Fermentation preparation method of active dandelion extract with functions of removing stasis and resisting inflammation

Abstract

The invention relates to the technical field of deep processing of natural products, in particular to a fermentation preparation method of an active dandelion extract with functions of removing stasis and resisting inflammation, which comprises the following steps: crushing dried whole dandelion, and carrying out synergistic enzymolysis by using a compound enzyme consisting of cellulase, pectinase and beta-glucanase; then carrying out primary anaerobic fermentation of lactobacillus plantarum and secondary aerobic fermentation of issatchenkia orientalis in sequence; and finally, centrifuging, filtering, concentrating and spray-drying to obtain the high-activity dandelion fermentation extract. Through a process chain of ternary composite enzymolysis and step-by-step fermentation, an anaerobic-aerobic alternating micro-ecological system is constructed, and synergistic enrichment and functional quantification of double active components are realized. Specifically, in the final fermentation extract, the content of chicoric acid is greater than or equal to 8.5%, and the content of small molecular glycopeptide is greater than or equal to 25%, which are far higher than those of the traditional aqueous extract.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of natural product deep processing, in particular to a method for efficiently preparing a fermented Taraxacum mongolicum Hand.-Mazz. extract rich in chlorogenic acid and small molecule glycopeptide by using composite enzyme synergistic pretreatment combined with specific probiotics and acid-tolerant yeast step-by-step fermentation technology. The extract has clear "dispersing" biological activity and can be widely applied in the fields of medicines, health foods and functional cosmetics. BACKGROUND

[0002] Taraxacum mongolicum Hand.-Mazz. is a traditional Chinese medicine, and its effects of clearing heat and resolving toxins, reducing swelling and dispersing nodules have been recorded in Chinese Pharmacopoeia and are widely used for the adjuvant treatment of "nodular" diseases such as breast hyperplasia, lymphadenitis and acne. Modern pharmacological studies have further confirmed that the core active ingredients chlorogenic acid and small molecule glycopeptide play a key role in antioxidant, anti-inflammatory and inhibition of abnormal cell proliferation.

[0003] However, the traditional preparation methods such as water extraction and alcohol extraction have the following significant defects: low extraction efficiency: the yield of chlorogenic acid and small molecule glycopeptide is not high. Single active ingredient: it is difficult to achieve the synergistic enrichment of the two key active ingredients. Process limitations: the cell wall structure is dense, and the conventional method cannot completely break the wall, and there are safety hazards in the use of organic solvents.

[0004] In recent years, microbial fermentation technology has been applied to Taraxacum mongolicum Hand.-Mazz. extraction, but the existing schemes still cannot solve the core contradiction of "high yield and high retention of chlorogenic acid" and "efficient generation of small molecule glycopeptide". Therefore, developing a green, efficient and industrialized preparation method has become a technical problem to be solved in the field. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application provides a fermentation preparation method of active Taraxacum mongolicum Hand.-Mazz. extract with dispersing and anti-inflammatory functions. The process can simultaneously achieve efficient enrichment of chlorogenic acid and small molecule glycopeptide, significantly enhance the dispersing activity, and has the advantages of green environmental protection, low energy consumption and suitability for large-scale production.

[0006] To achieve the above purpose, the present application is implemented by the following technical scheme: a fermentation preparation method of active Taraxacum mongolicum Hand.-Mazz. extract with dispersing and anti-inflammatory functions, comprising the following steps: S1. Raw material pretreatment The dry Taraxacum mongolicum Hand.-Mazz. whole plant is pulverized by a pulverizer, and Taraxacum mongolicum Hand.-Mazz. powder is obtained by passing through a 40-60 mesh Taylor sieve. Deionized water is added at a solid-liquid ratio of 1:10-1:15 (w / v), and stirring is performed to form a uniform suspension; S2. Composite enzyme synergistic enzymolysis Adjust the pH of the suspension to 4.5-5.5, add a complex enzyme preparation, and place in a constant temperature water bath shaker at 45-55℃, 50 rpm for enzymolysis for 2-4 hours; the complex enzyme preparation is compounded by cellulase, pectinase, and β-glucanase at an activity unit ratio of (2-4):(1-3):1, and the total amount of enzyme added is 0.5%-1.5% of the mass of the dandelion powder; S3. Primary anaerobic fermentation Transfer the enzyme solution to a fermentation tank, sterilize at 120℃, 0.1 MPa for 15 min, cool to 35-37℃ after sterilization, and inoculate with 5%-10% (v / v) of activated Lactobacillus plantarum seed liquid, and ferment at 37±1℃ under static or 50 rpm low-speed stirring, anaerobic conditions for 36-48 hours; S4. Secondary aerobic fermentation After primary fermentation, do not perform sterilization treatment, directly inoculate with 3%-5% (v / v) of activated Issatchenkia orientalis seed liquid, and aerobically ferment at 28-32℃, aeration amount of 1:0.8-1:1.2 vvm, stirring speed of 150-250 rpm for 60-75 hours; S5. Termination of fermentation and post-treatment After fermentation, transfer the fermentation liquid to a water bath, heat at 85℃ for 10 min to terminate the reaction, cool to room temperature, and then centrifuge at 4℃, 8000 rpm for 20 min using a high-speed centrifuge, filter the supernatant through a 0.45 μm microporous filter membrane to obtain a clear fermentation liquid; S6. Concentration and drying Place the clear fermentation liquid in a rotary evaporator, vacuum concentrate at ≤50℃, ≤-0.08 MPa to a solid content of 30%-40%, and then perform spray drying treatment on the concentrated liquid using a spray dryer, set the inlet air temperature to 170-190℃, the outlet air temperature to 80-90℃, and the atomization pressure to 0.2 MPa, and obtain dandelion fermented extract after drying.

