Alcohol-soluble polysaccharides from distiller's grains, and preparation method and application thereof

Abstract

The application discloses a kind of alcohol-soluble polysaccharide of vinasse and its preparation method and application, belong to polysaccharide extraction technical field.The application provides a kind of alcohol-soluble polysaccharide of vinasse and its preparation method and application to improve the alcohol-solubility and antioxidant property of vinasse polysaccharide, comprising: vinasse and water are mixed, are crushed and centrifuged, take supernatant, first join amylase and carry out enzymolysis, then join neutral protease and cellulase and carry out enzymolysis, the obtained vinasse enzymolysis liquid is concentrated after enzyme inactivation, after dialysis desalination, then make system total ethanol concentration reach 38~53%Vol, then through alcohol precipitation and centrifugation, the obtained alcohol precipitation supernatant is concentrated, using ultrafiltration membrane interception is obtained 4000~6000Da interception liquid, and freeze-drying is obtained alcohol-soluble polysaccharide of vinasse.The application significantly improves the activity, purity and alcohol-solubility of polysaccharide by optimizing enzymolysis technology, controlling alcohol precipitation condition and polysaccharide target molecular weight, with good antioxidant activity and in-vitro anti-liver injury activity.

Description

Technical Field

[0001] This invention belongs to the field of polysaccharide extraction technology, specifically relating to a distiller's grains alcohol-soluble polysaccharide, its preparation method, and its application. Background Technology

[0002] Baijiu (Chinese liquor) lees are a byproduct of the brewing industry. Limited by existing solid-state fermentation processes, lees contain a large number of bioactive components, among which polysaccharides are one. Polysaccharides possess antioxidant activity, scavenging free radicals in the body, reducing oxidative stress damage to cells, thereby delaying aging and preventing various chronic diseases such as cardiovascular disease, diabetes, and cancer. The monosaccharide composition of polysaccharides is a key factor affecting their activity. Mannose, galactose, and arabinose have strong antioxidant activity; increasing the proportion of mannose, galactose, and arabinose in the polysaccharide structure can enhance its antioxidant activity. Molecular weight also leads to differences in polysaccharide activity. Smaller molecular weight polysaccharides are easily absorbed and exhibit better biological activity, but their structure is easily destroyed during processing. Larger molecular weight polysaccharides are structurally stable and can better maintain their activity during processing, but excessively high molecular weight makes them difficult to absorb and their activity difficult to express. Alcohol-soluble sugars are beneficial for addition to organic reagents and convenient for application in cosmetics, etc. Increased sugar purity and reduced impurities also facilitate the expression of activity in polysaccharide applications. Distillers' grains are abundant, but traditional processing methods have overlooked their potential nutritional value. In recent years, the extraction of sugars from distillers' grains has attracted researchers' attention.

[0003] However, current research on the extraction of alcohol-soluble polysaccharides from baijiu lees is relatively limited, and studies on their antioxidant activity are lacking. Reference document 1 discloses a microwave-assisted enzymatic extraction method for baijiu lees polysaccharides optimized based on response surface methodology, comprising the following steps: A. Extraction: Baijiu lees are dried (moisture content ≤5%), pulverized, and sieved to obtain baijiu lees powder; the baijiu lees powder is mixed with water, microwaved, and then a complex enzyme is added for enzymatic hydrolysis. After the reaction is complete, the enzyme is inactivated, and then the mixture is centrifuged. The supernatant is collected and concentrated, and anhydrous ethanol is added to the concentrate for alcohol precipitation. The precipitated solution is then centrifuged, and the precipitate is collected. The precipitate is subsequently reconstituted with an appropriate amount of water, concentrated, and frozen. After drying, crude polysaccharide from baijiu lees is obtained; B. Impurity removal: The crude polysaccharide from baijiu lees obtained in step A is decolorized using macroporous adsorption resin to obtain a decolorized crude polysaccharide solution; the decolorized polysaccharide solution is then deproteinized using an enzymatic method combined with the Sevag method to obtain a deproteinized crude polysaccharide solution; the deproteinized crude polysaccharide solution is then dialyzed to remove inorganic salts and small molecules, followed by concentration and freeze-drying to obtain desalted crude polysaccharide; C. Separation and purification: The desalted crude polysaccharide obtained in step C is dissolved in water, first separated using an aqueous filter membrane, then separated using an ultrafiltration membrane, and finally concentrated and freeze-dried to obtain baijiu lees polysaccharide. However, this method does not disclose the alcohol solubility of the polysaccharide, nor does it study the effect of molecular weight on the properties of the polysaccharide.

[0004] Therefore, developing a new extraction process for baijiu lees and exploring its in vitro antioxidant activity is beneficial for the resource utilization of lees polysaccharides and improves the high-value utilization of baijiu lees. Summary of the Invention

[0005] To address the problems of existing technologies, this invention prepares alcohol-soluble polysaccharides from baijiu lees with antioxidant activity by enzymatic hydrolysis and ethanol extraction, while controlling the molecular weight.

