CN113416611A - Preparation method of food-grade detergent containing sugar substrate fermentation product - Google Patents
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D1/00—Detergent compositions based essentially on surface-active compounds; Use of these compounds as a detergent
- C11D1/66—Non-ionic compounds
- C11D1/662—Carbohydrates or derivatives
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- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
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- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/02—Acyclic radicals, not substituted by cyclic structures
- C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2445—Beta-glucosidase (3.2.1.21)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
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- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01021—Beta-glucosidase (3.2.1.21)
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Abstract
The invention relates to the technical field of cleaning agents, in particular to a preparation method of a food-grade cleaning agent containing a sugar substrate fermentation product. A preparation method of a food-grade detergent containing a sugar substrate fermentation product is characterized by comprising the following steps: s1, inoculating glycerol bacteria; s2, culturing glycerol bacteria; s3, fermenting by using glycerol bacteria; s4, inducing glycerol bacteria; s5, catalytic synthesis; and S6, separating and purifying. Compared with the prior art, the method has the advantages that the beta-glucosidase expressed by the induced glycerobacteria is used for catalyzing and preparing the alkyl glycoside, the purity of the alkyl glycoside is guaranteed, the yield of the alkyl glycoside is increased, and the alkyl glycoside is prepared by the catalysis of the glycerobacteria and the beta-glucosidase through a biological method, so that the method is safe, non-toxic, green and environment-friendly.
Description
Technical Field
The invention relates to the technical field of cleaning agents, in particular to a preparation method of a food-grade cleaning agent containing a sugar substrate fermentation product.
Background
The alkyl glycoside is safe and nontoxic, has remarkable detergency, and can be used as a food-grade detergent. In the preparation of alkyl glycosides, the desired product is usually formed by a reverse hydrolysis reaction between a monosaccharide, an alcohol with various carbon chains, and water. However, due to thermodynamic limitations, the reverse hydrolysis reaction reaches thermodynamic equilibrium very quickly, resulting in lower yields of the alkylglycoside. To increase the yield of the alkyl glycoside and to promote the equilibrium forward, the concentration of the reactants may be increased or the water activity may be decreased. However, too high a concentration of the reactants may result in the production of glycans during the reaction, reducing the purity of the alkylglycoside. Too low water activity can reduce the solubility of the monosaccharide and cannot ensure the yield of the alkyl glycoside.
At present, the alkyl glucoside adopted in the industry mainly adopts a chemical synthesis method which is divided into a one-step method and a two-step method. The one-step process is a direct glycosidation process by direct condensation of longer chain alcohols with glucose, which has high requirements on reaction conditions. The product produced by the one-step method has higher recovery rate, is a common method for foreign industry and has wide application. The one-step method has to strictly control the key factors such as temperature, catalyst type and the like, needs excessive fatty alcohol in the reaction process, and is difficult to separate. The two-step process is transglycosylation, which produces short-chain alkyl glycoside by condensing lower-chain fatty alcohol with glucose. Then the product is reacted with long-chain fatty alcohol to form long-chain alkyl glycoside. Compared with the direct method, the method is relatively mild, and the reaction speed is also improved. However, the separation of the products is difficult, two alkyl glucosides cannot be completely separated, and the process is complicated.
Therefore, the preparation method of the food-grade detergent containing the sugar substrate fermentation product is designed, so that the purity of the alkyl glycoside is ensured, and the yield of the alkyl glycoside is increased.
Disclosure of Invention
The invention overcomes the defects of the prior art, provides a preparation method of a food-grade cleaning agent containing a sugar substrate fermentation product, prepares the alkyl glycoside by utilizing beta-glucosidase catalysis expressed by induced glycerol bacteria, ensures the purity of the alkyl glycoside and simultaneously improves the yield of the alkyl glycoside, and prepares the alkyl glycoside by utilizing biological methods of the glycerol bacteria and the beta-glucosidase catalysis, thereby being safe, nontoxic, green and environment-friendly.
