Preparation method and application of artemisia apiacea fermentation extracting solution

Abstract

The invention discloses a preparation method and application of an artemisia apiacea fermentation extracting solution. The artemisia annua fermentation extracting solution is prepared from artemisia annua, ultrapure water, probiotics and the like. The preparation method comprises the following steps: picking fresh artemisia annua, cleaning, drying and sterilizing; crushing, sieving, and selecting artemisia annua particles with proper particle sizes; adding the selected artemisia annua particles, ultrapure water and probiotics into a temperature-controlled closed stirring reaction device, and carrying out strict anaerobic fermentation; separating a liquid phase part and a solid phase part from the obtained fermentation product through five filtering processes, performing microfiltration on the liquid phase part to remove thalli, and adding 1, 2-hexanediol and p-hydroxyacetophenone to obtain the artemisia apiacea fermentation extracting solution. The extracting solution prepared by fermenting natural plant raw materials and probiotics is high in molecular weight uniformity and high in physiological activity, retains the functional effect of sweet wormwood, is safe, low in sensitivity and low in irritation, and is suitable for being added into various daily chemical products.

Description

Technical Field

[0001] The invention relates to a method for extracting highly active artemisinin from Artemisia annua by fermentation, and in particular to a method for preparing an extract by fermenting Artemisia annua with probiotics, belonging to the field of biotechnology. Background Art

[0002] Artemisia annua is an annual herb that mainly contains artemisinin, sesquioxane, flavonoids, volatile oils, polysaccharides, etc. Artemisia annua extract is fermented by scientific biotechnology. It is safe, hypoallergenic, and low-irritating. It has good antibacterial and anti-inflammatory effects and is a good antibacterial agent for daily chemical products.

[0003] At present, most of the patents involving Artemisia annua mainly focus on the extraction of artemisinin, and the extraction methods mostly adopt physical solvent extraction, and a large amount of organic solvents are used in the extraction process, which is not convenient to directly add to daily chemical products. At the same time, there are a series of problems such as cumbersome repeated purification process and low raw material extraction rate. Through patent review, only patent CN 107522708A involves a production process for extracting artemisinin by enzymatic hydrolysis. After enzymatic hydrolysis of Artemisia annua, petroleum ether is still used for extraction, and it does not involve the preparation of Artemisia annua fermentation extract by probiotic fermentation.

[0004] At present, the research on extracting Artemisia annua through fermentation is still relatively limited. Based on the increasing demand for natural daily chemical raw materials in the market, there is great room for expansion in the research of such bio-fermentation plant extracts. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a preparation method and application of an Artemisia annua fermentation extract. In order to solve the above technical problem, the present invention provides an Artemisia annua fermentation extract, characterized in that it is prepared by temperature-controlled anaerobic fermentation of Artemisia annua, ultrapure water and probiotics as raw materials, and 1,2-hexanediol and p-hydroxyacetophenone are added after the fermentation product is filtered, and the components of the Artemisia annua fermentation extract are as follows: Artemisia annua extract ≥ 2.5%, 1,2-hexanediol 0.5%, p-hydroxyacetophenone 0.5%, and ultrapure water as the balance.

[0006] The present invention also provides a method for preparing the above-mentioned Artemisia annua fermentation extract, which is characterized by comprising the following steps: (1) Pick fresh Artemisia annua, wash, dry and sterilize; (2) Grinding, sieving, and selecting Artemisia annua particles with appropriate particle size; (3) Add the selected Artemisia annua granules, ultrapure water, and probiotics into a temperature-controlled, sealed stirring reaction device for strict anaerobic fermentation; (4) The obtained fermented product is separated into a liquid phase and a solid phase through a five-pass filtration process, the liquid phase is microfiltered to remove the bacterial cells, and 1,2-hexanediol and p-hydroxyacetophenone are added to obtain the Artemisia annua fermentation extract.

[0007] Preferably, in step (2), the artemisia annua particles are sieved through a 40-100 mesh sieve.

[0008] Preferably, in step (3), the fermentation temperature is controlled at 30°C-40°C, and the fermentation period is 2-14 days.

[0009] Preferably, the probiotic used for fermentation in step (3) is one of bifidobacterium, lactobacillus and yeast.

[0010] Preferably, the five filtering processes in step (4) are five of: soaking, distillation, precipitation, extraction, solvent extraction, and filtration.

