Glucose oxidase-based essence stock solution and preparation method thereof

Abstract

The invention provides an essence stock solution based on glucose oxidase. A preparation method of the essence stock solution comprises the following steps: step 1, selecting aspergillus niger or penicillium as a strain; step 2, planting the strain in a glucose agar culture medium; step 3, cleaning the fermentation tank; step 4, inoculating the activated strain into a fermentation tank for fermentation; 5, filtering and collecting the fermentation liquor, and acidifying and precipitating the fermentation liquor; step 6, centrifuging the precipitated fermentation product; step 7, removing redundant protein by a salting-out method, and sequentially performing concentration, centrifugation, dialysis and chromatography; and step 8, diluting the glucose oxidase with deionized water. The glucose oxidase provided by the invention is prepared by performing fermentation culture on excellent strains, then performing salting-out, concentration, centrifugation, dialysis, chromatography and refining, the conversion rate of the produced glucose oxidase is high, and the prepared high-purity glucose oxidase is directly diluted with deionized water, so that the essence stock solution of the glucose oxidase can be prepared.

Description

Technical Field

[0001] The present invention relates to the technical field of essence stock solutions, in particular to an essence stock solution based on glucose oxidase and a preparation method thereof. Background Art

[0002] Essence liquid and essence liquid are different. Essence liquid usually contains only 1-2 high-purity active ingredients, with a very simple formula and avoids unnecessary additives. Due to its single ingredient, the efficacy of essence liquid is very clear; while essence liquid contains multiple active ingredients, focuses on synergistic effects to achieve multiple effects, and has more internal ingredients.

[0003] Glucose oxidase also has antioxidant properties, inhibits bacterial growth, and enhances skin defense mechanisms. Furthermore, the enzyme can scavenge free radicals on the skin's surface, reducing oxidative stress damage and slowing the aging process. Currently, there are no glucose oxidase-based serums on the market. Therefore, we propose a glucose oxidase-based serum and its preparation method. Summary of the Invention

[0004] The present invention provides a glucose oxidase-based essence solution and a preparation method thereof, which has the advantage of high glucose oxidase conversion rate and solves the problems raised in the above-mentioned background technology.

[0005] The technical solution of the present invention is achieved as follows: a method for preparing a glucose oxidase-based essence solution, comprising the following steps: Step 1: Select Aspergillus niger or Penicillium as the strain; Step 2: Plant the strain in glucose agar medium and culture it at 28-35°C until the hyphae grow into white flocs; Step 3: Clean the fermentation tank to ensure it is free of contamination, pour the nutrient medium into the fermentation tank, and add an appropriate amount of yeast; Step 4: Inoculate the activated bacteria cultured in step 2 into a fermentation tank for fermentation, maintain the temperature at 28-35°C, and ferment for 24-72 hours; Step 5: Filter and collect the fermentation liquid in step 4, then add an appropriate amount of acid to the fermentation liquid to acidify and precipitate the fermentation liquid to obtain a precipitated fermentation product; Step 6: centrifuging the precipitated fermentation product to collect the precipitated bacteria; Step 7: Add the precipitated cells to a buffer solution, remove excess protein by salting out, and then sequentially perform concentration, centrifugation, dialysis, and chromatography to obtain glucose oxidase; Step 8: Dilute glucose oxidase with deionized water and then add recombinant human collagen to prepare the glucose oxidase essence solution.

[0006] Preferably, in step 2, the culture bottle is shaken regularly to promote uniform growth of mycelium.

[0007] Preferably, in step three, the nutrient medium uses corn steep liquor and soybean meal as nitrogen sources, glucose as carbon source, the pH is controlled at 4-6, and the DH value is controlled at 4-5.

[0008] Preferably, the ratio of carbon source to nitrogen source is 25:1.

[0009] Preferably, in step 4, the activated bacteria are inoculated into the liquid culture medium at a ratio of 5-12%.