[0006] This invention first provides a method for preparing alcohol-soluble polysaccharides from distiller's grains, which includes the following steps:

[0007] A. Mix the lees and water at a mass-volume ratio of 1g:8~25ml, then crush and centrifuge, and take the supernatant to obtain the lees pretreatment solution.

[0008] B. Adjust the temperature of the pretreated lees solution obtained in step A to 90~95℃, add amylase for enzymatic hydrolysis, then adjust the temperature to 40~45℃, add neutral protease and cellulase for enzymatic hydrolysis, and obtain lees hydrolysate.

[0009] C. Heat the enzymatic hydrolysate obtained in step B to 100-105℃ to inactivate the enzyme and then concentrate it. After dialysis and desalting, mix 50-70% Vol ethanol with the dialyzed enzymatic hydrolysate to make the total ethanol concentration in the system reach 38-53% Vol. Then, after alcohol precipitation and centrifugation, obtain the alcohol precipitation supernatant.

[0010] D. Concentrate the alcohol precipitation supernatant obtained in step C to remove ethanol. First, use a 6000Da ultrafiltration membrane to remove the portion not higher than 6000Da, and then use a 4000Da ultrafiltration membrane to remove the portion not lower than 4000Da, to obtain a retentate of 4000~6000Da. After freeze-drying the retentate, obtain alcohol-soluble polysaccharide from distiller's grains.

[0011] The monosaccharide composition of the alcohol-soluble polysaccharide from distillers' grains includes glucose, mannose, arabinose, and galactose, with the molar percentage of mannose not less than 15 mol%, the molar percentage of galactose not less than 15 mol%, the molar percentage of arabinose not less than 8 mol%, and the molar percentage of glucose not more than 38 mol%; the proportion of components with a Da of 4000~6000 in the alcohol-soluble polysaccharide from distillers' grains is not less than 85%.

[0012] In the preparation method of the above-mentioned alcohol-soluble polysaccharide from distiller's grains, in step A, the water content of the distiller's grains is ≤5wt%, the crude protein content is 13.2~16.5wt%, the starch content is 9.7~15.5wt%, and the cellulose content is 11.2~14.1wt%.

[0013] In the preparation method of the above-mentioned alcohol-soluble polysaccharide from distiller's grains, in step B, the amount of amylase added is 600~1000U / mL of distiller's grains pretreatment solution.

[0014] In the preparation method of the above-mentioned alcohol-soluble polysaccharide from distiller's grains, in step B, the amount of neutral protease added is 600~1200 U / mL of distiller's grains pretreatment solution.

[0015] In the preparation method of the above-mentioned alcohol-soluble polysaccharide from distiller's grains, in step B, the amount of cellulase added is 300~900 U / mL of distiller's grains pretreatment solution.

[0016] In the preparation method of the above-mentioned alcohol-soluble polysaccharide from distiller's grains, in step B, the amylase hydrolysis time is 1-5 hours.

[0017] In the preparation method of the above-mentioned alcohol-soluble polysaccharide from distiller's grains, in step B, the enzymatic hydrolysis time of neutral protease and cellulase is 1~5h.

[0018] In the preparation method of the above-mentioned alcohol-soluble polysaccharide from distiller's grains, in step C, the enzyme inactivation time is 10-30 min.

[0019] In the preparation method of the above-mentioned alcohol-soluble polysaccharide from distiller's grains, in step C, the concentration is to concentrate to 1 / 4 to 1 / 3 of the original volume.

[0020] In the preparation method of the above-mentioned alcohol-soluble polysaccharide from distiller's grains, in step C, the dialysis desalting is performed using a 500Da dialysis bag with deionized water.

[0021] In the preparation method of the above-mentioned alcohol-soluble polysaccharide from distiller's grains, in step C, the temperature of alcohol precipitation is 4~8℃.

[0022] In the preparation method of the above-mentioned alcohol-soluble polysaccharide from distiller's grains, in step C, the alcohol precipitation time is 12-15 hours.

[0023] In the preparation method of the above-mentioned alcohol-soluble polysaccharide from distiller's grains, step D involves concentrating the polysaccharide to 1 / 4 to 1 / 3 of its original volume.

[0024] This invention also provides the application of the alcohol-soluble polysaccharide obtained by the above method in the preparation of antioxidants or drugs for the prevention and treatment of liver damage.

[0025] Preferably, in the above applications, the antioxidant is an anti-ABTS antioxidant or an anti-DPPH antioxidant.

[0026] The beneficial effects of this invention are:

[0027] This invention optimizes the enzymatic hydrolysis technology and alcohol precipitation process of distiller's grains, which improves the modification of distiller's grains fiber and the dissolution of alcohol-soluble polysaccharides, increases extraction efficiency, avoids the use of non-food grade reagents, ensures product safety, and facilitates industrialization. It also helps to increase the molar proportion of mannose, galactose, and arabinose in the polysaccharides, further enhancing their antioxidant activity. By targeting polysaccharides with a molecular weight of 4000-6000 Da, the activity and purity of the polysaccharides are significantly improved. The resulting alcohol-soluble polysaccharides exhibit good solubility, dissolving in 38-53% Vol ethanol with a solubility greater than 85% and a purity exceeding 85%. They possess excellent antioxidant activity and in vitro anti-liver damage activity, enabling applications in numerous fields. Attached Figure Description

[0028] Figure 1 This is a flowchart illustrating the extraction process of alcohol-soluble polysaccharides from distiller's grains according to the present invention.

