Vinasse alcohol-soluble polysaccharide as well as preparation method and application thereof

Abstract

The invention discloses vinasse alcohol-soluble polysaccharide as well as a preparation method and application thereof, and belongs to the technical field of polysaccharide extraction. In order to improve the alcohol solubility and oxidation resistance of vinasse polysaccharides, the invention provides a vinasse alcohol-soluble polysaccharide and a preparation method and application thereof, and the preparation method comprises the following steps: mixing vinasse and water, crushing and centrifuging, taking supernate, firstly adding amylase for enzymolysis, then adding neutral protease and cellulase for enzymolysis, performing enzyme deactivation on the obtained vinasse enzymatic hydrolysate, and concentrating to obtain the vinasse alcohol-soluble polysaccharide. The method comprises the following steps: dissolving vinasse in distilled water, dialyzing and desalting, enabling the total ethanol concentration of the system to reach 38-53% Vol, then carrying out alcohol precipitation and centrifugation, concentrating the obtained alcohol precipitation supernatant, intercepting by adopting an ultrafiltration membrane to obtain 4000-6000 Da trapped fluid, and freeze-drying to obtain the vinasse alcohol-soluble polysaccharide. By optimizing the enzymolysis technology and controlling the alcohol precipitation condition and the target molecular weight of the polysaccharide, the activity, purity and alcohol solubility of the polysaccharide are remarkably improved, and the polysaccharide has good antioxidant activity and in-vitro anti-liver injury activity.

Description

Technical Field

[0001] The invention belongs to the technical field of polysaccharide extraction, and particularly relates to a vinasse alcohol-soluble polysaccharide and a preparation method and application thereof. Background Art

[0002] Liquor lees are byproducts of the brewing industry. Limited by the existing solid-state fermentation process, lees contain a large number of bioactive ingredients, among which polysaccharides are one of the active ingredients of lees. Polysaccharides have antioxidant activity, which can remove free radicals in the body and reduce the damage of oxidative stress to cells, thereby delaying aging and preventing the occurrence of various chronic diseases, such as cardiovascular disease, diabetes and cancer. The monosaccharide composition of polysaccharides is a key factor affecting the activity of polysaccharides. Among them, mannose, galactose and arabinose have strong antioxidant activity. Increasing the proportion of mannose, galactose and arabinose in the polysaccharide structure can improve the antioxidant activity of polysaccharides. Molecular weight can also lead to differences in polysaccharide activity. Polysaccharides with small molecular weight are easy to absorb and can exert better biological activity, but the structure is easily destroyed during processing; polysaccharides with large molecular weight are stable in deconstruction and can better maintain activity during processing, but the molecular weight is too high and it is not easy to absorb, and the activity is difficult to express. Alcohol-soluble sugars can be added to organic reagents for easy application in cosmetics, wine and other processes. Improving sugar purity and reducing impurities are also conducive to the expression of activity in polysaccharide applications. The reserves of distiller's grains are large, and traditional distiller's grains disposal ignores the potential nutritional value of distiller's grains. In recent years, the extraction of sugar substances from distiller's grains has attracted the attention of researchers.

[0003] However, there are relatively few studies on the extraction of alcohol-soluble polysaccharides from liquor lees, and there is a lack of research on its antioxidant activity. Comparative Document 1 discloses a method for microwave-assisted enzyme extraction of polysaccharides from liquor lees based on response surface methodology optimization, comprising the following steps: A. Extraction: liquor lees are dried (water content ≤ 5%), crushed and sieved to obtain liquor lees powder; liquor lees powder and water are mixed, microwave-treated, and a composite enzyme is added for enzymatic hydrolysis. After the reaction is completed, the enzyme is inactivated, and then centrifuged. The supernatant is collected and concentrated, and anhydrous ethanol is added to the concentrate for alcohol precipitation. The alcohol precipitation solution is then centrifuged to collect the precipitate. The precipitate is then re-dissolved, concentrated and frozen in appropriate amounts of water. After drying, crude polysaccharides from discarded liquor lees are obtained; B. impurity removal: the crude polysaccharides from discarded liquor lees obtained in step A are decolorized by macroporous adsorption resin to obtain a decolorized crude polysaccharide solution; the decolorized polysaccharide solution is deproteinized by enzyme method combined with Sevag method to obtain a deproteinized crude polysaccharide solution; the deproteinized crude polysaccharide solution is dialyzed to remove inorganic salts and small molecules, and then concentrated and freeze-dried to obtain desalted crude polysaccharides; C. separation and purification: the desalted crude polysaccharide obtained in step C is dissolved in water, first separated by aqueous filter membrane, then separated by ultrafiltration membrane, and finally concentrated and freeze-dried to obtain the polysaccharides from discarded liquor lees. However, this method does not disclose the alcohol solubility of polysaccharides, nor does it study the effect of molecular weight on the performance of polysaccharides.

[0004] Therefore, a new extraction process for liquor lees was developed and its in vitro antioxidant activity was explored, which is beneficial to the resource exploration of lees polysaccharides and improving the high-value utilization of liquor lees. Summary of the invention

[0005] In order to solve the problems of the prior art, the present invention prepares alcohol-soluble polysaccharides from liquor lees with antioxidant activity by enzymolysis and ethanol extraction of liquor lees and controlling the molecular weight.

