, and food therapy is one of the effective disease prevention methods for China since ancient times. The chewable tablet is a common oral tablet, can be used as a medicament and a health-care product, has increased surface area after being chewed, is convenient for the tablet to be quickly dissolved and absorbed, can reduce the burden of the medicament on the gastrointestinal tract, and is particularly suitable for patients with poor gastrointestinal function.

Disclosure of Invention

Aiming at the prior art, the invention provides sturgeon and dragon bone chewable tablets, a preparation method thereof and application thereof in preparing health-care products with the effects of relieving joint inflammation pain and promoting osteoblast differentiation.

The invention is realized by the following technical scheme:

a sturgeon and dragon bone chewable tablet comprises the following components: 10 parts of sturgeon longjin powder, 0 part or 45-60 parts of milk powder, 0 part or 30-60 parts of coconut powder and 0 part or 1-5 parts of magnesium stearate.

The sturgeon keel powder is prepared by the following method: drying sturgeon longjin at 105-110 ℃ for 60-110 min, then adding water, treating at 121 ℃ for 15-25 min to obtain sturgeon longjin liquid, filtering to obtain filtrate, and performing spray drying to obtain sturgeon longjin powder.

Further, the sturgeon keel powder is prepared by the following method: taking sturgeon keel, cleaning, and drying at 105-110 ℃ for 60-110 min, wherein the sturgeon keel is golden yellow and has a strong scorched aroma; cutting sturgeon longjin into small pieces, adding water (the feed liquid ratio is 1: 8-12), treating for 15-25 min at 103.43 kPa and 121 ℃ to obtain sturgeon longjin liquid, standing to normal temperature, filtering to obtain filtrate, spray drying to obtain sturgeon longjin powder, and sieving with a 60-mesh sieve.

The milk powder is selected from whole milk powder; the whole milk powder is a commercial product in the prior art, and is a commercial product in the prior art.

The coconut powder is a commercial product in the prior art.

Further, the sturgeon and dragon bone chewable tablet comprises the following components: 10 parts of sturgeon longjin powder, 45-60 parts of milk powder, 30-60 parts of coconut powder and 1-5 parts of magnesium stearate.

Further, the composition comprises the following components: 10 parts of sturgeon longjin powder, 60 parts of milk powder, 30 parts of coconut powder and 3 parts of magnesium stearate.

The preparation method of the sturgeon and dragon bone chewable tablet comprises the following steps:

(1) mixing and granulating: mixing sturgeon longjin powder, milk powder and coconut powder, adding appropriate amount of ethanol solution to obtain wet mixture, sieving the wet mixture, granulating, and drying to obtain granules;

(2) tabletting: adding magnesium stearate into the granules, mixing, tabletting by a tabletting machine to obtain the sturgeon and dragon tendon chewable tablet, sterilizing by ultraviolet irradiation, and packaging.

Further, the volume concentration of the ethanol solution is 70-90%.

Further, the drying conditions are as follows: drying for 1-2 hours at 45-65 ℃.

The sturgeon and dragon tendon chewable tablet is applied to the preparation of health care products with the effects of relieving joint inflammation and pain or promoting osteoblast differentiation.

The sturgeon keel powder prepared by the method is applied to preparing sturgeon keel chewable tablets and health-care products with the effects of relieving joint inflammation pain or promoting osteoblast differentiation.

The sturgeon-dragon tendon chewable tablet mainly comprises milk powder and sturgeon-dragon tendon powder, has the effects of relieving joint inflammation pain and promoting osteoblast differentiation, and can be eaten as a health-care product with the effects of relieving joint inflammation pain and promoting osteoblast differentiation. The coconut powder is used for seasoning, so that the coconut-flavored bone-strengthening milk is a bone-strengthening food which is suitable for people of all ages and has good flavor, and can partially replace milk powder, milk tablets and other forms of milk powder and milk tablets eaten by teenagers of 5-15 years old.

The sturgeon longeron is dried at 105-110 ℃ for 60-110 min, so that moisture in the sturgeon longeron can be removed, the sturgeon longeron is endowed with unique flavor (scorched aroma), seasonings (soy sauce, cassia bark, green Chinese onion, galangal, aniseed, fennel, pepper and the like) are not required to be added, and the sturgeon longeron is soft in texture. Compared with the traditional charcoal baking (the temperature is over 600 ℃) and high-temperature baking (the temperature is over 200 ℃), the temperature is lower, active peptides and other ingredients in sturgeon longjin are not damaged by high temperature (the chewable tablet of the invention still has obvious promotion effect on proliferation and differentiation of osteoblasts after passing through a digestive system in vivo), and can not be gelatinized to generate fishy smell and stink. Compared with low-temperature drying (below 100 ℃), the low-temperature drying cannot remove the internal bound water and cannot enable the sturgeon keel to generate unique flavor.