[0010] Preferably, in step 4, during the fermentation process, sterile air is introduced to maintain the dissolved oxygen content.

[0011] Preferably, in step 3, the fermentation tank is sterilized with high-pressure steam at 110-130° C. for 20-30 minutes.

[0012] Preferably, in step seven, the bacterial cells are subjected to cell wall breaking treatment before salting out.

[0013] Another technical problem to be solved by the present invention is to provide a glucose oxidase-based essence solution prepared by the above preparation method.

[0014] Compared with the prior art, the glucose oxidase provided in the present invention is prepared by fermentation and cultivation of superior bacterial strains, followed by purification through salting out, concentration, centrifugation, dialysis, and chromatography. The produced glucose oxidase has a high conversion rate. The prepared high-purity glucose oxidase can be directly diluted with deionized water to obtain an essence stock solution of glucose oxidase. DETAILED DESCRIPTION

[0015] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0016] The present invention specifically adopts the following method to prepare the glucose oxidase essence solution, which is specifically divided into eight steps, each of which is as follows: Step 1: Select Aspergillus niger or Penicillium as the strain; Step 2: Plant the strain in glucose agar medium and culture it in an environment of 28-35℃ until the mycelium grows into white flocs. Because the mold will produce white mycelium in the early growth stage, these mycelium gradually mature and form black spores as they absorb nutrients and carry out metabolic activities. During this period, shake the culture bottle regularly to promote uniform growth of mycelium. Step 3: Clean the fermentation tank and sterilize it with high-pressure steam at 110-130°C for 20-30 minutes to ensure it is contaminant-free. Pour the nutrient medium into the fermentation tank and add an appropriate amount of yeast. The nutrient medium uses corn steep liquor and soybean meal as nitrogen sources, glucose as carbon source, pH controlled at 4-6, DH value controlled at 4-5, and carbon source to nitrogen source ratio of 25:1. Step 4: Inoculate the activated bacteria cultured in step 2 into a fermentation tank for fermentation. The activated bacteria are inoculated into the liquid culture medium at a ratio of 5-12%. The temperature is maintained at 28-35°C and the fermentation time is 24-72 hours. During the fermentation process, sterile air is introduced to maintain the dissolved oxygen content. Continuous intermittent stirring is required during this process to help dissipate heat and cool the temperature, and also promote uniform growth of the bacteria. Step 5: Filter and collect the fermentation broth from step 4, then add an appropriate amount of acid (such as hydrochloric acid) to the broth to acidify and precipitate the fermentation broth to obtain a precipitated fermentation product. Acidification precipitation is achieved by adjusting the pH value of the solution to change the charge on the molecular surface, thereby affecting its solubility and ultimately causing precipitation. The precipitate can be collected by filtration after precipitation; Step 6: Centrifuge the precipitated fermentation product to collect the precipitated bacteria, and centrifuge at 2000-8000 rpm for 10-15 minutes; Step 7: Add the precipitated cells to a buffer solution and remove excess protein by salting out. Before salting out, the cells are broken into pieces, such as by ultrasonic cell wall breaking, surfactant or enzymatic cell wall breaking, so that the protein can be better precipitated, thereby better releasing the enzyme in the cells. After concentration, centrifugation, dialysis, and chromatography, glucose oxidase can be obtained. Step 8. Dilute glucose oxidase with deionized water, and then add recombinant human collagen to prepare the glucose oxidase essence solution. In the essence solution, glucose oxidase accounts for 1-18%, and recombinant human collagen accounts for 3-25%. In this way, glucose oxidase and recombinant human collagen are more easily absorbed by the skin, and the essence solution can be mixed with other formulas to prepare essence. Other formulas include plant extracts, plant extracts include honeysuckle hydrosol, wormwood hydrosol, honeysuckle hydrosol, etc., and recombinant human collagen mainly helps delay aging and improve skin texture by promoting skin repair, enhancing moisturizing ability and improving elasticity.

