Chitooligosaccharides from squid cartilage, and preparation method and application thereof

Abstract

The application discloses chitooligosaccharide from squid cartilage, and a preparation method and application thereof, and belongs to the technical field of amino polysaccharide extraction and application. The chitooligosaccharide is prepared from chitin obtained by desalting and deproteinization of squid cartilage, and a deproteinization process for obtaining the chitin is also provided. The chitin is prepared into beta-chitosan through deacetylation, and the beta-chitosan is prepared into chitooligosaccharide through cellulose hydrolysis. The preparation process of the chitooligosaccharide is simple and easy to implement. The characterization information of the prepared chitooligosaccharide is analyzed through experiments, the chitooligosaccharide contains oligomers with a polymerization degree of 2-6, and is a positively charged oligosaccharide. The application also provides application of the chitooligosaccharide in treatment and relief of type II diabetes. A large number of experiments prove that the chitooligosaccharide from squid cartilage has biological functions such as antioxidant, anti-diabetic, cholesterol-lowering, immune regulation and antibacterial activity, the prepared chitooligosaccharide from squid cartilage has relatively comprehensive biological functions, the preparation process is simple, and the chitooligosaccharide has wide application value in medicines and foods.

Description

Technical Field

[0001] This invention belongs to the technical field of squid cartilage polysaccharide extraction and application, specifically relating to a squid cartilage-derived chitosan oligosaccharide, its preparation method, and its application. Background Technology

[0002] Currently, diabetes is classified into type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM). Type 2 is more common than type 1, and the diagnosis rate of T2DM is constantly rising due to high-calorie diets and sedentary lifestyles. The core pathological feature of T2DM is the disruption of the glucose and lipid metabolism network caused by decreased insulin sensitivity, and its occurrence and development are closely related to abnormalities in the PI3K / Akt signaling pathway. This pathway, as the core hub of insulin signaling, directly affects physiological processes such as glucose transport and gluconeogenesis inhibition.

[0003] Squid cartilage is rich in β-chitin, whose molecular chain interactions are weak, exhibiting superior chemical modifiability compared to α-chitin. Chitin and its derivatives, chitosan and chitosan oligosaccharides, as natural alkaline amino polysaccharides, show broad application prospects in biomedicine, functional foods, cosmetics, agriculture, and environmental protection due to their unique biocompatibility, biodegradability, antibacterial, antioxidant, and immunomodulatory effects.

[0004] In addition, chitosan oligosaccharides have many beneficial effects on health, such as regulating blood glucose homeostasis, lowering blood cholesterol, controlling blood pressure, controlling arthritis, and improving calcium absorption. It is worth noting that COS contained in squid cartilage, a marine biological resource, has not been fully developed. Systematic research on squid cartilage COS remains lacking, and key questions such as its specific pathways for regulating blood glucose homeostasis and lowering cholesterol, the molecular mechanisms of its anti-arthritis activity, and its calcium ion transport-promoting effects urgently need to be elucidated. Future research should focus on resolving its structure-activity relationship, establishing standardized preparation processes, and verifying its bioactivity through cell experiments and animal models, providing a scientific basis for the development of marine-derived functional foods and novel biomaterials. This exploration will not only help expand the pathways for the high-value utilization of marine organisms but may also open up new directions for the prevention and treatment of chronic metabolic diseases.

[0005] The chitosan industry currently faces multiple challenges, including limited raw material sources, environmentally unfriendly traditional extraction processes, the need to improve product performance, and insufficient development of high-value-added applications. In particular, a comprehensive, efficient, clean, and high-value utilization technology system for β-chitin resources found in marine processing waste (such as squid cartilage) has not yet been established. Therefore, developing a novel, environmentally friendly chitosan oligosaccharide extraction process for squid cartilage and exploring its new applications in high-end fields is of significant scientific and industrial value for promoting the upgrading of the chitin industry, achieving full utilization of marine resources, and enhancing the international competitiveness of products. Summary of the Invention

[0006] Based on the above-mentioned technical deficiencies, the purpose of this invention is to provide a squid cartilage-derived chitosan oligosaccharide, its preparation method, and its application. This invention uses squid cartilage as raw material, and obtains β-chitin through desalting and enzymatic hydrolysis, and further processes it to prepare chitosan oligosaccharide. The prepared chitosan oligosaccharide meets the application standards through composition and characterization information verification, has targeted improvement on type II diabetes, has a wide range of applications, and has promotional value.

[0007] To achieve the above objectives, the present invention is implemented through the following solution: This invention provides a method for preparing squid cartilage shell oligosaccharides derived from squid cartilage, the method comprising the following steps: (1) Raw material processing: Wash the squid cartilage thoroughly with water, dry it overnight at 25~30℃, grind and sieve to obtain powder; (2) Desalination treatment: The powder is washed with hydrochloric acid and water in sequence until the washing effluent is neutralized, and then washed with distilled water until neutral. After washing, it is dried to obtain squid cartilage powder. (3) Enzymatic decomposition of protein: The squid cartilage powder is mixed with a protease solution and fully enzymatically hydrolyzed. After enzymatic hydrolysis, it is dried to obtain β-chitin powder. (4) Mix the β-chitosan powder with NaOH solution and react fully at 80~100℃. After the reaction is completed, wash and dry the mixture to prepare squid cartilage β-chitosan. (5) The squid cartilage β-chitosan was fully dissolved in acetic acid solution, and then an acetate-sodium acetate buffer solution with pH 5-6 was added to prepare a β-chitosan solution. Cellulase was added to the β-chitosan solution and fully enzymatically hydrolyzed at 50-55℃. After the enzymatic hydrolysis was completed, the enzyme was inactivated. Then, the pH of the enzyme-inactivated solution was adjusted to 7.0-7.5 with NaOH solution to precipitate the unreacted β-chitosan. After the hydrolysis was completed, the supernatant was collected by centrifugation. The supernatant was dialyzed and dried to obtain squid cartilage chitosan oligosaccharide.

[0008] Furthermore, in step (3), the protease solution is a solution composed of alkaline protease and trypsin, the mass ratio of alkaline protease to trypsin is 1:1~3, the amount of compound enzyme added is 3~5% based on the mass of squid cartilage powder, and the material-liquid ratio of squid cartilage powder to protease solution is 1:8~10.

[0009] Furthermore, the enzyme activity of the alkaline protease and trypsin is 1000 U / g to 100000 U / g.

[0010] Furthermore, the conditions for enzymatic hydrolysis in step (3) are: pH 7.0~8.0, hydrolysis temperature 40~45℃, and hydrolysis time 5~6 h.

[0011] Furthermore, the optimized preparation method includes the following: the protease is a complex enzyme composed of alkaline protease and trypsin, with a mass ratio of 1:2; the ratio of squid cartilage powder to protease solution is 1:8; the amount of protease added is 3% based on the mass of squid cartilage powder; and the enzymatic hydrolysis conditions in step (3) include: pH 7.9, hydrolysis temperature 40℃, and hydrolysis time 6 h.

[0012] Furthermore, the β-chitosan solution contains β-chitosan at a mass concentration of 15~20 mg / mL.

[0013] Furthermore, the specific activity of the cellulase is 500~1000 U / g.

[0014] The present invention also provides squid cartilage oligosaccharides prepared by the aforementioned preparation method.

[0015] Furthermore, the squid cartilage shell oligosaccharide contains oligomers with a degree of polymerization of 2 to 6.

[0016] Furthermore, the squid cartilage shell oligosaccharide is a positively charged oligosaccharide.

[0017] The present invention also provides the application of the aforementioned squid cartilage oligosaccharide in the preparation of drugs or foods for relieving or treating type II diabetes.

[0018] Furthermore, the drug or food contains squid cartilage oligosaccharide in a mass content of 100-400 mg / kg.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention utilizes squid cartilage powder to prepare chitosan oligosaccharide (COS) through an optimized process. The prepared squid cartilage COS not only retains the bioactivity of chitosan but also, due to its unique basic amino structure and water-soluble properties, becomes a rare positively charged oligosaccharide in nature. The squid cartilage COS possesses antioxidant, anti-diabetic, cholesterol-lowering, immunomodulatory, and antibacterial activities, and can be applied in functional foods, medicines, and other fields, broadening the application scope of squid cartilage COS, increasing the economic value of squid cartilage, and has promising prospects for promotion.

[0020] 2. The squid cartilage COS prepared in this invention can reduce the levels of TNF-α and IL-6 in the serum of patients with type 2 diabetes mellitus (T2DM) to alleviate inflammation. It can also improve abnormal liver function in T2DM by reducing serum ALT and AST levels. Squid cartilage COS can alleviate insulin resistance (IR) by activating the PI3K / Akt signaling pathway, promote hepatocyte uptake of glucose and glycogen synthesis, and regulate intestinal function. This further verifies that COS derived from squid cartilage has comprehensive biological functions and improves type 2 diabetes mellitus from different levels, providing a new approach and method for the prevention and treatment of type 2 diabetes mellitus, which is of great significance. Attached Figure Description