The sturgeon longjin is treated in a high-temperature drying mode, is spray-dried (the grain size of the directly crushed longjin is large, the taste is poor), and is prepared into a chewable tablet form, so that the form of sturgeon longjin products is enriched, and the sturgeon longjin which is a traditional precious food material can be eaten by common people. In addition, the sturgeon longjin chewable tablet has good health care efficacy, can inhibit arthritis, relieve pain, maintain and protect knee joint cartilage, and has obvious promotion effect on the proliferation and differentiation of osteoblasts. The invention has important significance for developing and utilizing sturgeon keel products.

Drawings

FIG. 1: a schematic diagram for measuring basic components of sturgeon longjin.

FIG. 2 is a schematic diagram: schematic diagram for measuring basic components of sturgeon fishbone.

FIG. 3: schematic diagram of influence of drying time on sturgeon keel.

FIG. 4 is a schematic view of: schematic diagram of influence of drying time on sturgeon fishbone.

FIG. 5 is a schematic view of: schematic diagram of influence of drying temperature and time on sturgeon keel.

FIG. 6: schematic representation of the effect of magnesium stearate addition on chewable tablet hardness.

FIG. 7: sensory scoring results for the different samples are shown schematically.

FIG. 8: schematic representation of the effect of different concentrations of samples on the proliferative activity of MC3T3-E1 cells.

FIG. 9: light microscopy analysis of MC3T3-E1 cells is shown.

FIG. 10: AKP activity in MC3T3-E1 cells is shown.

FIG. 11: VG staining of knee joint sections of rats in each experimental group at week 4.

FIG. 12: VG staining of knee sections of rats in each experimental group at week 12.

FIG. 13: mean fluorescence intensity of NGF is shown.

FIG. 14: mean fluorescence intensity of SP.

Detailed Description

The present invention will be further described with reference to the following examples. However, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the invention.

The instruments, reagents and materials used in the following examples are conventional instruments, reagents and materials known in the art and are commercially available. Unless otherwise specified, the experimental methods and the detection methods in the following examples are conventional experimental methods and detection methods in the prior art.

The whole milk powder used in the invention is purchased from imported New Zealand Saka, and the product number is 16371792258. The coconut powder used in the present invention is coconut powder purchased from Hainan Chunguo food Co., Ltd, and its production license is SC 11346900500128.

Experiment 1: determination of basic ingredients of sturgeon keel and sturgeon fishbone

The experimental method comprises the following steps: the sturgeon keel and sturgeon fishbone are washed by deionized water to remove surface impurities for later use, the water content in the sample is measured by adopting a method in GB 5009.3-2016 food water content measurement, the ash content in the sample is measured by adopting a method in GB 5009.4-2016 food ash content measurement, the protein content in the sample is measured by adopting a method in GB 5009.5-2016 food protein measurement, the fat content in the sample is measured by adopting a method in GB 5009.6-2016 food fatty acid measurement, and the total sugar content in the sample is measured by referring to a method in GB/T9695.31 meat product total sugar content measurement. The results are shown in fig. 1 and fig. 2, the moisture in the ingredients of the sturgeon keel and the sturgeon bone occupies most of the ingredients, so that the removal of the moisture in the two ingredients during the processing is one of the key control points for improving the product quality; the data show that although the sturgeon longjin and the sturgeon bones jointly form a spine of the sturgeon, the main component content of the sturgeon longjin and the sturgeon bones has larger difference, the bones contain more saccharides, and the saccharides in the longjin only occupy a small part.

Experiment 2: influence of different drying temperatures and time on sturgeon keel and fishbone

The experimental method comprises the following steps: washing sturgeon keel and sturgeon bone with deionized water to remove surface impurities, cutting into certain length, and oven drying at different temperatures for 120 min. Oven drying sturgeon keel of certain length at 110 deg.C for 0 min, 30 min, 60 min, 90 min, 120 min, 150 min, 180 min, observing, taking pictures, and recording the drying state.

