Preparation method of corncob polysaccharide and application thereof as lipid inhibitor

Abstract

The present application relates to a kind of preparation method of corncob polysaccharide and its application as lipid inhibitor.A kind of preparation method of corncob polysaccharide includes the steps of preparing defatted corncob dry powder, alkali extraction crude polysaccharide, enzymatic hydrolysis purification, alcohol precipitation and refining, etc., through defatting, ultrasonic alkali extraction, enzymatic hydrolysis purification, low-temperature alcohol precipitation and freeze-drying etc., realize the efficient preparation of corncob polysaccharide from corncob, improve the comprehensive utilization value of corncob, promote the high-value utilization of bulk agricultural and sideline products;The application of polysaccharide component extracted from corncob as lipid inhibitor is also disclosed, the corncob polysaccharide component of the present application can effectively improve the weight gain and fat deposition of obese mice, providing a new solution for the prevention and treatment of obesity, which can be used as fat substitute or functional food ingredient of low-fat food.

Description

Technical Field

[0001] This invention relates to a method for preparing corn cob polysaccharides, and the application of polysaccharide components extracted from corn cobs as lipid inhibitors. Background Technology

[0002] Corn, scientifically known as Zea mays L., is an annual tall herbaceous plant belonging to the genus Zea in the family Poaceae. It originated in Latin America and is one of the world's three major staple crops.

[0003] Maize is widely distributed throughout China and is one of the country's important food, feed, and industrial crops, ranking among the top in terms of both planting area and yield. As one of the crops with strong drought resistance, it has high adaptability and cultivation flexibility.

[0004] The kernels of corn are spherical or oblate caryopsis-shaped grains arranged in an ear, and are its main economic part. The corn cob is the axis of the corn ear after the kernels have been removed. In corn processing, the corn cob is a major by-product, with only a small amount used in pig feed, cardboard, cement board, and cement brick production, resulting in low overall utilization and resource waste. Despite the abundance of corn cob resources, research on its polysaccharide components is relatively limited.

[0005] In terms of existing technology, Chinese patent application number 201110082417.3 discloses a method for extracting polysaccharides from corn cobs, including steps such as drying corn cobs, crushing, extracting supernatant, and ethanol extraction. The product obtained by this patent is "crude polysaccharide", which has a complex composition and needs further purification before it can be used in medicine or food.

[0006] Chinese patent application number 202011165396.7 discloses a method for extracting functional sugars from corn cobs. The steps are as follows: soaking corn cobs in a first tank for half an hour, then draining the water; transferring the drained corn cobs to a second tank and dehydrating them with 0.5-5% sulfuric acid for 5 hours; drying the dehydrated corn cobs at 50°C for 12 hours; placing the dried corn cobs in a steam explosion tank for steam explosion; adding alkaline solution to the obtained supernatant to make the pH greater than 7; adding a certain amount of deionized water to the product after steam explosion and centrifuging it at a certain speed; and decolorizing, desalting, concentrating, and crystallizing the liquid to obtain a functional sugar product with high purity. This technical solution mainly relies on steam explosion + acid treatment. The steam explosion conditions fluctuate greatly, making it difficult to guarantee product consistency; and the acid treatment process easily leads to sugar degradation and impaired product activity.

[0007] Chinese patent application number 202211706516.9 discloses a method for producing polysaccharides using corn cob fermentation. The method involves mixing pretreated corn cobs with cellulase, *Lactobacillus acidophilus* powder, *Streptococcus thermophilus* powder, and distilled water; adjusting the pH; stirring thoroughly; sealing; and incubating for fermentation. The fermented corn cobs are then mixed with distilled water, stirred thoroughly, filtered to extract the fermentation broth, and then concentrated. The concentrated broth is mixed with ethanol, stirred thoroughly, and allowed to stand to precipitate the polysaccharides. The precipitated mixture is centrifuged, and the supernatant is removed. This step is repeated 3-5 times to remove impurities. The precipitate after impurity removal is dried to obtain the fermented polysaccharides. While this method utilizes microbial fermentation and achieves waste utilization, the products are mixed, have low purity, and have a long production cycle. The product is a mixed sugar, mainly used as a feed additive, with low added value.

[0008] Based on the existing technological background, this invention conducts a comprehensive and in-depth study on corn cob polysaccharides and designs a new preparation process, aiming to improve the process controllability, technology integration, and product added value to a certain extent, and provide further scientific basis and technical support for the development and utilization of corn cob polysaccharides. Summary of the Invention

[0009] To address the shortcomings of existing technologies, this invention proposes a method for preparing corn cob polysaccharides, and also discloses the application of corn cob polysaccharides extracted by this method as lipid inhibitors.

[0010] The technical solution adopted in this invention is as follows:

[0011] A method for preparing corn cob polysaccharide includes the following steps:

[0012] S1. Prepare defatted corn cob powder;

[0013] S11. Drying and pulverizing: Select corn cobs without mold, wash them and dry them in an oven. After pulverizing them, sieve them to remove coarse particles and obtain fine corn cob powder.