[0011] Preferably, the pores of the filter membrane used for filtration in step (4) are 40 μm; the pores of the filter membrane used for microfiltration are 0.22 μm; and the amount of 1,2-hexanediol and p-hydroxyacetophenone added to the liquid phase is 0.5% of the mass of the liquid phase.

[0012] The present invention also provides the use of the Artemisia annua fermentation extract in daily chemical products, characterized in that the addition amount of the Artemisia annua fermentation extract is 0.1%-50% of the total mass of all raw materials.

[0013] The present invention provides a method for preparing an Artemisia annua fermented extract, which uses natural plant raw materials supplemented with probiotic fermentation, and makes full use of the degradation of macromolecular compounds by bacterial metabolism to prepare an extract, which is safe, low-sensitivity, low-irritation, has good antibacterial effect, and can be widely used in daily chemical products. First, probiotics can more efficiently dissolve the active compounds of Artemisia annua, and the contents of artemisinin, polysaccharides, amino acids, proteins, etc. are improved. Second, the fermentation filtrate retains the natural pleasant breath of plant-derived herbs, and realizes the mutual benefit of plant sources and probiotics. Third, the separation and sterilization processes of production are both treated at room temperature, which greatly reduces the loss and destruction of artemisinin active factors. In addition, the process also greatly reduces energy consumption compared with the extraction method. Fourth, the fermentation sterilization filtrate can be directly added to natural raw materials used in daily chemical products, etc., to provide antibacterial function. In summary, the process technology for preparing a fermentation extract by fermenting Artemisia annua provided by the present invention has no wastewater discharge during the process, and the production process adopts strict anaerobic fermentation control, and basically no waste gas is discharged, which effectively realizes the green and environmental protection of the production technology. DETAILED DESCRIPTION

[0014] In order to make the present invention more clearly understood, preferred embodiments are described in detail as follows.

[0015] Example 1 A method for preparing an Artemisia annua fermented extract: 1) Activation of fermentation bacteria Weigh 0.6 g of sodium acetate, 4 g of soy peptone, 4 g of glucose, 1 g of yeast extract, 0.4 g of dipotassium hydrogen phosphate, 0.12 g of magnesium sulfate, 0.05 g of manganese sulfate, and 200 ml of distilled water, adjust the pH value to 6.2±0.2, sterilize with high-pressure steam for 15 min, cool to room temperature, inoculate bifidobacteria for activation and culture for 48 h, and prepare an activated seed culture medium.

[0016] 2) Preparation of fermentation medium The washed and dried Artemisia annua was taken and crushed to obtain 40 mesh Artemisia annua particles. 0.1 wt% Artemisia annua particles, 0.1 wt% glucose, 0.001 wt% casein, 0.001 wt% vitamin C were added to the fermentation system, and the rest was supplemented with ultrapure water, and then the mixture was fully shaken and sterilized at 121° C. for 15 min.

[0017] 3) Fermentation process After the seed culture medium is activated, the fermentation extract is diluted to a quantitative value of 1×109 cfu / mL. The fermentation extract is added to the fermentation medium at a mass of 0.5 wt% of the mass of the fermentation system and fermented at 37°C for 24 hours.

[0018] 4) Post-fermentation treatment After the fermentation is completed, the supernatant is soaked, distilled, precipitated, extracted, and the solvent is extracted. The plant residues are removed by a 40 μm filter membrane, and the bacteria are removed by a 0.22 μm filter membrane. 1,2-hexanediol and p-hydroxyacetophenone are added to prepare the Artemisia annua fermentation extract.

[0019] Example 2 A method for preparing an Artemisia annua fermented extract: 1) Activation of fermentation bacteria Weigh 0.6 g of sodium acetate, 4 g of soy peptone, 4 g of glucose, 1 g of yeast extract, 0.4 g of dipotassium hydrogen phosphate, 0.12 g of magnesium sulfate, 0.05 g of manganese sulfate, and 200 ml of distilled water, adjust the pH value to 6.2±0.2, sterilize with high-pressure steam for 15 min, cool to room temperature, inoculate lactic acid bacteria for activation culture for 48 h, and prepare the activated seed culture medium.

[0020] 2) Preparation of fermentation medium The washed and dried Artemisia annua was taken and crushed to obtain 80 mesh Artemisia annua particles. 0.1wt% Artemisia annua particles, 1wt% glucose, 0.001wt% casein, 0.001wt% vitamin C were added to the fermentation system, and the rest was supplemented with water, and the mixture was fully shaken and sterilized at 121°C for 15 minutes.

