CN113372463B - Method for extracting probiotic functional sugar from distilled rice wine distillation residual liquid - Google Patents
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Abstract
The invention belongs to the field of polysaccharide extraction, and particularly relates to a method for extracting probiotic functional sugar from distilled rice wine distillation residual liquid. The method comprises the following steps: concentrating the distillation residue of distilled rice wine; adding ethanol solution into the concentrated distilled rice wine distillation residual liquid until the final concentration of ethanol is 30%, precipitating with ethanol, and centrifuging; taking supernatant, adding ethanol solution until the final concentration of ethanol is 75-85%, precipitating with ethanol, and centrifuging; concentrating the supernatant obtained by centrifugation, and freeze-drying to obtain rice wine crude polysaccharide; dissolving the rice wine crude polysaccharide to obtain a rice wine crude polysaccharide sample liquid, and removing protein by adopting a Sevage reagent, wherein the dosage ratio of the Sevage reagent to the rice wine crude polysaccharide sample liquid is 1: 3-6; and putting the deproteinized rice wine polysaccharide sample liquid into a dialysis bag with molecular weight cutoff of 3500Da for dialysis in ultrapure water, and then concentrating, freezing and drying to obtain the target probiotic functional sugar. The polysaccharide extracted by the method has high yield and high purity, and has better effect than inulin in the process of promoting the proliferation of the bifidobacterium breve.
Description
Technical Field
The invention belongs to the technical field of polysaccharide extraction, and particularly relates to a method for extracting probiotic functional sugar from distilled rice wine distillation residual liquid.
Background
The rice wine is a traditional Chinese alcoholic beverage prepared by taking glutinous rice as a raw material, steaming, inoculating and mixing with yeast, and carrying out microbial saccharification and fermentation, and is generally divided into brewed rice wine and distilled rice wine. The distilled rice wine is rice white spirit with high alcohol content, such as Guilin three-flower rice-flavor white spirit and the like, which is obtained by distilling rice wine on the basis of brewing rice wine. During the fermentation process, under the comprehensive action of microorganisms such as mould and saccharomycetes in the distiller's yeast and metabolites of various enzymes, various biomacromolecules such as protein, polysaccharide and fat are degraded into a large number of small molecular substances such as monosaccharide, oligosaccharide, short-chain peptide, amino acid and organic acid. The comprehensive influence of the substances endows the rice wine with unique nutrition and flavor.
The glutinous rice starch is degraded and converted by the enzyme produced by the microorganism in the koji, so that the brewed rice wine contains abundant saccharide substances. These saccharides not only give unique taste to rice wine, but also are main energy substances of human body. In recent years, more and more researchers and consumers begin to pay attention to the nutritional and health functions of rice wine. The research on functional factors of rice wine is reported to be insufficient, for example, Deng Shihui and the like are obtained by measuring the oxidation resistance and fatigue resistance of Jiang river rice wine, compared with VC, the oxidation resistance of Jiang river rice wine is better, the DPPH clearance rate of Jiang river rice wine is 49.65%, and in addition, Jiang river rice wine also has certain fatigue resistance, so that the content of liver glycogen can be increased, and the levels of serum urea nitrogen and serum lactic acid after exercise can be reduced. However, the research on the biological activity and active ingredients of rice wine is still in the first stage, and deep and systematic research is lacked.
A large amount of microorganisms exist in the intestinal tract of a human body, and the microorganisms in the intestinal tract keep dynamic balance at all times under normal conditions. However, some external or internal factors affect the abundance and composition of intestinal microorganisms, resulting in intestinal microbial imbalance, which may threaten human health in severe cases. It has been found that diet is one of the key pathways for altering intestinal microorganisms. The regulation of gut health by modification of the diet has attracted a great deal of human attention. Researches show that the edible prebiotics can effectively regulate and control the imbalance of intestinal microorganisms, thereby improving human diseases and promoting the human body to return to a healthy state. Prebiotics can exert these effects, primarily because they can regulate the ph of the gut, regulate the composition and abundance of gut microbes, and promote the formation of beneficial metabolites, after they are fermented in the gut. Ideally, prebiotics should be able to lower intestinal pH, promote the growth and activity of beneficial bacteria, and increase the production of short chain fatty acids.
In order to obtain higher alcoholic strength, alcohol is usually separated from these polysaccharides in the distillation process, resulting in the polysaccharides being dissolved in the distillation residue, resulting in waste of raw materials. In the prior art, no report of extracting the probiotic functional sugar from distilled rice wine distillation residual liquid is found.