[0014] S12. Degreasing: Add n-hexane to the corn cob powder and stir to degrease;

[0015] S13. Centrifugal drying: After centrifugation, discard the supernatant and air-dry the resulting precipitate at room temperature to obtain defatted corn cob powder;

[0016] S2. Alkali extraction of crude polysaccharides:

[0017] S21. Alkali extraction: The defatted corn cob powder obtained in step S1 is added to a 2% sodium hydroxide solution and extracted by ultrasonic extraction to obtain an ultrasonic extract.

[0018] S22. Centrifugation collection: After centrifuging the ultrasonic extract using a high-speed centrifuge, the supernatant is collected, which is the corn cob crude polysaccharide alkaline extract;

[0019] S23. Concentration: The corn cob crude polysaccharide alkaline extract is concentrated by rotary evaporation to obtain a concentrated corn cob crude polysaccharide solution;

[0020] S3. Enzymatic hydrolysis and purification: Adjust the concentrated corn cob crude polysaccharide solution obtained in step S2 to pH 7 for enzymatic hydrolysis. After enzymatic hydrolysis, inactivate the enzyme by boiling water bath for 30-60 min, cool to room temperature and centrifuge to collect the supernatant.

[0021] S4. Precipitation and Refining:

[0022] S41. Alcohol precipitation: Place the supernatant obtained in step S3 in an ice-water bath to pre-cool to a low temperature of 0-4℃, and slowly add 4 times the volume of 95% ethanol while stirring at a speed of 500-800 rpm. After the addition is complete, stir slowly at a speed of 100-200 rpm for 20-30 minutes, then stop stirring and let it stand at room temperature for 24 hours.

[0023] S42. Centrifuge and collect the precipitate: After centrifugation, discard the supernatant and collect the precipitate;

[0024] S43. Redissolution and concentration: The precipitate is redissolved with an appropriate amount of pure water and stirred in a constant temperature water bath at 90℃ for 2 hours to ensure that the polysaccharide is completely dissolved. Finally, the solution is concentrated by rotary evaporation, and after dialysis and freeze-drying, corn cob polysaccharide is obtained.

[0025] In step S12, the degreasing step includes a two-step degreasing step. First, n-hexane is added to the corn cob powder at a material-to-liquid ratio of 1:10. After stirring for 30 minutes to degrease, the mixture is centrifuged and the supernatant is discarded. Then, n-hexane is added to the precipitate at a material-to-liquid ratio of 1:15. The mixture is stirred and extracted for another 15 minutes, centrifuged again, and then air-dried at room temperature.

[0026] In step S12, the degreasing process is carried out by stirring in a constant temperature water bath at 40-45℃.

[0027] In step S3, papain is used for enzymatic hydrolysis. The amount of papain added is 0.4% of the total mass of the concentrated corn cob crude polysaccharide solution, and the hydrolysis conditions are 60℃ and 2h.

[0028] In the alkaline extraction step S21, the specific steps of ultrasonic extraction are as follows: the mixture of defatted corn cob powder obtained in step S1 and sodium hydroxide solution is placed in an ultrasonic-assisted device, the ultrasonic frequency is set to 40 kHz, and ultrasonic extraction is performed at 60-70℃ with a power of 200-500W for 15-20 minutes; the ultrasonic process is in intermittent mode, working for 2 seconds and then resting for 2 seconds, and the material-liquid ratio of defatted corn cob powder to sodium hydroxide solution is 1:15-25.

[0029] The ultrasonic extraction time was 15 minutes, with an ultrasonic power of 450W for the first 5 minutes and 250W for the next 10 minutes.

[0030] The composition of the corn cob polysaccharide, by mass fraction, is as follows: neutral sugar content is 89.20±1.93%, uronic acid content is 4.02±0.75%, and protein content is 6.89±0.26%.

[0031] The composition of the neutral sugars, by mass fraction, is as follows: xylose content is 57.37%, arabinose content is 23.15%, galactose content is 9.16%, and glucose content is 8.26%.

[0032] The present invention also discloses the application of corn cob polysaccharide prepared by the present invention as a lipid inhibitor.

[0033] Beneficial effects of the invention:

[0034] 1. This invention achieves efficient preparation of corn cob polysaccharides from corn cobs through a multi-step process including defatting, ultrasonic alkaline extraction, enzymatic purification, low-temperature alcohol precipitation and freeze drying. The purity of the obtained corn cob polysaccharides reaches 90%, which improves the comprehensive utilization value of corn cobs and promotes the high-value utilization of bulk agricultural by-products.

[0035] 2. The corn cob polysaccharide component of the present invention can effectively improve weight gain and fat deposition in obese mice, providing a new solution for the prevention and treatment of obesity;

[0036] 3. The weight loss effect is not achieved by suppressing appetite, but mainly by inhibiting lipase activity and adsorbing dietary fat. This can reduce discomfort during the weight loss process and can be used as a fat substitute for low-fat foods or as an ingredient in functional foods. Attached Figure Description

[0037] Figure 1 The molecular weight of a corn cob polysaccharide of the present invention is obtained based on gel permeation chromatography analysis.