[0021] Figure 1 The effect of a single enzyme on the residual amount of β-chitosan protein; Figure 2 The effect of the ratio of complex enzymes on the residual amount of β-chitoxin protein; Figure 3 The effect of enzymatic decomposition process on the residual amount of β-chitosan protein is shown in the figure, where a is the material-to-liquid ratio, b is the enzymatic hydrolysis time, c is the pH, d is the temperature, and e is the amount of enzyme added. Figure 4 The response surface plot shows the effect of the interaction of enzyme deproteinization factors on the residual amount of β-chitosan protein, where a represents pH and enzyme dosage, b represents temperature and enzyme dosage, and c represents temperature and pH. Figure 5 This is a diagram illustrating the extraction process of β-CS and COS from squid cartilage. Figure 6 The graph shows the results of the degree of deacetylation determination of β-CS in squid cartilage; Figure 7 Fourier transform infrared spectroscopy results for β-CS in squid cartilage; Figure 8 Fourier transform infrared spectroscopy results for COS (coarse cartilage) of squid; Figure 9 X-ray diffraction results for β-CS of squid cartilage; Figure 10 The results of differential scanning calorimetry determination of β-CS in squid cartilage; Figure 11 Mass spectrometry results for COS in squid cartilage; Figure 12 The results of thin-layer chromatography determination of COS in squid cartilage; Figure 13 The changes in body weight and fasting blood glucose in mice after COS intervention with squid cartilage are shown in Figure a, where a represents the change in body weight and b represents the change in fasting blood glucose. Figure 14The graph shows the changes in blood glucose levels in mice during the OGTT and the area under the curve for each group. In the graph, a represents the change in blood glucose levels and b represents the area under the curve for each group. Figure 15 The changes in GSP and liver glycogen levels in mice after COS intervention with squid cartilage are shown in Figure a, where GSP changes and liver glycogen changes are shown in Figure b. Figure 16 The results show the evaluation of insulin levels and insulin resistance homeostasis model in mice after COS intervention with squid cartilage, where a represents the change in insulin levels and b represents the evaluation of the insulin resistance homeostasis model. Figure 17 The images show the results of pancreas (45×) and liver (30×) sections in mice after COS intervention with squid cartilage. The left image shows the pancreas, and the right image shows the liver. Figure 18 The results show the serum lipid levels in mice after COS intervention with squid cartilage, where a represents changes in TC, b represents changes in TG, c represents changes in LDL-C, and d represents changes in HDL-C levels. Figure 19 The changes in serum IL-6 and TNF-α levels in mice after COS intervention with squid cartilage are shown in Figure a, where a represents the change in IL-6 level and b represents the change in TNF-α level. Figure 20 The graph shows the changes in oxidative stress in mice after COS intervention with squid cartilage, where a represents serum SOD, b represents serum MDA, c represents serum T-AOC level, d represents liver SOD, and e represents changes in liver MDA content. Figure 21 The changes in serum ALT and AST levels in mice after COS intervention with squid cartilage; Figure 22 The expression levels of p-Akt / Akt and PI3K / β-actin, proteins in the insulin signaling pathway, in mice after COS intervention with squid cartilage are shown. In this figure, a represents the expression level of p-Akt / Akt, and b represents the expression level of PI3K / β-actin. Figure 23 The graph shows the changes in fatty acid and total short-chain fatty acid content in mouse feces after COS intervention with squid cartilage. In the graph, a represents acetic acid, b represents propionic acid, c represents isobutyric acid, d represents butyric acid, e represents isovaleric acid, f represents valeric acid, g represents isohexanoic acid, h represents hexanoic acid, and i represents total short-chain fatty acids. Figure 24 The results show the gut microbiota of mice after COS intervention with squid cartilage, where a represents the results of Chao index analysis, b represents the results of Ace index analysis, and c represents the results of Shannon index analysis. Figure 25 The figure shows the results of PCA and PCoA analysis of the gut microbiota in mice after COS intervention with squid cartilage, where a represents PCA and b represents PCoA. Figure 26 The gut microbiota composition at the OTU level in mice after COS intervention with squid cartilage; Figure 27 The table shows the gut microbiota composition and Firmicutes / Bacteroidetes ratio at the phylum level in mice after COS intervention with squid cartilage, where a represents gut microbiota composition and b represents Firmicutes / Bacteroidetes ratio. Figure 28 The gut microbiota composition at the genus level in mice after COS intervention with squid cartilage; Figure 29 LEfSe analysis of gut microbiota after COS intervention in squid cartilage; Figure 30 LDA discriminant analysis of gut microbiota after COS intervention in squid cartilage; Figure 31 A heatmap showing the correlation between gut microbiota abundance and T2DM biomarkers in mice after COS intervention with squid cartilage; Figure 32 A heatmap showing the correlation between gut microbiota abundance and T2DM biomarkers in mice after COS intervention with squid cartilage; Note: This means P < 0.05. The letter represents P < 0.01; different letters represent P < 0.05, and the same letter represents P > 0.05. Detailed Implementation

[0022] To better illustrate the objectives, technical solutions, and advantages of this invention, the technical solutions in the embodiments of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0023] Unless otherwise specified in the embodiments of the present invention, the conditions shall be performed in accordance with conventional conditions or conditions recommended by the manufacturer.

[0024] In the embodiments of this invention, reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0025] The data processing and analysis involved in this invention used IBM SPSS Statistics 27 and Origin 2021 for data analysis and plotting. All experiments were repeated three times, and the data are expressed as mean ± standard error. Post-hoc Tukey-Kramer test was used for one-way ANOVA to analyze significant differences in multiple comparisons. P < 0.05 indicated statistical significance; marking the same letter indicated no significant difference between the two groups (P > 0.05), and marking different letters indicated a significant difference between the two groups (P < 0.05).

[0026] Example 1: Optimization of enzymatic protein desorption process in the preparation of β-chitin from squid cartilage. This embodiment optimizes the enzymatic protein desorption process in the preparation of squid cartilage β-chitin. The specific operations include the following steps: 1. Effect of a single enzyme on the efficiency of enzymatic protein decomposition Alkaline protease, papain, and trypsin were selected for enzymatic hydrolysis experiments, with an enzyme dosage of 10000 U / g. Enzymatic hydrolysis was carried out under strictly controlled isothermal conditions according to the optimal temperature and pH listed in Table 1 for each protease. 2.00 g of squid cartilage powder was taken, and the substrate solution concentration was adjusted to 0.02 g / mL. Enzymatic hydrolysis was performed for 5 h according to the optimal reaction parameters for each enzyme. After the reaction, the enzymes were inactivated by boiling in a 100 ℃ water bath for 10 min, centrifuged (5000 r / min, 15 min), and the precipitate was washed with water until the OD of the supernatant was [value missing]. 600 The result was 0. The precipitate was dried in a 60 °C oven for 24 h and then tested.

[0027] Table 1. Optimal reaction temperature and pH of enzymes

[0028] 2. Effect of the ratio of compound enzymes on the enzymatic decomposition effect of proteins Take 2.00 g of squid cartilage powder, with a substrate concentration of 0.02 g / mL. Combine the two enzymes with the best reaction performance in the single enzyme experiments to form a complex enzyme, with addition ratios of 1:1, 1:1.5, 1:2, 1:2.5, and 1:3, and an enzyme addition amount of 1.0%. React at 60 ℃ and pH 8.5 for 4 h. After the reaction, inactivate the enzyme in a 100 ℃ boiling water bath for 10 min, centrifuge at 5000 r / min for 15 min, and wash the precipitate with water until the OD of the supernatant is [value missing]. 600 The result was 0. Finally, the precipitate was transferred to a constant temperature drying oven and dried at 60 °C for 24 h before being transferred to a desiccator for storage.

[0029] 3. Effects of enzymatic hydrolysis conditions on the efficiency of enzymatic protein decomposition Weigh 2.00 g of squid cartilage powder. Using the compound enzyme ratio determined in the previous step, the enzymatic hydrolysis reaction was carried out under different conditions: different material-to-liquid ratios (1:6, 1:8, 1:10, 1:12, 1:14, where the material-to-liquid ratio is the ratio of the mass of squid cartilage powder to the volume of the compound protease solution, where the mass unit is g and the volume unit is mL); different enzyme dosages (1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, where the enzyme dosage is the percentage of the mass of the compound enzyme to the mass of the squid cartilage powder); different temperatures (35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃); and different pH values ​​(7.6, 7.9, 8.2, 8.5, 8.8, 9.1). The reaction was then terminated by water bath incubation at 100 ℃ for 10 min. Finally, the reaction was carried out at 5000 mL / min. Centrifuge continuously at r / min for 15 min, repeatedly washing the precipitate until the OD of the supernatant reaches a certain value. 600 The final precipitate was transferred to a constant temperature drying oven and dried at 60 ℃ for 24 h before being transferred to a desiccator for storage. When one of the conditions changed, the fixed conditions were: enzymatic hydrolysis temperature 60 ℃, pH 8.5, material-to-liquid ratio 1:10, enzyme dosage 2.0%, and enzymatic hydrolysis time 4 h.

[0030] 4. Response surface methodology optimization of enzymatic protein decomposition process Based on single-factor experiments, a three-factor, three-level response surface methodology was designed, using the protein content in chitin after enzymatic hydrolysis as the evaluation index to optimize the enzymatic deproteinization process. Design-Expert 13 statistical analysis software was used to analyze and process the response surface data obtained from each experimental batch.

[0031] 5. Determination of protein content Add 0.10 g of sample to 100 mL of 5% (v / v) sodium hydroxide solution, stir magnetically at 95 °C for 2.5 h, then centrifuge at 4000 r / min for 5 min, collect the supernatant, and add physiological saline to 100 mL for later use. The protein content in the 100 mL liquid containing the supernatant was determined using the Bradford reagent kit.

[0032] 6. Optimization Results and Analysis (1) Effect of a single enzyme on the residual amount of β-chitosan protein To investigate the effects of alkaline protease, papain, and trypsin on protein deproteinization, the residual protein content in β-chitin samples after deproteinization was quantitatively measured and compared. Based on... Figure 1The results showed that the protein concentrations were lowest at alkaline protease and trypsin, at 304.91 ± 40.19 and 406.70 ± 53.24 μg / mL, respectively. Therefore, these two enzymes were selected as the complex enzymes for the following experiments.

[0033] (2) Effect of the ratio of complex enzymes on the residual amount of β-chitosan protein To determine the effectiveness of each enzyme combination in deproteinization, the residual protein content in the β-chitosan samples after deproteinization was quantitatively measured and compared. Based on... Figure 2 It can be seen that the protein concentration is lowest when the mass ratio of alkaline protease to trypsin is 1:2, which is 295.90 ± 18.64 μg / mL. Therefore, this ratio was used for the following experiments.