[0006] The present invention first provides a method for preparing vinasse alcohol-soluble polysaccharide, which comprises the following steps:

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

[0008] B. Adjusting the temperature of the vinasse pretreatment solution obtained in step A to 90-95° C., adding amylase for enzymolysis, then adjusting the temperature to 40-45° C., adding neutral protease and cellulase for enzymolysis, and obtaining vinasse enzymolysis solution;

[0009] C. The vinasse enzymatic hydrolysate obtained in step B is heated to 100-105° C. to inactivate the enzyme and then concentrated. The concentrate is dialyzed for desalination, and then 50-70% Vol ethanol is mixed with the dialyzed enzymatic hydrolysate to make the total ethanol concentration in the system reach 38-53% Vol., and then alcohol precipitation and centrifugation are performed to obtain an alcohol precipitation supernatant;

[0010] D. The alcohol precipitation supernatant obtained in step C is concentrated to remove ethanol, and a portion not higher than 6000Da is firstly intercepted with a 6000Da ultrafiltration membrane, and then a portion not lower than 4000Da is intercepted with a 4000Da ultrafiltration membrane to obtain a retentate of 4000 to 6000Da, and the retentate is freeze-dried to obtain a vinasse alcohol-soluble polysaccharide.

[0011] Among them, in the preparation method of the above-mentioned vinasse alcohol-soluble polysaccharide, in step A, the water content of the vinasse 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%.

[0012] Wherein, in the method for preparing the above-mentioned vinasse alcohol-soluble polysaccharide, in step B, the amount of amylase added is 600-1000U / mL vinasse pretreatment solution.

[0013] Wherein, in the method for preparing the above-mentioned vinasse alcohol-soluble polysaccharide, in step B, the amount of the neutral protease added is 600-1200 U / mL vinasse pretreatment solution.

[0014] Wherein, in the method for preparing the above-mentioned vinasse alcohol-soluble polysaccharide, in step B, the added amount of the cellulase is 300-900 U / mL vinasse pretreatment solution.

[0015] Wherein, in the method for preparing the above-mentioned wine lees alcohol-soluble polysaccharide, in step B, the time of amylase hydrolysis is 1 to 5 hours.

[0016] Wherein, in the method for preparing the above-mentioned wine lees alcohol-soluble polysaccharide, in step B, the enzymatic hydrolysis time of neutral protease and cellulase is 1 to 5 hours.

[0017] Wherein, in the method for preparing the above-mentioned wine lees alcohol-soluble polysaccharide, in step C, the enzyme inactivation time is 10 to 30 minutes.

[0018] Wherein, in the method for preparing the above-mentioned wine lees alcohol-soluble polysaccharide, in step C, the concentration is concentrated to 1 / 4 to 1 / 3 of the original volume.

[0019] Wherein, in the method for preparing the above-mentioned wine lees alcohol-soluble polysaccharide, in step C, the dialysis desalination adopts 500Da dialysis bag deionized water for desalination.

[0020] Wherein, in the method for preparing the above-mentioned wine lees alcohol-soluble polysaccharide, in step C, the temperature of the alcohol precipitation is 4-8°C.

[0021] Wherein, in the method for preparing the above-mentioned wine lees alcohol-soluble polysaccharide, in step C, the alcohol precipitation time is 12 to 15 hours.

[0022] Wherein, in the method for preparing the above-mentioned wine lees alcohol-soluble polysaccharide, in step D, the concentration is concentrated to 1 / 4 to 1 / 3 of the original volume.

[0023] The present invention also provides a distiller's grains alcohol-soluble polysaccharide, which is prepared by the above method. The monosaccharide composition of the distiller's grains alcohol-soluble polysaccharide includes glucose, mannose, arabinose and glucose, and the molar proportion of mannose is not less than 15 mol%, the molar proportion of galactose is not less than 15 mol%, the molar proportion of arabinose is not less than 8 mol%, and the molar proportion of glucose is not higher than 38 mol%; the proportion of components with a range of 4000 to 6000 Da in the distiller's grains alcohol-soluble polysaccharide is not less than 85%.

[0024] The present invention also provides the obtained distiller's grains alcohol-soluble polysaccharide prepared by the above method or the use of the above distiller's grains alcohol-soluble polysaccharide in the preparation of antioxidants, the preparation of drugs for preventing and treating liver damage, and the blending of liquor or the preparation of liqueur.

[0025] Preferably, in the above application, the antioxidant is an ABTS antioxidant or a DPPH antioxidant.

[0026] Beneficial effects of the present invention:

[0027] The invention optimizes the vinasse enzymatic hydrolysis technology and the alcohol precipitation process, which is beneficial to improving the modification of vinasse fiber and the dissolution of alcohol-soluble polysaccharides, improving the extraction efficiency, avoiding the use of non-food grade reagents, and ensuring the safety of the product. The invention is easy to industrialize, and is also beneficial to providing the molar ratio of mannose, galactose and arabinose in the polysaccharide, further improving the antioxidant activity of the polysaccharide. The activity and purity of the polysaccharide are significantly improved by intercepting the polysaccharide with a target molecular weight of 4000 to 6000 Da. The obtained alcohol-soluble polysaccharide has good solubility, can be dissolved in 38 to 53% Vol of ethanol, has a solubility of more than 85%, a purity of more than 85%, has good antioxidant activity and in vitro anti-liver damage activity, and can be applied in many fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The present invention is a process route for extracting alcohol-soluble polysaccharides from vinasse.