[0021] 3) Fermentation process After the seed culture medium is activated, the fermentation extract is diluted to a quantitative value of 1×109 cfu / mL. The fermentation extract is added to the fermentation medium at a mass of 2 wt% of the mass of the fermentation system and fermented at 37° C. for 3 days.

[0022] 4) Post-fermentation treatment After the fermentation is completed, the supernatant is soaked, distilled, precipitated, extracted, and the solvent is extracted. The plant residues are removed by a 40 μm filter membrane, and the bacteria are removed by a 0.22 μm filter membrane. 1,2-hexanediol and p-hydroxyacetophenone are added to prepare the Artemisia annua fermentation extract.

[0023] Example 3 A method for preparing an Artemisia annua fermented extract: 1) Activation of fermentation bacteria Weigh 0.6 g of sodium acetate, 4 g of soy peptone, 4 g of glucose, 1 g of yeast extract, 0.4 g of dipotassium hydrogen phosphate, 0.12 g of magnesium sulfate, 0.05 g of manganese sulfate, and 200 ml of distilled water, adjust the pH value to 6.2±0.2, sterilize with high-pressure steam for 15 min, cool to room temperature, inoculate lactic acid bacteria for activation culture for 48 h, and prepare the activated seed culture medium.

[0024] 2) Preparation of fermentation medium The washed and dried Artemisia annua was taken and crushed to obtain 100 mesh Artemisia annua particles. 0.1wt% Artemisia annua particles, 3wt% glucose, 0.001wt% casein, 0.001wt% vitamin C were added to the fermentation system, and the rest was supplemented with water, and the mixture was fully shaken and sterilized at 121°C for 15 minutes.

[0025] 3) Fermentation process After the seed culture medium is activated, the fermentation extract is diluted to a quantitative value of 1×109 cfu / mL. The fermentation extract is added to the fermentation culture medium at a mass of 3 wt% of the mass of the fermentation system, and fermented in an incubator at 37°C for 7 days.

[0026] 4) Post-fermentation treatment After the fermentation is completed, the supernatant is soaked, distilled, precipitated, extracted, and the solvent is extracted. The plant residues are removed by a 40 μm filter membrane, and the bacteria are removed by a 0.22 μm filter membrane. 1,2-hexanediol and p-hydroxyacetophenone are added to prepare the Artemisia annua fermentation extract.

[0027] The fermentation extracts prepared in Examples 1-3 were subjected to relevant tests.

[0028] The detection method is as follows: 1. Artemisinin concentration Refer to Chinese Pharmacopoeia 2015 Edition 2. Amino Acid Content Refer to QB / T 2409-1998 3. Polysaccharide content Refer to SN / T 4260-2015 4. Protein content Refer to GB / T 6432-2018 The specific data of the above test are shown in Table 1.

[0029] Table 1 sample Protein (g / L) Amino Acid(g / L) Total sugar (g / L) Artemisinin (g / L) Example 1 80 120 70 100 Example 2 90 160 80 200 Example 3 70 115 69 150 Example

[0030] An antibacterial laundry detergent comprising the following components in percentage by mass: Element content Pure water 63.52wt% Artemisia annua fermented extract 1.00wt% AES 14.00wt% AEO-7 10.00wt% APG0810 4.00wt% APGIC06 2.00wt% CAB 3.00wt% Sodium Citrate 2.00wt% essence 0.20wt% preservative 0.08wt% Protease 0.20wt% Preparation method: add water, AES, AEO-7, APG0810, APGIC06, sodium citrate, CAB, flavor, preservative and protease into a water phase pot, keep stirring to dissolve evenly, add Artemisia annua fermented extract and stir evenly to obtain the antibacterial laundry detergent.

[0031] Example 5 An antibacterial dishwashing detergent comprising the following components in percentage by mass: Element content Pure water 71.11wt% Artemisia annua fermented extract 1.00wt% AES 15.00wt% APG0810 6.00wt% 6501 3.00wt% XP-70 1.50wt% Citric acid monohydrate 0.23wt% essence 0.08wt% KF-88 0.08wt% Sodium chloride 2.00wt% Preparation method: add water, AES, APG0810, 6501, XP-70, citric acid monohydrate, essence, preservative and sodium chloride into the water phase pot, keep stirring to dissolve evenly, add Artemisia annua fermented extract and stir evenly to obtain the antibacterial dishwashing detergent.