[0034] (3) Effect of enzymatic hydrolysis conditions on the residual amount of β-chitin protein The residual protein content in β-chitin samples after deproteinization was quantitatively determined and compared to analyze the deproteinization effect under different material-to-liquid ratios, enzymatic hydrolysis times, pH, temperatures, and enzyme dosages. Experiments were conducted using the optimal compound enzyme ratio described above, with a material-to-liquid ratio of 1:8 (…). Figure 3 The lowest protein concentration was observed (as shown in a), at 207.38 ± 5.01 μg / mL; the protein concentration was highest after 6 h of enzymatic hydrolysis (…). Figure 3 The lowest protein concentration was observed (as shown in b), at 185.28 ± 4.38 μg / mL; the concentration was highest at enzymatic hydrolysis at pH 7.9. Figure 3 The lowest protein concentration was observed (as shown in c), at 165.65 ± 8.71 μg / mL; the protein concentration was highest at an enzymatic hydrolysis temperature of 45 ℃. Figure 3 The lowest protein concentration was observed (as shown in d), at 135.90 ± 7.81 μg / mL; when the enzyme concentration was 3.0% ( Figure 3 The lowest protein concentration (shown in figure e) was 168.98 ± 6.94 μg / mL. The optimal reaction conditions for enzymatic protein desorption were determined through single-factor experiments. Further optimization of process parameters will be achieved using response surface methodology.

[0035] (4) Optimization of enzymatic deproteinization process using response surface methodology Based on the results of the single-factor experiments, three factors—enzymatic hydrolysis pH, enzymatic hydrolysis temperature, and enzyme dosage—were selected for response surface optimization to determine the optimal solution for the enzymatic deproteinization process. The specific factor level design is shown in Table 2.

[0036] Table 2 Response Surface Design Factor Level Table

[0037] Following a response surface methodology, the residual protein content in β-chitosan under corresponding conditions was determined. The results are shown in Table 3, and the response surface plot derived from these results is shown in [Table 3]. Figure 4When the mass ratio of alkaline protease to trypsin is 1:2, the optimal process parameters predicted by the response surface methodology are 3.0% enzyme dosage, pH 7.9, and temperature 40 ℃, at which the protein concentration is 170.61 μg / mL.

[0038] Table 3. Response Surface Experiment Design and Results

[0039] The results of the regression model variance analysis are shown in Table 4. The three key process parameters, enzyme dosage, pH, and temperature, made significant contributions to the model. P <0.05, where the overall model reached a statistically significant level ( P <0.01). Analysis of variance data from the regression model showed no significant difference between the lack-of-fit term and the pure error ( ). P The value >0.05 indicates that the model has a good fit and high reliability. Furthermore, the model's R-value is... 2 The coefficient of determination is 0.9916, R0. Adj 2 = 0.9808, which proves that the model can reflect the relationship between the factors and has a high degree of fit.

[0040] Validation experiment: At an enzyme dosage of 3.0%, pH 7.9, and temperature of 40 ℃, the protein concentration was 162.38 μg / mL, which matched the predicted value well, indicating that the model can predict the optimal response value. Therefore, squid cartilage β-chitin was prepared according to the optimized enzymatic deproteinization process.

[0041] Table 4. Analysis of Variance of Regression Model

[0042] Note: represent P <0.05, represent P <0.01.

[0043] 7. Based on the optimization of the above-mentioned protease-mediated protein desorption process, an optimal preparation method for β-chitin is obtained, the preparation method comprising the following steps: (1) Raw material processing: The squid cartilage was thoroughly washed with water, dried overnight at 30 ℃, and then ground with a grinder. After that, the powder smaller than 0.30 mm was separated by passing it through a 50-mesh sieve; (2) β-Chitosan desalting treatment: The powder was washed with hydrochloric acid (0.55 M, 10 mL hydrochloric acid per gram of powder) at room temperature for desalting treatment, repeated twice for 1.5 h each time. Then it was washed with distilled water until the washing effluent was neutralized. Then it was washed with distilled water until neutral and dried in a vacuum oven at 50 °C for 24 h to obtain desalted squid cartilage powder; (3) Enzymatic hydrolysis process: Squid cartilage powder is hydrolyzed using a combined protease solution. The protease solution is composed of alkaline protease and trypsin in a mass ratio of 1:2. The ratio of squid cartilage powder to protease solution is 1:8. The amount of enzyme added is 3.0%. β-chitin is obtained by hydrolysis for 6 h at a pH of 7.9 and a hydrolysis temperature of 40°C.

[0044] Example 2: Preparation of β-chitosan and chitosan oligosaccharides from squid cartilage The β-chitosan powder described in this embodiment is prepared by drying the β-chitosan obtained after process optimization in Example 1. Based on this, this embodiment provides the preparation of squid cartilage β-chitosan and its oligosaccharides, and analyzes their extraction rates. 1. This embodiment provides the preparation of squid cartilage β-chitosan and its oligosaccharides, and the specific operation includes the following steps: (1) A 50% (w / v) NaOH solution was mixed with β-chitosan powder at a volume-to-mass ratio of 8:1 (the volume-to-mass ratio is the ratio of the volume of NaOH solution to the mass of β-chitosan powder, where the volume unit is L and the mass unit is g), and the mixture was reacted at 100℃ for 12 h. The mixture was washed with pure water and dried in an oven to obtain squid cartilage β-CS.

[0045] (2) Weigh a certain amount of squid cartilage β-CS, dissolve it completely in 0.2 mol / L acetic acid solution, add pH 5.2 acetate-sodium acetate buffer to prepare a chitosan solution with a concentration of 20 mg / mL, add cellulase solution at a ratio of 500 U / g enzyme activity, and react in a constant temperature water bath at 55℃ for 72 h. After the reaction is completed, inactivate the enzyme in a boiling water bath for 10 min, adjust the pH to 7.0~7.5 with 200 mg / mL NaOH solution to precipitate the unreacted β-CS, centrifuge at 6000 r / min for 10 min, filter, and take the supernatant. After dialyzing at 100 Da, concentrate and freeze dry to obtain squid cartilage COS.

[0046] 2. Investigate the effect of the preparation method described above on the extraction rate of squid cartilage powder. β-Chitosan was prepared by chemical desalting and enzymatic deproteinization. Then, squid cartilage β-CS was prepared by chemical deacetylation. Finally, COS was obtained by enzymatic degradation of β-CS. The mass of solids obtained in each step of the extraction process and the final extraction rate are shown in Table 5. The extraction process flow diagram is detailed in [link to flow chart]. Figure 5 .

[0047] Table 5 Extraction rates of β-chitoxin, β-CS, and COS from squid cartilage

[0048] Example 3: Basic composition and characterization analysis of squid cartilage β-CS and squid cartilage COS The squid cartilage β-CS and squid cartilage COS described in this embodiment were prepared and provided in Example 2.

[0049] 1. pH Measurement The pH of β-CS in squid cartilage was determined according to the method shown in General Chapter 0631 of the Pharmacopoeia of the People's Republic of China (2020 Edition). 0.50 g of β-CS sample was weighed, added to 50 mL of water, and stirred until completely dissolved before testing. A calibrated pH meter was used, calibrated with a standard buffer solution. The temperature of the test solution was kept consistent with the standard solution. The electrode was directly immersed in the test solution for measurement, and a stable value was read.

[0050] 2. Protein content determination Weigh 0.10 g of β-CS sample, dissolve it in 1% glacial acetic acid solution, and finally dilute to 10 mL. Determine the protein content of β-CS in squid cartilage according to Method 5 of General Chapter 0731 in the Pharmacopoeia of the People's Republic of China (2020 Edition). The principle of the assay is that the protein binds to Coomassie brilliant blue dye to form a blue complex, and the absorbance is measured at a wavelength of 595 nm. Quantification is achieved by comparing with a standard protein reference.

[0051] 3. Determination of loss on drying The loss on drying of β-CS from squid cartilage was determined according to the method shown in General Chapter 0831 of the Pharmacopoeia of the People's Republic of China (2020 Edition). 1.00 g of β-CS sample was placed in a flat weighing bottle and dried in an oven at 105 ℃ until constant weight. The percentage loss was calculated by subtracting the weight loss from the sample size.

[0052] 4. Determination of residue on ignition The weight of β-CS residue from squid cartilage was determined according to the method shown in General Chapter 0841 of the Pharmacopoeia of the People's Republic of China (2020 Edition). 1.00 g of β-CS sample was accurately weighed and placed in a constant-weight crucible. After carbonization, sulfuric acid was added, and the mixture was ignited at 700–800 °C until completely ashed. After constant weight, the weight percentage of the residue was calculated.

[0053] The test results are shown in Table 6. The squid cartilage β-CS prepared in Example 2 meets the corresponding index requirements for CS in the Chinese Pharmacopoeia.

[0054] Table 6. Basic Composition Analysis of β-CS

[0055] 5. Degree of deacetylation determination Weigh 0.20 g of squid cartilage β-CS sample and determine the degree of deacetylation of squid cartilage β-CS by potentiometric titration according to the national food safety standard GB 29941—2013, Food Additives, Deacetylated Chitosan.

[0056] The results are as follows Figure 6 As shown, the solubility of β-CS increases with increasing degree of deacetylation. The degree of deacetylation of the β-CS sample was determined by potentiometric titration, and the pH changes during the titration process are shown in the figure. Figure 6 As shown, the calculated degree of deacetylation of squid cartilage CS is 95.77%, which meets the requirement of CS deacetylation degree (≥85.00%) in "GB 29941—2013 National Food Safety Standard Food Additives Deacetylated Chitosan".

[0057] 6. Fourier transform infrared spectroscopy determination Potassium bromide was thoroughly ground and then placed in a 105 °C oven overnight with samples of squid cartilage β-chitin, β-CS, and COS to remove moisture completely. A trace amount of the sample was then thoroughly mixed with potassium bromide at a ratio of 1:200 (w / w), ground, pressed into a thin sheet, and subjected to infrared spectroscopy. The results were recorded at 4000 cm⁻¹. -1 ~400 cm -1 The spectral data was scanned 32 times with a resolution of 4 cm⁻¹. -1 .

[0058] (1) The infrared spectra of the obtained squid cartilage β-CS sample were determined by analyzing the primary structure to determine whether it has the β configuration. The infrared spectra are shown in the figure. Figure 7 .

[0059] The specific peak values ​​and corresponding functional groups are shown in Table 7. 1645.47 cm⁻¹ -1 and 1322.93 cm -1 The low peak intensity indicates a reduction in residual N-acetyl groups and a high degree of deacetylation. The results of this example show that amide II and III bands correspond to 1590.99 cm⁻¹, respectively. -1 and 1419.35 cm -1The characteristic peaks are located at the CS sample, and from the overall infrared spectrum, the CS sample has similar peak values ​​to β-chitin, which indicates that the prepared squid cartilage CS conforms to the structural characteristics of β-CS.