[0029] Figure 2 This is a graph showing the effects of the vinasse alcohol-soluble polysaccharide obtained in Example 1 on hepatocyte growth and antioxidant enzyme levels; wherein A represents the effect of the polysaccharide on cell growth, and B to D represent the effect of the polysaccharide on the antioxidant enzyme levels of hepatocytes; a to d represent significant differences between the groups, p<0.05.

[0030] Figure 3 This is a graph showing the effects of the high molecular weight alcohol-soluble polysaccharide obtained in Comparative Example 2 on hepatocyte growth and antioxidant enzyme levels; wherein A is the effect of the polysaccharide on cell growth, and B to D are the effects of the cells on the antioxidant enzyme levels of hepatocytes; a to d represent significant differences between the groups, p<0.05.

[0031] Figure 4 This is a graph showing the effects of the small molecular weight alcohol-soluble polysaccharide obtained in comparative example 3 on hepatocyte growth and antioxidant enzyme levels; wherein A is the effect of the polysaccharide on cell growth, and B to D are the effects of the cells on the antioxidant enzyme levels of hepatocytes; a to d represent significant differences between the groups, p<0.05. DETAILED DESCRIPTION

[0032] The present invention is further described in detail below by way of examples, but the protection scope of the present invention is not limited to the scope of the examples.

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

[0034] Table 1 Dry liquor lees index (wt%)

[0035]

[0036]

[0037] 2. Calculation of purity of alcohol-soluble polysaccharides from vinasse

[0038] The polysaccharide was dissolved in deionized water to obtain the substance concentration, and the total sugar content was determined by the phenol-sulfuric acid method to obtain the sugar concentration. The total sugar concentration was then divided by the substance concentration to obtain the purity of the vinasse polysaccharide.

[0039] 3. Determination of polysaccharide alcohol solubility

[0040] Weigh the dry 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°C for 12 hours, then centrifuge, discard the supernatant, put the centrifuge tube with the precipitate in a 100°C oven for 2 hours, and then measure the weight (M3).

[0041] Polysaccharide alcohol solubility = (1-(M3-M1) / M2)*100%.

[0042] 4. Determination of antioxidant activity of alcohol-soluble polysaccharides from vinasse

[0043] (1) Determination of DPPH free radical scavenging ability

[0044] Referring to the national standard "DPPH and ABTS method for the determination of the antioxidant activity of polypeptides" (GB / T 39100-2020), the alcohol-soluble polysaccharide was first prepared into an aqueous solution with a concentration of 2 mg / mL, and DPPH was prepared into an ethanol solution with a concentration of 50 μg / mL. 1 mL of alcohol-soluble polysaccharide solution was mixed with 3 mL of DPPH solution as the experimental group As; 1 mL of alcohol-soluble polysaccharide solution was mixed with 3 mL of ethanol solution as the control group Ac; 3 mL of DPPH solution was mixed with 1 mL of water as the blank group Ab. After each group was mixed, it was reacted at room temperature in the dark for 30 minutes, and the absorbance at a wavelength of 517 nm was detected by an enzyme marker.

[0045] DPPH free radical scavenging rate = (1-(As-Ac) / Ab)*100%.

[0046] (2) ABTS + Free radical scavenging ability determination method

[0047] Referring to the national standard "Determination of Antioxidant Activity of Peptides by DPPH and ABTS Method" (GB / T 39100-2020), first prepare the alcohol-soluble polysaccharide into an aqueous solution with a concentration of 2 mg / mL. Take 200 mg of ABTS, dissolve it in 50 mL of water, and adjust the absorbance to 0.7 with water after 24 hours of exposure to light to prepare ABTS. + Assay solution. Take 0.4 mL of alcohol-soluble polysaccharide solution and 3.6 mL of ABTS + The test solution was mixed as the experimental group As; 0.4 mL of water and 3.6 mL of ABTS were taken +The test solution mixture was used as the blank group Ab; after being mixed evenly, the mixture was reacted at room temperature in the dark for 5 minutes, and the absorbance at a wavelength of 734 nm was detected by an enzyme marker.

[0048] ABTS + Free radical scavenging rate = (Ab-As) / Ab*100%.

[0049] 5. Polysaccharide molecular weight distribution determination method

[0050] The molecular weight distribution of polysaccharide samples was determined by high performance gel permeation chromatography (Waters 2695; Waters) with a differential refractometer Waters 2414. The mobile phase was 0.02% NaNO3 aqueous solution, the flow rate was 0.5 mL / min, and the chromatographic column was Ulterahydroge TM (7.8×300 mm, Waters), column temperature 35° C. Glucose, sucrose, and dextran standards with molecular weights of 10 kDa, 40 kDa, 70 kDa, 150 kDa, 500 kDa, and 2000 kDa were used to determine the peak time, and a sample molecular weight-peak time standard curve was drawn to calculate the sample molecular weight distribution.