[0038] Figure 2 The Fourier transform infrared spectrum of corn cob polysaccharide obtained by the preparation method of corn cob polysaccharide according to the present invention is shown.

[0039] Figure 3 X-ray diffraction pattern of the physicochemical properties of corn cob polysaccharides;

[0040] Figure 4 Thermogravimetric curve of corn cob polysaccharide;

[0041] Figure 5 The thermal decomposition curve of corn cob polysaccharide;

[0042] Figure 6 The graph shows the relationship between the viscosity and shear rate of corn cob polysaccharide.

[0043] Figure 7 A schematic diagram showing the experimental results of the oil-holding capacity of several polysaccharides;

[0044] Figure 8 The graph shows the relationship between the inhibition rate of corn cob polysaccharides on lipase in vitro;

[0045] Figure 9 The graph shows the relationship between the yield of mouse body weight in each group and the experimental time.

[0046] Figure 10 A graph showing the relationship between food intake and experimental time for each group of mice;

[0047] Figure 11 The results are the area under the curve (AUC) of each group of mice in the oral glucose tolerance test (OGTT);

[0048] Figure 12 The graph shows the weight gain rate of mice in each group at the end of the experiment;

[0049] Figure 13 The results of HE staining of white fat after dissection of three groups of mice are shown.

[0050] Figure 14 The changes in triglyceride content in the feces of mice in each group;

[0051] Figure 15 This shows the changes in lipase activity in the contents of different intestinal segments;

[0052] Figure 16 This is a corn cob polysaccharide sample prepared using the method of the present invention. Detailed Implementation

[0053] To make the invention's objectives, technical concepts, and advantages clearer, the following description, in conjunction with the appendix, is provided. Figures 1-16 The technical solution of the present invention will be described in further detail below. It should be understood that the following embodiments are only for explaining and illustrating preferred embodiments of the present invention, and should not be regarded as, nor constitute a limitation on the scope of patent protection claimed by the present invention.

[0054] Example 1:

[0055] This embodiment discloses a method for preparing corn cob polysaccharide, including the following steps:

[0056] S1. Prepare defatted corn cob powder;

[0057] S11. Drying and pulverizing: Select corn cobs without mold, wash them and dry them in an oven at 50℃. After pulverizing them, pass them through an 80-mesh sieve to remove coarse particles and obtain fine corn cob powder.

[0058] S12. Degreasing: Take 50g of fine corn cob powder and add 500ml of n-hexane to the corn cob powder at a material-to-liquid ratio of w / v=1:10. Stir in a constant temperature water bath at 45℃ for 30 minutes to defatting. The boiling point of n-hexane is approximately 69℃, and degreasing should be carried out in a well-ventilated area. Appropriate heating relative to room temperature can significantly improve the solubility and diffusion rate of lipids.

[0059] S13. Centrifugal drying: Centrifuge at 4500 r / min for 10 minutes. After centrifugation, discard the supernatant and air-dry the obtained precipitate at room temperature to obtain defatted corn cob powder.

[0060] S2. Alkali extraction of crude polysaccharides:

[0061] S21. Alkali extraction: The defatted corn cob powder obtained in step S1 is added to a 2% sodium hydroxide solution at a material-to-liquid ratio of 1:15. Extraction is performed using ultrasonic extraction. The mixture of defatted corn cob powder and sodium hydroxide solution is placed in an ultrasonic-assisted device and ultrasonically extracted at 65°C and 400W power for 15 minutes to obtain the ultrasonic extract. The ultrasonic process is intermittent, with a 2-second work cycle followed by a 2-second pause to prevent overheating.

[0062] S22. Centrifugation collection: After passing the ultrasonic extract through a high-speed centrifuge, centrifuge at 4500 r / min for 10 minutes and collect the supernatant, which is the corn cob crude polysaccharide alkaline extract;

[0063] S23. Concentration: The corn cob crude polysaccharide alkaline extract is concentrated by rotary evaporation to obtain a concentrated corn cob crude polysaccharide solution;

[0064] S3: Enzymatic hydrolysis and purification: The concentrated corn cob crude polysaccharide solution obtained in step S2 was adjusted to pH 7 for enzymatic hydrolysis. Papain was added for enzymatic hydrolysis. The amount of papain added was 0.4% of the total mass of the concentrated corn cob crude polysaccharide solution. The enzymatic hydrolysis conditions were 60℃ for 2 hours. After enzymatic hydrolysis, the enzyme was inactivated by boiling water bath for 30-60 minutes. After cooling to room temperature, the solution was centrifuged at 10000r / min for 10 minutes to remove impurities such as protein and starch. The supernatant was collected.

[0065] S4: Precipitation and Refining:

[0066] S41. Alcohol precipitation: Place the supernatant obtained in step S3 in an ice-water bath to pre-cool to a low temperature of 0-4℃, stir at 600 rpm for 10 min and slowly add 4 times the volume of 95% ethanol. After the addition is complete, stir at 200 rpm for 20-30 min, then stop stirring and let stand at room temperature for 24 hours.