[0029] Figure 2 The graph shows the effect of the alcohol-soluble polysaccharide obtained in Example 1 on hepatocyte growth and antioxidant enzyme levels; where A represents the effect of polysaccharide on cell growth, and B~D represent the effect of polysaccharide on hepatocyte antioxidant enzyme levels; a~d represent significant differences between groups, p < 0.05.

[0030] Figure 3 The figure shows the effect of the high molecular weight alcohol-soluble polysaccharide obtained in Comparative Example 2 on hepatocyte growth and antioxidant enzyme levels; where A represents the effect of polysaccharide on cell growth, and B~D represent the effect of cells on hepatocyte antioxidant enzyme levels; a~d represent significant differences between groups, p < 0.05.

[0031] Figure 4 The figure shows the effect of the low molecular weight alcohol-soluble polysaccharide obtained in Comparative Example 3 on hepatocyte growth and antioxidant enzyme levels; where A represents the effect of polysaccharide on cell growth, and B~D represent the effect of cells on hepatocyte antioxidant enzyme levels; a~d represent significant differences between groups, p < 0.05. Detailed Implementation

[0032] The present invention will be further described in detail below through embodiments, but the scope of protection of the present invention is not limited to the embodiments described herein.

[0033] 1. The physicochemical properties of the dried distiller's grains used are shown in Table 1.

[0034] Table 1. Indicators of dried baijiu lees (wt%)

[0035]

[0036] 2. Calculation of purity of alcohol-soluble polysaccharides from distiller's grains

[0037] The polysaccharide was dissolved in deionized water to obtain the concentration of the substance. The total sugar content was determined by the phenol-sulfuric acid method to obtain the sugar concentration. The purity of the polysaccharide was then obtained by dividing the total sugar concentration by the concentration of the substance.

[0038] 3. Determination of polysaccharide alcohol solubility

[0039] Weigh the dried centrifuge tube (M1), accurately weigh 40 mg of polysaccharide (M2), then add 60% Vol ethanol aqueous solution to make the polysaccharide concentration reach 2 mg / mL, let stand at 4℃ for 12 h, then centrifuge, discard the supernatant, put the centrifuge tube with precipitate into a 100℃ oven to dry for 2 h and then measure the weight (M3).

[0040] .

[0041] 4. Determination method for antioxidant activity of alcohol-soluble polysaccharides from distiller's grains

[0042] (1) Method for determining the scavenging ability of DPPH free radicals

[0043] Referring to the national standard "Determination of Antioxidant Activity of Peptides by DPPH and ABTS Methods" (GB / T 39100-2020), the alcohol-soluble polysaccharide was first prepared into an aqueous solution with a concentration of 2 mg / mL, and the DPPH was prepared into an ethanol solution with a concentration of 50 μg / mL. 1 mL of the alcohol-soluble polysaccharide solution was mixed with 3 mL of the DPPH solution as the experimental group As; 1 mL of the alcohol-soluble polysaccharide solution was mixed with 3 mL of the ethanol solution as the control group Ac; and 3 mL of the DPPH solution was mixed with 1 mL of water as the blank group Ab. After mixing each group thoroughly, the reaction was carried out at room temperature in the dark for 30 min, and the absorbance at a wavelength of 517 nm was detected using a microplate reader.

[0044] .

[0045] (2) ABTS + Methods for determining free radical scavenging ability

[0046] Referring to the national standard "Determination of Antioxidant Activity of Peptides - DPPH and ABTS Methods" (GB / T 39100-2020), the alcohol-soluble polysaccharide was first prepared into an aqueous solution with a concentration of 2 mg / mL. 200 mg of ABTS was dissolved in 50 mL of water, and after 24 hours in the dark, the absorbance was adjusted to 0.7 with water to prepare the ABTS solution. + Assay solution. Take 0.4 mL of alcohol-soluble polysaccharide solution and 3.6 mL of ABTS. + The test solution was mixed to form experimental group As; 0.4 mL of water and 3.6 mL of ABTS were taken. +The assay solution was mixed to serve as the blank control (Ab); after thorough mixing, the mixture was reacted at room temperature in the dark for 5 min, and the absorbance at a wavelength of 734 nm was measured using an ELISA reader.

[0047] .

[0048] 5. Methods for determining the molecular weight distribution of polysaccharides

[0049] The molecular weight distribution of polysaccharide samples was determined using high-performance gel permeation chromatography (Waters 2695; Waters) with a Waters 2414 differential refractive index detector. The mobile phase was 0.02% NaNO3 aqueous solution, the flow rate was 0.5 mL / min, and the chromatographic column was an Ultraahydroge TM (7.8 × 300 mm, Waters), column temperature 35 ℃. The elution times were determined using glucose, sucrose, and dextran standards with molecular weights of 10 kDa, 40 kDa, 70 kDa, 150 kDa, 500 kDa, and 2000 kDa. A standard curve of sample molecular weight versus elution time was plotted to calculate the molecular weight distribution of the samples.