[0017] The following are descriptions of different recipes: Example 1 Step 1: Select Aspergillus niger or Penicillium as the strain; Step 2: Plant the strain in glucose agar medium and culture it at 28°C until the hyphae grow into white flocs; Step 3: Clean the fermenter and sterilize it with high-pressure steam at 110°C for 20 minutes to ensure it is contaminant-free. Pour the nutrient medium into the fermenter and add an appropriate amount of yeast. The nutrient medium uses corn steep liquor and soybean meal as nitrogen sources, glucose as a carbon source, and is controlled at a pH of 4, a DH value of 4, and a carbon source to nitrogen source ratio of 25:1. Step 4: inoculate the activated bacteria cultured in step 2 into a fermentation tank for fermentation, inoculate the activated bacteria into the liquid culture medium at a ratio of 5%, maintain the temperature at 28°C, and ferment for 24 hours. During the fermentation process, introduce sterile air to maintain the dissolved oxygen content; Step 5: Filter and collect the fermentation liquid in step 4, then add an appropriate amount of acid to the fermentation liquid to acidify and precipitate the fermentation liquid to obtain a precipitated fermentation product; Step 6: centrifuging the precipitated fermentation product to collect the precipitated bacteria; Step 7: Add the precipitated cells to a buffer solution and remove excess protein by salting out. Before salting out, the cells are broken into pieces. The cells are then concentrated, centrifuged, dialyzed, and chromatographed to obtain glucose oxidase. Step 8: Dilute glucose oxidase with deionized water and then add recombinant human collagen to prepare the glucose oxidase essence solution.

[0018] Example 2

[0019] Step 1: Select Aspergillus niger or Penicillium as the strain; Step 2: Plant the strain in glucose agar medium and culture it at 29°C until the hyphae grow into white flocs; Step 3: Clean the fermenter and sterilize it with high-pressure steam at 115°C for 22 minutes to ensure it is contaminant-free. Pour the nutrient medium into the fermenter and add an appropriate amount of yeast. The nutrient medium uses corn steep liquor and soybean meal as nitrogen sources, glucose as a carbon source, and is controlled at a pH of 4.5, a DH value of 4, and a carbon-to-nitrogen ratio of 25:1. Step 4: inoculate the activated bacteria cultured in step 2 into a fermentation tank for fermentation, inoculate the activated bacteria into the liquid culture medium at a ratio of 6%, maintain the temperature at 30°C, and ferment for 30 hours. During the fermentation process, introduce sterile air to maintain the dissolved oxygen content; Step 5: Filter and collect the fermentation liquid in step 4, then add an appropriate amount of acid to the fermentation liquid to acidify and precipitate the fermentation liquid to obtain a precipitated fermentation product; Step 6: centrifuging the precipitated fermentation product to collect the precipitated bacteria; Step 7: Add the precipitated cells to a buffer solution and remove excess protein by salting out. Before salting out, the cells are broken into pieces. The cells are then concentrated, centrifuged, dialyzed, and chromatographed to obtain glucose oxidase. Step 8: Dilute glucose oxidase with deionized water and then add recombinant human collagen to prepare the glucose oxidase essence solution.