As shown in fig. 3 and 4, when sturgeon cartilage and sturgeon bone are dried at different temperatures, the length of the sturgeon cartilage and sturgeon bone shrink to different degrees due to moisture loss, the two sturgeon cartilage and sturgeon bone have significant differences in color between above 105 ℃ and below 100 ℃, the two sturgeon cartilage and sturgeon bone begin to yellow when above 105 ℃, and the two flavors change with the change of color, the sturgeon bone can smell fishy smell and stink significantly when above 105 ℃, which may be related to the composition of sturgeon bone and the oxidation of fat, and the bone after drying is hard, forms a firm shell, and is not beneficial to processing and utilization. The sturgeon longeron presents a burnt flavor with good flavor, which may cause the release of flavor-developing factors in the sturgeon longeron due to high temperature, and the protein in the sturgeon longeron occupies most of dry substances, and does not generate complex reaction similar to fishbone, thereby having no fishy smell and stink; and the dried sturgeon keel has soft texture, is not as hard as sturgeon bones, has good edible mouthfeel, and is convenient to process and utilize.

Taking 110 ℃ as an example, the influence of the drying time on the product quality is further researched, and the result is shown in fig. 5, as the drying time increases, the length of the sturgeon keel is obviously reduced, the change of the length of the sturgeon keel is large before the sturgeon keel is dried at 110 ℃ for 60 min, and the change of the length of the sturgeon keel after the sturgeon keel is 60 min, which indicates that most of the moisture is evaporated before the sturgeon keel is 60 min. Meanwhile, the color of the sturgeon longeron is also changed from white at the beginning to yellow, but the sturgeon longeron is blackish when being dried for 120 min, and the blackish becomes more serious along with the time, and the blackish is probably caused by the carbonization of protein in the longeron due to excessive high temperature, so that the generation of black is caused. Therefore, the drying temperature and time of the dragon tendons are finally determined to be 105-110 ℃ for drying for 60-110 min, and the optimal temperature and time are as follows: oven drying at 110 deg.C for 90 min.

Experiment 3: amino acid composition of sturgeon dragon tendon

The experimental method comprises the following steps: referring to GB5009.124-2016, an amino acid analyzer is used to determine the content and distribution of amino acids in sturgeon keel. Calculating an Essential Amino Acid Index (EAAI) according to an FAO/WHO recommended amino acid scoring standard mode and a chemical scoring benchmark of the whole egg protein, wherein the formula is as follows:

the measurement results are shown in table 1.

TABLE 1 amino acid composition of sturgeon Triticum, different letters in each column indicate significant differences between groups (P < 0.05)

Name(s)	Content (mg/g)	Name (R)	Content (mg/g)	Name(s)	Content (mg/g)
						Asp	7.55±0.14^d	Glu	12.72±1.6^b	Cys	0.00±0.00^g
Thr	2.92±0.36^f	Gly	16.55±2.03^a	Val	2.52±0.34^f
						Ser	3.65±0.43^f	Ala	5.62±0.73^e	Met	1.26±0.17^g
Ile	1.87±0.51^g	Tyr	0.92±0.81^g	Lys	3.13±0.43^f
						Leu	5.52±1.16^e	Phe	1.87±0.54^g	His	1.39±0.18^g
Arg	6.79±1.04d^e	Pro	9.01±0.92^c

As can be seen from table 1, the sturgeon longjin contains 16 of 20 amino acids (tryptophan cannot be detected by this method), includes 8 amino acids essential to human body, the index score of the essential amino acid is 41.25, which is a high-quality protein source, the most abundant amino acid is Gly, which is related to collagen on the surface of sturgeon longjin, and the more Glu, which is a known umami amino acid, is a direct source of special umami presented when the sturgeon longjin is stewed, and it is presumed that Glu is one of the factors that the high-temperature dried longjin presents scorched aroma.

Experiment 4: influence of magnesium stearate addition on sturgeon and dragon bone chewable tablets

Preparing sturgeon longjin powder: taking sturgeon dragon bones, cleaning, and drying for 90 min at 110 ℃; cutting sturgeon longjin into small pieces, adding water at a feed-liquid ratio of 1:10, treating at 103.43 kPa and 121 ℃ for 20 min to obtain sturgeon longjin liquid, standing to normal temperature, filtering to obtain filtrate, spray drying to obtain sturgeon longjin powder, and sieving with a 60-mesh sieve.