[0067] Combining high-speed and low-speed stirring is an optimized strategy to address the physicochemical requirements at different stages of the alcohol precipitation process. First, high-speed mechanical stirring rapidly disperses ethanol into the entire polysaccharide solution upon addition. This avoids localized areas of excessively high ethanol concentration near the ethanol inlet, ensuring a synchronous and uniform supersaturation state throughout the system. This enhances the mixing efficiency of ethanol and the polysaccharide solution, prevents localized high concentrations from trapping impurities, and provides external shear force to promote the collision, growth, and sedimentation of polysaccharide particles. It fundamentally overcomes the slow process of static diffusion, creating optimal conditions for the formation of numerous fine crystal nuclei, laying the foundation for obtaining polysaccharide precipitates with uniform particle size. Simultaneously, it prevents impurity trapping. Insufficient stirring (such as low-speed or static ethanol addition) can cause localized high ethanol concentrations to rapidly precipitate polysaccharides, forming large and dense clumps. These clumps easily trap impurities such as water, salts, and pigments, leading to decreased product purity and difficulty in subsequent washing.

[0068] After crystal nuclei form, the system requires a relatively stable environment to allow dissolved polysaccharide molecules to migrate orderly and deposit on the surface of the crystal nuclei. The strong shear force generated by high-speed stirring will break up the newly formed, fragile micro-crystal nuclei and aggregates, hindering their growth. Switching to low-speed stirring provides sufficient gentle agitation, allowing polysaccharide molecules to collide and combine with the crystal nuclei, while avoiding destructive shearing. Therefore, after a period of high-speed stirring, low-speed stirring at 100-200 rpm can continuously provide gentle mixing without hindering crystal nuclei growth, promoting the maturation and enlargement of the precipitate particles, resulting in a more uniform overall particle size distribution and a denser structure. This leads to higher efficiency in subsequent centrifugation and a clearer supernatant.

[0069] S42. Centrifuge to collect precipitate: After centrifuging at 4500 r / min for 20 minutes, discard the supernatant and collect the precipitate;

[0070] S43. Redissolution and concentration: The precipitate was redissolved with an appropriate amount of pure water and stirred in a 90℃ constant temperature water bath for 2 hours to ensure that the polysaccharide was completely dissolved. Finally, the solution was concentrated by rotary evaporation, dialyzed with distilled water, and the water was changed every 2 hours. The solution was dialyzed in a 4℃ refrigerator for 48 hours and then freeze-dried to obtain corn cob polysaccharide.

[0071] In this embodiment, corn cobs of Suyu 28 were selected as the raw material for the preparation of corn cob polysaccharides, with a yield of 6.5%.

[0072] Example 2: This example describes a method for preparing corn cob polysaccharides. The similarities to Example 1 are not repeated here, but the difference lies in step S12, which includes a secondary defatting step. Specifically, n-hexane is added to the corn cob powder at a material-to-liquid ratio of 1:10, and the mixture is stirred for 30 minutes to defatted. After stirring and centrifugation, the supernatant is discarded. Then, n-hexane is added to the precipitate again at a material-to-liquid ratio of 1:15, and the extraction is continued for 15 minutes. The mixture is then centrifuged again and air-dried at room temperature. Advantages: The defatting effect is significantly improved, with only a slight increase in total solvent usage (total 1:15), but the effect is far better than a one-time 1:10 ratio. Equipment requirements are low, requiring only conventional laboratory equipment. It offers high cost-effectiveness and is suitable for small to medium-scale production.

[0073] In the alkali extraction step of S21, the ultrasonic extraction time is 15 minutes. The ultrasonic power is 450W for the first 5 minutes to rapidly disrupt the cell structure, and the ultrasonic power is 250W for the next 10 minutes for mass transfer extraction. This helps to reduce energy consumption and component damage while ensuring the extraction rate. The ultrasonic process is in intermittent mode, with a 2-second working interval followed by a 2-second rest interval.

[0074] In this embodiment, corn cobs of variety Suyu 28 were selected as the raw material for preparing corn cob polysaccharides. The final corn cob polysaccharide sample is shown below. Figure 16 As shown, the yield was 7.95±0.3%, and by mass fraction, the content of neutral sugars was 89.20±1.93%, the content of uronic acid was 4.02±0.75%, and the content of protein was 6.89±0.26%. Neutral sugars refer to monosaccharides that do not contain acidic groups such as carboxyl groups and are electrically neutral overall. The purity of the polysaccharide was characterized by the neutral sugar content. The corn cob polysaccharide prepared in this example had a purity of approximately 90% and was almost ash-free.

[0075] The experimental procedure for characterizing the structure of corn cob polysaccharides is as follows:

[0076] Determination of neutral sugar content using the phenol-sulfuric acid method:

[0077] Accurately weigh 5 mg of standard dextran into a 50 ml volumetric flask to prepare a 0.1 mg / ml standard glucose solution. Sequentially pipette 0, 0.2, 0.4, 0.6, 0.8, and 1.0 ml of the solution into separate test tubes, and add distilled water to each tube to a final volume of 1.0 ml. Then, add 1.0 ml of 3% phenol to each test tube and mix well. Slowly add 4.0 ml of concentrated sulfuric acid, shake well, and let stand for 30 minutes. Measure the absorbance at 490 nm. Perform instrument calibration using 1.0 ml of water and the same colorimetric method. Plot a standard curve of polysaccharide micrograms versus optical density values ​​on the x-axis and y-axis.