[0060] Table 7 Infrared bands and corresponding functional groups of β-CS

[0061] (2) Infrared spectroscopy was performed on the prepared squid cartilage COS samples to determine their primary structure, such as... Figure 8 As shown. Specific peak values ​​and corresponding functional groups are shown in Table 8, 1076.09 cm⁻¹. -1 The peaks at 1641.13, 1564.95, and 1411.64 cm⁻¹ represent the contraction peaks of the CO vibration in the sugar ring. -1 The area represents amide bands I, II, and III. Therefore, the prepared sample exhibits the structural characteristics of COS.

[0062] Table 8 Infrared bands and corresponding functional groups of COS

[0063] 7. X-ray diffraction analysis The crystal structures of β-chitin and β-CS were analyzed using X-ray diffraction. The samples were placed on the sample cell surface, and a Cu target Kα beam was used with a wavelength of 0.154 nm and a scanning speed of 5° / min. -1 The scanning range is 5~40°. The formula for calculating the crystallinity of the sample is as follows:

[0064] In the formula: I 110 Represents the crystalline region (2θ = 20). o The diffraction intensity value of I am Represents the amorphous region (2θ = 16) o The diffraction intensity value of ).

[0065] according to Figure 9It is known that both chitin and CS exhibit two diffraction peaks. The diffraction peaks for β-chitin are at 8.7° and 20.1°, respectively. The diffraction peaks for CS are at 10.8° and 20°, respectively. This indicates that the 2θ values ​​of the first diffraction peak for β-chitin and CS are different, while the 2θ values ​​of the second diffraction peak are similar. The diffraction intensity of β-chitin at 16° is 933, and at 20° it is 4167, with a crystallinity of 77.61%. The diffraction intensity of CS at 16° is 959, and at 20° it is 2330, with a crystallinity of 58.84%. The results show that when chitin is further deacetylated to CS, its crystal structure is completely destroyed, and CS with a deacetylation degree of 95.77% is amorphous. Therefore, the prepared CS sample belongs to the β configuration and has low crystallinity and an amorphous structure.

[0066] 8. Differential scanning calorimetry determination The thermal properties of β-CS and commercially available CS were compared using differential scanning calorimetry (DSC). 5.00 mg of CS was weighed into a crucible, with a blank crucible used as a control. The DSC parameters were set as follows: N2 flow rate 25 mL / min, heating rate 10 °C / min, and temperature scan range 25–500 °C.

[0067] Differential scanning calorimetry (DSC) has provided crucial thermodynamic data for the study of the structure and properties of squid cartilage β-CS. For example... Figure 10 As shown, the thermal stability of squid cartilage β-CS changes compared to commercially available CS. The endothermic peak reveals the energy required to remove water from β-CS, reflecting the hydration capacity of β-CS molecules. The exothermic peak characterizes the polymer's thermal decomposition behavior and the oxidative crosslinking reaction process. Experimental results show that the endothermic peaks of squid cartilage β-CS and commercially available CS are located at 90 ℃ and 83 ℃, respectively, while the exothermic peaks are located at 309 ℃ and 316 ℃, respectively. Therefore, the squid cartilage β-CS prepared in this invention exhibits a stronger water-binding capacity and a lower decomposition temperature than commercially available CS.

[0068] 9. Mass spectrometry analysis Dissolve 7 μL of trifluoroacetic acid in 7 mL of ultrapure water to prepare a 0.1% trifluoroacetic acid solution. Add 3 mL of acetonitrile and mix well. This solution is designated as solution T. Dissolve 20.00 mg of matrix 2,5-dihydroxybenzoic acid in 1 mL of solution T to prepare a 20 mg / mL matrix solution. Place the squid cartilage COS sample target into a matrix-assisted laser desorption / ionization-time-of-flight mass spectrometer and detect it using positive ion reflectance mode.

[0069] The degree of polymerization of squid cartilage (COS) samples was determined using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALA-TFS). The peak of chitosan oligosaccharide was [M+Na]. + The ion peak. For example... Figure 11As shown, the m / z value is below 1300, indicating that the sample mainly consists of COS with a degree of polymerization ranging from 2 to 6. The peaks at 363.02, 523.54, 684.34, 845.56, and 1006.47 represent the signal peaks for chitobiose, chitotriose, chitotetraose, chitopentose, and chitohexaose, respectively. The difference between two adjacent chitosan oligosaccharide monomers is 161 mass units, which is precisely the molecular weight of one glucosamine residue.

[0070] Experimental results show that β-CS can be effectively converted into COS, which is consistent with the thin-layer chromatography results. Chitosan derived from squid cartilage can also be degraded into chitosan oligosaccharides using cellulase, indicating that the degradation effect of chitosan derived from shrimp shell and squid cartilage using cellulase is similar. The reason for the large number of disaccharides analyzed in this example is that the enzymatic hydrolysis time is longer, resulting in more complete enzymatic hydrolysis.

[0071] 10. Thin-layer chromatography The hydrolysis products were centrifuged at 8000 r / min for 10 min. After collecting the supernatant, the supernatant was precisely spotted onto a silica gel thin-layer chromatography plate using micro-spotting technology (spotting interval 5 mm, baseline 10 mm from the bottom). A 5% glucosamine and squid cartilage COS standard aqueous solutions with a degree of polymerization of 2–6 were used as controls. Chromatographic separation was performed in a closed developing chamber using isopropanol:water:ammonia = 30:15:2 (v / v / v) as the developing solvent. The chromatographic development was performed using the ascending method. After development, the plates were dried in an oven at 100 °C, then sprayed with a 0.5% (w / v) ninhydrin ethanol solution as a colorimetric reagent, and finally developed in a drying oven at 105 °C for 20 min.

[0072] The degree of polymerization distribution of COS in the enzymatic hydrolysate was qualitatively determined using thin-layer chromatography. For example... Figure 12 As shown, the resulting squid cartilage COS is mainly composed of oligosaccharides with a degree of polymerization ranging from 2 to 6, with disaccharides being the most abundant. The sample also contains sugars with a degree of polymerization > 6, indicating that cellulase can successfully hydrolyze β-CS to prepare squid cartilage COS.

[0073] As described above, chitin is obtained from squid cartilage after desalting and deproteinization. This invention provides a preparation process for extracting chitin, using a mixture of alkaline protease and trypsin as a complex enzyme. The mass ratio of alkaline protease to trypsin is 1:2, the material-to-liquid ratio is 1:8, the enzymatic hydrolysis time is 6 h, the enzyme dosage is 3.0%, the pH is 7.9, and the hydrolysis temperature is 40 ℃. Furthermore, the structure of the squid cartilage chitin sample was characterized, and its infrared spectrum showed a depth of 1630.14 cm⁻¹. -1The characteristic peaks are β-configuration peaks. X-ray diffraction shows characteristic diffraction peaks at 8.7° and 20.1°, with a crystallinity of 77.61%. Characterization results indicate that squid cartilage chitin conforms to the structural characteristics of β-chitin. Chitin is deacetylated to CS. Basic index determination and structural characterization of squid cartilage CS were performed, and its degree of deacetylation reached 95.77%, with a peak value at 1590.99 cm⁻¹ in the infrared spectrum. -1 and 1419.35 cm -1 The peaks at 10.8° and 20° are characteristic of amide II and III bands, and X-ray diffraction shows characteristic diffraction peaks of the β configuration at 10.8° and 20°, with a crystallinity decrease to 58.84%, indicating that squid cartilage CS is β-CS. Differential scanning calorimetry analysis shows that the decomposition temperature of β-CS is 309 ℃. Cos prepared by cellulase degradation of β-CS was analyzed by thin-layer chromatography and matrix-assisted laser desorption / ionization-time-of-flight mass spectrometry, revealing that the COS sample mainly contains oligomers with a degree of polymerization of 2–6.

[0074] This invention provides data supporting the use of squid cartilage as a high-quality raw material for the production of β-CS and COS, and the structural characteristics of the prepared squid cartilage β-CS and COS meet the application standards.

[0075] Example 4: Investigating the bioactivity and function of squid cartilage COS 1. Experimental preparation and methods (1) Animal preparation Four-week-old SPF-grade male C57BL / 6J mice were purchased from Jicui Pharmaceutical (Jiangsu) Biotechnology Co., Ltd. The experimental animal number was No. 20230810C576J2400127002. The experiment was approved by the Experimental Animal Welfare and Ethics Committee of Qingdao University. Mice were housed separately in cages under a 12-hour light-dark cycle (8:00~20:00), at a room temperature of 23 ± 2 ℃, a relative humidity of approximately 40%, and good ventilation. They had free access to food and water during the experiment.

[0076] (2) Construction of a type II diabetic mouse model After 7 days of acclimatization, mice were randomly divided into two groups based on body weight: a normal control group (NC, n = 10) fed a basal diet, and a model group fed a high-fat diet (casein 23.31%, L-cysteine ​​0.35%, corn starch 8.48%, maltodextrin 11.65%, sucrose 20.14%, cellulose 5.83%, soybean oil 2.91%, lard 20.68%, complex minerals 5.23%, complex vitamins 1.16%, and tartrate choline 0.23%), for 4 weeks. After four weeks of high-fat feeding, mice were fasted for 12 hours. The model group received intraperitoneal injections of 60 mg / kg·BW streptozotocin for three consecutive days, while the normal control group received the same volume of citrate buffer (pH = 4). On day 7 of modeling, mice were subjected to a 6-hour fasting period, with normal water intake. Blood samples were collected from the tail vein for blood glucose testing. Mice with fasting blood glucose levels exceeding 11.1 mmol / L and exhibiting typical T2DM symptoms (polydipsia, polyphagia, polyuria, and weight loss) were selected for further use. Mice that did not successfully develop the model were given additional streptozotocin injections.