In order to achieve the purpose, the preparation method of the food-grade detergent containing the sugar substrate fermentation product is characterized by comprising the following steps:
s1, inoculating glycerol bacteria: inoculating glycerol strain refrigerated at-20 deg.C to slant culture medium without mixed bacteria, activating at 37 deg.C for 2 days, and storing in 4 deg.C refrigerator;
s2, culturing glycerol: inoculating the strain in the slant culture medium of the step S1 in a seed culture medium on a sterilized clean workbench, and culturing in a constant temperature oscillator at 37 ℃ and 200r/min for 12 h;
s3, fermenting glycerol: inoculating 3% of strains in the seed culture medium in the step S2 into a liquid fermentation culture medium on a sterilized clean workbench, culturing in a constant temperature oscillator at 37 ℃ and 200r/min for 48h, taking the fermentation liquor, performing centrifugal filtration, and putting the obtained liquid part into a refrigerator at-20 ℃ for later use;
s4, induction of glycerol bacteria: adding lactose with the concentration of 2% into the liquid part of the step S3, controlling the lactose induction time to be 2h, the induction temperature to be 34 ℃ and the culture time to be 4 days, after the culture is finished, taking the fermentation liquor to carry out centrifugal filtration, washing the obtained precipitate part with a buffer solution to obtain beta-glucosidase, determining whether the enzyme activity meets the requirement, if so, carrying out the step S5, and if not, repeating the step S4;
s5, catalytic synthesis: adding 12-14% of glucose, 71-73% of fatty alcohol, 10-15% of water and 1.5-2.5% of beta-glucosidase into a constant-temperature oscillator at 52 ℃ and 200r/min, adjusting the pH =6 by using a disodium hydrogen phosphate-citric acid buffer solution, and reacting for 48-50 h;
s6, separation and purification: filtering the reaction solution of step S5 to remove enzyme, standing for layering, separating water layer from organic layer, distilling organic layer under reduced pressure to remove fatty alcohol to obtain crude extract, and purifying the crude extract by membrane separation to obtain alkyl glycoside, i.e. food grade detergent.
The slant culture medium in step S1 comprisesThe raw materials with the following weight percentages: 0.2-0.3% of glucose, 0.4-0.6% of yeast extract powder, 0.4-0.6% of sodium chloride, 0.8-1.2% of tryptone, 1-3% of agar and 0.2-0.3% of K2HPO494-97% of deionized water, and the sum of the contents of all the components is 100%.
The preparation method of the slant culture medium in the step S1 is specifically as follows: adding the required raw materials into blank culture medium, sterilizing at 121 deg.C for 20min, placing the hot inclined plane after sterilization, cooling and fixing the inclined plane, culturing at 37 deg.C for 2-3 days, detecting whether there is mixed bacteria after culturing, and placing in 4 deg.C refrigerator for use.
The seed culture medium in the step S2 comprises the following raw materials in percentage by weight: 0.3-0.5% of glucose, 0.5-1.5% of peptone, 0.5-1.5% of sodium chloride, 0.4-0.6% of yeast extract powder and 95.9-98.3% of deionized water, wherein the sum of the contents of all the components is 100%; the seed medium was sterilized at 121 ℃ for 20min after the preparation.
The liquid fermentation culture medium in the step S3 comprises the following raw materials in percentage by weight: 0.4-0.6% yeast extract, 0.4-0.6% Na2HPO40.4 to 0.6 percent of glycerin, 0.03 to 0.07 percent of glucose and 98.13 to 98.77 percent of deionized water, wherein the sum of the contents of all the components is 100 percent.
The preparation method of the liquid fermentation medium in the step S3 is specifically as follows: soaking 0.5% yeast and 0.5% Na2HPO4Preparing liquid fermentation medium from 0.5% of glycerol, 0.05% of glucose and 98.13-98.77% of deionized water, sterilizing at 121 deg.C for 20min, adding 0.2% of lactose, stirring to dissolve, sealing, sterilizing at 121 deg.C for 20min, and adjusting pH with disodium hydrogen phosphate-citric acid buffer solution to 7-7.5.