Disclosure of Invention
The polysaccharide substances in the distillation raffinate are excavated, so that the distillation raffinate is recycled, and the production cost is reduced, which is urgently needed by the industry at present.
The first purpose of the invention is to provide a method for extracting probiotic functional sugar from distilled rice wine distillation raffinate, which comprises the following steps:
(1) concentrating the distillation residue of distilled rice wine;
(2) adding an ethanol solution into the concentrated distilled rice wine distillation residual liquid obtained in the step (1) until the final concentration of ethanol is 25-30%, precipitating with ethanol, and centrifuging;
(3) taking the supernatant obtained by the centrifugation in the step (2), adding an ethanol solution until the final concentration of ethanol is 75-85%, precipitating with ethanol, and centrifuging;
(4) concentrating the supernatant obtained by the centrifugation in the step (3), and freeze-drying to obtain rice wine crude polysaccharide;
(5) dissolving the rice wine crude polysaccharide to obtain a rice wine crude polysaccharide sample liquid, and removing protein by adopting a Sevage reagent, wherein the dosage ratio of the Sevage reagent to the rice wine crude polysaccharide sample liquid is 1: 3-6;
(6) and (3) putting the deproteinized rice wine polysaccharide sample liquid obtained in the step (5) into a dialysis bag with molecular weight cutoff of 3500 Da-5000 Da for dialysis in ultrapure water, and then concentrating, freezing and drying to obtain the target probiotic functional sugar.
As an embodiment of the present invention, step S1 is further included after step (3) and before step (4);
s1: taking the precipitate obtained by the centrifugation in the step (3), adding 25-30% ethanol for redissolution, standing and centrifuging;
the step (4) is specifically as follows: and (4) concentrating the supernatant obtained by the centrifugation in the step (3) and/or the step S1, and freeze-drying to obtain the rice wine crude polysaccharide.
Wherein the standing in the step S1 specifically includes: standing for 10-20 h at 4-10 ℃.
In one embodiment of the present invention, the final concentration of ethanol in step (3) is 80%.
In one embodiment of the invention, the concentration in the step (1) is specifically carried out at 45-55 ℃ under reduced pressure until the original volume is 1/5-1/3.
In one embodiment of the present invention, the ethanol solution used in the step (2) and/or the step (3) is 95% ethanol.
In one embodiment of the present invention, the dialysis time in the step (6) is 48 to 72 hours. The purpose of dialysis is to remove impurities such as inorganic salts and oligosaccharides.
As an embodiment of the invention, the alcohol precipitation temperature in the step (2) and/or the step (3) is 4-10 ℃, and the alcohol precipitation time is 12-24 h.
As an embodiment of the present invention, the centrifugation conditions in the step (2) and/or the step (3) are: the rotating speed is 5000r/min to 6000r/min, and the centrifugation time is 10min to 15 min.
As an embodiment of the present invention, the specific steps of removing protein by using Sevage reagent in step (5) are as follows:
mixing the rice wine crude polysaccharide sample liquid with a Sevage reagent, placing the mixture in a shaking table, fully shaking, centrifuging to obtain a supernatant, and repeating the steps for multiple times until no protein intermediate layer appears; the supernatant was collected and concentrated under reduced pressure to remove the organic solvent. Wherein the temperature of the reduced pressure concentration is 45-55 ℃.
The second purpose of the invention is to provide a rice wine probiotic functional sugar product obtained by the method.
The third purpose of the invention is to provide the application of the rice wine probiotic functional sugar product in probiotic culture.
The fourth purpose of the invention is to provide an enlarged culture method of probiotics, which adopts the rice wine probiotic functional sugar product as a culture medium of an external carbon source to culture probiotics; the probiotic bacteria include but are not limited to bifidobacterium breve and lactobacillus rhamnosus.
The invention has the beneficial effects that:
(1) the invention adopts a fractional precipitation method, and sequentially adds solvents with different proportions according to different solubilities of the polysaccharide in lower alcohols with different concentrations to achieve the aim of distributed precipitation of the polysaccharide. Therefore, the step precipitation method is selected to separate polysaccharides with different molecular weights to obtain the polysaccharides required by us.
(2) The polysaccharide sample is obtained by freeze drying, compared with other drying methods, the polysaccharide sample has low heat consumption, the loss of some volatile components in the product is low, the dried substance is loose and porous and is in a spongy shape, the substance is quickly and completely dissolved after being added with water, and more than 95 to 99 percent of water can be removed, so that the dried product can be stored for a long time without deterioration.
(3) The method has the advantages of simple operation, mild condition, difficult damage to polysaccharide structure and no denaturation of polysaccharide.