[0077] (3) Squid cartilage shell oligosaccharide intervention program Based on the establishment of the model in mice, the diabetic mice were divided into five groups (n = 10): model group (DC), positive control group (MET), low-dose COS group (LCOS), medium-dose COS group (MCOS), and high-dose COS group (HCOS). The mice were administered the appropriate treatment via gavage daily for 28 days. Specific groupings and dosages are shown in Table 9. Sterilized feed and drinking water were provided throughout the experiment, and clean bedding was changed daily.

[0078] Table 9 Experimental Groups and Test Substance Doses

[0079] (4) Measurement of body weight, fasting blood glucose and oral glucose tolerance The measurement method includes the following steps: S1: All mice were weighed once a day to determine their health status.

[0080] S2: Blood glucose was monitored regularly on a weekly basis. Fasting blood glucose levels in mice were determined by tail vein blood collection. Mice were fasted for 6 hours before the test (free access to water was allowed).

[0081] S3: Oral Glucose Tolerance Test (OGTT): After 4 weeks of intervention with squid cartilage COS, mice in each experimental group were fasted for 12 hours (with free access to water). The following day, 6 mice meeting the criteria were randomly selected from each experimental group and orally administered a glucose solution at a dose of 2.0 g / kg. Blood glucose concentration changes were continuously measured at the baseline time point (0 min) and at four subsequent time points (30, 60, 90, and 120 min) using tail vein blood sampling. Based on the blood glucose test results at each time point, OGTT curves were plotted, and the area under the curve (AUC) for each group was accurately calculated using the integral method.

[0082] (5) Sample collection and biochemical parameter determination After fasting overnight, blood was drawn from the eye socket using capillary blood and placed in an anticoagulant centrifuge tube. The blood sample was then centrifuged to separate the serum and frozen for later use in an ultra-low temperature freezer at -80 ℃.

[0083] The liver, pancreas, and colon were rapidly removed, washed in PBS, and placed in liquid nitrogen. Colonic contents were collected and placed in sterile cryovials, then frozen and stored at -80 °C for later use. A portion of the pancreatic and liver tissue was fixed in 4% paraformaldehyde for 48 h, then embedded in paraffin, sectioned, and analyzed.

[0084] Serum insulin (INS), total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), alanine aminotransferase (ALT), aspartate aminotransferase (AST), glycated serum protein (GSP), TNF-α, IL-6, total antioxidant capacity (T-AOC), malondialdehyde (MDA), superoxide dismutase (SOD) levels, and liver glycogen, SOD, and MDA levels were measured using appropriate kits.

[0085] Homeostasis model assessment of insulin resistance (HOMA-IR) is calculated using the following formula:

[0086] (6) Histopathological examination The hematoxylin-eosin (HE) staining method was employed and further improved. The specific steps included: Fresh liver and pancreas sections were immersed in 4% paraformaldehyde solution, mounted in paraffin, and then cut into 5 μm thick sections. After dewaxing to water, these sections were immersed in hematoxylin staining solution for staining. After briefly rinsing off excess stain with running water, they were differentiated with 1% hydrochloric acid-ethanol and rinsed with running water to regain their blue color. The blue-regained sections were then immersed in eosin staining solution for staining. After briefly rinsing off excess stain with running water, they underwent a gradient alcohol dehydration process using 70%, 80%, 90%, 95% and anhydrous ethanol, followed by clearing with xylene. Finally, the sections were mounted with neutral resin and covered with coverslips, completing the HE staining process. The HE-stained sections were then imaged under a microscope.

[0087] (7) Determination of short-chain fatty acid content Mouse feces were collected and placed in cryovials, stored at -80 °C for later use. A certain amount of feces was weighed, homogenized, and then centrifuged and filtered to remove particulate matter. After derivatization, the content of short-chain fatty acids was determined by gas chromatography-mass spectrometry.

[0088] (8) Determination of gut microbiota composition Fecal microbiota DNA was collected from mice, and 16S rRNA gene sequencing was performed on the DNA. Clustering and species classification analyses were then performed on the sequencing results.

[0089] (9) Western Blotting detection Proteins were extracted from mouse liver tissue using RIPA lysis buffer, and the extracted proteins were quantified using a BCA kit. Proteins were then separated by electrophoresis and transferred to a transfer membrane. The membrane was incubated overnight at 4 °C with antibodies against PI3K (1:1000), Akt (1:1000), and phosphorylated protein kinase B (p-Akt) (1:1000). After washing with phosphate buffer containing Tween 20, the membrane was incubated with secondary antibody (1:100000) at room temperature for 1 hour. Finally, developing solution was added, and protein bands were observed using a chemiluminescence detection system.

[0090] 2. Experimental Results and Analysis 2.1 Oligosaccharides from squid cartilage shell regulate glucose metabolism homeostasis (1) Effects of squid cartilage oligosaccharides on body weight and fasting blood glucose in T2DM mice Changes in body weight can reflect whether squid cartilage (COS) has a significant regulatory effect on metabolism in mice. For example... Figure 13As shown in Figure a, after intervention with squid cartilage COS, the body weight of mice in the NC, MET, MCOS, and HCOS groups showed an increasing trend, while the body weight of mice in the DC and LCOS groups showed a decreasing trend. However, the body weight decrease trend in the LCOS group was slower than that in the DC group. At week 4, compared with the DC group, the body weight of mice in the LCOS group was significantly lower. P <0.05), MCOS ( P <0.01), HCOS ( P <0.01) and MET ( P The body weight of mice in the <0.01 group increased significantly in a dose-dependent manner. This result indicates that LCOS, MCOS, and HCOS have a significant regulatory effect on mouse body weight, which is attributed to the regulation of energy metabolism and fat accumulation.

[0091] Type 2 diabetes mellitus (T2DM) can cause elevated blood glucose levels in mice, leading to abnormal glucose metabolism. This study aims to preliminarily determine whether squid cartilage (COS) can alleviate glucose metabolism disorders by measuring fasting blood glucose levels in mice. Figure 13 As shown in Figure b, after COS intervention, the blood glucose level in the DC group mice was significantly higher than that in the NC group. P <0.05%, blood glucose levels were significantly lower in the LCOS, MCOS, and HCOS groups compared to the DC group ( P <0.05), and with increasing doses of LCOS, MCOS, and HCOS, blood glucose levels gradually decreased. This indicates that the hypoglycemic effects of LCOS, MCOS, and HCOS are dose-dependent, and that COS may lower blood glucose levels by improving insulin sensitivity and promoting glucose uptake and utilization.

[0092] (2) Effect of squid cartilage shell oligosaccharides on oral glucose tolerance in T2DM mice The oral glucose tolerance test (OGTT) aimed to evaluate the regulatory effect of squid cartilage (COS) on glucose metabolism in mice. During the OGTT, blood glucose levels in all groups of mice rose sharply within 30 minutes after oral glucose administration, and then decreased between 30 and 120 minutes. Compared with the DC group, improved blood glucose levels were observed after administration of squid cartilage (COS). Figure 14 As shown in Figure a). Compared with the NC group, the area under the curve (AUC) of the DC group was significantly increased (P<0.05), indicating that the glucose tolerance of mice with T2DM was significantly reduced. Figure 14 (As shown in b). Notably, after MET and HCOS intervention, AUC decreased by 33.95% and 30.14% respectively within 120 min (P<0.05), indicating that glucose tolerance in T2DM mice was significantly improved after squid cartilage COS intervention, and this phenomenon was analyzed to be related to the recovery of pancreatic function.

[0093] (3) Effects of squid cartilage oligosaccharides on glycated serum protein and liver glycogen content in T2DM mice The effects of squid cartilage COS on glucose metabolism were investigated by measuring glycated serum protein (GSP) and liver glycogen levels in mice. GSP levels were correlated with the rate of non-enzymatic glycation reactions in glucose metabolism, while liver glycogen levels represented the liver's ability to uptake and utilize glucose. Results showed that GSP levels in T2DM mice (…) Figure 15 (As shown in a) significantly increased liver glycogen levels ( Figure 15 (As shown in b) significantly reduced ( P <0.05. GSP and liver glycogen levels were significantly restored by squid cartilage MCOS and HCOS intervention ( P <0.05). This indicates that MCOS and HCOS can delay the rate of non-enzymatic glycosylation in T2DM mice, reduce liver glycogenolysis and promote liver glycogen synthesis, and improve glucose metabolism disorders.

[0094] 2.2 Oligosaccharides from squid cartilage shell improve insulin resistance (1) Effect of squid cartilage oligosaccharide on insulin levels in T2DM mice Insulin resistance (IR), a core pathological feature of type 2 diabetes mellitus (T2DM), manifests as compensatory hyperinsulinemia caused by decreased insulin sensitivity in peripheral tissues. While this state can maintain glycemic homeostasis in the short term, its long-term persistence leads to the gradual decline of pancreatic β-cell function. Notably, persistent hyperinsulinemia not only accelerates the depletion of pancreatic β-cell function but also triggers a vicious cycle of compensatory glucose and lipid metabolism. Therefore, reversing IR is a crucial step in T2DM intervention.

[0095] like Figure 16 As shown in Figure a, the serum insulin level in T2DM mice was significantly higher than that in the NC group mice ( P <0.05. After 4 weeks of intervention with MET, MCOS, and HCOS, insulin levels in mice decreased by 32.60%, 14.52%, and 22.51%, respectively. P <0.05). Furthermore, the steady-state model evaluation of IR (HOMA-IR) is as follows: Figure 16 As shown in Figure b, the HOMA-IR of the DC group increased significantly ( P <0.05). Compared with the DC group, the MET, LCOS, MCOS, and HCOS groups significantly reduced the HOMA-IR in T2DM mice. P <0.05). This indicates that squid cartilage MCOS and HCOS can effectively reduce insulin levels and improve insulin resistance (IR).

[0096] (2) Effects of squid cartilage shell oligosaccharides on the main effector organs of insulin in T2DM mice Diabetes is caused by an absolute or relative deficiency of insulin; the pancreas's ability to produce insulin depends on its structure and the number of islet cells in the pancreas. Figure 17 In the NC group, the islets were oval-shaped with a regular structure, uniform boundaries, and oval-shaped, neatly arranged nuclei. Conversely, in the DC group, the islets were atrophied, the number of islet cells decreased, the cell distribution was disordered, and the nuclei were irregularly shaped. In the LCOS and MCOS groups, the number of islet cells increased, while in the HCOS group, the islets returned to normal.