[0078] To prepare a 0.1 mg / ml sample of corn cob polysaccharide, take 1 ml of the sample, add 1.0 ml of 3% phenol, and quickly add 4.0 ml of concentrated sulfuric acid. Shake the sample on a vortex mixer to mix thoroughly. Let it stand for 30 minutes and measure the absorbance at 490 nm. Substitute the measured absorbance into the standard curve to obtain the polysaccharide concentration.

[0079] 1) Carbazole-sulfuric acid method for the determination of galacturonic acid: Using a 0.1 mg / ml galacturonic acid standard solution, pipette 0, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mL into each test tube, respectively. Add distilled water to each tube to a final volume of 1.0 mL. Slowly add 6 mL of pure sulfuric acid to each tube in an ice bath, shaking continuously. Then, incubate the tubes in a water bath at 85°C for 20 min, and then cool to room temperature. Add 0.2 mL of 0.1% carbazole-ethanol to each tube and maintain the solution at room temperature for 2 h. Measure the absorbance at 530 nm. Use 1.0 mL of water as a blank, following the same colorimetric procedure.

[0080] 2) Prepare a corn cob polysaccharide sample of 0.1 mg / ml. Take 1 ml of the sample and add 6 ml of pure sulfuric acid. Shake the sample on a vortex mixer to mix thoroughly. After incubating in a water bath at 85℃ for 20 min, remove the sample and cool it to room temperature. Then add 0.2 ml of 0.1% carbazole-ethanol and keep the sample at room temperature for 2 h. Measure the absorbance at 530 nm.

[0081] 3) Coomassie Brilliant Blue Method for Protein Content Determination: Using bovine serum albumin as a standard, the protein content was determined using the Bradford Protein Assay Kit. The procedure was performed according to the instructions.

[0082] 4) Molecular weight determination:

[0083] The molecular weight of PCP samples was determined by high-performance gel permeation chromatography (HPGPC) using an Agilent 1260HPL high-performance liquid chromatography system. A mobile phase of 0.02% (w:v) NaN and ultrapure water was prepared, and standard curves were plotted using dextran T standards (molecular weights of 10 kDa, 40 kDa, 50 kDa, 70 kDa, 500 kDa, and 2000 kDa) and glucose. Corn cob polysaccharide samples and standards (concentration 1 mg / mL) were prepared using the mobile phase, completely dissolved and filtered through a 0.22 μm membrane before being injected into the sample system for analysis.

[0084] 5) Monosaccharide composition analysis:

[0085] Based on the electrochemical activity of sugar molecules and their ionization in strongly alkaline solutions, the monosaccharide composition of corn cob polysaccharides was analyzed using high-performance anion exchange chromatography (HPAEC) and pulsed amperometric detection (PAD). For the determination of monosaccharide composition (common sugars) using the sulfuric acid method, accurately weigh 5.0 mg of sample into a stoppered test tube. Under ice bath conditions, add 0.5 ml of 12M concentrated sulfuric acid (sulfuric acid:water = 2:1) using a pipette. Stir with a magnetic stirrer for half an hour, then add 2.5 ml of distilled water. After cooling in a 100°C oil bath for 2-4 hours, pour the sample solution into a 50 ml volumetric flask. Rinse the stoppered test tube containing the sample solution with distilled water, and finally dilute to 50 ml. Shake well. Take 2 ml of the diluted solution and dilute to 10 ml. Shake well. Use a 2 ml syringe to draw up the diluted solution and pass it through a 0.22 μm membrane. First rinse the sample vial, then fill it with the sample solution (you can first add a small amount of sample solution to cover the bottom of the vial, tap the bottom of the vial to remove air bubbles, and then slowly add until it overflows the mouth of the vial, ensuring that there are no air bubbles in the vial). Stopper the vial and inject the sample. Chromatographic analysis results of the monosaccharide composition of corn cob polysaccharide obtained by the preparation method of corn cob polysaccharide of the present invention: The monosaccharide components of the corn cob polysaccharide obtained by the present invention, by mass fraction, are mainly composed of xylose (Xyl, 57.37%) and arabinose (Ara, 23.15%), and also contain galactose (Gal, 9.16%), glucose (Glc, 8.26%), glucuronic acid (GlcA, 1.49%) and galacturonic acid (GalA, 0.57%). The results indicate that the polysaccharide belongs to a heteropolysaccharide mainly composed of xylose and arabinose, among which xylose, arabinose, galactose and glucose are neutral sugars.

[0086] 6) Fourier transform infrared (FT-IR) spectroscopy:

[0087] The structural characteristics of corn cob polysaccharide samples were recorded using a Nicolet 5700 FTIR spectrometer (USA). In short, freeze-dried corn cob polysaccharide was thoroughly mixed with KBr at a ratio of 1:100 (w / w) and ground into powder. The powder was then pressed into thin sheets, and the mixture was immediately placed in the optical path and observed at 4000-400 cm⁻¹. -1 It can scan within the range, up to 64 times, with a resolution of 4cm. -1 .