(4) The polysaccharide prepared by the method is easy to operate, low in production cost, high in yield and high in purity, and the obtained polysaccharide is stable in structure, not easy to damage and high in probiotic activity. The method provided by the invention can obtain high-purity polysaccharide, wherein the content of protein is very low, and the method is favorable for the next purification.
(5) The polysaccharide extracted by the method has high yield and high purity, and has better effect than inulin particularly in the process of promoting the proliferation of the bifidobacterium breve.
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FIG. 1 shows the results of comparative experiments of prebiotics activity of polysaccharide and inulin as prebiotics for Bifidobacterium breve in sake prepared in example 1 of the present invention;
FIG. 2 shows the results of comparative experiments of prebiotics activity of polysaccharide and inulin as prebiotics for Lactobacillus rhamnosus in sake of example 1.
Detailed Description
The present invention is further described below with reference to specific examples so that those skilled in the art can better understand the present invention, but the present invention is not limited thereto.
The test method comprises the following steps:
1. testing of polysaccharide content: measuring polysaccharide content by phenol-sulfuric acid method
2. And (3) determining the molecular weight of the rice wine polysaccharide: determining relative molecular mass of sake polysaccharide by High Performance Gel Filtration Chromatography (HPGFC), dissolving appropriate amount of sake polysaccharide in ultrapure water to final concentration of 5mg/mL, filtering with 0.22 μm filter membrane, and analyzing; the chromatographic conditions were as follows: the instrument comprises the following steps: LC-1200 high performance liquid chromatograph; a detector: a RID detector; a chromatographic column: superose12(1.0 cm. times.30 cm); mobile phase: 0.01mol/L NaOH; flow rate: 0.5 mL/min; column temperature: 30 ℃; sample introduction amount: 20 μ L. Standard curve equations were obtained from dextran standards (Mw: 3.65kDa, 21kDa, 55.5kDa, 123.5kDa and 326.6 kDa).
3. Determination of monosaccharide composition: and (3) measuring the monosaccharide composition of the rice wine polysaccharide by adopting an ICS-5000 ion chromatograph. Taking 5mg polysaccharide samples, adding 300 mu L of 2mol/L trifluoroacetic acid (TFA) solution, uniformly mixing, and hydrolyzing at 100 ℃ for 6 hours; after cooling to room temperature, the mixture is blown dry by nitrogen, then 300 mul of methanol is added for blowing dry again, the operations are repeated for 4-5 times, TFA is completely removed, 25mL of ultrapure water is added for dissolution, and the mixture is diluted by 10 times and then is subjected to sample loading and measurement.
The source of the biological material is as follows:
inulin: purchased from Shanghai national drug group, and other reagents were of analytical grade.
MRS culture medium: peptone 10 g/L; 10g/L of beef extract; 5g/L of yeast extract; 2g/L of sodium acetate; 2g/L of diammonium hydrogen citrate; magnesium sulfate 0.58 g/L; 0.25g/L of manganese sulfate; 5g/L of carbon source; 0.05% (w/v) L-cysteine.
Bifidobacterium breve with the preservation number of CGMCC1.3001, the preservation unit is China general microbiological culture Collection center (CGMCC), the preservation address of No. 3 West Lu 1 of the North Chen of the sunward area in Beijing, the institute of microbiology, China academy of sciences.
Lactobacillus rhamnosus, the preservation number is CGMCC1.8882, the preservation unit is China general microbiological culture Collection center (CGMCC), the preservation address is No. 3 of Xilu No. 1 of Beijing university of Tokyo sunward, and the institute of microbiology of China academy of sciences.
Example 1
A method for extracting probiotic functional sugar from distilled rice wine distillation residual liquid comprises the following steps:
(1) collecting 500ml distilled rice wine distillation residual liquid (fermented glutinous rice as raw material by distiller's yeast to obtain brewed rice wine, and the residual liquid after distillation), and concentrating under reduced pressure at 50 deg.C to original volume of 1/3;
(2) adding 95% ethanol into the distilled rice wine distillation residual liquid after the reduced pressure concentration in the step (1) until the final concentration of the ethanol is 30%, fully stirring, and precipitating with ethanol at 4 ℃ for 12 h; then 6000r/min, centrifugation for 15 min.
(3) Taking the supernatant obtained by the centrifugation in the step (2), adding 95% ethanol until the final concentration of the ethanol is 80%, fully stirring, and precipitating with ethanol at 4 ℃ for 12 h; then 6000r/min, centrifugation for 15 min.