Mixing the whole milk powder, the coconut powder and the sturgeon longjin powder (the weight ratio of the whole milk powder, the coconut powder and the sturgeon longjin powder is 6:3: 1) uniformly, slowly adding an ethanol solution with the concentration of 90% into the mixed material, continuously stirring by hands until the mixture can be slightly kneaded into a ball, stopping stirring to obtain a wet mixed material, sieving and granulating to obtain a 60-mesh wet mixed material, drying for 2 hours at the temperature of 55 ℃, sieving with a 100-mesh sieve first, and then sieving with a 200-mesh sieve to obtain 200-mesh granules. Adding magnesium stearate with different contents into the 200-mesh granules, specifically: 0%, 1%, 2%, 3%, 4%, 5%; after tableting and molding, the hardness was measured using a texture analyzer, and as a result, as shown in fig. 6, when the addition amount of magnesium stearate was 1% or less, the hardness was significantly lower than the high addition amount (P < 0.05), and tableting was substantially not molded, and it was difficult to maintain the tablet form even after molding; the appearance, glossiness and hardness of the chewable tablet are gradually increased along with the increase of the addition amount of the magnesium stearate, when the addition amount exceeds 4%, the hardness of the chewable tablet is not obviously changed (P is more than 0.05), the difference between the appearance and glossiness of the chewable tablet and the addition amount of 3% is not large, and the addition amount of the magnesium stearate is 3% which is considered to be an optimal proportion.

Experiment 5: influence of milk powder and coconut powder addition ratio on sturgeon and dragon tendon chewable tablets

The experimental method comprises the following steps: uniformly mixing sturgeon longjin powder (the preparation method is same as experiment 4), whole milk powder and coconut powder according to a certain proportion (the specific proportion is detailed in table 2), slowly adding 90% ethanol solution into the mixed material, continuously stirring by hands until the mixture can be slightly kneaded into a mass, stopping to obtain a wet mixed material, sieving and granulating to obtain a 60-mesh wet mixed material, drying for 2 hours at 55 ℃, sieving by a 100-mesh sieve, and then sieving by a 200-mesh sieve to obtain 200-mesh granules; adding 3% of magnesium stearate, uniformly mixing, tabletting by a tablet machine, sterilizing by ultraviolet irradiation, and packaging to obtain the sturgeon and dragon tendon chewable tablet product.

20 trained sensory evaluators were invited to perform sensory evaluation of the products in the food sensory evaluation room and sensory scores were recorded, and after removing the highest score and the lowest score, the average was taken as a sensory score result, and the sensory score detailed is shown in table 3, and the result is shown in fig. 7. As can be seen from fig. 7, when the milk powder is added in an amount of 60% (based on 100% of the total weight of the whole milk powder, the coconut powder and the sturgeon keel powder) and the coconut powder is added in an amount of 30%, the score is the highest, and the score of sample 4 reaches 96.

TABLE 2

	Whole milk powder (gram)	Coconut powder (gram)	Dragon tendon powder (gram)	Magnesium stearate
					Sample
1	60	30	10	0%
					Sample
2	60	30	10	1%
					Sample 3	60	30	10	2%
Sample No. 4	60	30	10	3%
					Sample No. 5	60	30	10	4%
Sample No. 6	60	30	10	5%
					Sample 7	70	20	10	3%
Sample
	8	65	35	10	3%
Sample 9						55	35	10	3%
	Sample
10		50	40	10	3%
	Sample 11					45	45	10	3%

TABLE 3 sensory rules of Scoring

Experiment 6: in vitro experiment of sturgeon keel simulated digestion product

6.1 simulated digestion

The experimental method comprises the following steps: taking 10 g of a sample (the sample is respectively the sturgeon keel chewable tablet sample 4 prepared in example 5 and the sturgeon bone chewable tablet) (the preparation method of the sturgeon bone chewable tablet is the same as the sample 4 prepared in example 5, except that sturgeon bone powder is used for replacing sturgeon keel powder) as an example, the sample is added into 40 mL of simulated gastric juice electrolyte solution, pepsin 8000U is added, hydrochloric acid is added to adjust the pH value to 2.0, the reaction is carried out for 2 h at 37 ℃, and sodium hydroxide solution is used for adjusting the pH value to 7.0 to terminate the reaction. Then adding the mixture into 40 mL of simulated intestinal juice electrolyte solution, adding 4000U of trypsin and 800U of lipase, reacting for 2 h at 37 ℃, inactivating for 10 min in a 100 ℃ boiling water bath, standing to normal temperature, and freeze-drying to obtain a simulated digested fishbone sample and a dragon bone sample. The compositions of the simulated gastric electrolyte solution and the simulated intestinal electrolyte solution are shown in table 4.