[0097] Type 2 diabetes mellitus (T2DM) is often associated with alterations in hepatic lipid metabolism and fatty liver disease. Therefore, after COS intervention, HE staining was performed on mouse livers to observe any physiological abnormalities. Normal mouse liver tissue structure was intact, with hepatocytes arranged radially and orderly with clear nucleocytoplasm, while the DC group exhibited typical characteristics of metabolic liver injury, including blurred hepatic sinusoidal structure, hepatocyte steatosis and disordered arrangement, and increased inflammatory cell infiltration. After 4 weeks of COS intervention, hepatocyte steatosis was alleviated in the LCOS and MCOS groups, and inflammatory cell infiltration was improved. In the HCOS group, liver structure nearly returned to normal, and the regularity of hepatocyte arrangement was significantly improved. Therefore, the experimental results indicate that squid cartilage (COS) has a restorative and protective effect on pancreatic islet cells and liver tissue, and can improve histological damage.

[0098] (3) Oligosaccharides from squid cartilage shell regulate lipid metabolism Dyslipidemia is a key characteristic of patients with type 2 diabetes mellitus (T2DM). High levels of total cholesterol (TC), triglycerides (TG), and LDL-C, along with low levels of high-density lipoprotein cholesterol (HDL-C), can lead to atherosclerosis and cardiovascular disease. Serum TC (TC) in T2DM mice... Figure 18 a), TG ( Figure 18 (b) and LDL-C ( Figure 18 The level of c in the middle group was significantly higher than that in the NC group mice. P <0.05). Conversely, HDL-C levels were significantly reduced ( Figure 18 The results (d) indicate that T2DM mice have severe lipid metabolism disorders. Furthermore, after squid cartilage COS intervention, the levels of TC, TG, and LDL-C in MCOS and HCOS groups were significantly reduced (d). P <0.05), while HDL-C levels were significantly elevated ( P <0.05). This indicates that squid cartilage MCOS and HCOS effectively reduced TC, TG, and LDL-C levels and increased HDL-C levels in T2DM mice, thereby improving lipid metabolism regulation and reducing the risk of cardiovascular diseases associated with T2DM.

[0099] (4) Oligosaccharides from squid cartilage shell regulate the inflammatory environment In type 2 diabetes mellitus (T2DM), abnormal macrophage activation and excessive T cell differentiation lead to the over-secretion of inflammatory factors (such as IL-6 and TNF-α), which in turn causes local and even systemic inflammation, resulting in insulin resistance. Serum IL-6 levels in T2DM mice are significantly higher than normal. Figure 19 (a) and TNF-α ( Figure 19 The level of b in the middle group was significantly higher than that in the NC group mice. P <0.05 indicates a strong inflammatory response in T2DM mice. After intervention with squid cartilage COS, the expression levels of IL-6 and TNF-α in MCOS and HCOS group mice were significantly reduced ( P <0.05). This indicates that squid cartilage MCOS and HCOS significantly reduced IL-6 and TNF-α levels in T2DM mice, and the underlying mechanism may be related to the inhibition of macrophage-mediated inflammatory responses or the regulation of immune homeostasis.

[0100] (5) Oligosaccharides from squid cartilage shell enhance antioxidant capacity. During type 2 diabetes mellitus (T2DM), the body experiences excessive levels of reactive oxygen species (ROS), leading to oxidative stress damage, lipid peroxidation, increased MDA levels, and heightened discomfort. Superoxide dismutase (SOD) can scavenge ROS, and its higher activity indicates stronger antioxidant properties. This study evaluated the antioxidant effect of squid cartilage COS on T2DM mice by measuring serum MDA, SOD, and T-AOC levels, as well as liver MDA and SOD levels. Compared with the control group, serum and liver SOD activity in T2DM mice (…) Figure 20 As shown in a, and Figure 20 (as shown in d) and T-AOC level ( Figure 20 As shown in c), serum and liver MDA levels were significantly reduced (P<0.05). Figure 20 b and Figure 20 The levels of MDA (as shown in e) were significantly increased (P<0.05). This indicates that lipid peroxidation occurred in the body, disrupting normal physiological functions and significantly reducing antioxidant capacity. Squid cartilage COS intervention significantly improved MDA content, SOD activity, and T-AOC levels in T2DM mice (P<0.05), enhanced oxidative stress, and the effects of MCOS and HCOS were dose-dependent (P<0.05). This suggests that squid cartilage MCOS and HCOS can restore the antioxidant defense system in high-fat diet-streptozotocin-induced T2DM mice.

[0101] (6) Oligosaccharides from squid cartilage shell improve liver function Diabetes mellitus leads to disordered glucose and lipid metabolism, steatosis, and oxidative stress in the liver. Liver disease further exacerbates liver dysfunction (IR) and blood glucose dysregulation. The release of liver enzymes such as ALT and AST reflects the damage to hepatocyte membranes caused by oxidative stress. According to... Figure 21Data showed that serum ALT and AST levels in the DC group were significantly higher than those in the NC group. P <0.05. However, intervention with squid cartilage MCOS and HCOS in T2DM mice significantly reduced serum ALT and AST levels ( P <0.05). This indicates that squid cartilage MCOS and HCOS can improve abnormal liver function and enhance liver metabolism in T2DM mice, thereby regulating blood glucose homeostasis.

[0102] (7) Oligosaccharides from squid cartilage shell activate the insulin signaling pathway To further elucidate the potential pathway by which squid cartilage COS alleviates type 2 diabetes mellitus (T2DM), western blotting was used to determine the protein expression of the PI3K / Akt pathway in mouse liver. Among numerous insulin signaling pathways, PI3K / Akt is the most important. PI3K is located upstream of Akt, and PI3K activation is fundamental to Akt activation. Activated Akt then participates in and activates the pathway of liver glycogen synthesis, promoting glycogen production. The expression of PI3K, p-Akt, and Akt is shown below. Figure 22 As shown. Compared with the NC group mice, the expression of p-Akt / Akt and PI3K / β-actin proteins in T2DM mice was significantly reduced (as shown). P <0.05%. Compared with the DC group, the positive drug intervention led to a significant upregulation ( P <0.05. Furthermore, LCOS, MCOS, and HCOS significantly reversed the decrease in p-Akt / Akt and PI3K / β-actin protein expression in the DC group ( P <0.05). Therefore, it can be inferred that squid cartilage COS can alleviate IR by activating the expression of the PI3K / Akt pathway that affects T2DM, and promote hepatocyte uptake of glucose and glycogen synthesis.

[0103] (8) Oligosaccharides from squid cartilage shell regulate short-chain fatty acid levels Short-chain fatty acids are products of fermentation by major gut microbes. They play a role in combating type 2 diabetes mellitus (T2DM) by protecting the intestinal barrier, regulating the secretion of gastrointestinal hormones, regulating glucose and lipid metabolism, and improving insulin sensitivity. Short-chain fatty acids mainly fall into two categories: one is primarily produced through the fermentation of dietary fiber by anaerobic gut microbiota, such as acetic acid, propionic acid, butyric acid, and valeric acid; the other mainly originates from the catabolism of branched-chain amino acids, such as isobutyric acid and isovaleric acid.

[0104] like Figure 23As shown, the DC group exhibited a significant decrease in both individual short-chain fatty acids and total short-chain fatty acids compared to the NC group (P<0.05). This indicates that T2DM causes intestinal microecological dysbiosis, thereby reducing the production of short-chain fatty acids. Compared to the DC group, squid cartilage COS significantly reversed the decrease in short-chain fatty acid content in the feces of T2DM mice (P<0.001), and promoted the production of higher concentrations of acetic acid (P = 0.007), propionic acid (P = 0.002), butyric acid (P<0.001), isovaleric acid (P<0.05), valerate (P<0.05), and isohexanoic acid (P = 0.003), while no significant differences were observed in isobutyric acid and hexanoic acid. This suggests that squid cartilage COS may increase short-chain fatty acid content by enhancing intestinal flora metabolism and promoting the growth of beneficial bacteria. Acetic acid can serve as a major energy source for ATP production by intestinal mucosal cells, ensuring the integrity of the intestinal wall barrier and helping to prevent intestinal inflammation. Furthermore, acetic acid has been shown to activate the G protein-coupled receptor 43 signaling pathway, increasing skeletal muscle glucose transporter protein 4 translocation and glucose uptake, thus improving glucose metabolism. Propionate has the function of regulating appetite and lowering blood lipids. The regulation of glucose and lipid metabolism by squid cartilage (COS) is associated with increased levels of acetic acid and propionate. Butyrate is a major energy source for colonic cells and can enhance the absorption of sodium and water in the colon; butyrate plays a key role in lowering blood glucose, improving weight control, and insulin sensitivity. Increased butyrate helps inhibit the acetylation of two subunits of nuclear factor κB, P50 and P65, and reduces the secretion of pro-inflammatory cytokines such as TNF-α and IL-6, further demonstrating that squid cartilage (COS) can regulate inflammation and alleviate insulin resistance by promoting butyrate production.

[0105] 2.3 Oligosaccharides from squid cartilage shell improve gut microbiota The gut microbiota is a key element of the intestinal barrier, significantly influencing various pathophysiological processes, including maintaining intestinal barrier integrity, immune system stability, and metabolism. This study analyzes the α-diversity of squid cartilage shell oligosaccharides at the operational taxonomic level.

[0106] (1) α-diversity analysis at the operational taxonomic level Alpha-diversity analysis was used to determine the abundance of microbial diversity at the operational taxonomic unit (OTU) level. The Chao and Ace indices quantify the number of gut microbiota species, while the Shannon index is used to assess the evenness of species distribution. Figure 24 As shown, the Chao, Shannon, and Ace indices of mice in the DC group were significantly lower than those in the NC group. P <0.01), compared with the DC group, the Chao, Shannon, and Ace indices of mice in the COS group were significantly increased ( P<0.01). Notably, there was no significant difference in Shannon index between the COS group and the NC group. This indicates that squid cartilage (COS) can improve the abundance and evenness of gut microbiota distribution in T2DM mice.