[0050] 6. Analysis of the polysaccharide and monosaccharide composition of distiller's grains

[0051] 5 mg of sample was hydrolyzed in ampoules with 2 mL of trifluoroacetic acid (3.00 mol / L) at 120 °C for 3 h. After hydrolysis, the sample was dried under nitrogen, and 0.2 mL of methanol was added and purged three times under nitrogen to remove residual trifluoroacetic acid. After drying, the sample was filtered through a 0.22 µm filter membrane after adding deionized water. The filtered liquid was transferred to a liquid chromatography vial and detected using a high-performance ion chromatography (HPLC) system ICS-5000+SP-5. The system was equipped with a pulsed amperometric detector, a Dionex carbopactmpa 20 anion exchange column (3 mm × 150 mm), and a PA20 guard column (3 mm × 50 mm). The sample loading volume was 20 µL, and the column temperature was 30 °C during detection. The mobile phases were deionized water (phase A), 250 mmol / L NaAc deionized water (phase B), and 250 mmol / L NaOH deionized water (phase C). The mobile phase operation flow was as follows: 99.1% A, 0.9% C (20 min), 19.1% A, 80% B, 0.9% C (20.1~27 min), 20% A, 80% C (27.1~38 min), with a flow rate of 0.5 mL / min.

[0052] 7. Cell viability assay

[0053] Polysaccharides were dissolved in complete culture medium solution to prepare polysaccharide intervention reagents with concentrations ranging from 0 to 120 μg / mL. After the polysaccharides were fully dissolved, they were filtered through a sterile 0.1 μm syringe filter membrane for later use. The experiment included a polysaccharide intervention group, a control group, and a blank group, with 6 replicates per group. After 24 h of cell culture, the supernatant from all 96 wells was carefully aspirated and removed. 100 μL of the polysaccharide intervention reagents at the above concentrations were added to the polysaccharide intervention group. 100 μL of complete culture medium without polysaccharide samples was added to the wells containing the cells in the control group. No cells were seeded in the blank group; only 100 μL of complete culture medium was added. The 96-well plates were then placed in a cell culture incubator and allowed to stand for 24 h. After standing, the 96-well plates were removed from the incubator, and 10 μL of CCK-8 solution was added to each well using a pipette before transferring the plates to the cell culture incubator. The plates were then allowed to stand for another 4 h. After standing, the absorbance at 450 nm was measured using a microplate reader. Cell viability was calculated using the following formula:

[0054] ;

[0055] In the formula, OD1 represents the absorbance of cells under polysaccharide intervention; OD2 represents the absorbance of cells without polysaccharide intervention; and OD0 represents the absorbance of the complete culture medium.

[0056] 8. Determination of antioxidant enzyme levels in cell secretions

[0057] Hepatocytes treated with polysaccharides were removed, cell culture medium was aspirated, the culture medium was centrifuged, and the supernatant was obtained. The levels of antioxidant enzymes in the supernatant, including superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT), were measured according to the kit instructions.

[0058] Example 1

[0059] (1) Take dried lees and deionized water, mix them at a ratio of W / V=1:8, crush them, centrifuge and filter to obtain the supernatant, and obtain the lees pretreatment solution.

[0060] (2) Adjust the temperature of the pretreated lees solution obtained in step (1) to 90°C, add 1000 U / mL of amylase, and hydrolyze for 1 h. Then control the temperature to 40°C, add 1200 U / mL of neutral protease and 900 U / mL of cellulase, and hydrolyze for 1 h to obtain lees hydrolysate.

[0061] (3) Heat the enzyme hydrolysate obtained in step (2) to 100°C to inactivate the enzyme for 10 min, concentrate it to 1 / 4 of the original volume, desalt it with deionized water using a 500 Da dialysis bag, and then mix it with 50% Vol ethanol to make the final ethanol concentration reach 38% Vol. After ethanol precipitation at 4°C for 12 h, centrifuge and take the supernatant.

[0062] (4) Concentrate the supernatant from step (3) to 1 / 4 of its original volume, remove ethanol, and retain it with an ultrafiltration membrane. First, use a 6000 Da ultrafiltration membrane to remove the portion not higher than 6000 Da, and then use a 4000 Da ultrafiltration membrane to remove the portion not lower than 4000 Da. Finally, obtain a retentate with a 4000-6000 Da. Freeze-dry the retentate to obtain alcohol-soluble polysaccharide from distillers' grains.

[0063] The alcohol solubility, purity, molecular weight, monosaccharide composition and antioxidant activity of alcohol-soluble polysaccharides from distiller's grains were determined, and the results are shown in Tables 2 and 3.

[0064] Example 2

[0065] (1) Take dried lees and deionized water, mix them at a ratio of W / V=1:25, crush them, centrifuge and filter to obtain the supernatant, and obtain the lees pretreatment solution.