The preparation method of the sturgeon fishbone powder comprises the following steps: decocting sturgeon skull and spine at 100 ℃ for 10 min, taking out, cleaning, cutting into small pieces, adding purified water according to a material-liquid ratio of 1:5, adding compound protease (composed of papain and lactalbumin at a weight ratio of 1: 3) with the mass of 2% of the sturgeon bone, adjusting pH to 7.0, and performing enzymolysis at 55 ℃ for 3 h to obtain enzymolysis liquid; and then treating the enzymolysis liquid at 103.43 kPa and 121 ℃ for 20 min to obtain sturgeon bone liquid, standing to normal temperature, filtering to remove impurities to obtain clear filtrate, spray-drying to obtain sturgeon bone powder, and sieving with a 60-mesh sieve.

TABLE 4 simulated gastric electrolyte solution and simulated intestinal electrolyte solution formulations

Name of medicine	Concentration of drug/mol	Simulated gastric juice electrolyte	Simulated intestinal fluid electrolyte
				Potassium chloride	0.5M	16.09%	8.84%
Dipotassium hydrogen phosphate	0.5M	2.10%	1.04%
				Sodium bicarbonate	1M	29.15%	55.25%
Magnesium chloride	0.15M	0.93%	1.43%
				Ammonium bicarbonate	0.5M	1.17%	0.00%
Calcium chloride	0.3M	0.01%	0.05%
				Hydrochloric acid	6M	3.03%	0.91%
Sodium hydroxide (NaOH)	2M	27.52%	12.48%

6.2 cell culture

The experimental method comprises the following steps: MC3T3-E1 cells (mouse embryonic osteoblast precursor cells) were cultured in T cells containing 5mL of complete alpha-MEM medium (containing 10% fetal bovine serum and 1% penicillin, streptomycin antibodies)₂₅Placing in a cell bottle at 37 deg.C and 5% CO₂The culture medium was changed every 2 days. After the cells grow to T₂₅The cells were passaged at 90% of the culture flask, the original medium in the flask was aspirated at the time of passaging, gently washed with 2 mL of sterile PBS (1X), the PBS was aspirated, treated with pancreatin (containing 0.25% of EDTA) at 37 ℃ for 3 min, neutralized with 2 mL of complete alpha-MEM medium to terminate the reaction, centrifuged at 1000 g for 5min, the supernatant was discarded, the cells were resuspended in 1 mL of complete alpha-MEM medium, and after gently homogenized, 0.5 mL of the supernatant was added to a new complete alpha-MEM medium T containing 4.5 mL of complete alpha-MEM medium₂₅Placing the cell culture flask in an incubator to continue culture (37 ℃, 5% CO)₂）。

6.3 MTT assay for the Effect of samples on MC3T3-E1 cells

Selecting MC3T3-E1 cells in 5-20 generations, when the cells are cultured to 90% of the cell bottles, digesting and centrifuging by pancreatin, gently blowing 1 mL of cell suspension uniformly, counting the number of the cells in the cell suspension by using a blood counting plate, and adjusting the concentration of the cell suspension to be 1 x 10⁵one/mL, 100. mu.L per well was inoculated into a 96-well plate and cultured for 24 h, the original medium in the well was aspirated, washed 2 times with sterile PBS, and finally 100. mu.L of a sample (meaning a fish after simulated digestion) was addedBone samples, tendon samples) of 0. mu.g/mL, 25. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL, 200. mu.g/mL, 400. mu.g/mL, respectively, and cultured in an incubator for 20 hours (37 ℃, 5% CO)₂) Adding 10 mu L of MTT with the concentration of 5 mg/mL into each hole, and incubating for 4 h at 37 ℃; after incubation, the well medium was aspirated, 150. mu.L of DMSO was added, the absorbance A was measured at 490nm after shaking the plate for 15 min, and the cell proliferation activity was calculated according to the following formula (wherein A is_{Blank space}Absorbance at sample concentration of 0. mu.g/mL): cell proliferation activity = (a)_{Sample (I)}-A_{Blank space}）/（A_Control-A_{Blank space}). The results are shown in FIG. 8.

As can be seen from fig. 8, the simulated digestion products of the fishbone component and the keel component both have a promoting effect on the proliferation of MC3T3-E1 cells, and the proliferation promotion gradually increases with the increase of the concentration, but when the concentration exceeds 100 μ g/mL, the proliferation promoting effect is reduced compared with the prior art, but the proliferation promoting effects of the two simulated digestion products tend to be stable, the proliferation promoting effect of the keel simulated digestion product on MC3T3-E1 cells reaches 18.28 ± 2.52% at the maximum when the concentration exceeds 100 μ g/mL, at the concentration, the proliferation promoting effect of the keel simulated digestion product is obviously higher than that of the fishbone simulated digestion group (P < 0.05), and under the condition of in-vitro simulated digestion, the proliferation promoting effect of sturgeon MC3T3-E1 cells of the sturgeon the fishbone is better than that of the sturgeon.