(4) And (4) taking the precipitate obtained by centrifugation in the step (3), adding 30% ethanol for redissolving, standing at 4 ℃ for 12h, and then centrifuging at 6000r/min for 15 min.
(5) Taking the supernatant obtained by the centrifugation in the step (3) and/or the step (4), concentrating under reduced pressure, and freeze-drying to obtain rice wine crude polysaccharide;
(6) dissolving a certain amount of rice wine crude polysaccharide, removing protein by adopting a Sevage reagent (trichloromethane: n-butyl alcohol is 4: 1; v/v), adding the Sevage reagent of sample liquid 1/5(v/v), putting into a shaking table, fully oscillating, centrifuging at 5000r/min for 10min, taking supernatant, and repeating the operation for more than 5 times until no protein layer appears in the middle; collecting the supernatant; concentrating under reduced pressure at 50 deg.C to remove organic reagent;
(7) dialyzing the mixture in ultrapure water for 72 hours by using a dialysis bag with a molecular weight cutoff of 3500Da, concentrating the dialyzed mixture under reduced pressure, and freeze-drying the concentrated mixture to obtain a sample 1.
The amount of polysaccharide extracted in sample 1, as tested, from example 1 was: 5.23g/L, purity: 87.23 percent. Color: white. The weight average molecular weight of the sake polysaccharide is 8554Da according to a standard curve drawn by standard molecular weight glucan by an HPGFC method, wherein the sake polysaccharide is mainly composed of glucose (91.56%), galactose (2.13%), mannose (1.61%), glucuronic acid (1.84%), galactaric acid (1.34%), a small amount of trehalose (0.67%), arabinose (0.12%) and the like according to ion chromatography.
Example 2
The purpose of this example is to test the probiotic activity of sample 1 extracted in example 1: specifically, the rice wine polysaccharide extracted by the invention has the effect of promoting the proliferation of probiotics (bifidobacterium breve). MRS culture medium is used as basic culture medium, and 0.5g/L L-cysteine is added in the basic culture medium. On the basis of the modified MRS medium, a comparative experiment was designed, using 5g/L of sake polysaccharide (sample 1 obtained in example 1) as an external carbon source as an experimental group, 5g/L of inulin (known by those skilled in the art as a commonly used probiotic carbon source substance) as an external carbon source as a probiotic reference group, and no external carbon source as a blank control group.
Placing glycerol tube at normal temperature, thawing, coating 100 μ L of glycerol tube on MRS solid culture medium, performing anaerobic culture at 37 deg.C for 48h for activation, preparing probiotic seed solution, adding 5mL of seed solution into 30mL of sterile PBS solution to obtain bacterial suspension, inoculating 0.5mL of bacterial suspension into anaerobic tube (5mL), performing anaerobic culture at 37 deg.C for 48h, sampling at 0, 24, 48h, and determining biomass OD600。
As shown in fig. 1, the bacterial concentration of bifidobacterium breve with rice wine polysaccharide as an external carbon source was higher compared with inulin: increasing from 0.089 + -0.004 to 0.697 + -0.013 indicates that the sake polysaccharides have better probiotic activity against Bifidobacterium breve than inulin.
Example 3
With reference to example 2, the only difference is that bifidobacterium breve is replaced by lactobacillus rhamnosus. The results are shown in FIG. 2.
As shown in FIG. 2, the bacterial concentration of Lactobacillus rhamnosus with sake polysaccharides as carbon source increased from 0.109. + -. 0.001 to 0.214. + -. 0.005 and the inulin group increased from 0.109. + -. 0.001 to 0.235. + -. 0.005, but the difference was very small, compared to inulin. The rice wine polysaccharide has basically equivalent probiotic activity to inulin for lactobacillus rhamnosus.
Comparative example 1
Referring to example 1, sample 2 was obtained except that the drying manner in step (7) was replaced by freeze-drying.
The test shows that the extraction of polysaccharide in sample 2 prepared in comparative example 1 is: 3.51g/L, purity: 79.4 percent, light yellow color, obvious color difference with a sample obtained by freeze drying, and reduced purity.
Comparative example 2
Referring to example 1, the difference is that the step (6) of removing protein from sevage reagent is omitted, and the specific operation is as follows: and (7) after a certain amount of rice wine crude polysaccharide is dissolved, dialyzing in ultrapure water for 72 hours by using a dialysis bag with molecular weight cutoff of 3500Da, concentrating under reduced pressure, and freeze-drying to obtain a sample 3.
The test shows that the extraction of polysaccharide in sample 3 prepared in comparative example 2 is: 6.12g/L, purity: 65.3 percent, white color and low purity of polysaccharide without protein removal.