[0066] (2) Adjust the temperature of the pretreated lees solution obtained in step (1) to 95℃, add 600 U / mL of amylase, and hydrolyze for 5h. Then control the temperature to 45℃, add 600 U / mL of neutral protease and 300 U / mL of cellulase, and hydrolyze for 5h to obtain lees hydrolysate.

[0067] (3) Heat the enzyme hydrolysate obtained in step (2) to 100°C to inactivate the enzyme for 10 min, concentrate it to 1 / 4 of the original volume, desalt it with deionized water using a 500 Da dialysis bag, and then mix it with 50% Vol ethanol to make the final ethanol concentration reach 38% Vol. After ethanol precipitation at 6°C for 13 h, centrifuge and take the supernatant.

[0068] (4) Concentrate the supernatant from step (3) to 1 / 4 of its original volume, remove ethanol, and retain it with an ultrafiltration membrane. First, use a 6000 Da ultrafiltration membrane to remove the portion not higher than 6000 Da, and then use a 4000 Da ultrafiltration membrane to remove the portion not lower than 4000 Da. Finally, obtain a retentate with a retentate of 4000~6000 Da. Freeze-dry the retentate to obtain alcohol-soluble polysaccharide from distiller's grains.

[0069] The alcohol solubility, purity, molecular weight, monosaccharide composition and antioxidant activity of alcohol-soluble polysaccharides from distiller's grains were determined, and the results are shown in Tables 2 and 3.

[0070] Example 3

[0071] (1) Take dried lees and deionized water, mix them at a ratio of W / V=1:15, crush them, centrifuge and filter to obtain the supernatant, and obtain the lees pretreatment solution.

[0072] (2) Adjust the temperature of the pretreated lees solution obtained in step (1) to 93℃, add 800 U / mL of amylase, and hydrolyze for 3h. Then control the temperature to 43℃, add 1000 U / mL of neutral protease and 600 U / mL of cellulase, and hydrolyze for 3h to obtain lees hydrolysate.

[0073] (3) Heat the enzyme hydrolysate obtained in step (2) to 100°C to inactivate the enzyme for 10 min, concentrate it to 1 / 4 of the original volume, desalt it with deionized water using a 500 Da dialysis bag, and then mix it with 50% Vol ethanol to make the final ethanol concentration reach 38% Vol. After ethanol precipitation at 8°C for 15 h, centrifuge and take the supernatant.

[0074] (4) Concentrate the supernatant from step (3) to 1 / 4 of its original volume, remove ethanol, and retain it with an ultrafiltration membrane. First, use a 6000 Da ultrafiltration membrane to remove the portion not higher than 6000 Da, and then use a 4000 Da ultrafiltration membrane to remove the portion not lower than 4000 Da. Finally, obtain a retentate with a retentate of 4000~6000 Da. Freeze-dry the retentate to obtain alcohol-soluble polysaccharide from distiller's grains.

[0075] The alcohol solubility, purity, molecular weight, monosaccharide composition and antioxidant activity of alcohol-soluble polysaccharides from distiller's grains were determined, and the results are shown in Tables 2 and 3.

[0076] Example 4: Setting the ethanol concentration during the alcohol precipitation process

[0077] Example 4.1: In step (3) of Example 1, the method was changed to use 60% Vol ethanol to mix with the enzymatic hydrolysate after dialysis for alcohol precipitation, so that the final concentration of ethanol reaches 45% Vol. Other conditions or parameters are the same as in Example 1.

[0078] Example 4.2: In step (3) of Example 1, the mixture of 70% Vol ethanol and the enzymatic hydrolysate after dialysis was used for alcohol precipitation, so that the final concentration of ethanol reached 53% Vol. Other conditions or parameters were the same as in Example 1.

[0079] The alcohol solubility and antioxidant activity of the alcohol-soluble polysaccharide from distiller's grains were calculated and compared with those of Example 1. The results are shown in Table 4.

[0080] Example 5: Using alcohol-soluble polysaccharides from distillers' grains for hepatocyte repair

[0081] The alcohol-soluble polysaccharide extracted from distillers' grains in Example 1 was dissolved in cell culture medium to achieve final polysaccharide concentrations of 10, 20, 40, 60, 80, 100, and 120 μg / mL. Cell growth was measured, and polysaccharide toxicity was analyzed. Results showed that final polysaccharide concentrations of 10–80 μg / mL had no inhibitory or toxic effect on cell growth, while concentrations of 40, 60, and 80 μg / mL promoted cell growth. Subsequently, HepG2 hepatocytes damaged by H2O2 were treated with polysaccharide concentrations of 40, 60, and 80 μg / mL. Results showed that compared to hepatocytes without polysaccharide intervention (Mod group), when the intervention concentration reached 60 μg / mL, the activities of antioxidant enzymes SOD, CAT, and GSH-Px in the secretory fluid of polysaccharide-treated hepatocytes were significantly increased by 80.1%, 50.0%, and 85.7%, respectively. This indicates that distillers' grains polysaccharide can reduce cellular oxidative stress and promote cell repair. Related results are shown in [link to relevant data]. Figure 2 .