The buffer solution in step S4 is a potassium hydrogen phosphate solution or a sodium hydrogen phosphate solution.
The carbon chain length of the fatty alcohol in the step S5 is 8-12.
In the step S5, the mass ratio of the glucose to the fatty alcohol is 1: 5-1: 6.
compared with the prior art, the method has the advantages that the beta-glucosidase expressed by the induced glycerobacteria is used for catalyzing and preparing the alkyl glycoside, the purity of the alkyl glycoside is guaranteed, the yield of the alkyl glycoside is increased, and the alkyl glycoside is prepared by catalysis of the glycerobacteria and the beta-glucosidase by a biological method, so that the method is safe, non-toxic, green and environment-friendly.
Drawings
FIG. 1 is a chemical reaction equation for preparing alkylglycoside according to step S5 of the present invention.
Detailed Description
Example 1:
the embodiment provides a preparation method of a food-grade detergent containing a sugar substrate fermentation product, which specifically comprises the following steps:
s1, inoculating glycerol bacteria: inoculating glycerol strain refrigerated at-20 deg.C to slant culture medium without mixed bacteria, activating at 37 deg.C for 2 days, and storing in 4 deg.C refrigerator;
s2, culturing glycerol: inoculating the strain in the slant culture medium of the step S1 in a seed culture medium on a sterilized clean workbench, and culturing in a constant temperature oscillator at 37 ℃ and 200r/min for 12 h;
s3, fermenting glycerol: inoculating strain in the seed culture medium of step S2 into a liquid fermentation culture medium through an inoculating loop on a sterilized clean workbench, culturing in a constant temperature oscillator at 37 ℃ and 200r/min for 48h, taking the fermentation liquid, performing centrifugal filtration, and putting the obtained liquid part into a refrigerator at-20 ℃ for later use;
s4, induction of glycerol bacteria: adding lactose with the concentration of 2% into the liquid part of the step S3, controlling the lactose induction time to be 2h, the induction temperature to be 34 ℃ and the culture time to be 4 days, after the culture is finished, taking the fermentation liquor to carry out centrifugal filtration, washing the obtained precipitate part with a buffer solution to obtain beta-glucosidase, determining whether the enzyme activity meets the requirement, if so, carrying out the step S5, and if not, repeating the step S4;
s5, catalytic synthesis: adding 12-14% of glucose, 71-73% of fatty alcohol, 13% of water and 2% of beta-glucosidase into a constant temperature oscillator at 52 ℃ and 180r/min, adjusting the pH to be =6 by using a disodium hydrogen phosphate-citric acid buffer solution, and reacting for 36 h. As shown in fig. 1, glucose reacts with fatty alcohol under the catalysis of beta-glucosidase to obtain alkyl glycoside and water;
s6, separation and purification: filtering the reaction solution of step S5 to remove enzyme, standing for layering, separating water layer from organic layer, distilling organic layer under reduced pressure to remove fatty alcohol to obtain crude extract, and purifying the crude extract by membrane separation to obtain alkyl glycoside, i.e. food grade detergent.
The slant culture medium in the step S1 comprises the following raw materials by weight percent: 0.25% glucose, 0.5% yeast extract powder, 0.5% sodium chloride, 1% tryptone, 2% agar, 0.25% K2HPO495.5% deionized water. The preparation method of the slant culture medium comprises the following steps: adding the required raw materials into a blank culture medium, sterilizing at 121 deg.C for 20min, placing the slant while it is hot after sterilization, cooling and fixing the slant, culturing at 37 deg.C for 2 days, detecting whether there is mixed bacteria after culturing, and placing in a refrigerator at 4 deg.C for use.
The seed culture medium in the step S2 comprises the following raw materials in percentage by weight: 0.4% of glucose, 1% of peptone, 1% of sodium chloride, 0.5% of yeast extract powder and 97.1% of deionized water. The seed medium was sterilized at 121 ℃ for 20min after the preparation.