[0032] Example 6 An antibacterial hand sanitizer comprising the following components in percentage by mass: Element content MES 2.00wt% APG0810 7.00wt% K12A 5.00wt% Glycerol 1.00wt% Citric acid monohydrate 0.11wt% essence 0.15wt% Sodium Hyaluronate 0.0001wt% Phenoxyethanol 0.5wt% water 84.24wt% Preparation method: Add pure water, MES, APG0810, K12A, propylene glycol, citric acid monohydrate, essence, sodium hyaluronate and phenoxyethanol into the water phase pot, keep stirring to dissolve evenly, add Artemisia annua fermented extract and stir evenly to obtain the antibacterial hand sanitizer.

[0033] Examples 4-6 were subjected to relevant tests.

[0034] The detection method is as follows: 1. Sterilization rate of Staphylococcus aureus: Refer to QB / T 2738-2012 Evaluation method for antibacterial and antimicrobial effects of daily chemical products 7.2.4 Test method for bactericidal effect of antibacterial daily chemical products (suspension quantitative method), and time for 30 minutes.

[0035] 2. Escherichia coli sterilization rate Refer to QB / T 2738-2012 Evaluation method for antibacterial and antimicrobial effects of daily chemical products 7.2.4 Test method for bactericidal effect of antibacterial daily chemical products (suspension quantitative method), and time for 30 minutes.

[0036] The specific data of the above test are shown in Table 2.

[0037] Table 2 sample Sterilization rate of Staphylococcus aureus (%) Escherichia coli sterilization rate (%) Example 4 >99.992 >99.997 Example 5 >99.990 >99.995 Example 6 >99.995 >99.998

Claims

1. A method for preparing an Artemisia annua fermentation extract and its application, characterized in that: Artemisia annua, ultrapure water and probiotics are used as raw materials for temperature-controlled anaerobic fermentation, and 1,2-hexanediol and p-hydroxyacetophenone are added after the fermentation product is filtered to obtain the artemisia annua fermentation extract. Calculated by weight percentage, the components of the artemisia annua fermentation extract are: artemisia annua extract ≥2.5%, 1,2-hexanediol 0.5%, p-hydroxyacetophenone 0.5%, and ultrapure water as the balance.

2. The method for preparing the Artemisia annua fermentation extract according to claim 1, characterized in that: The steps include: (1) Pick fresh Artemisia annua, wash, dry and sterilize; (2) Grinding, sieving, and selecting Artemisia annua particles with appropriate particle size; (3) Add the selected Artemisia annua granules, ultrapure water, and probiotics into a temperature-controlled, sealed stirring reaction device for strict anaerobic fermentation; (4) The obtained fermented product is separated into a liquid phase and a solid phase through a five-pass filtration process, the liquid phase is microfiltered to remove the bacterial cells, and 1,2-hexanediol and p-hydroxyacetophenone are added to obtain the Artemisia annua fermentation extract.

3. The method for preparing the Artemisia annua fermentation extract according to claim 2, characterized in that: In the step (2), the artemisia annua particles are sieved through a 40-100 mesh sieve.

4. The method for preparing the Artemisia annua fermentation extract according to claim 2, characterized in that: In step (3), the fermentation temperature is controlled at 30°C-40°C, and the fermentation period is 2-14 days.

5. The method for preparing the Artemisia annua fermentation extract according to claim 2, characterized in that: The probiotic in step (3) is one of bifidobacterium, lactobacillus and yeast.

6. The method for preparing the Artemisia annua fermentation extract according to claim 2, characterized in that: The five filtering processes in step (4) are: soaking, distillation, precipitation, extraction, solvent extraction, and filtration.

7. The method for preparing the Artemisia annua fermentation extract according to claim 2, characterized in that: The pore size of the filter membrane used for filtration in step (4) is 40 μm; the pore size of the filter membrane used for microfiltration is 0.22 μm; and the amount of 1,2-hexanediol and p-hydroxyacetophenone added to the liquid phase is 0.5% of the mass of the liquid phase.

8. The use of the Artemisia annua fermented extract according to claim 1 in daily chemical products, characterized in that: The amount of the Artemisia annua fermentation extract added is 0.1%-50% of the total mass of all raw materials.