[0082] Comparative Example 1: No enzymatic hydrolysis treatment

[0083] The polysaccharide enzymatic hydrolysis step in Example 1 is omitted, while the other steps and conditions are the same as in Example 1. The method includes the following steps:

[0084] (1) Take dried lees and deionized water, mix them at a ratio of W / V=1:8, crush them, centrifuge and filter to obtain the supernatant, and obtain the lees pretreatment solution.

[0085] (2) After heating the pretreated lees obtained in step (1) to 100℃ to inactivate the enzyme for 10 min, concentrate it to 1 / 4 of the original volume, desalt it with deionized water using a 500 Da dialysis bag, and then mix it with 50% Vol ethanol to make the final ethanol concentration reach 38% Vol. After ethanol precipitation at 4℃ for 12 h, centrifuge and take the supernatant.

[0086] (3) Concentrate the supernatant from step (2) to 1 / 4 of its original volume, remove ethanol, and retain it with an ultrafiltration membrane. First, use a 6000 Da ultrafiltration membrane to remove the portion not higher than 6000 Da, and then use a 4000 Da ultrafiltration membrane to remove the portion not lower than 4000 Da. Finally, obtain a retentate with a retentate of 4000~6000 Da. Freeze-dry the retentate to obtain alcohol-soluble polysaccharide from distillers' grains.

[0087] The alcohol solubility, purity, molecular weight, monosaccharide composition and antioxidant activity of alcohol-soluble polysaccharides from distiller's grains were determined, and the results are shown in Tables 2 and 3.

[0088] Comparative Example 2: Retention of the portion with a molecular weight greater than 6000 Da

[0089] The ultrafiltration membrane retention step in Example 1 is adjusted by using a 6000 Da ultrafiltration membrane to remove the portion of the concentrated supernatant exceeding 6000 Da. Other steps and conditions remain the same as in Example 1. The method includes the following steps:

[0090] (1) Take dried wine lees and deionized water, mix them at a ratio of W / V=1:8, crush them, centrifuge and filter to obtain the supernatant, and obtain the wine lees pretreatment solution.

[0091] (2) Adjust the temperature of the pretreated lees solution obtained in step (1) to 90°C, add 1000 U / mL of amylase, and hydrolyze for 1 h. Then control the temperature to 40°C, add 1200 U / mL of neutral protease and 900 U / mL of cellulase, and hydrolyze for 1 h to obtain lees hydrolysate.

[0092] (3) Heat the enzyme hydrolysate obtained in step (2) to 100℃ to inactivate the enzyme for 10 min, concentrate it to 1 / 4 of the original volume, desalt it with deionized water using a 500Da dialysis bag, and then mix it with 50% Vol ethanol to make the final ethanol concentration reach 38% Vol. After ethanol precipitation at 4℃ for 12 h, centrifuge and take the supernatant.

[0093] (4) Concentrate the supernatant from step (3) to 1 / 4 of its original volume, remove ethanol, and retain it with an ultrafiltration membrane. Use a 6000Da ultrafiltration membrane to remove the portion of the concentrated supernatant that is greater than 6000Da. Freeze-dry the retained liquid to obtain a high molecular weight alcohol-soluble polysaccharide (>6000 Da).

[0094] The alcohol solubility, purity, molecular weight, monosaccharide composition and antioxidant activity of alcohol-soluble polysaccharides from distiller's grains were determined, and the results are shown in Tables 2 and 3.

[0095] The alcohol-soluble polysaccharide extracted from distillers' grains in Comparative Example 2 was dissolved in cell culture medium. The final polysaccharide concentrations in the cell culture medium were 10, 20, 40, 60, 80, 100, and 120 μg / mL. Cell growth was measured, and polysaccharide toxicity was analyzed. The results showed that a final polysaccharide concentration exceeding 80 μg / mL significantly inhibited cell growth. HepG2 hepatocytes damaged by H2O2 were treated with concentrations of 40, 60, and 80 μg / mL. The results showed that compared with hepatocytes without polysaccharide intervention (Mod group), the increase in the activity levels of antioxidant enzymes SOD, CAT, and GSH-Px in the cell secretions of hepatocytes treated with high molecular weight alcohol-soluble polysaccharides (> 6000 Da) was not significant (p > 0.05). Figure 3 High molecular weight alcohol-soluble polysaccharides (> 6000 Da) have low ability to regulate cellular antioxidant enzymes and failed to exhibit biological activity in regulating cellular oxidative stress response at the experimentally set dosage.

[0096] Comparative Example 3: Retention of the molecular weight range smaller than 4000 Da

[0097] The ultrafiltration membrane retention step in Example 1 is adjusted by using a 4000 Da ultrafiltration membrane to remove the portion of the concentrated supernatant smaller than 4000 Da. Other steps and conditions remain the same as in Example 1. The method includes the following steps:

[0098] (1) Take dried lees and deionized water, mix them at a ratio of W / V=1:8, crush them, centrifuge and filter to obtain the supernatant, and obtain the lees pretreatment solution.