Comparative example 3
Referring to example 1, only with the difference that the dialysis step in step (7) was omitted, sample 4 was obtained.
The test shows that the extraction of polysaccharide in sample 4 prepared in comparative example 3 is: 4.85g/L, purity: 77.7%, low purity, impure color and yellowish white.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A method for extracting probiotic functional sugar from distilled rice wine distillation residual liquid is characterized by comprising the following steps:
(1) concentrating the distillation residue of distilled rice wine;
(2) adding an ethanol solution into the concentrated distilled rice wine distillation residual liquid obtained in the step (1) until the final concentration of ethanol is 25-30%, precipitating with ethanol, and centrifuging;
(3) taking the supernatant obtained by the centrifugation in the step (2), adding an ethanol solution until the final concentration of ethanol is 75-85%, precipitating with ethanol, and centrifuging;
(4) concentrating the supernatant obtained by the centrifugation in the step (3), and freeze-drying to obtain rice wine crude polysaccharide;
(5) dissolving the rice wine crude polysaccharide to obtain a rice wine crude polysaccharide sample liquid, and removing protein by adopting a Sevage reagent, wherein the dosage ratio of the Sevage reagent to the rice wine crude polysaccharide sample liquid is 1: 3-6;
(6) and (3) putting the deproteinized rice wine polysaccharide sample liquid obtained in the step (5) into a dialysis bag with molecular weight cutoff of 3500 Da-5000 Da for dialysis in ultrapure water, and then concentrating, freezing and drying to obtain the target probiotic functional sugar.
2. The method for extracting prebiotic functional sugar from distilled rice wine distillation residue as claimed in claim 1, further comprising step S1 after the step (3) and before the step (4);
s1: taking the precipitate obtained by the centrifugation in the step (3), adding 25-30% ethanol for redissolution, standing and centrifuging;
the step (4) is specifically as follows: and (4) concentrating the supernatant obtained by the centrifugation in the step (3) and/or the step S1, and freeze-drying to obtain the rice wine crude polysaccharide.
3. The method for extracting prebiotic functional sugar from distilled rice wine distillation residue as claimed in claim 1 or 2, wherein the final concentration of ethanol in the step (2) is 80%.
4. The method for extracting the probiotic functional sugar from the distilled rice wine distillation residual liquid according to claim 1 or 2, wherein the concentration in the step (1) is specifically reduced pressure concentration at 45-55 ℃ to 1/5-1/3 of the original volume.
5. The method for extracting probiotic functional sugar from distilled rice wine distillation residue according to claim 1 or 2, wherein the ethanol solution used in the step (2) and/or the step (3) is 95% ethanol.
6. The method for extracting probiotic functional sugar from distilled rice wine distillation residue according to claim 1 or 2, wherein the dialysis time in the step (6) is 48-72 h.
7. The method for extracting probiotic functional sugar from distilled rice wine distillation residue according to claim 1 or 2, wherein the alcohol precipitation temperature in the step (2) and/or the step (3) is 4-10 ℃, and the alcohol precipitation time is 12-24 h.
8. The method for extracting probiotic functional sugar from distilled rice wine distillation residue liquid as claimed in claim 1 or 2, wherein the step (5) of removing protein by Sevage reagent comprises the following specific steps:
mixing the rice wine crude polysaccharide sample liquid with a Sevage reagent, placing the mixture in a shaking table, fully shaking, centrifuging to obtain a supernatant, and repeating the steps for multiple times until no protein intermediate layer appears; the supernatant was collected and concentrated under reduced pressure to remove the organic solvent.
9. A rice wine probiotic functional sugar product obtained by the method of any one of claims 1-8.
10. The use of the rice wine probiotic functional sugar product of claim 9 in probiotic culture.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103897934A (en) * | 2014-04-16 | 2014-07-02 | 孝感麻糖米酒有限责任公司 | Preparation method of functional rice wine |
| CN109369822A (en) * | 2018-12-06 | 2019-02-22 | 辽宁大学 | A kind of extracting method of prebiotic bacterium exopolysaccharide |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103897934A (en) * | 2014-04-16 | 2014-07-02 | 孝感麻糖米酒有限责任公司 | Preparation method of functional rice wine |
| CN109369822A (en) * | 2018-12-06 | 2019-02-22 | 辽宁大学 | A kind of extracting method of prebiotic bacterium exopolysaccharide |
Non-Patent Citations (1)
| Title |
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| "Thermal inactivation kinetics of Bacillus cereus in Chinese rice wine and in simulated media based on wine components";Ruiling Lv等;《Food Control》;20180203;308-313页 * |
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