CN114231427A - Fermentation method capable of effectively improving quality of rose cell sap - Google Patents
Fermentation method capable of effectively improving quality of rose cell sap Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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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
- C12N1/20—Bacteria; Culture media therefor
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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
- C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
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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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
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- C—CHEMISTRY; METALLURGY
- 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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
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- C—CHEMISTRY; METALLURGY
- 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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/62—Carboxylic acid esters
Abstract
The invention discloses a fermentation method capable of effectively improving the quality of rose cellular fluid, which comprises the following steps: inoculating torula or enterobacter from agar slant culture medium for storing strain into liquid seed culture medium, and culturing at 20-30 deg.C for 12-48 hr to activate strain to obtain seed solution; inoculating the seed solution into a liquid fermentation culture medium prepared by taking Damascus rose hydrosol as a matrix according to the volume percentage of 4-8%, and culturing at 29-33 ℃ for 24-168 hours to obtain fermentation liquor. The invention improves the polyphenol content in the rose cell sap by 1-10%, the phenethyl alcohol content by 5-80%, the eugenol by 10-70%, the methyl eugenol by 30-85%, and simultaneously generates new aroma substances such as benzyl acetate, phenethyl acetate, citronellol and the like. The method has important market value for improving the quality of the rose hydrosol and the cell sap by utilizing the thallus fermentation.
Description
Technical Field
The invention relates to a fermentation method capable of effectively improving the quality of rose hydrosol and cell sap, belonging to the technical field of microbial fermentation.
Background
The rose hydrosol, also called water essential oil, is a condensed water solution separated from the rose essential oil in the distillation extraction process, and is a byproduct of the essential oil. The properties of the essential oil are similar to those of the essential oil, but are not completely the same. The pure dew contains small amount of essential oil components, and also contains water soluble substances in plant body. The rose cellular fluid is a liquid obtained by drying and distilling fresh rose flowers (crowns) at low temperature and in sub-vacuum condition in the process of preparing rose tea, wherein volatile substances are taken out by circulating hot air and condensed into the liquid by a condenser, and the liquid is a byproduct in the process of preparing the rose tea. The two products belong to essential oil extraction byproducts and have single application value, and the quality of hydrolat and cell sap used for daily chemical products in the market is uneven, so that a method for improving the application value of the two products is urgently needed to expand the efficacy and the market value of the two products. Research shows that the rose extract is rich in various phenols, alcohols, esters and other aroma substances, and microorganisms serving as a 'cell factory' can potentially metabolize the rose extract to generate functional components, so that the application value is improved.
The fermentation of the rose hydrosol by using the microorganism is a technical method with mild condition, simple and convenient operation and environmental protection. The substrate can be effectively converted into other potential beneficial components through the natural metabolic conversion mechanism of the microorganism, and the method is widely applied to other fields and has a mature operation basis.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: provides a fermentation method capable of effectively improving the quality of rose hydrosol and cell sap.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a fermentation method capable of effectively improving the quality of rose cell sap comprises the following steps:
step 1): inoculating torula or enterobacter from agar slant culture medium for storing strain into liquid seed culture medium, and culturing at 20-30 deg.C for 12-48 hr to activate strain to obtain seed solution;
step 2): inoculating the seed solution into a liquid fermentation culture medium prepared by taking Damascus rose hydrosol as a matrix according to the volume percentage of 4-8%, and culturing at 29-33 ℃ for 24-168 hours to obtain fermentation liquor.
Preferably, the yeast or enterobacter in step 1) is Debaryomyces seeberinservi, Enterobacter reuteri, Kluyveromyces marxianus or Zygosaccharomyces bailii.
Preferably, the seed culture medium in step 1) contains: 1-3g/L of dipotassium phosphate trihydrate, 0.5-2g/L of monopotassium phosphate, 0.2-0.7g/L of anhydrous magnesium sulfate, 4-6g/L of sodium chloride, 8-11g/L of ammonium sulfate, 4-6g/L of glucose, and distilled water for fixing the volume and adjusting the pH value to 4.8-7.2.