[0020] Example 3

[0021] Step 1: Select Aspergillus niger or Penicillium as the strain; Step 2: Plant the strain in glucose agar medium and culture it at 30°C until the hyphae grow into white flocs; Step 3: Clean the fermenter and sterilize it with high-pressure steam at 120°C for 25 minutes to ensure it is contaminant-free. Pour the nutrient medium into the fermenter and add an appropriate amount of yeast. The nutrient medium uses corn steep liquor and soybean meal as nitrogen sources, glucose as a carbon source, and the pH and DH value are controlled at 5. The ratio of carbon source to nitrogen source is 25:1. Step 4: inoculate the activated bacteria cultured in step 2 into a fermentation tank for fermentation, inoculate the activated bacteria into the liquid culture medium at a ratio of 5-12%, maintain the temperature at 30°C, and ferment for 48 hours. During the fermentation process, introduce sterile air to maintain the dissolved oxygen content; Step 5: Filter and collect the fermentation liquid in step 4, then add an appropriate amount of acid to the fermentation liquid to acidify and precipitate the fermentation liquid to obtain a precipitated fermentation product; Step 6: centrifuging the precipitated fermentation product to collect the precipitated bacteria; Step 7: Add the precipitated cells to a buffer solution and remove excess protein by salting out. Before salting out, the cells are broken into pieces. The cells are then concentrated, centrifuged, dialyzed, and chromatographed to obtain glucose oxidase. Step 8: Dilute glucose oxidase with deionized water and then add recombinant human collagen to prepare the glucose oxidase essence solution.

[0022] Example 4

[0023] Step 1: Select Aspergillus niger or Penicillium as the strain; Step 2: Plant the strain in glucose agar medium and culture it at 30°C until the hyphae grow into white flocs; Step 3: Clean the fermenter and sterilize it with high-pressure steam at 125°C for 26 minutes to ensure it is contaminant-free. Pour the nutrient medium into the fermenter and add an appropriate amount of yeast. The nutrient medium uses corn steep liquor and soybean meal as nitrogen sources, glucose as a carbon source, and the pH and DH values ​​are controlled at 5. The ratio of carbon source to nitrogen source is 25:1. Step 4: inoculate the activated bacteria cultured in step 2 into a fermentation tank for fermentation, inoculate the activated bacteria into the liquid culture medium at a ratio of 7%, maintain the temperature at 30°C, and ferment for 52 hours. During the fermentation process, introduce sterile air to maintain the dissolved oxygen content; Step 5: Filter and collect the fermentation liquid in step 4, then add an appropriate amount of acid to the fermentation liquid to acidify and precipitate the fermentation liquid to obtain a precipitated fermentation product; Step 6: centrifuging the precipitated fermentation product to collect the precipitated bacteria; Step 7: Add the precipitated cells to a buffer solution and remove excess protein by salting out. Before salting out, the cells are broken into pieces. The cells are then concentrated, centrifuged, dialyzed, and chromatographed to obtain glucose oxidase. Step 8: Dilute glucose oxidase with deionized water and then add recombinant human collagen to prepare the glucose oxidase essence solution.

[0024] Example 5

[0025] Step 1: Select Aspergillus niger or Penicillium as the strain; Step 2: Plant the strain in glucose agar medium and culture it at 3°C ​​until the hyphae grow into white flocs; Step 3: Clean the fermenter and sterilize it with high-pressure steam at 125°C for 25 minutes to ensure it is contaminant-free. Pour the nutrient medium into the fermenter and add an appropriate amount of yeast. The nutrient medium uses corn steep liquor and soybean meal as nitrogen sources, glucose as a carbon source, and the pH and DH value are controlled at 5. The ratio of carbon source to nitrogen source is 25:1. Step 4: inoculate the activated bacteria cultured in step 2 into a fermentation tank for fermentation, inoculate the activated bacteria into the liquid culture medium at a ratio of 8%, maintain the temperature at 30°C, and ferment for 65 hours. During the fermentation process, introduce sterile air to maintain the dissolved oxygen content; Step 5: Filter and collect the fermentation liquid in step 4, then add an appropriate amount of acid to the fermentation liquid to acidify and precipitate the fermentation liquid to obtain a precipitated fermentation product; Step 6: centrifuging the precipitated fermentation product to collect the precipitated bacteria; Step 7: Add the precipitated cells to a buffer solution and remove excess protein by salting out. Before salting out, the cells are broken into pieces. The cells are then concentrated, centrifuged, dialyzed, and chromatographed to obtain glucose oxidase. Step 8: Dilute glucose oxidase with deionized water and then add recombinant human collagen to prepare the glucose oxidase essence solution.