The liquid fermentation medium in the step S3 comprises the following raw materials in percentage by weight: 0.5% yeast extract, 0.5% Na2HPO40.5% of glycerol, 0.05% of glucose and 98.45% of deionized water.
The preparation method of the liquid fermentation medium in the step S3 is specifically as follows: soaking 0.5% yeast and 0.5% Na2HPO4Preparing a liquid fermentation medium from 0.5% of glycerol, 0.05% of glucose and 98.45% of deionized water, sterilizing at 121 ℃ for 20min, adding 0.2% of lactose, stirring to dissolve, sealing, sterilizing at 121 ℃ for 20min, and adjusting the pH to =7 by using a disodium hydrogen phosphate-citric acid buffer solution.
The buffer solution in step S4 is a potassium hydrogen phosphate solution or a sodium hydrogen phosphate solution.
The carbon chain length of the fatty alcohol in the step S5 is 8-12.
The type of the fatty alcohol selected in this example is cotinine C8 alcohol or C12 alcohol. .
The mass ratio of glucose to fatty alcohol in step S5 was 1: 5.
In step S6, the glucose content in the separated aqueous layer was detected, and the glucose conversion rate was 60-80%, which indicates that the method of the present invention has high glucose conversion rate and high yield of the prepared alkylglycoside.
The food-grade detergent prepared by the embodiment has the components of alkyl glycoside, can clean food, and is safe and nontoxic. In the embodiment, the alkyl glycoside is prepared by catalyzing beta-glucosidase expressed by induced glycerol bacteria, so that the yield of the alkyl glycoside is improved while the purity of the alkyl glycoside is ensured, and the alkyl glycoside is prepared by catalyzing the glycerol bacteria and the beta-glucosidase by a biological method, so that the method is safe, non-toxic, green and environment-friendly.
Claims (9)
1. A preparation method of a food-grade detergent containing a sugar substrate fermentation product is characterized by comprising the following steps:
s1, inoculating glycerol bacteria: inoculating glycerol strain refrigerated at-20 deg.C to slant culture medium without mixed bacteria, activating at 37 deg.C for 2 days, and storing in 4 deg.C refrigerator;
s2, culturing glycerol: inoculating the strain in the slant culture medium of the step S1 in a seed culture medium on a sterilized clean workbench, and culturing in a constant temperature oscillator at 37 ℃ and 200r/min for 12 h;
s3, fermenting glycerol: inoculating 3% of strains in the seed culture medium in the step S2 into a liquid fermentation culture medium on a sterilized clean workbench, culturing in a constant temperature oscillator at 37 ℃ and 200r/min for 48h, taking the fermentation liquor, performing centrifugal filtration, and putting the obtained liquid part into a refrigerator at-20 ℃ for later use;
s4, induction of glycerol bacteria: adding lactose with the concentration of 2% into the liquid part of the step S3, controlling the lactose induction time to be 2h, the induction temperature to be 34 ℃ and the culture time to be 4 days, after the culture is finished, taking the fermentation liquor to carry out centrifugal filtration, washing the obtained precipitate part with a buffer solution to obtain beta-glucosidase, determining whether the enzyme activity meets the requirement, if so, carrying out the step S5, and if not, repeating the step S4;
s5, catalytic synthesis: adding 12-14% of glucose, 71-73% of fatty alcohol, 10-15% of water and 1.5-2.5% of beta-glucosidase into a constant-temperature oscillator at 52 ℃ and 200r/min, adjusting the pH =6 by using a disodium hydrogen phosphate-citric acid buffer solution, and reacting for 48-50 h;
s6, separation and purification: filtering the reaction solution of step S5 to remove enzyme, standing for layering, separating water layer from organic layer, distilling organic layer under reduced pressure to remove fatty alcohol to obtain crude extract, and purifying the crude extract by membrane separation to obtain alkyl glycoside, i.e. food grade detergent.