Preferably, the liquid fermentation medium in step 2) contains: 1-3g/L of dipotassium phosphate trihydrate, 0.5-1g/L of monopotassium phosphate, 0.2-0.7g/L of anhydrous sulfuric acid, 2-7g/L of sodium chloride, 8-11g/L of ammonium sulfate and 9-12g/L of glycerol, taking Damascus rose hydrosol as a matrix to fix the volume, and adjusting the pH to 5.3-6.8.
Preferably, the culturing in step 1) is performed in a constant temperature shaker at a rotation speed of 150-220 rpm.
Preferably, the culturing in step 2) is performed in a constant temperature shaker at a rotation speed of 180-220 rpm.
Preferably, the fermentation liquor obtained in the step 2) is filtered by a nylon filter membrane with the diameter of 0.2-0.3 μm and then is stored at the temperature of 3-6 ℃.
Preferably, the fermentation liquor obtained in the step 2) is measured, the mass content of polyphenol is improved by 1-10%, the mass content of phenethyl alcohol is improved by 5-80%, the mass content of eugenol is improved by 10-70%, the mass content of methyl eugenol is improved by 30-85%, and meanwhile, new aroma substances are generated.
More preferably, the new aroma substances include benzyl acetate, phenethyl acetate and citronellol.
More preferably, in the method for measuring, the total phenol content is measured by: (1) adding 1-3mL of Folin phenol reagent with volume concentration of 10% into 0.3-0.5mL of supernatant of the centrifuged fermentation liquor, uniformly mixing, standing for 4-6min, then adding 1.4-1.8mL of sodium carbonate solution with mass concentration of 7.5%, standing for 60min, measuring the light absorption value at 765nm, and calculating the total phenol content according to a standard curve; the method for measuring the new aroma substances comprises the following steps: and (3) taking gamma-decalactone dissolved in n-hexane as an internal standard substance, and determining the volatile components of the fermentation liquor by adopting GC-MS.
Compared with the prior art, the invention has the beneficial effects that:
(1) the strain used by the invention is from damascus rose field soil, and can improve the polyphenol content and increase the aroma components of rose extract;
(2) the fermentation process of the invention has mild reaction conditions, is environment-friendly and has good development and application prospects.
Drawings
FIG. 1 shows four species of bacteria used in the fermentation process; wherein (a) is Seebergine Denah's yeast Cyberlindera saturnus, and (b) is Enterobacter reuteri ludwigii, and (c) is Kluyveromyces marxianus, and (d) is Zygosaccharomyces bailii;
FIG. 2 shows the polyphenol content of the rose hydrosol of example 3 after fermentation for five days; wherein mud A represents Damascus rose field soil, the middle ones represent numbers, and 5d represents fermentation for 5 days;
FIG. 3 is a graph showing the relationship between gallic acid concentration and absorbance.
Detailed Description
In order to make the invention more comprehensible, preferred embodiments are described in detail below with reference to the accompanying drawings.
Examples
Screening Rosa damascena fermentation bacteria from Rosa damascena field soil
(1) The formula of the enrichment medium is 1-3g/L potassium dihydrogen phosphate, 0.2-0.7g/L anhydrous magnesium sulfate, 3-6g/L sodium chloride, 4-6g/L ammonium sulfate, 4-6g/L glucose, 9-11g/L peptone, distilled water and rose hydrosol which are prepared according to a certain proportion to fix the volume and adjust the pH value to 6-7 +/-0.2. Sterilizing at 121 deg.C and 0.1MPa for 20 min.
(2) Soaking 8-12g soil in 80-100mL sterile normal saline for 0.75-1.25h, shaking in a greenhouse for 25-35min, standing for 25-35min, sucking 8-12mL supernatant in 88-92mL enrichment medium, and shake culturing at 28-32 deg.C for 46-50 h.
(3) YPDA culture medium was prepared from Rosa damascena hydrolat. Sterilizing at 121 deg.C and 0.1MPa for 20min, cooling to 20-50 deg.C, adding 15-20mL culture medium into the culture dish on clean bench, standing for solidification, and volatilizing residual water.
(4) Diluting the soil bacterium liquid of the rose damascus rosea field to 10 percent by using 0.9 percent of sterile normal saline-1～10-6Adding 0.5mL of bacterial liquid into each plate, uniformly coating with a sterile coater, and culturing at 28-35 deg.C for 24-50 h. The colonies obtained are shown in FIG. 1.