6.4 light microscopy analysis of MC3T3-E1 cells

Selecting MC3T3-E1 cells in 5-20 generations, when the cells are cultured to 90% of a cell bottle, digesting and centrifuging by using pancreatin, then lightly blowing 1 mL of cell suspension uniformly, counting the number of the cells in the cell suspension by using a blood counting plate, and adjusting the concentration of the cell suspension to be 1 x 10⁵The seed/mL is inoculated into a 6-well plate in an amount of 2500 mu L per well and cultured for 24 h; the original medium in the wells was aspirated, gently washed with sterile PBS 2 times, added to 2500. mu.L of a medium containing a sample (the sample was dissolved in a medium containing no fetal calf serum) at a concentration of 100. mu.g/mL (while the medium containing no sample was used as a blank), and cultured in an incubator for 48 hours (37 ℃, 5% CO)₂) The images were recorded at 0 h, 12 h, 24 h, and 48 h by taking pictures at the same positions using a common light microscope, and the results are shown in FIG. 9.

As can be seen from FIG. 9, both samples had a promoting effect on the proliferation of MC3T3-E1 cells at different time periods, which is consistent with the results of the above MTT assay. The largest effect of the digestion product of the dragon tendon analogue on the proliferation promotion of the MC3T3-E1 cells was observed visually at 12 h, and the number of the cells was much higher than that of the other two groups at 24 h and 48 h. The fish bone simulated digestion product has no obvious proliferation promoting effect on MC3T3-E1 cells at 12 h, but also has a certain proliferation promoting effect at 48 h.

6.5 determination of alkaline phosphatase Activity (AKP) in MC3T3-E1 cells

Selecting MC3T3-E1 cells in 5-20 generations, when the cells are cultured to 90% of the cell bottles, digesting and centrifuging by pancreatin, gently blowing 1 mL of cell suspension uniformly, counting the number of the cells in the cell suspension by using a blood counting plate, and adjusting the concentration of the cell suspension to be 1 x 10⁵The seed/mL is inoculated into a 6-well plate in an amount of 2500 mu L per well and cultured for 24 h; the original culture medium in the well was aspirated, gently washed 2 times with sterile PBS, 2500. mu.L of a sample-containing culture medium with a concentration of 100. mu.g/mL (the sample was dissolved in a culture medium without fetal bovine serum), incubated for 24 hours, aspirated, gently washed 2 times with sterile PBS, 1 mL of sterile PBS was added, the cells in the well plate were collected with a cell scraper, centrifuged at 1000 g for 5min, the supernatant was discarded, 200. mu.L of lysate was added, lysed on ice for 30 min, and the AKP activity in the cells was measured as described in the alkaline phosphatase assay kit (Nanjing institute) with the results shown in FIG. 10.

Alkaline phosphatase is one of the representative markers of osteoblast differentiation, and it can directly reflect the differentiation of cells into osteoblasts, and as can be seen from fig. 10, both samples have a promoting effect on osteoblast differentiation, and the dragon's tendon-simulated digestion products have a significant effect on osteoblast differentiation promotion (P < 0.05).

Combining the above in vitro experiments, it can be concluded that: after the sturgeon keel and the sturgeon keel are subjected to in-vitro simulated digestion, the growth and differentiation of osteoblasts are obviously promoted.

Experiment 7: in vivo test for relieving osteoarthritis pain of sturgeon longjin chewable tablets

7.1 animal culture

The animals used in the experiment were female SD rats (150 + -5 g, 8 weeks old) purchased from Jinan Pengyue laboratory animal Breeding Co., Ltd, and all procedures were performed according to the guidelines provided by the ethical Committee of laboratory animals of the university of oceanic (Qingdao) in China. The rat feed used in the experiment was a rod-like AIN-93G basal feed provided by nantong troffe fodder science and technology limited. The experimental mouse drinking water is deionized water. Animal feeding conditions: SD rats are separately fed in cages, the lighting of an animal room is maintained in a light/dark cycle for 12 hours, the temperature is constant at 22 +/-1 ℃, the humidity is 50% +/-5%, the ventilation is good, the air exchange is 20 times per hour, and the animal room is periodically sterilized. The animal cage box and the rat drinking water bottle used in the experiment are both made of stainless steel and polycarbonate plastic, and the sterilized corncob padding is used, and the padding is replaced every 2 days. After adaptive feeding for 1 week, 48 SD rats were randomly divided into 8 rats per group, a blank control group (Normal), a Model group (Model), a sturgeon longjin chewable tablet Product group (Product), a positive control group (Drug), a Collagen group (Collagen), and a chondroitin sulfate group (CS), according to the weight "zigzag" distribution. And (3) performing intragastric administration intervention according to the conversion coefficient of the administration dose of the rat and the administration dose of the normal person of 6.3 and the reference stomach volume of the rat of 0.9 mL per hundred grams.