[0107] (2) β-diversity analysis at the genus level PCA and PCoA were used to analyze the similarity between bacterial communities at the genus level, revealing intergroup differences in the overall structure of the bacterial community. Figure 25 As shown, the microbial community structure was completely separated between the DC and NC groups, and squid cartilage COS improved the abundance of gut microbiota, making it closer to that of the NC group. This indicates that squid cartilage COS can reverse the disrupted composition of the gut microbiota, bringing it closer to a normal physiological state.

[0108] (3) Composition of gut microbiota at the operational taxonomic level Venn diagram analysis was used to analyze the overall gut microbiota composition at the operational taxonomic unit (OTU) level. Overlapping regions reflect the proportion of shared OTUs, thus clarifying the similarities and uniqueness between different gut microbiota groups. For example... Figure 26 As shown, the number of OTUs in the NC, DC, and COS groups were 607, 386, and 541, respectively. A total of 323 OTUs were found in all samples, indicating the presence of similar gut microbiota in the NC, DC, and COS groups. Analyzing the number of shared OTUs between each pair of groups, the COS group had 515 OTUs, and the NC and DC groups had 341 OTUs. This suggests that after COS intervention with squid cartilage, the gut microbiota composition was closer to that of the NC group, showing a significant difference from the gut microbiota of the DC group.

[0109] (4) Composition of gut microbiota at the phylum level To investigate the regulatory role of squid cartilage COS at the phylum level on gut microbiota composition, a heatmap analysis was performed. The gut microbiome mainly belongs to six phyla: Firmicutes (… Firmicutes Bacteroidetes ( Bacteroidota The gut microbiota are categorized into four phyla: Actinobacteria, Proteobacteria, Clostridium, and Verrucous Microbes. Among these, Firmicutes and Bacteroidetes are the most prevalent, accounting for 90% of the gut microbiota. Figure 27 As shown in Figure a, the main phyla were Firmicutes and Bacteroidetes, which is consistent with the basic composition of the gut microbiota. After 4 weeks of intervention with squid cartilage (COS), its relative abundance was over 92%. Compared with the DC group, the relative abundance of Firmicutes in the COS group was significantly reduced ( P <0.01), while the relative abundance of Bacteroidetes was significantly increased ( P<0.01). A decreased Firmicutes to Bacteroidetes (F / B) ratio predicts improved gut homeostasis, and this decrease promotes the production of short-chain fatty acids, which are involved in carbohydrate metabolism. In colonic tissue, carbohydrates are metabolized into short-chain fatty acids with the participation of gut microbiota, which can reduce fat deposition in the liver, activate receptors in the liver and pancreas, thereby improving glucose and lipid metabolism. Figure 27 The results showed that the F / B ratio in the DC group was significantly higher than that in the NC group. P <0.05), while COS intervention can significantly reduce the F / B ratio ( P <0.05). Experiments show that squid cartilage COS can reduce the F / B ratio, which may promote the production of short-chain fatty acids and improve glucose and lipid metabolism.

[0110] (5) Composition of gut microbiota at the genus level Thermographic analysis further clarifies the regulatory role of squid cartilage (COS) on gut microbiota composition at the genus level. Figure 28 The display shows that, compared to the NC group, the DC group Dubosiella ,norank_f__Desulfovibrionaceae, Turicibacter , Coriobacteriaceae UCG-002 Akkermansia , Bifidobacterium , Mucispirillum and Blautia It has a significantly higher abundance, while Allobaculum , Bacteroides , Helicobacter , Ruminococcus , Candidatus_Arthromitus , Muribaculum , Prevotellaceae UCG-001 Lachnospiraceae_ UCG-001 Enterorhabdus , Parabacteroides , Lachnospiraceae NK4A136 group Alistipes and Roseburia Abundance was significantly reduced. Conversely, compared to the DC group, the COS group showed a significant increase. Candidatus_Arthromitus , Muribaculum , Prevotellaceae UCG-001 Lachnospiraceae_ UCG-001 Enterorhabdus , Parabacteroides , Lachnospiraceae NK4A136 group Alistipes and RoseburiaThe abundance of [unclear - possibly referring to a specific type of microbial activity or component]. Experiments showed that most microbial changes in the DC group were significantly reversed after squid cartilage (COS) intervention. Furthermore, carbohydrates are an important energy source for the gut microbiota; therefore, the anti-T2DM efficacy of squid cartilage (COS) may be primarily attributed to the remodeling of the gut microbiota.

[0111] Dubosiella High abundance of COS can reflect elevated blood sugar levels in individuals with type 2 diabetes mellitus (T2DM). Squid cartilage (COS) can significantly reduce... Dubosiella The abundance of COS corroborates the finding that COS can regulate glucose metabolism. This example demonstrates that... Turicibacter The abundance of bacteria was higher in the DC group than in the NC group, and higher abundance indicated greater inhibition of acetic acid synthesis. Squid cartilage COS could reduce... Turicibacter The bacterial abundance is reduced to the lowest level to regulate the acetic acid synthesis process. Mucispirillum Bacterial abundance was positively correlated with inflammation. In this embodiment, squid cartilage COS could reduce... Mucispirillum Abundance regulates the inflammatory environment of the body.

[0112] It is worth noting that in this embodiment, the DC group produced shorter-chain fatty acids compared to the NC group. Lachnospiraceae NK4A136 group Alistipes , Lachnospiraceae_ UCG-001 and Roseburia The abundance of squid cartilage (COS) decreases, while its abundance increases, thereby improving hyperglycemia, lipid metabolism, and inflammation. Enterorhabdus It is a bacterium that can degrade amino acids to produce butyric acid. The COS intervention in squid cartilage reversed the progression of T2DM in mice. Enterorhabdus The decreasing abundance of butyrate promotes butyrate production, which in turn regulates inflammation and alleviates inflammation-related inflammation (IR).

[0113] Combination Figure 28 As shown, Akkermansia The high abundance in the DC group may be related to self-protective mechanisms occurring in the gut. Akkermansia It is considered a "next-generation probiotic" because it can reduce inflammation, inhibit hepatocyte death, promote the integrity of intestinal epithelial cells, and alleviate glucose intolerance associated with type 2 diabetes mellitus (T2DM). This indicates that... Akkermansia This may be beneficial for the corresponding disease models. Therefore, in the DC group Akkermansia The relatively high abundance of this genus may be an adaptation of mice to type 2 diabetes mellitus (T2DM), which is beneficial to their health. Furthermore, research indicates that... Akkermansia The abundance of [a substance] was positively correlated with the concentration of mucin in the cecum. In the DC group... Akkermansia The high bacterial abundance was attributed to the low amylose content in the high-fat diet.

[0114] In this embodiment, the DC group BifidobacteriumHigher bacterial abundance is associated with self-protective mechanisms in the gut and higher availability of glucose and oligosaccharides in the large intestine and colon. Parabacteroides It is associated with reducing inflammation and IR, as well as enhancing intestinal integrity. In this embodiment, squid cartilage COS intervention increased... Parabacteroides Abundance, in order to achieve anti-T2DM effects through anti-inflammatory and IR reduction.

[0115] (6) Classification abundance and LDA discriminant analysis by LEfSe analysis The LEfSe method is used to compare the microbial composition of specific bacterial taxa among groups. Figure 29 The LEfSe results indicated that the important microbial communities of the COS group were o__Bacteroidales, o__Lachnospirales, o__Peptococcales, and o__Monoglobales, extending to... g__Odoribacter , g__Alistipes , g__Rikenella , g__ Rikenellaceae_ RC9_gut_group, g__Lachnospiraceae_ NK4A136_group g__ Roseburia , g__GCA-900066575 , g__Eubacterium_xylanophilum _group、 g__ASF356 , g__ Lachnospiraceae_ UCG-001 g__A2、g__Eisenbergiella , g__norank_f__Peptococcaceae and g__Mon-globe LDA discriminant analysis can identify characteristic differences in gut microbiota under different physiological states, screen key microbiota biomarkers, and assist in disease diagnosis and microbiota function research. An LDA > 3.5 is considered to indicate a significant difference.

[0116] LDA discriminant analysis ( Figure 30 The results showed that several significant categories with LDA > 4 were identified in the DC group, belonging to the phylum Firmicutes c__Bacilli, f__Erysipelotrichaceae, o__Erysipelotrichales, and g__DubosiellaThe following phyla, belonging to the phylum *Desulfovibrionia*, *Desulfovibrionaceae*, *Desulfovibrionales*, and *gnorankf__Desulfovibrionaceae*, were identified. This indicates that Firmicutes and *Desulfovibrionia* phyla were significantly enriched in the DC group. In the COS group, several significant categories with LDA > 4 were identified, belonging to the phylum *Bacteroidetes*, including *c__Bacteroidia*, *p__Bacteroidota*, *o__Bacteroidales*, and *f__Rikenellaceae*. g__ Odoric bacteria and g__Alistipes ; Belongs to the phylum Firmicutes, f__Lachnospiraceae, g__ Lachnospiraceae_ NK4A136_group and o__Lachnospirales; belonging to the phylum Proteobacteria, f__Marinifilaceae. This indicates that Bacteroidetes, Firmicutes, and Proteobacteria are significantly enriched in the COS group. Bacteroidetes are beneficial bacteria in the gut, helping to maintain normal immune status. Furthermore, the DC group had a higher abundance of Firmicutes, while the COS group had a higher abundance of Bacteroidetes, suggesting that Firmicutes and Bacteroidetes are key gut microbiota influencing T2DM, which corroborates the results of the gut microbiota composition analysis.

[0117] The above results illustrate g__Dubosiella f__Desulfovibrionaceae are the main factors contributing to T2DM dysbiosis, while g__Odoribacter , g__Lachnospiraceae_ NK4A136_group and g__Alistipes This is the key reason why T2DM has been improved. g__Dubosiella High levels of lipopolysaccharide (LPS) accumulation in patients with type 2 diabetes mellitus (T2DM) can reflect their hyperglycemia. Furthermore, in T2DM patients, a high-fat diet and impaired gastrointestinal motility lead to the abnormal proliferation of Gram-negative bacteria containing LPS. Simultaneously, disruption of the intestinal barrier and increased intestinal permeability promote the absorption of more LPS into the bloodstream.