[0099] (2) Adjust the temperature of the pretreated lees solution obtained in step (1) to 90°C, add 1000 U / mL of amylase, and hydrolyze for 1 h. Then control the temperature to 40°C, add 1200 U / mL of neutral protease and 900 U / mL of cellulase, and hydrolyze for 1 h to obtain lees hydrolysate.

[0100] (3) Heat the enzyme hydrolysate obtained in step (2) to 100℃ to inactivate the enzyme for 10 min, concentrate it to 1 / 4 of the original volume, desalt it with deionized water using a 500 Da dialysis bag, and then mix it with 50% Vol ethanol to make the final ethanol concentration reach 38% Vol. After ethanol precipitation at 4℃ for 12 h, centrifuge and take the supernatant.

[0101] (4) Concentrate the supernatant from step (3) to 1 / 4 of its original volume, remove ethanol, and retain it with an ultrafiltration membrane. Use a 4000 Da ultrafiltration membrane to remove the portion of the concentrated supernatant that is less than 4000 Da. Freeze-dry the retained liquid to obtain a low molecular weight alcohol-soluble polysaccharide (< 4000 Da).

[0102] The alcohol-soluble polysaccharide extracted from distillers' grains in Comparative Example 3 was dissolved in cell culture medium at final concentrations of 10, 20, 40, 60, 80, 100, and 120 μg / mL. Cell growth was measured, and polysaccharide toxicity was analyzed. Results showed that a final polysaccharide concentration exceeding 80 μg / mL significantly inhibited cell growth. HepG2 hepatocytes damaged by H2O2 were treated with concentrations of 40, 60, and 80 μg / mL. Compared to hepatocytes without polysaccharide intervention (Mod group), hepatocytes treated with low molecular weight alcohol-soluble polysaccharides (< 4000 Da) showed a significant increase in the activity levels of antioxidant enzymes SOD, CAT, and GSH-PX in their cell secretions (p < 0.05). Figure 4Among the interventions, when the concentration reached 60 μg / mL, the activities of antioxidant enzymes SOD, CAT, and GSH-Px in the secretory fluid of hepatocytes treated with polysaccharides were significantly increased by 52.0%, 31.2%, and 42.8%, respectively (p < 0.05). However, under the same concentration conditions, compared with alcohol-soluble polysaccharides with a molecular weight of 4000-6000 Da, low molecular weight alcohol-soluble polysaccharides (< 4000 Da) had a weaker effect on promoting the activity of cellular antioxidant enzymes and a lower effect on regulating cellular oxidative stress. The biological activity of low molecular weight alcohol-soluble polysaccharides (< 4000 Da) was lower than that of alcohol-soluble polysaccharides with a molecular weight of 4000-6000 Da.

[0103] Comparative studies of Example 5, Comparative Example 2, and Comparative Example 3 revealed that molecular weight significantly affects the bioactivity of alcohol-soluble polysaccharides. Components with excessively large (> 6000 Da) or excessively small (< 4000 Da) molecular weights of the alcohol-soluble polysaccharides from distiller's grains failed to exhibit good regulatory capacity for cellular antioxidant enzyme levels. In contrast, alcohol-soluble polysaccharides with the target molecular weight (4000~6000 Da) exhibited strong bioactivity, significantly improved cellular antioxidant enzyme levels, and demonstrated a strong effect on regulating cellular oxidative stress.

[0104] The alcohol solubility, purity, molecular weight, monosaccharide composition and antioxidant activity of alcohol-soluble polysaccharides from distiller's grains were determined, and the results are shown in Tables 2 and 3.

[0105] Table 2. Effects of different treatment methods on the alcohol solubility, purity, and molecular weight of alcohol-soluble polysaccharides from distiller's grains.

[0106]

[0107] Note: Different lowercase letters in the same column represent significant differences between groups, p < 0.05; alcohol solubility determination was performed in 60% Vol ethanol aqueous solution.

[0108] Table 2 shows that enzymatic hydrolysis is a crucial step that significantly improves the purity and antioxidant activity of alcohol-soluble polysaccharides from distiller's grains. This is primarily because enzymatic hydrolysis reduces impurities such as protein and starch in the hydrolysate and alters the polysaccharide structure, facilitating the release of polysaccharides during the process and effectively increasing purity. Simultaneously, the molecular weight of the polysaccharides is also affected by the hydrolysis process; unhydrolyzed polysaccharides have larger molecular weights. Furthermore, efficient molecular weight cutoff yields the target polysaccharide components, increasing the proportion of polysaccharides with a molecular weight of 4000-6000 Da, which further enhances polysaccharide purity.

[0109] Table 3. Effects of different treatment methods on the antioxidant activity and monosaccharide composition of alcohol-soluble polysaccharides from distiller's grains.

[0110]

[0111] Note: Different lowercase letters in the same column indicate significant differences between groups, p < 0.05.