Preparing a stomach-irrigation agent of the sturgeon and dragon tendon chewable tablets: mixing sturgeon and dragon tendon chewable tablet powder (specific component composition corresponds to the sample 4 in the experiment 5) with water, controlling the concentration of the gavage agent to be 1 mg/mL by using distilled water, and converting the gavage amount of each animal to be 250 mg/kg per day according to a dosage formula of rats and human bodies.

Preparing a collagen group gastric lavage agent: the collagen freeze-dried powder extracted from the sturgeon keel is dissolved by distilled water, and the concentration and the dosage are the same as above.

Preparing a chondroitin sulfate gastric lavage agent: the chondroitin sulfate lyophilized powder extracted from sturgeon longjin is dissolved by distilled water, and the concentration and the dosage are the same as above.

Preparing a positive control group gastric lavage agent: grinding the Jianli type II collagen glucosamine cartilage tablet into powder, and dissolving with distilled water, wherein the concentration and the dosage are the same as above.

Preparing a gastric lavage agent for a blank control group and a model group: the stomach was perfused with the same volume of 0.9% normal saline.

After administering 48 SD rats by gastric gavage with the test substance for 2 weeks, left knee osteoarthritis was induced in rats by anterior resection of medial meniscus and medial collateral ligament in addition to the blank control group. Rats were anesthetized by intraperitoneal injection of 10% chloral hydrate, and the left knee joints of the rats were sterilized by swabbing with an alcohol cotton ball and the left knee hairs were removed with a shaver. Laying a sterile towel, incising skin and fascia layer by layer, cutting off medial collateral ligament, making a 0.5 cm incision at the joint along the inner side of knee to expose medial meniscus, picking, washing joint cavity with antibiotic injection, and suturing layer by layer. Rats were fed with the test substance for 12 weeks after surgery and were given 30 min of exercise per day to induce osteoarthritis according to the osteoarthritis induction program. At 4 weeks after surgery, rat specimens were collected randomly for histological analysis, and other rat specimens were collected at 12 weeks after surgery for analysis.

7.2 histological observations

The food intake, hair, wound condition, mobility and the like of rats were observed and recorded every day before gastric lavage of the test subjects, rat knee joint specimens were randomly collected after 4 weeks and 12 weeks, respectively, the specimens were immersed in 4% formaldehyde solution, left to stand for 48 hours, decalcified with 10% EDTA decalcifying solution for 6 weeks, liquid was changed every week, embedded with normal paraffin and sliced, the slice height was 5 μm, xylene was dewaxed, dehydrated in anhydrous, 95%, 85%, 75% ethanol solution gradient, and stained with acid reddish (van gieseon, VG) staining solution, observed under a microscope and photographed and recorded, and the results are shown in fig. 11 and 12.

As can be seen from fig. 11 and 12, the collagen fibers of the blank control group were arranged in order, and the network structure was intact and uniformly distributed. In the osteoarthritis model group, collagen fibers were disorganized and the network structure was destroyed, compared to the normal group. At week 12, except for the blank control group, the collagen fiber structure of the knee joint of rats in each of the other groups was more severe than that at week 4. Compared with the model group, the collagen fiber net structures of the sturgeon longjin chewable tablet product group, the collagen protein group and the chondroitin sulfate group are complete, which shows that the sturgeon longjin chewable tablet product has the function of maintaining and protecting the collagen fiber structures at the knee joint cartilage.

7.3 immunofluorescence assay for NGF and SP expression

Rat knee joint tissue sections are manufactured by the method of 7.2, the tissue sections are washed by PBS washing liquor, 4% paraformaldehyde solution is added, the tissue sections are kept stand for 15 min, the tissue sections are washed by PBS, 0.5% polyethylene glycol octyl phenyl ether solution is added, the tissue sections are permeated for 20 min at 25 ℃, after the PBS is washed, sealing solution is added for soaking for 0.5 h, primary Antibody (NGF Antibody, Affinity) is added after the liquid is discarded, the tissue sections are kept stand for 24 h at 4 ℃, the washing liquor is washed for more than 3 times, secondary Antibody (rabbit Antibody, Affinity) is added, the tissue sections are kept stand for 1 h at 25 ℃, the washing liquor is used for washing the tissue sections, sealing liquid (containing fluorescence quenching agent) is added, the dyeing result of the tissue sections is observed by using an inverted fluorescence microscope, the average immunofluorescence intensity is measured by Image J software, and the results of NGF and SP detection of rats in each group are shown in figures 13 and 14.