[0118] Lipopolysaccharide can induce metabolic endotoxemia, increase pro-inflammatory factors, and exacerbate obesity and the development of type 2 diabetes mellitus. f__Desulfovibrionaceae It is a Gram-negative bacterium that releases lipopolysaccharides in the intestine. In this embodiment, squid cartilage (COS) can reduce the levels of [certain substances] in the feces of T2DM mice. f__Desulfovibrionaceae To block the production of lipopolysaccharide, thereby inhibiting the worsening of T2DM symptoms. g__AlistipesIt can produce acetic acid and butyric acid, improve glucose utilization, promote M2 polarization of macrophages, reduce inflammatory responses, and reverse the adverse effects of a high-fat diet. In this embodiment, squid cartilage COS may be utilized... g__Odoribacter , g__Lachnospiraceae_ NK4A136_group and g__Alistipes The gut microbiota can reduce inflammatory responses, improve insulin resistance (IR), enhance glucose metabolism, and increase short-chain fatty acid levels, thereby achieving the effect of resisting type 2 diabetes mellitus (T2DM).

[0119] (7) Correlation between gut microbiota abundance and type 2 diabetes biomarkers To elucidate the association between different microorganisms and T2DM-related metabolic indicators (including glucose and lipid metabolism, oxidative stress, inflammatory factors, and short-chain fatty acids), Spearman correlation analysis was performed. Figure 31 The results showed that at the phylum level, Bacteroides were significantly positively correlated with SOD, HDL-C, T-AOC, single short-chain fatty acids, and total short-chain fatty acids, while they were significantly negatively correlated with TNF-α, GSP, TG, ALT, AST, IL-6, TC, homeostasis model assessment of insulin resistance (HOMA-IR), LDL-C, and MDA. Dethiobacteria (…) Desulfobacterium ), phylum deferrobacteria ( Deferribacterota Firmicutes ( Firmicutes ) and Actinobacteria ( Actinobacteria The results were the opposite. This indicates that Bacteroidetes, Dethiobacteria, Deferobacteria, Firmicutes, and Actinobacteria are closely related to the occurrence and development of T2DM.

[0120] Bacteroides, a genus within the Bacteroidetes phylum, is one of the major bacterial groups producing short-chain fatty acids, with propionic acid being its primary fermentation product. Propionic acid significantly inhibits fatty acid synthesis and cholesterol production in the liver. This invention experimentally demonstrates that Bacteroides promotes the production of short-chain fatty acids. Furthermore, Dethiobacterium, a microorganism that reduces sulfur compounds via sulfite reductase and degrades them to produce butyrate, plays a crucial role in energy metabolism. This invention also concludes that Dethiobacterium is detrimental to butyrate production. In addition, there is a positive correlation between Firmicutes and the pro-inflammatory cytokine IFN-γ, as well as between Firmicutes and IL-6. Firmicutes stimulate macrophages to secrete pro-inflammatory markers, leading to chronic inflammation. This invention also demonstrates the pro-inflammatory properties of Firmicutes.

[0121] Spearman correlation analysis clarified the relationship between various bacterial species and T2DM-related metabolic indicators at the genus level. Figure 32 The data shows that at the genus level, norank_f__Muribaculaceae, Mouse stick , Allobacillus , Candidate_ Arthromyoma and Odoric bacteria There is a certain positive correlation between biochemical indicators related to the improvement of T2DM, such as glucose and lipid metabolism, antioxidant capacity, inflammatory response, short-chain fatty acids, and IR. Mucispirillum and norank_f__ Desulfovibrionaceae There is a negative correlation with this. Furthermore, other bacteria do not exhibit such a uniform pattern, but they are correlated with changes in some of these indicators. Enterorhabdus , Rikenellaceae_ RC9_gut_group, Lachnospiraceae_ NK4A136_group Common grebe and Eubacterium_xylanophilum_ There is a positive correlation between the group and the formation of short-chain fatty acids (acetic acid, propionic acid, butyric acid, isovaleric acid, valeric acid, and isohexanoic acid). Prevotellaceae_ UCG-001 Eubacterium_xylanophilum _group Desulfovibrio , Lachnospiraceae_ NK4A136_group and Alistipe It has a positive correlation with SOD and T-AOC, and a negative correlation with MDA. Enterorhabdus , Lachnospiraceae_ NK4A136_group and Alistipe It is associated with elevated HDL-C and has a negative correlation with TG, TC and LDL-C. Lachnospiraceae_ NK4A136_group Prevotellaceae_ UCG-001 Rikenella , Alistipe and Eubacterium_xylanophilum_ The group showed a negative correlation with pro-inflammatory factors TNF-α and IL-6. Lachnospiraceae_ NK4A136_group and Alistipe A negative correlation was found with HOMA-IR. A significant correlation was found between the gut microbiota and T2DM biomarkers. Therefore, squid cartilage (COS) can exert a synergistic effect by regulating glucose and lipid metabolism, increasing short-chain fatty acid content, and modulating gut microbiota composition to alleviate T2DM symptoms.

[0122] The squid cartilage COS prepared in this invention can alleviate abnormal glucose and lipid metabolism and insulin resistance (IR) in T2DM mice induced by a high-fat diet and streptozotocin. The squid cartilage COS significantly reduces fasting blood glucose levels in T2DM mice, improves glucose tolerance, slows the rate of non-enzymatic glycation in T2DM mice, reduces liver glycogenolysis and promotes liver glycogen synthesis, thus improving glucose metabolism disorders. T2DM patients often have varying degrees of lipid metabolism disorders; squid cartilage COS regulates lipid metabolism in T2DM mice by reducing TC, TG, and LDL-C levels and increasing HDL-C levels.

[0123] Squid cartilage (COS) can alleviate inflammation by reducing serum TNF-α and IL-6 levels in type 2 diabetes mellitus (T2DM) mice. The release of liver enzymes such as ALT and AST indicates oxidative stress damage to hepatocyte membranes, and COS can improve abnormal liver function in T2DM mice by reducing serum ALT and AST levels. The PI3K / Akt signaling pathway is one of the key pathways regulating insulin levels and is closely related to the pathogenesis of insulin resistance (IR). It plays a very important role in insulin-regulated glucose and lipid metabolism. Squid cartilage (COS) can alleviate IR by activating the PI3K / Akt signaling pathway, promoting hepatocyte uptake of glucose and glycogen synthesis. Squid cartilage (COS) intervention can significantly increase the Chao1, Shannon, and Ace indices in mice, making the abundance and composition of gut microbiota closer to the normal physiological state.

[0124] The above examples only represent the technical solutions of this invention and are not intended to limit the experiments. Although we have improved the experimental scheme, researchers in the same field can still further improve the experimental scheme described above or make scientifically equivalent substitutions for the experimental steps. These changes do not cause the substance of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed in this invention.

Claims

1. A method for preparing squid cartilage chitin oligosaccharides derived from squid cartilage, characterized in that, The preparation method of the squid cartilage shell oligosaccharide includes the following steps: (1) Raw material processing: Wash the squid cartilage with water, dry it overnight at 25~30℃, grind and sieve to obtain powder; (2) Desalination treatment: The powder is washed with hydrochloric acid and water in sequence until the washing effluent is neutralized, and then washed with distilled water until neutral. After washing, it is dried to obtain squid cartilage powder. (3) Enzymatic decomposition of protein: The squid cartilage powder is mixed with a protease solution and fully enzymatically hydrolyzed. After enzymatic hydrolysis, it is dried to obtain β-chitin powder. (4) The β-chitosan powder is mixed with NaOH solution and reacted at 80~100℃. After the reaction is completed, it is washed and dried to prepare squid cartilage β-chitosan. (5) The squid cartilage β-chitosan was fully dissolved in acetic acid solution, and then an acetate-sodium acetate buffer solution with pH 5-6 was added to prepare a β-chitosan solution. Cellulase was added to the β-chitosan solution and fully enzymatically hydrolyzed at 50-55℃. After the enzymatic hydrolysis was completed, the enzyme was inactivated. Then, the pH of the enzyme-inactivated solution was adjusted to 7.0-7.5 with NaOH solution to precipitate the unreacted β-chitosan. After the hydrolysis was completed, the supernatant was collected by centrifugation. The supernatant was dialyzed and dried to obtain squid cartilage chitosan oligosaccharide.

2. The preparation method according to claim 1, characterized in that, In step (3), the protease is a complex enzyme composed of alkaline protease and trypsin, with the mass ratio of alkaline protease to trypsin being 1:1~3; the amount of the complex enzyme added is 3~5% based on the mass of squid cartilage powder, and the material-liquid ratio of squid cartilage powder to protease solution is 1:8~10.

3. The preparation method according to claim 2, characterized in that, The conditions for enzymatic hydrolysis in step (3) include: pH 7.0~8.0, hydrolysis temperature 40~45℃, and hydrolysis time 5~6 h.

4. The preparation method according to claim 2, characterized in that, The optimized preparation method includes the following: the protease is a complex enzyme composed of alkaline protease and trypsin, with a mass ratio of 1:2; the ratio of squid cartilage powder to protease solution is 1:8; the amount of the complex enzyme added is 3% based on the mass of squid cartilage powder; the enzymatic hydrolysis conditions in step (3) include: pH 7.9, hydrolysis temperature 40℃, and hydrolysis time 6 h.

5. The preparation method according to claim 1, characterized in that, The cellulase activity is 500~1000 U / g.

6. The squid cartilage oligosaccharide prepared by any one of the preparation methods according to claims 1 to 5.

7. The squid cartilage chitin oligosaccharide according to claim 6, characterized in that, The squid cartilage shell oligosaccharide contains oligomers with a degree of polymerization of 2 to 6.

8. The squid cartilage shell oligosaccharide according to claim 6, characterized in that, The squid cartilage shell oligosaccharide is a positively charged oligosaccharide.

9. The use of the squid cartilage shell oligosaccharide according to claim 6 in the preparation of a medicine or food for relieving or treating type II diabetes.

10. The application according to claim 9, characterized in that, The drug or food contains squid cartilage oligosaccharide at a mass content of 100-400 mg / kg.

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