Second, fermented rose hydrosol of yeast.
(1) The formula of the culture medium used for activating fermentation is 1-3g/L of dipotassium phosphate trihydrate, 0.5-2g/L of monopotassium phosphate, 0.2-0.7g/L of anhydrous magnesium sulfate, 4-6g/L of sodium chloride, 8-11g/L of ammonium sulfate, 4-6g/L of glucose and distilled water for constant volume, and the pH value is adjusted to 5.0-7.0 +/-0.2.
(2) A100 plus 500 mu L specification sterile gun head is dipped with a needle tail bacterial colony and is injected into a culture medium for fermentation activation with the volume of 8-15mL, a sample bottle with the specification of 20-50mL is placed in a constant temperature oscillator for shaking culture at 170 plus 220rpm for 18-24 h.
(3) The formula of the fermentation medium prepared from the rosa damascena extract comprises 1-3g/L of dipotassium hydrogen phosphate trihydrate, 0.5-1g/L of monopotassium phosphate, 0.2-0.7g/L of anhydrous sulfuric acid, 2-7g/L of sodium chloride, 8-11g/L of ammonium sulfate, 9-12g/L of glycerol and hydrosol of rosa damascena as a matrix, wherein the pH value is 5.5-6.6 +/-0.2.
(4) 4 to 8 percent of culture medium bacterial liquid for fermentation activation is added into the culture medium for fermentation activation for culture based on a constant temperature shaker. The fermentation conditions are 29-33 ℃, and the constant temperature shaking culture at 180-.
Third, analysis of functional ingredients in fermentation broth
(1) Centrifuging the fermentation liquid, leaving supernatant, filtering with 0.2-0.3 μm nylon filter membrane, and storing in 3-6 deg.C refrigerator. And drawing a standard curve by using gallic acid as a standard substance to serve as a conversion standard, and using fermentation liquor without adding bacteria as a control group. Adding 0.3-0.5mL of centrifuged supernatant fermentation liquor into a test tube, adding 1-3mL of a 10% forskolin phenol reagent by volume concentration, uniformly mixing, standing for 4-6min, adding 1.4-1.8mL of a 7.5% sodium carbonate solution by mass concentration, standing for 60min, measuring the light absorption value at 765nm, and calculating the total phenol content according to a standard curve.
As shown in FIG. 2, the strains used in group C, D, E, F, G, H were Enterobacter lewisii and Kluyveromyces marxianus. In the polyphenol content, they were all higher than the control group for 5 days, and therefore, these 6 strains showed an increasing tendency in the polyphenol content compared to the strain at the same transformation time, and the polyphenol content was increased by 9.80% compared to the control group.
And (3) detection process: preparation of 10% forinophenol reagent: precisely weighing 10.0mL of forrin phenol, transferring the forrin phenol into a 100mL volumetric flask, carrying out repeated lubrication and washing, and carrying out constant volume to a scale mark to prepare a solution of the forrin phenol with the volume concentration of 10%.
Preparation of 7.5% sodium carbonate solution: 7.5g of sodium carbonate was weighed precisely, and a small amount of distilled water was added to dissolve it completely. Transferring the solution into a 100mL volumetric flask, carrying out multiple times of moistening and washing, and fixing the volume to a scale mark to prepare a 7.5% sodium carbonate solution.
And (3) measuring the polyphenol content of the sample:
taking 0.4mL of supernatant, adding 2mL of forskolin phenol reagent with volume concentration of 10% into a 5mL centrifuge tube, uniformly mixing, standing for 5min, adding 1.6mL of sodium carbonate solution with mass concentration of 7.5%, standing for 60min, measuring the light absorption value at 765nm to obtain a standard curve as shown in figure 3, and calculating the total phenol content according to the standard curve.