[0051] 6. Analysis of monosaccharide composition of distiller’s grains polysaccharides

[0052] 5 mg of sample was hydrolyzed with 2 mL of trifluoroacetic acid (3.00 mol / L) in an ampoule at 120°C for 3 h. After the hydrolysis was completed, the sample was dried with nitrogen and 0.2 mL of methanol was added and nitrogen was blown 3 times to remove the residual trifluoroacetic acid. After the sample was dried, deionized water was added and filtered through a 0.22 μm filter membrane. The liquid that passed through the membrane was transferred to a liquid phase vial and detected by a high-pressure ion chromatography ICS-5000+SP-5 device, which was equipped with a pulsed amperometric detector, an anion exchange column Dionex carbopactmpa 20 (3 mm×150 mm) and a guard column PA20 (3 mm×50 mm). The sample loading amount was 20 μL, and the column temperature was 30°C during detection. The mobile phases were deionized water (phase A), 250mmol / LNaAc deionized water solution (phase B) and 250mmol / L NaOH deionized water solution (phase C), and the mobile phase operation flow was: 99.1% A, 0.9% C (20min), 19.1% A, 80% B, 0.9% C (20.1-27min), 20% A, 80% C (27.1-38min), and the flow rate was 0.5mL / min.

[0053] 7. Cell viability determination

[0054] The polysaccharide was dissolved in the complete culture medium solution, and the polysaccharide intervention reagent with a concentration of 0-120 μg / mL was prepared. After the polysaccharide was fully dissolved, it was filtered with a sterile 0.1 μm needle filter membrane for use. The experiment set up a polysaccharide intervention group, a control group and a blank group, and each group was made into 6 duplicate wells. After 24 hours of cell culture, all wells were carefully aspirated to remove the supernatant of the 96 wells. 100 μL of the polysaccharide intervention reagent of each concentration was added to the polysaccharide intervention group. In the control group, 100 μL of complete culture medium without polysaccharide samples was added to the wells where cells were planted. The blank group did not inoculate cells, and only 100 μL of complete culture medium was added. The 96-well plate was then placed in a cell culture incubator for 24 hours. After the standing period, the 96-well plate was taken out from the incubator, and 10 μL of CCK-8 solution was added to each well with a spray gun and transferred to the cell culture incubator. It was allowed to stand for 4 hours again. After the standing period, the 96-well plate was taken out, and the absorbance of each well at UV 450nm was measured with an ELISA reader. The formula for calculating the cell survival rate was:

[0055]

[0056] In the formula, OD1 represents the absorbance value of cells under polysaccharide intervention; OD2 represents the absorbance value of cells without polysaccharide intervention; OD0 represents the absorbance value of complete culture medium.

[0057] 8. Determination of antioxidant enzyme levels in cell secretions

[0058] The hepatocytes treated with polysaccharides were removed, the cell culture medium was aspirated, the culture medium was centrifuged, and the supernatant was obtained. According to the instructions of the kit, the levels of antioxidant enzymes in the supernatant were measured, including superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT).

[0059] Example 1

[0060] (1) Dry vinasse and deionized water are mixed at a solid-liquid ratio of W / V=1:8, and then crushed. The supernatant is collected by centrifugal filtration to obtain a vinasse pretreatment solution.

[0061] (2) The temperature of the lees pretreatment solution obtained in step (1) was adjusted to 90° C., 1000 U / mL of amylase was added, and enzymatic hydrolysis was performed for 1 hour. Then, the temperature was controlled to 40° C., 1200 U / mL of neutral protease and 900 U / mL of cellulase were added, and enzymatic hydrolysis was performed for 1 hour to obtain lees enzymatic hydrolyzate.

[0062] (3) The lees enzymatic hydrolysate obtained in step (2) is heated to 100° C. to inactivate the enzyme for 10 min, then concentrated to 1 / 4 of the original volume, desalted using a 500 Da dialysis bag with deionized water, and then mixed with 50% Vol ethanol and the dialyzed enzymatic hydrolysate to achieve a final ethanol concentration of 38% Vol. After alcohol precipitation at 4° C. for 12 h, the supernatant is centrifuged.

[0063] (4) The supernatant of step (3) is concentrated to 1 / 4 of the original volume, ethanol is removed, and the supernatant is retained by an ultrafiltration membrane. A 6000Da ultrafiltration membrane is first used to capture a portion not higher than 6000Da, and then a 4000Da ultrafiltration membrane is used to capture a portion not lower than 4000Da, and finally a retentate of 4000-6000Da is obtained. The retentate is freeze-dried to obtain a vinasse alcohol-soluble polysaccharide.

[0064] The alcohol solubility, purity, molecular weight, monosaccharide composition and antioxidant activity of alcohol-soluble polysaccharides from vinasse were determined. The results are shown in Tables 2 and 3.

[0065] Example 2

[0066] (1) Dry vinasse and deionized water are mixed at a solid-liquid ratio of W / V=1:25, and then crushed. The supernatant is collected by centrifugal filtration to obtain a vinasse pretreatment solution.