[0112] Table 3 shows that the activity and monosaccharide composition of distiller's grains polysaccharides are affected by enzymatic hydrolysis and retention steps. Enzymatic hydrolysis can improve the purity of distiller's grains polysaccharides and enhance their antioxidant activity, yielding polysaccharides with higher molar ratios of mannose, galactose, and arabinose, which further enhances their antioxidant activity. On the other hand, polysaccharides with the target molecular weight (4000~6000 Da) exhibit higher activity than those with high molecular weight (> 6000 Da) and low molecular weight (< 4000 Da), indicating that retention has a significant impact on the activity of the target polysaccharides, and extracting the target component can yield distiller's grains polysaccharides with higher activity.

[0113] Table 4. Effects of final ethanol concentration on polysaccharide alcohol solubility, antioxidant properties, and monosaccharide composition.

[0114]

[0115] Note: Different lowercase letters in the same column represent significant differences between groups, p < 0.05; alcohol solubility determination was performed in 60% Vol ethanol aqueous solution.

[0116] Table 4 shows that the final ethanol concentration during the alcohol precipitation process significantly affects the alcohol solubility of polysaccharides from distiller's grains. Polysaccharides from distiller's grains precipitated with a higher final ethanol concentration exhibit better alcohol solubility. The final ethanol concentration has no significant effect on the antioxidant activity and monosaccharide composition of the polysaccharides.

[0117] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing alcohol-soluble polysaccharides from distiller's grains, characterized in that: Includes the following steps: A. Mix the lees and water at a mass-volume ratio of 1g:8~25ml, then crush and centrifuge, and take the supernatant to obtain the lees pretreatment solution. B. Adjust the temperature of the pretreated lees solution obtained in step A to 90~95℃, add amylase for enzymatic hydrolysis, then adjust the temperature to 40~45℃, add neutral protease and cellulase for enzymatic hydrolysis, and obtain lees hydrolysate. C. Heat the enzymatic hydrolysate obtained in step B to 100-105℃ to inactivate the enzyme and then concentrate it. After dialysis and desalting, mix 50-70% Vol ethanol with the dialyzed enzymatic hydrolysate to make the total ethanol concentration in the system reach 38-53% Vol. Then, after alcohol precipitation and centrifugation, obtain the alcohol precipitation supernatant. D. Concentrate the alcohol precipitation supernatant obtained in step C to remove ethanol. First, use a 6000Da ultrafiltration membrane to remove the portion not higher than 6000Da, and then use a 4000Da ultrafiltration membrane to remove the portion not lower than 4000Da, to obtain a retentate of 4000~6000Da. After freeze-drying the retentate, obtain alcohol-soluble polysaccharide from distiller's grains. The monosaccharide composition of the alcohol-soluble polysaccharide from distillers' grains includes glucose, mannose, arabinose, and galactose, with the molar percentage of mannose not less than 15 mol%, the molar percentage of galactose not less than 15 mol%, the molar percentage of arabinose not less than 8 mol%, and the molar percentage of glucose not more than 38 mol%; the proportion of components with a Da of 4000~6000 in the alcohol-soluble polysaccharide from distillers' grains is not less than 85%.

2. The method for preparing alcohol-soluble polysaccharides from distiller's grains according to claim 1, characterized in that: In step A, the water content of the distiller's grains is ≤5wt%, the crude protein content is 13.2~16.5wt%, the starch content is 9.7~15.5wt%, and the cellulose content is 11.2~14.1wt%.

3. The method for preparing alcohol-soluble polysaccharides from distiller's grains according to claim 1, characterized in that: In step B, the amount of amylase added is 600~1000 U / mL of distiller's grains pretreatment solution.

4. The method for preparing alcohol-soluble polysaccharides from distiller's grains according to claim 1, characterized in that: In step B, the amount of neutral protease added is 600~1200 U / mL of distiller's grains pretreatment solution.

5. The method for preparing alcohol-soluble polysaccharides from distiller's grains according to claim 1, characterized in that: In step B, the amount of cellulase added is 300~900 U / mL of distiller's grains pretreatment solution.

6. The method for preparing alcohol-soluble polysaccharides from distiller's grains according to claim 1, characterized in that: At least one of the following must be met: In step B, the amylase hydrolysis time is 1~5 hours; In step B, the hydrolysis time for neutral protease and cellulase is 1-5 hours.

7. The method for preparing alcohol-soluble polysaccharides from distiller's grains according to claim 1, characterized in that: At least one of the following must be met: In step C, the enzyme inactivation time is 10-30 min; In step C, the concentration is to concentrate to 1 / 4 to 1 / 3 of the original volume; In step C, the dialysis desalination is performed using a 500Da dialysis bag with deionized water. In step C, the temperature of the alcohol precipitation is 4~8℃; In step C, the alcohol precipitation time is 12-15 hours.

8. The method for preparing alcohol-soluble polysaccharides from distiller's grains according to any one of claims 1 to 7, characterized in that: In step D, the concentration is to concentrate to 1 / 4 to 1 / 3 of the original volume.

9. The use of the alcohol-soluble polysaccharide prepared by the preparation method according to any one of claims 1 to 8 in the preparation of antioxidants or drugs for the prevention and treatment of liver damage.

10. The application according to claim 9, characterized in that: The antioxidant is an anti-ABTS antioxidant or an anti-DPPH antioxidant.

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