[0088] Figure 1 The molecular weight of a corn cob polysaccharide of the present invention was determined by gel permeation chromatography; the average molecular weight of the sample was approximately 2.5 × 10⁻⁶. 4 Da;

[0089] Figure 2The Fourier transform infrared (FTIR) spectrum of corn cob polysaccharide obtained by the preparation method of corn cob polysaccharide according to the present invention is shown below. The structural characteristics of the corn cob polysaccharide sample were recorded using a Nicolet 5700 FTIR spectrometer, and the results are as follows: [The image shows a peak at 3744.3 cm⁻¹]. -1 3401.9cm -1 The broad peak at 2919.5 cm⁻¹ corresponds to the hydrogen-bonded -OH group, while the peak at 2919.5 cm⁻¹ corresponds to the hydrogen-bonded -OH group. -1 The weaker absorption band at 1602.7 cm⁻¹ is attributed to the -CH stretching vibration in the -CH₂ and -CH₃ groups. -1 The absorption band at 663 cm⁻¹ indicates that part of the signal originates from the bending vibration of the -OH groups in the adsorbed water. Furthermore, at 663 cm⁻¹... -1 475cm -1 The absorption band at that location confirmed the presence of the pyranose structure.

[0090] Figure 3 The X-ray diffraction pattern of corn cob polysaccharide shows the physicochemical properties. The figure shows that a broad peak appears in the range of 10–40℃. The polysaccharide is mainly amorphous, but there are some fluctuations in the broad peak. It has a locally ordered amorphous structure with low crystallinity, good water solubility, and poor stability.

[0091] Figure 4 The thermogravimetric curve of corn cob polysaccharide is shown. At 235℃, due to the volatilization and release of water and small molecules in the early stage, the weight of the polysaccharide is 87%, and the upper limit of heat resistance is about 235℃.

[0092] Figure 5 The graph shows the thermal decomposition curve of corn cob polysaccharide. In the range of 200-350℃, the curve continues to decline in the positive range, with both exothermic and endothermic reactions occurring, but gradually transitioning to endothermic reactions. In the range of 350-600℃, there is a significant negative trough, and the decomposition process is mainly endothermic bond breaking.

[0093] Figure 6 This is a graph showing the relationship between the viscosity and shear rate of corn cob polysaccharide. As the shear rate increases, the viscosity of corn cob polysaccharide gradually decreases, indicating that the solution exhibits shear-thinning non-Newtonian fluid characteristics. Its viscosity is thick at rest but increases in fluidity under stress; applying this property to food and pharmaceuticals could improve taste. The viscosity changes significantly with concentration, and this property can be precisely controlled by adjusting the concentration.

[0094] Figure 7 This is a schematic diagram showing the experimental results of oil-holding capacity of several polysaccharides. In the figure, the horizontal axis represents different polysaccharide samples, in order: aloe vera polysaccharide, Dendrobium officinale polysaccharide, corn cob polysaccharide, and peanut shell polysaccharide. Oil-holding capacity is an important indicator for evaluating the functional properties of polysaccharides. Figure 7It can be seen that corn cob polysaccharides have a relatively good oil retention capacity and can better absorb oil. This characteristic makes corn cob polysaccharides a suitable fat substitute for low-fat foods or an ingredient in functional foods.

[0095] Figure 8 This graph shows the relationship between the inhibition rate of corn cob polysaccharides on lipase in vitro. The trend in the graph indicates that the inhibition rate of lipase significantly increases with increasing corn cob polysaccharide concentration, demonstrating a good lipase inhibitory effect.

[0096] Example 3:

[0097] This embodiment discloses the application of corn cob polysaccharide prepared by the method of the present invention as a lipid inhibitor, and demonstrates the ameliorative effect of corn cob polysaccharide on HFD obese mice induced by a high-fat diet through experiments.

[0098] Test method:

[0099] 1. Forty healthy male mice aged six weeks (SPF grade C57BL / 6J, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were selected and randomly divided into four groups of 20 mice each. The groups were named control group, HFD model group, low-dose corn cob polysaccharide group, and high-dose corn cob polysaccharide group, respectively. Each group was acclimatized for one week. During the acclimatization period, all groups were fed SPF maintenance feed (Jiangxi Qianchong Scientific Instrument Co., Ltd.). The above maintenance feed and water were provided randomly.

[0100] Adaptive feeding conditions: All mice were housed in specific pathogen-free (SPF) conditions, with alternating periods of 12 hours of light and 12 hours of darkness.

[0101] 2. Model making:

[0102] Control group: also known as the blank group, which was fed under the previous adaptive feeding conditions for eight weeks;

[0103] HFD model group: The group was fed under the previous adaptive feeding conditions for eight weeks. During this period, a high-fat diet (model M10160) was used, which was purchased from Chengdu Parker Biotechnology Co., Ltd. The nutrient composition (mass fraction) was 26.2% protein, 26.3% carbohydrates, and 34.9% fat. The high-fat diet and drinking water were provided at will during the feeding period.