[0067] (2) The temperature of the lees pretreatment solution obtained in step (1) was adjusted to 95° C., 600 U / mL of amylase was added, and enzymatic hydrolysis was performed for 5 h. The temperature was then controlled to 45° C., 600 U / mL of neutral protease and 300 U / mL of cellulase were added, and enzymatic hydrolysis was performed for 5 h to obtain lees enzymatic hydrolyzate.

[0068] (3) The vinasse hydrolysate obtained in step (2) is heated to 100° C. to inactivate the enzyme for 10 min, then concentrated to 1 / 4 of the original volume, desalted with deionized water in a 500 Da dialysis bag, and then mixed with 50% Vol ethanol to achieve a final ethanol concentration of 38% Vol. After alcohol precipitation at 6° C. for 13 h, the supernatant is collected by centrifugation.

[0069] (4) The supernatant of step (3) is concentrated to 1 / 4 of the original volume, ethanol is removed, and the supernatant is retained by an ultrafiltration membrane. A portion not higher than 6000Da is firstly captured by a 6000Da ultrafiltration membrane, and then a portion not lower than 4000Da is captured by a 4000Da ultrafiltration membrane, to finally obtain a retentate of 4000 to 6000Da. The retentate is freeze-dried to obtain a vinasse alcohol-soluble polysaccharide.

[0070] The alcohol solubility, purity, molecular weight, monosaccharide composition and antioxidant activity of alcohol-soluble polysaccharides from vinasse were determined. The results are shown in Tables 2 and 3.

[0071] Example 3

[0072] (1) Dry vinasse and deionized water are mixed at a solid-liquid ratio of W / V=1:15, and then crushed. The supernatant is collected by centrifugal filtration to obtain a vinasse pretreatment solution.

[0073] (2) The temperature of the lees pretreatment solution obtained in step (1) was adjusted to 93° C., 800 U / mL of amylase was added, and enzymatic hydrolysis was carried out for 3 h. The temperature was then controlled to 43° C., 1000 U / mL of neutral protease and 600 U / mL of cellulase were added, and enzymatic hydrolysis was carried out for 3 h to obtain lees enzymatic hydrolyzate.

[0074] (3) The vinasse hydrolysate obtained in step (2) is heated to 100° C. to inactivate the enzyme for 10 min, then concentrated to 1 / 4 of the original volume, desalted using a 500 Da dialysis bag with deionized water, and then mixed with 50% Vol ethanol to obtain a final ethanol concentration of 38% Vol. After alcohol precipitation at 8° C. for 15 h, the supernatant is collected by centrifugation.

[0075] (4) The supernatant of step (3) is concentrated to 1 / 4 of the original volume, ethanol is removed, and the supernatant is retained by an ultrafiltration membrane. A portion not higher than 6000Da is firstly captured by a 6000Da ultrafiltration membrane, and then a portion not lower than 4000Da is captured by a 4000Da ultrafiltration membrane, to finally obtain a retentate of 4000 to 6000Da. The retentate is freeze-dried to obtain a vinasse alcohol-soluble polysaccharide.

[0076] The alcohol solubility, purity, molecular weight, monosaccharide composition and antioxidant activity of alcohol-soluble polysaccharides from vinasse were determined. The results are shown in Tables 2 and 3.

[0077] Example 4: Setting of ethanol concentration during alcohol precipitation

[0078] Example 4.1: Step (3) in Example 1 was adjusted to use 60% Vol ethanol and the dialyzed enzymatic hydrolysate for alcohol precipitation, so that the final ethanol concentration reached 45% Vol. Other conditions or parameters were consistent with Example 1.

[0079] Example 4.2: Step (3) in Example 1 was adjusted to use 70% Vol ethanol and the dialyzed enzymatic hydrolysate for alcohol precipitation, so that the final ethanol concentration reached 53% Vol. Other conditions or parameters were consistent with Example 1.

[0080] The alcohol solubility and antioxidant activity of the alcohol-soluble polysaccharide of vinasse were calculated and compared with those in Example 1. The results are shown in Table 4.

[0081] Example 5: Using alcohol-soluble polysaccharides from vinasse for liver cell repair

[0082] The alcohol-soluble polysaccharide extracted from the lees in Example 1 was dissolved in the cell culture medium, and the final concentration of the polysaccharide in the cell culture medium was 10, 20, 40, 60, 80, 100 and 120 μg / mL, the cell growth was measured, and the toxicity of the polysaccharide was analyzed. The results showed that the polysaccharide final concentration of 10-80 μg / mL had no inhibitory effect on cell growth and no toxic effect, and the concentration of 40, 60 and 80 μg / mL had a promoting effect on cell growth. Subsequently, the HepG2 hepatocytes damaged by H2O2 were intervened under the conditions of polysaccharide concentrations of 40, 60 and 80 μg / mL. The results showed that compared with the hepatocytes without excessive sugar intervention (Mod group), when the intervention concentration reached 60 μg / mL, the enzyme activities of the antioxidant enzymes SOD, CAT and GSH-Px in the cell secretion fluid of the hepatocytes intervened by polysaccharides were significantly increased by 80.1%, 50.0% and 85.7%, respectively, indicating that lees polysaccharides can reduce the level of cellular oxidative stress and are beneficial to cell repair. The relevant results are shown in Figure 2 .