(2) Performing GC-MS analysis on the volatile components of the fermentation liquor: and (3) taking gamma-decalactone dissolved by n-hexane as an internal standard substance, and determining the volatile components of the fermentation liquor of the screened strain by adopting GC-MS. The content of phenethyl alcohol is improved by 5.66 percent, the content of eugenol is improved by 12.14 percent, the content of methyl eugenol is improved by 32.26 percent, and new aroma substances such as benzyl acetate, phenethyl acetate and the like are generated. The results are shown in tables 1 and 2.
TABLE 1 volatile composition table of control group Damascus rose hydrolat fermentation liquor
TABLE 2 table of volatile components of the fermentation broth of Rosa damascena of sample group
Claims (10)
1. A fermentation method capable of effectively improving the quality of rose cellular fluid is characterized by comprising the following steps:
step 1): inoculating torula or enterobacter from agar slant culture medium for storing strain into liquid seed culture medium, and culturing at 20-30 deg.C for 12-48 hr to activate strain to obtain seed solution;
step 2): inoculating the seed solution into a liquid fermentation culture medium prepared by taking Damascus rose hydrosol as a matrix according to the volume percentage of 4-8%, and culturing at 29-33 ℃ for 24-168 hours to obtain fermentation liquor.
2. The fermentation method according to claim 1, wherein the yeast or enterobacter in step 1) is Seebergine Denay, Enterobacter reuteri, Kluyveromyces marxianus or Zygosaccharomyces bailii.
3. The fermentation method for improving the quality of rose cellular fluid according to claim 1, wherein the seed culture medium in step 1) comprises: 1-3g/L of dipotassium phosphate trihydrate, 0.5-2g/L of monopotassium phosphate, 0.2-0.7g/L of anhydrous magnesium sulfate, 4-6g/L of sodium chloride, 8-11g/L of ammonium sulfate, 4-6g/L of glucose, and distilled water for fixing the volume and adjusting the pH value to 4.8-7.2.
4. The fermentation method for improving rose cellular fluid quality according to claim 1, wherein the liquid fermentation medium in step 2) comprises: 1-3g/L of dipotassium phosphate trihydrate, 0.5-1g/L of monopotassium phosphate, 0.2-0.7g/L of anhydrous sulfuric acid, 2-7g/L of sodium chloride, 8-11g/L of ammonium sulfate and 9-12g/L of glycerol, taking Damascus rose hydrosol as a matrix to fix the volume, and adjusting the pH to 5.3-6.8.
5. The fermentation method for improving the quality of rose cellular fluid according to claim 1, wherein the culturing in step 1) is performed in a constant temperature shaker at a rotation speed of 150-220 rpm.
6. The fermentation method according to claim 1, wherein the culturing in step 2) is performed in a constant temperature shaker at a rotation speed of 180-220 rpm.
7. The fermentation method of claim 1, wherein the fermentation broth obtained in step 2) is filtered through a 0.2-0.3 μm nylon filter membrane and stored at 3-6 ℃.
8. The fermentation method capable of effectively improving the quality of rose cellular fluid according to claim 1, wherein the fermentation broth obtained in the step 2) is measured, the mass content of polyphenol is improved by 1-10%, the mass content of phenethyl alcohol is improved by 5-80%, the mass content of eugenol is improved by 10-70%, the mass content of methyl eugenol is improved by 30-85%, and new aroma substances are generated.
9. The fermentation method of claim 8, wherein the new aromatic substances include benzyl acetate, phenethyl acetate and citronellol.
10. The fermentation method for effectively improving the quality of rose cellular fluid according to claim 8, wherein the total phenol content in the measurement method is determined by: (1) adding 1-3mL of Folin phenol reagent with volume concentration of 10% into 0.3-0.5mL of supernatant of the centrifuged fermentation liquor, uniformly mixing, standing for 4-6min, then adding 1.4-1.8mL of sodium carbonate solution with mass concentration of 7.5%, standing for 60min, measuring the light absorption value at 765nm, and calculating the total phenol content according to a standard curve; the method for measuring the new aroma substances comprises the following steps: and (3) taking gamma-decalactone dissolved in n-hexane as an internal standard substance, and determining the volatile components of the fermentation liquor by adopting GC-MS.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115531232A (en) * | 2022-06-28 | 2022-12-30 | 银谷芳香科技有限公司 | Preparation method and application of rose composite fermentation liquor rich in ellagic acid |
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