[0104] Low-dose corn cob polysaccharide group: The mice were fed the same adaptive feeding conditions for eight weeks, during which time they were fed a high-fat diet and were given corn cob polysaccharide solution by gavage at a dose of 200 mg / kg BW per day.

[0105] High-dose corn cob polysaccharide group: The mice were fed the same adaptive feeding method for eight weeks, during which time they were fed a high-fat diet and were given corn cob polysaccharide solution by gavage at a dose of 400 mg / kg body weight per day.

[0106] For each group of mice, body weight and body temperature were recorded from week 1 to week 8 to track the progression of obesity.

[0107] Each group consisted of n=10 mice. Data were expressed as mean ± standard deviation (SD). Statistical analysis was performed using SPSS 22.0 and Graphpad Prism 10. The t-test was used to assess differences between the two groups. A p-value less than 0.05 was considered statistically significant and was marked with "*"; a p-value less than 0.01 was considered highly statistically significant and was marked with "**"; and a p-value less than 0.001 was considered highly statistically significant and was marked with "***", without a specific numerical value.

[0108] Figure 9 The graph shows the relationship between the yield of body weight in each group of mice and the experimental time, used to evaluate the effect of corn cob polysaccharide on body weight gain in live animals. The graph shows that both the low-dose and high-dose corn cob polysaccharide groups significantly inhibited body weight gain.

[0109] Figure 10 The graph shows the relationship between food intake and experimental time in each group of mice. The graph shows that there was no significant difference in food intake between the low-dose corn cob polysaccharide group, the high-dose corn cob polysaccharide group and the HFD model group. This means that the inhibitory effect of corn cob polysaccharide on weight gain is not achieved by suppressing appetite, but mainly through the previously verified mechanism of inhibiting lipase activity and good adsorption of oil, which can reduce the discomfort during the weight loss process.

[0110] Figure 11 The figure shows the area under the curve (AUC) of the oral glucose tolerance test (OGTT) in each group of mice, used to comprehensively evaluate the body's ability to regulate blood glucose. The vertical axis represents the area under the glucose tolerance curve, in mmol / L*min; a higher value indicates poorer blood glucose regulation. The figure shows that the HFD model group had the highest AUC, indicating significant glucose tolerance abnormalities in the model group mice. The AUCs of the low-dose corn cob polysaccharide group and the high-dose corn cob polysaccharide group were both lower than those of the HFD model group, indicating that corn cob polysaccharide intervention improved glucose tolerance.

[0111] Figure 12 The graph shows the weight gain rates of mice in each group at the end of the experiment. Compared with the HFD model group, the weight gain rate of mice in the low-dose and high-dose corn cob polysaccharide groups was significantly slower, indicating that corn cob polysaccharide can effectively improve obesity. This weight data is consistent with previous experimental results.

[0112] In vitro: Corn cob polysaccharides effectively inhibit lipase activity and reduce fat absorption; corn cob polysaccharides also have good oil adsorption capacity, encapsulating dietary fat in the intestine. In vivo: Corn cob polysaccharide intervention does not reduce food intake, indicating that weight control is not achieved by suppressing appetite, but by reducing energy absorption. In other words, corn cob polysaccharides effectively control weight gain caused by a high-fat diet by inhibiting dietary fat absorption, rather than suppressing appetite.

[0113] Figure 13 The results of HE staining of white adipose tissue from three groups of mice after dissection are shown in the figure. In the control group, adipocytes were regular in morphology and uniform in size. Compared with the control group, the adipocytes in the white adipose tissue of the HFD model group were significantly larger, with reduced intercellular spaces, exhibiting typical hypertrophic obesity characteristics. After intervention with corn cob polysaccharide, the adipocytes in the low-dose corn cob polysaccharide group were significantly smaller, and the cells were more tightly packed, indicating that corn cob polysaccharide can effectively inhibit adipocyte hypertrophy and improve morphological changes in adipose tissue.

[0114] Figure 14 The changes in triglyceride content in the feces of mice in each group were shown. Compared with the control group, the triglyceride content in the feces of the HFD model group was significantly lower, indicating increased lipid absorption. After intervention with corn cob polysaccharide, the TG content in the feces of mice was significantly increased, indicating that corn cob polysaccharide can promote lipid excretion and reduce intestinal fat absorption.

[0115] Figure 15 The study showed changes in lipase activity in the contents of different intestinal segments. Compared with the control group, the HFD model group exhibited significantly increased lipase activity in the contents of the duodenum, jejunum, and ileum, indicating enhanced lipid digestion and absorption. After intervention with corn cob polysaccharide, lipase activity in all of the above intestinal segments was significantly reduced. This further demonstrates that corn cob polysaccharide can reduce lipid hydrolysis and absorption by inhibiting intestinal lipase activity, thereby exerting an anti-obesity effect to some extent.

[0116] Therefore, the corn cob polysaccharide prepared by the method disclosed in this invention has anti-obesity and metabolic regulation effects by inhibiting lipase activity, adsorbing oil, and reducing fat cell volume.