[0083] Comparative Example 1: No enzymatic treatment

[0084] The polysaccharide enzymatic hydrolysis step in Example 1 is not implemented, and the other steps and conditions are consistent with those in Example 1. The method comprises the following steps:

[0085] (1) Dry vinasse and deionized water are mixed at a solid-liquid ratio of W / V=1:8, and then crushed. The supernatant is collected by centrifugal filtration to obtain a vinasse pretreatment solution.

[0086] (2) The vinasse obtained in step (1) was pretreated and heated to 100° C. to inactivate the enzyme for 10 min, then concentrated to 1 / 4 of the original volume, desalted with deionized water in a 500 Da dialysis bag, and then mixed with 50% Vol ethanol and the dialyzed enzymatic hydrolyzate to make the final ethanol concentration reach 38% Vol. After alcohol precipitation at 4° C. for 12 h, the supernatant was collected by centrifugation.

[0087] (3) The supernatant of step (2) is concentrated to 1 / 4 of the original volume, ethanol is removed, and the supernatant is retained by an ultrafiltration membrane. A portion not higher than 6000Da is firstly captured by a 6000Da ultrafiltration membrane, and then a portion not lower than 4000Da is captured by a 4000Da ultrafiltration membrane, to finally obtain a retentate of 4000 to 6000Da. The retentate is freeze-dried to obtain a vinasse alcohol-soluble polysaccharide.

[0088] The alcohol solubility, purity, molecular weight, monosaccharide composition and antioxidant activity of alcohol-soluble polysaccharides from vinasse were determined. The results are shown in Tables 2 and 3.

[0089] Comparative Example 2: Retention of the molecular weight segment greater than 6000Da

[0090] The ultrafiltration membrane interception step in Example 1 was adjusted, and the concentrated supernatant was intercepted with a 6000Da ultrafiltration membrane to obtain a portion larger than 6000Da. The other steps and conditions were the same as those in Example 1. The method comprises the following steps:

[0091] (1) Dry vinasse and deionized water are mixed at a solid-liquid ratio of W / V=1:8, and then crushed. The supernatant is collected by centrifugal filtration to obtain a vinasse pretreatment solution.

[0092] (2) The temperature of the lees pretreatment solution obtained in step (1) was adjusted to 90° C., 1000 U / mL of amylase was added, and enzymatic hydrolysis was performed for 1 hour. Then, the temperature was controlled to 40° C., 1200 U / mL of neutral protease and 900 U / mL of cellulase were added, and enzymatic hydrolysis was performed for 1 hour to obtain lees enzymatic hydrolyzate.

[0093] (3) The lees enzymatic hydrolysate obtained in step (2) is heated to 100° C. to inactivate the enzyme for 10 min, then concentrated to 1 / 4 of the original volume, desalted using a 500 Da dialysis bag with deionized water, and then mixed with 50% Vol ethanol and the dialyzed enzymatic hydrolysate to achieve a final ethanol concentration of 38% Vol. After alcohol precipitation at 4° C. for 12 h, the supernatant is centrifuged.

[0094] (4) The supernatant of step (3) is concentrated to 1 / 4 of the original volume, ethanol is removed, and the supernatant is retained by an ultrafiltration membrane. The portion larger than 6000Da is retained by a 6000Da ultrafiltration membrane, and the retained solution is freeze-dried to obtain a high molecular weight alcohol-soluble polysaccharide (>6000Da).

[0095] The alcohol solubility, purity, molecular weight, monosaccharide composition and antioxidant activity of alcohol-soluble polysaccharides from vinasse were determined. The results are shown in Tables 2 and 3.

[0096] The alcohol-soluble polysaccharide extracted from the lees in Comparative Example 2 was dissolved in the cell culture medium, and the final concentration of the polysaccharide in the cell culture medium was 10, 20, 40, 60, 80, 100 and 120 μg / mL. The cell growth was measured and the toxicity of the polysaccharide was analyzed. The results showed that when the final concentration of the polysaccharide exceeded 80 μg / mL, it had a significant inhibitory effect on cell growth. Concentrations of 40, 60 and 80 μg / mL were selected to intervene in HepG2 liver cells damaged by H2O2. The results showed that compared with the liver cells without excessive sugar intervention (Mod group), the effect of improving the activity levels of antioxidant enzymes SOD, CAT and GSH-Px in the cell secretion fluid of the liver cells intervened by high molecular weight alcohol-soluble polysaccharides (>6000Da) was not significant (p>0.05, Figure 3 ). Large molecular weight alcohol-soluble polysaccharides (>6000Da) have low ability to regulate cellular antioxidant enzymes and failed to show biological activity in regulating cellular oxidative stress response at the experimental setting dose.

[0097] Comparative Example 3: Retaining the molecular weight segment less than 4000Da

[0098] The ultrafiltration membrane interception step in Example 1 was adjusted, and the concentrated supernatant was intercepted with a 4000Da ultrafiltration membrane to obtain a portion less than 4000Da. The other steps and conditions were the same as those in Example 1. The method comprises the following steps:

[0099] (1) Dry vinasse and deionized water are mixed at a solid-liquid ratio of W / V=1:8, and then crushed. The supernatant is collected by centrifugal filtration to obtain a vinasse pretreatment solution.