2. The method of claim 1, wherein the sugar substrate fermentation product-containing food grade detergent comprises: the slant culture medium in the step S1 comprises the following raw materials in percentage by weight: 0.2-0.3% of glucose, 0.4-0.6% of yeast extract powder, 0.4-0.6% of sodium chloride, 0.8-1.2% of tryptone, 1-3% of agar and 0.2-0.3% of K2HPO494-97% of deionized water, and the sum of the contents of all the components is 100%.
3. The method of claim 1 or 2, wherein the sugar substrate fermentation product comprises a sugar substrate fermentation product, and the method comprises the following steps: the preparation method of the slant culture medium in the step S1 is specifically as follows: adding the required raw materials into blank culture medium, sterilizing at 121 deg.C for 20min, placing the hot inclined plane after sterilization, cooling and fixing the inclined plane, culturing at 37 deg.C for 2-3 days, detecting whether there is mixed bacteria after culturing, and placing in 4 deg.C refrigerator for use.
4. The method of claim 1, wherein the sugar substrate fermentation product-containing food grade detergent comprises: the seed culture medium in the step S2 comprises the following raw materials in percentage by weight: 0.3-0.5% of glucose, 0.5-1.5% of peptone, 0.5-1.5% of sodium chloride, 0.4-0.6% of yeast extract powder and 95.9-98.3% of deionized water, wherein the sum of the contents of all the components is 100%; the seed medium was sterilized at 121 ℃ for 20min after the preparation.
5. The method of claim 1, wherein the sugar substrate fermentation product-containing food grade detergent comprises: the liquid fermentation culture medium in the step S3 comprises the following raw materials in percentage by weight: 0.4-0.6% yeast extract, 0.4-0.6% Na2HPO40.4 to 0.6 percent of glycerin, 0.03 to 0.07 percent of glucose and 98.13 to 98.77 percent of deionized water, wherein the sum of the contents of all the components is 100 percent.
6. The method of claim 1 or 5, wherein the sugar substrate fermentation product comprises a sugar substrate fermentation product, and the method comprises the following steps: the preparation method of the liquid fermentation medium in the step S3 is specifically as follows: soaking 0.5% yeast and 0.5% Na2HPO4Preparing liquid fermentation medium from 0.5% of glycerol, 0.05% of glucose and 98.13-98.77% of deionized water, sterilizing at 121 deg.C for 20min, adding 0.2% of lactose, stirring to dissolve, sealing, sterilizing at 121 deg.C for 20min, and adjusting pH with disodium hydrogen phosphate-citric acid buffer solution to 7-7.5.
7. The method of claim 1, wherein the sugar substrate fermentation product-containing food grade detergent comprises: the buffer solution in step S4 is a potassium hydrogen phosphate solution or a sodium hydrogen phosphate solution.
8. The method of claim 1, wherein the sugar substrate fermentation product-containing food grade detergent comprises: the carbon chain length of the fatty alcohol in the step S5 is 8-12.
9. The method of claim 1, wherein the sugar substrate fermentation product-containing food grade detergent comprises: in the step S5, the mass ratio of the glucose to the fatty alcohol is 1: 5-1: 6.
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Citations (5)
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|---|---|---|---|---|
| CN104988125A (en) * | 2015-04-15 | 2015-10-21 | 浙江科技学院 | High tolerance beta-glucosidase and applications thereof |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104988125A (en) * | 2015-04-15 | 2015-10-21 | 浙江科技学院 | High tolerance beta-glucosidase and applications thereof |
| CN105255745A (en) * | 2015-10-29 | 2016-01-20 | 安徽工程大学 | Rhizopus for producing beta-glucosidase and method of producing enzyme through rhizopus fermentation |
| CN105907821A (en) * | 2016-06-26 | 2016-08-31 | 周荣 | Method for preparing alkyl glycoside |
| CN112094835A (en) * | 2020-09-24 | 2020-12-18 | 江南大学 | Application of beta-glucosidase mutant |
| CN112375755A (en) * | 2020-11-12 | 2021-02-19 | 湖北三峡职业技术学院 | Breeding method of aspergillus niger capable of producing beta-glucosidase at high yield |
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Application publication date: 20210921 |
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