[0117] The yield of corn cob polysaccharides extracted using the alkali extraction method reached 7.95±0.3%. This invention improves the comprehensive utilization value of corn cobs and promotes the high-value utilization of major agricultural by-products. The corn cob polysaccharide components of this invention can effectively improve weight gain and fat deposition in obese mice, providing a new solution for the prevention and treatment of obesity.

[0118] Of course, the above description is only a preferred embodiment of the present invention and does not constitute a limitation of the present invention. Those skilled in the art, guided by existing technology, can make other modifications to the implementation of the present invention without creative effort. Any modifications made within the spirit and principles of the present invention, or simple substitutions or equivalent replacements made using conventional techniques in the art, should be included within the protection scope of the present invention.

Claims

1. A method for preparing corn cob polysaccharide, characterized in that, Includes the following steps: S1. Prepare defatted corn cob powder; S11. Drying and pulverizing: Select corn cobs without mold, wash them and dry them in an oven. After pulverizing them in a pulverizer, sieve them to remove coarse particles and obtain fine corn cob powder. S12. Degreasing: Add n-hexane to the corn cob powder and stir to degrease; S13. Centrifugal drying: After centrifugation, discard the supernatant and air-dry the resulting precipitate at room temperature to obtain defatted corn cob powder; S2. Alkali extraction of crude polysaccharides: S21. Alkali extraction: The defatted corn cob powder obtained in step S1 is added to a 2% sodium hydroxide solution and extracted by ultrasonic extraction to obtain an ultrasonic extract. The specific steps of the ultrasonic extraction are as follows: the mixture of defatted corn cob powder obtained in step S1 and sodium hydroxide solution is placed in an ultrasonic-assisted device, the ultrasonic frequency is set to 40 kHz, and ultrasonic extraction is performed at 60-70℃ with a power of 200-500W for 15-20 minutes; the ultrasonic process is in intermittent mode, working for 2 seconds and then resting for 2 seconds, and the material-liquid ratio of defatted corn cob powder to sodium hydroxide solution is 1:15-25; the ultrasonic extraction time is 15 minutes, with an ultrasonic power of 450W for the first 5 minutes and 250W for the last 10 minutes; S22. Centrifugation and collection: After centrifuging the ultrasonic extract using a high-speed centrifuge, the supernatant is collected, which is the corn cob crude polysaccharide alkaline extract; S23. Concentration: The corn cob crude polysaccharide alkaline extract is concentrated by rotary evaporation to obtain a concentrated corn cob crude polysaccharide solution; S3. Enzymatic hydrolysis and purification: Adjust the concentrated corn cob crude polysaccharide solution obtained in step S2 to pH=7 for enzymatic hydrolysis. After enzymatic hydrolysis, inactivate the enzyme by boiling water bath for 30-60 min, cool to room temperature and centrifuge to collect the supernatant. S4. Precipitation and Refining: S41. Alcohol precipitation: Place the supernatant obtained in step S3 in an ice-water bath to pre-cool to a low temperature of 0-4℃, and slowly add 4 times the volume of 95% ethanol while stirring at a speed of 500-800 rpm. After the addition is complete, stir slowly at a speed of 100-200 rpm for 20-30 minutes, then stop stirring and let it stand at room temperature for 24 hours. S42. Centrifuge and collect the precipitate: After centrifugation, discard the supernatant and collect the precipitate; S43. Redissolution and concentration: The precipitate is redissolved with an appropriate amount of pure water and stirred in a 90℃ constant temperature water bath for 2 hours to ensure that the polysaccharide is completely dissolved. Finally, the solution is concentrated by rotary evaporation, and after dialysis and freeze drying, corn cob polysaccharide is obtained. The composition of the corn cob polysaccharide, by mass fraction, is as follows: neutral sugar content is 89.20±1.93%, uronic acid content is 4.02±0.75%, and protein content is 6.89±0.26%. The composition of the neutral sugars, by mass fraction, is as follows: xylose content is 57.37%, arabinose content is 23.15%, galactose content is 9.16%, and glucose content is 8.26%.

2. The method for preparing corn cob polysaccharide according to claim 1, characterized in that: In step S12, the degreasing step includes a two-step degreasing step. First, n-hexane is added to the corn cob powder at a material-to-liquid ratio of 1:

10. After stirring for 30 minutes to degrease, the mixture is centrifuged and the supernatant is discarded. Then, n-hexane is added to the precipitate at a material-to-liquid ratio of 1:

15. The mixture is stirred and extracted for another 15 minutes, centrifuged again, and then air-dried at room temperature.

3. The method for preparing corn cob polysaccharide according to claim 2, characterized in that: In step S12, the degreasing process is carried out by stirring in a constant temperature water bath at 40-45℃.

4. The method for preparing corn cob polysaccharide according to claim 1, characterized in that: In step S3, papain is used for enzymatic hydrolysis. The amount of papain added is 0.4% of the total mass of the concentrated corn cob crude polysaccharide solution, and the hydrolysis conditions are 60℃ for 2 hours.

5. The application of corn cob polysaccharide prepared by the method according to any one of claims 1-4 as a lipid inhibitor.

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