[0100] (2) The temperature of the lees pretreatment solution obtained in step (1) was adjusted to 90° C., 1000 U / mL of amylase was added, and enzymatic hydrolysis was performed for 1 hour. Then, the temperature was controlled to 40° C., 1200 U / mL of neutral protease and 900 U / mL of cellulase were added, and enzymatic hydrolysis was performed for 1 hour to obtain lees enzymatic hydrolyzate.

[0101] (3) The lees enzymatic hydrolysate obtained in step (2) is heated to 100° C. to inactivate the enzyme for 10 min, then concentrated to 1 / 4 of the original volume, desalted using a 500 Da dialysis bag with deionized water, and then mixed with 50% Vol ethanol and the dialyzed enzymatic hydrolysate to achieve a final ethanol concentration of 38% Vol. After alcohol precipitation at 4° C. for 12 h, the supernatant is centrifuged.

[0102] (4) The supernatant of step (3) is concentrated to 1 / 4 of the original volume, ethanol is removed, and the supernatant is retained by an ultrafiltration membrane. The portion less than 4000Da is retained by a 4000Da ultrafiltration membrane, and the retained liquid is freeze-dried to obtain a low molecular weight wine lees alcohol-soluble polysaccharide (<4000Da).

[0103] The alcohol-soluble polysaccharide extracted from the lees in comparative example 3 was dissolved in the cell culture medium, and the final concentration of the polysaccharide in the cell culture medium was 10, 20, 40, 60, 80, 100 and 120 μg / mL. The cell growth was measured and the toxicity of the polysaccharide was analyzed. The results showed that when the final concentration of the dry polysaccharide exceeded 80 μg / mL, it had a significant inhibitory effect on cell growth. Concentrations of 40, 60 and 80 μg / mL were selected to intervene in HepG2 liver cells damaged by H2O2. The results showed that compared with the liver cells without excessive sugar intervention (Mod group), the antioxidant enzymes SOD, CAT and GSH-PX enzyme activity levels in the cell secretion fluid of the liver cells intervened by small molecular weight alcohol-soluble polysaccharides (<4000Da) were significantly improved (p<0.05, Figure 4). Among them, when the intervention concentration reached 60μg / mL, the activity levels of antioxidant enzymes SOD, CAT and GSH-Px in the cell secretion fluid of hepatocytes intervened by 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 4000-6000Da, small molecular weight alcohol-soluble polysaccharides (<4000Da) had a weaker effect on promoting the activity level of cellular antioxidant enzymes and a lower effect on regulating cellular oxidative stress. The biological activity of small molecular weight alcohol-soluble polysaccharides (<4000Da) is lower than that of alcohol-soluble polysaccharides with a molecular weight of 4000-6000Da.

[0104] By comparing Example 5, Comparative Example 2 and Comparative Example 3, it is found that the molecular weight has a significant effect on the biological activity of alcohol-soluble polysaccharides. Components of vinasse alcohol-soluble polysaccharides with molecular weights that are too large (>6000Da) or too small (<4000Da) fail to show good ability to regulate the level of cellular antioxidant enzymes, while alcohol-soluble polysaccharides with target molecular weights (4000-6000Da) have strong biological activity, can significantly increase the level of cellular antioxidant enzymes, and have a strong effect on regulating cellular oxidative stress.

[0105] The alcohol solubility, purity, molecular weight, monosaccharide composition and antioxidant activity of alcohol-soluble polysaccharides from vinasse were determined. The results are shown in Tables 2 and 3.

[0106] Table 2 Effects of different treatment methods on alcohol solubility, purity and molecular weight of alcohol-soluble polysaccharides in vinasse alcohol-soluble polysaccharides

[0107]

[0108]

[0109] Note: Different lowercase letters in the same column represent significant differences among groups, p<0.05; alcohol solubility was determined in 60% Vol ethanol aqueous solution.

[0110] Table 2 shows that enzymatic hydrolysis is a very critical step, which can significantly improve the purity and antioxidant activity of alcohol-soluble polysaccharides from lees. The main reason may be that enzymatic hydrolysis reduces impurities such as protein and starch in the lees hydrolyzate and changes the structure of lees polysaccharides, which is conducive to the release of polysaccharides during the enzymatic hydrolysis process and effectively improves the purity of polysaccharides. At the same time, the molecular weight of polysaccharides is also affected by the enzymatic hydrolysis process, and the molecular weight of polysaccharides that have not been enzymatically hydrolyzed is larger. On the other hand, molecular weight interception can efficiently obtain the target polysaccharide component, increase the proportion of polysaccharides with a molecular weight of 4000-6000Da, and help improve the purity of polysaccharides.