As can be seen from fig. 13 and 14, the expression levels of NGF and SP in the knee joint tissues of the rats in the model group showed a significantly increased trend (P < 0.05) compared to the blank control group. Compared with the model group, the expression level of each group is obviously reduced (P < 0.05), which indicates that the expression of NGF and SP in the knee joint tissues of the animals can be effectively reduced through the intervention of the test substances. Except for the positive control group, the NGF expression of each group has no significant difference (P is more than 0.05), and the SP expression of the collagen group is obviously increased.

The main symptom of osteoarthritis is pain in joints, and the relief of joint pain becomes the key to the treatment of osteoarthritis. Nerve Growth Factor (NGF) is widely present in tissues of organisms, and when inflammation and tissue damage occur, NGF can generate pain by means of mediating sensitization of nerve endings and increase of pain receptors, and NGF can also cause further expression of partial inflammatory factors to aggravate inflammation. Substance P (SP) is a neuropeptide which is distributed around nerves and has a pro-inflammatory effect, and can improve vascular permeability and promote nerve sensitization, thereby causing pain. In the experiment, the sturgeon and dragon tendon chewable tablet can effectively inhibit the expression of NG and SP, thereby relieving arthralgia caused by osteoarthritis, controlling the further increase of inflammatory factors, and effectively inhibiting the pain caused by arthritis.

The above examples are provided to enable those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure. Modifications apparent to those skilled in the art are intended to be within the scope of the appended claims.

Claims

1. A sturgeon and dragon bone chewable tablet is characterized in that: comprises the following components: 10 parts of sturgeon longjin powder, 0 part or 45-60 parts of milk powder, 0 part or 30-60 parts of coconut powder and 0 part or 1-5 parts of magnesium stearate;

2. The sturgeon keel chewable tablet according to claim 1 is characterized in that: the sturgeon keel powder is prepared by the following method: taking sturgeon keel, cleaning, and drying at 110 ℃ for 90 min; cutting sturgeon longjin into small pieces, adding into water at a feed-liquid ratio of 1:10, treating at 103.43 kPa and 121 ℃ for 20 min to obtain sturgeon longjin liquid, standing to normal temperature, filtering to obtain filtrate, spray drying to obtain sturgeon longjin powder, and sieving with a 60-mesh sieve.

3. The sturgeon keel chewable tablet according to claim 1, characterized in that: the milk powder is selected from whole milk powder.

4. The sturgeon keel chewable tablet according to claim 1, characterized in that: the composition consists of the following components: 10 parts of sturgeon longjin powder, 45-60 parts of milk powder, 30-60 parts of coconut powder and 1-5 parts of magnesium stearate.

5. A sturgeon and dragon bone chewable tablet according to claim 4, characterized in that: the composition consists of the following components: 10 parts of sturgeon longjin powder, 60 parts of milk powder, 30 parts of coconut powder and 3 parts of magnesium stearate.

6. A method of preparing a sturgeon and dragon bone chewable tablet according to any one of claims 1 to 5, characterized in that: the method comprises the following steps:

(1) mixing and granulating: mixing sturgeon longjin powder, milk powder and coconut powder, adding ethanol solution to obtain wet mixture, sieving the wet mixture, granulating, and drying to obtain granules;

(2) tabletting: adding magnesium stearate into the granules, mixing uniformly, and tabletting to obtain the sturgeon and dragon bone chewable tablets.

7. The method of claim 6, wherein: the volume concentration of the ethanol solution is 70-90%; the drying conditions are as follows: drying for 1-2 hours at 45-65 ℃.

8. The sturgeon and dragon bone chewable tablet of any one of claims 1 to 5 is used for preparing health care products with effects of relieving joint inflammation pain or promoting osteoblast differentiation.

9. A sturgeon keel powder is characterized in that: is prepared by the following method: drying sturgeon longjin at 105-110 ℃ for 60-110 min, then adding water, treating at 121 ℃ for 15-25 min to obtain sturgeon longjin liquid, filtering to obtain filtrate, and performing spray drying to obtain sturgeon longjin powder.

10. The sturgeon spindle keel powder according to claim 9 is applied to preparation of sturgeon spindle keel chewable tablets or health care products with effects of relieving joint inflammation pain or promoting osteoblast differentiation.