[0026] The glucose oxidase prepared in Example 1 to Example 5 is tested one by one, and the testing process is as follows: 1. The concentration of gluconic acid and the biomass of the bacteria after the fermentation is ended are measured in Example 1 to Example 5, and the specific results are shown in Table 1: Table 1

[0027] As shown in Table 1, after the fermentation is ended, the biomass is the highest, and the concentration of gluconic acid is the lowest. The glucose oxidase produced by Aspergillus niger or Penicillium can metabolize glucose into gluconic acid. With the increase of the concentration of the metabolite, the synthesis of the glucose oxidase is inhibited by feedback.

[0028] 2. The enzyme activity of the precipitated bacteria obtained in the application is tested by using a spectrophotometer (460 nm absorbance), and the specific enzyme activity data is shown in Table 2: Table 2

[0029] As shown in Table 2, the enzyme activity of the precipitated bacteria prepared in Example 1 to Example 5 is high.

[0030] 3. The recovery rate of the glucose oxidase prepared in Example 1 to Example 5 is detected, and the results are shown in Table 3: Table 3

[0031] The enzyme activity recovery rate of the glucose oxidase obtained after the purification is increased to more than 90%.

[0032] The above only describes the preferred embodiments of the application and is not used to limit the application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the application shall be included in the protection scope of the application.

Claims

1. A method for preparing a glucose oxidase-based essence solution, characterized in that: The steps include: Step 1: Select Aspergillus niger or Penicillium as the strain; Step 2: Plant the strain in glucose agar medium and culture it at 28-35°C until the hyphae grow into white flocs; Step 3: Clean the fermentation tank to ensure it is free of contamination, pour the nutrient medium into the fermentation tank, and add an appropriate amount of yeast; Step 4: Inoculate the activated bacteria cultured in step 2 into a fermentation tank for fermentation, maintain the temperature at 28-35°C, and ferment for 24-72 hours; Step 5: Filter and collect the fermentation liquid in step 4, then add an appropriate amount of acid to the fermentation liquid to acidify and precipitate the fermentation liquid to obtain a precipitated fermentation product; Step 6: centrifuging the precipitated fermentation product to collect the precipitated bacteria; Step 7: Add the precipitated cells to a buffer solution, remove excess protein by salting out, and then sequentially perform concentration, centrifugation, dialysis, and chromatography to obtain glucose oxidase; Step 8: Dilute glucose oxidase with deionized water and then add recombinant human collagen to prepare the glucose oxidase essence solution.

2. The method for preparing the glucose oxidase essence solution according to claim 1, wherein: In step 2, the culture bottle is shaken regularly to promote uniform growth of mycelium.

3. The method for preparing the glucose oxidase essence solution according to claim 1, wherein: In step 3, the nutrient medium uses corn steep liquor and soybean cake powder as nitrogen sources, glucose as carbon source, pH is controlled at 4-6, and DH value is controlled at 4-5.

4. The method for preparing the glucose oxidase-based essence solution according to claim 3, wherein: The ratio of carbon source to nitrogen source is 25:

1.

5. The method for preparing the glucose oxidase-based essence solution according to claim 1, wherein: In step 4, the activated bacteria are inoculated into the liquid culture medium at a ratio of 5-12%.

6. The method for preparing the glucose oxidase-based essence solution according to claim 5, wherein: In step 4, during the fermentation process, sterile air is introduced to maintain the dissolved oxygen content.

7. The method for preparing the glucose oxidase-based essence solution according to claim 4, wherein: In step 3, the fermentation tank is sterilized with high pressure steam at 110-130° C. for 20-30 minutes.

8. The method for preparing the glucose oxidase-based essence solution according to claim 1, wherein: In step seven, the bacterial cells are broken before salting out.

9. A glucose oxidase-based essence solution, characterized in that: It is through claim 1 8 prepared by any one of the preparation methods.

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