[0111] Table 3 Effects of different treatments on the antioxidant activity and monosaccharide composition of alcohol-soluble polysaccharides in vinasse alcohol-soluble polysaccharides

[0112]

[0113] Note: Different lowercase letters in the same column represent significant differences between groups, p<0.05

[0114] Table 3 shows that the activity and monosaccharide composition of vinasse polysaccharides are affected by the enzymatic hydrolysis and interception steps. Enzymatic hydrolysis can improve the purity of vinasse polysaccharides and improve the antioxidant activity of polysaccharides. Polysaccharides with a high molar ratio of mannose, galactose and arabinose can be obtained, which further improves the antioxidant activity of polysaccharides. On the other hand, the activity of polysaccharides with a target molecular weight (4000-6000Da) is higher than that of polysaccharides with a large molecular weight (>6000Da) and polysaccharides with a small molecular weight (<4000Da), indicating that interception has a significant effect on the activity of the target polysaccharide, and extracting the target component can obtain vinasse polysaccharides with higher activity.

[0115] Table 4 Effect of final ethanol concentration on alcohol solubility, antioxidant capacity and monosaccharide composition of polysaccharides

[0116]

[0117]

[0118] Note: Different lowercase letters in the same column represent significant differences among groups, p<0.05; alcohol solubility was determined in 60% Vol ethanol aqueous solution.

[0119] Table 4 shows that the adjustment of the final ethanol concentration during the alcohol precipitation process has a significant effect on the alcohol solubility of the lees polysaccharide. The lees polysaccharide obtained from the alcohol precipitation with a higher final ethanol concentration has better alcohol solubility. The final ethanol concentration has no significant effect on the antioxidant activity and monosaccharide composition of the polysaccharide.

[0120] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing vinasse alcohol-soluble polysaccharides, characterized in that: The following steps are involved: A. Mix the vinasse and water in a mass volume ratio of 1 g: 8-25 ml, then crush and centrifuge, take the supernatant, and obtain a vinasse pretreatment solution; B. Adjusting the temperature of the vinasse pretreatment solution obtained in step A to 90-95° C., adding amylase for enzymolysis, then adjusting the temperature to 40-45° C., adding neutral protease and cellulase for enzymolysis, and obtaining vinasse enzymolysis solution; C. The vinasse enzymatic hydrolysate obtained in step B is heated to 100-105° C. to inactivate the enzyme and then concentrated. The concentrate is dialyzed for desalination, and then 50-70% Vol ethanol is mixed with the dialyzed enzymatic hydrolysate to make the total ethanol concentration in the system reach 38-53% Vol., and then alcohol precipitation and centrifugation are performed to obtain an alcohol precipitation supernatant; D. The alcohol precipitation supernatant obtained in step C is concentrated to remove ethanol, and a portion not higher than 6000Da is firstly intercepted with a 6000Da ultrafiltration membrane, and then a portion not lower than 4000Da is intercepted with a 4000Da ultrafiltration membrane to obtain a retentate of 4000 to 6000Da, and the retentate is freeze-dried to obtain a vinasse alcohol-soluble polysaccharide.

2. The method for preparing vinasse alcohol-soluble polysaccharide according to claim 1, characterized in that: In step A, the water content of the vinasse 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 vinasse alcohol-soluble polysaccharide according to claim 1, characterized in that: In step B, the amount of amylase added is 600-1000 U / mL of the lees pretreatment solution.

4. The method for preparing vinasse alcohol-soluble polysaccharide according to claim 1, characterized in that: In step B, the amount of the neutral protease added is 600-1200 U / mL of the lees pretreatment solution.

5. The method for preparing vinasse alcohol-soluble polysaccharide according to claim 1, characterized in that: In step B, the added amount of the cellulase is 300-900 U / mL of the lees pretreatment solution.

6. The method for preparing vinasse alcohol-soluble polysaccharide according to claim 1, characterized in that: At least one of the following is met: In step B, the time of amylase hydrolysis is 1 to 5 hours; In step B, the enzymatic hydrolysis time of neutral protease and cellulase is 1 to 5 hours.

7. The method for preparing vinasse alcohol-soluble polysaccharide according to claim 1, characterized in that: At least one of the following is met: In step C, the enzyme inactivation time is 10 to 30 minutes; In step C, the concentration is concentrated to 1 / 4 to 1 / 3 of the original volume; In step C, the dialysis desalination adopts 500Da dialysis bag deionized water desalination; In step C, the temperature of the alcohol precipitation is 4-8°C; In step C, the alcohol precipitation time is 12 to 15 hours.

8. The method for preparing vinasse alcohol-soluble polysaccharide 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 method for preparing the vinasse alcohol-soluble polysaccharide according to claim 1 is used to prepare the vinasse alcohol-soluble polysaccharide, characterized in that: The monosaccharide composition of the wine lees alcohol-soluble polysaccharide includes glucose, mannose, arabinose and glucose, and the molar proportion of mannose is not less than 15 mol%, the molar proportion of galactose is not less than 15 mol%, the molar proportion of arabinose is not less than 8 mol%, and the molar proportion of glucose is not higher than 38 mol%; the proportion of components with a size of 4000 to 6000 Da in the wine lees alcohol-soluble polysaccharide is not less than 85%.

10. Use of the alcohol-soluble polysaccharide of vinasse prepared by the preparation method described in any one of claims 1 to 8 and the alcohol-soluble polysaccharide of vinasse described in claim 9 in the preparation of antioxidants, the preparation of drugs for preventing and treating liver damage, and the blending of liquor or the preparation of liqueur; preferably, the antioxidant is an anti-ABTS oxidant or an anti-DPPH oxidant.

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