[0007] Preferably, the activation culture medium of the Lactobacillus plantarum seed liquid in step S3 is MRS culture medium, and the activation condition is 37℃ static culture for 24 hours. The formula of the MRS culture medium is as follows: 10 g / L of proteose peptone, 10 g / L of beef extract, 5 g / L of yeast extract, 20 g / L of glucose, 5 g / L of sodium acetate, 2 g / L of diammonium citrate, 1 mL / L of Tween 80, 0.58 g / L of MgSO4·7H2O, 0.25 g / L of MnSO4·4H2O, and pH 6.2-6.4. The Lactobacillus plantarum is a strain with a preservation number of SHBCC D25103 preserved by Shanghai Collection Center of Biological Technology (SHBCC).

[0008] Preferably, the activation medium for the *Issa mesasura* seed culture in step S4 is YPD medium, and the activation conditions are 30°C, 200 rpm shaking culture for 18 hours. The specific formulation of YPD medium is: yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L, pH 6.0. The *Issa mesasura* strain used is the one preserved by the Shanghai Biotechnology Collection Center (SHBCC) with accession number SHBCC D81754.

[0009] This invention provides a fermentation preparation method for an active dandelion extract with anti-inflammatory and nodule-dispersing functions. It possesses the following beneficial effects: This invention creates a ternary enzymatic hydrolysis + stepwise fermentation process chain, constructing an anaerobic-aerobic alternating micro-ecosystem, and achieving synergistic enrichment and functional quantification of dual active components. Specifically, in the final fermentation extract, the chicoric acid content is ≥8.5%, and the small molecule glycopeptide content is ≥25%, which is far higher than that of traditional water extracts.

[0010] This invention allows for direct transfer without sterilization after primary fermentation, avoiding energy waste and component loss caused by high-temperature sterilization; no organic solvents are used throughout the process, and the COD value of the wastewater is 1000–1500 mg / L, which is more than 70% lower than the 5000–6000 mg / L of the alcohol extraction method. Attached Figure Description

[0011] Figure 1 This is a schematic diagram of the preparation process of the present invention; Figure 2 This is a liquid chromatogram of the dandelion fermentation extract prepared in Example 1 of the present invention; Figure 3 This is a liquid chromatogram of the dandelion fermentation extract prepared in Comparative Example 1 of the present invention. Detailed Implementation

[0012] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example

[0013] like Figure 1 As shown, this embodiment of the invention provides a fermentation preparation method for an active dandelion extract with anti-inflammatory and nodule-dispersing functions, comprising the following steps: S1. Raw material pretreatment 1 kg of dried whole dandelion was ground by a pulverizer, and dandelion powder was obtained by passing through a 50-mesh Tyler screen. 12 L of deionized water was added to the dandelion powder at a solid-liquid ratio of 1:12 (w / v), and a uniform suspension was formed by stirring at 100 rpm for 15 min. S2. Complex enzyme synergistic enzymolysis Citric acid was added to adjust the pH of the suspension to 5.0, and a complex enzyme preparation was added. The enzyme solution was incubated in a constant-temperature water bath shaker at 50°C and 50 rpm for 3 hours. The complex enzyme preparation was composed of cellulase 6000 U, pectinase 4000 U, and β-glucanase 2000 U, and the total amount of enzyme added was 1.0% of the mass of the dandelion powder. S3. Primary anaerobic fermentation The enzyme solution was transferred to a fermentation tank, sterilized at 120°C and 0.1 MPa for 15 min, cooled to 37°C after sterilization, and inoculated with 10% (v / v) of activated Lactobacillus plantarum seed solution. The fermentation was carried out at 37°C under static or low-speed stirring at 50 rpm and anaerobic conditions for 48 hours. The pH value after fermentation was 4.0. S4. Secondary aerobic fermentation After primary fermentation, no sterilization was performed, and 5% (v / v) of activated Issatchenkia orientalis seed solution was inoculated. The aerobic fermentation was carried out at 30°C, with a ventilation rate of 1:1.0 vvm and a stirring speed of 200 rpm for 72 hours. S5. Termination of fermentation and post-treatment After fermentation, the fermentation solution was transferred to a water bath, heated at 85°C for 10 min to terminate the reaction, and cooled to room temperature. The solution was centrifuged at 4°C and 8000 rpm for 20 min using a high-speed centrifuge. The supernatant was filtered through a 0.45 μm microporous filter to obtain a clear fermentation solution. S6. Concentration and drying The clear fermentation solution was placed in a rotary evaporator and vacuum concentrated at ≤50°C and ≤-0.08 MPa to a solid content of 35%. The concentrated solution was then subjected to spray drying using a spray dryer. The inlet temperature was set to 180°C, the outlet temperature was set to 85°C, and the atomization pressure was set to 0.2 MPa. After drying, the dandelion fermentation extract was obtained.

[0014] The prepared dandelion fermentation extract was detected, and the detection results are as follows: The content of chicoric acid was measured by HPLC (chromatographic column: Agilent ZORBAX SB-C18, 250×4.6 mm, 5 μm; mobile phase: acetonitrile:0.1% phosphoric acid aqueous solution, 18:82; flow rate 1.0 mL / min; column temperature 30°C; detection wavelength 330 nm; reference substance: Sigma-Aldrich), and the content was 9.2% (w / w).

[0015] Small molecule glycopeptide content: SEC-MALS method (chromatographic column: Waters Ultrahydrogel 250; mobile phase: 0.1M NaNO3 aqueous solution; flow rate 0.5mL / min; standard: dextran series, 1kDa, 5kDa, 10kDa; detector: Waters2414 differential refractive index detector + Wyatt Dawn Heleos II laser light scattering detector), the proportion of components with a molecular weight of <10kDa was 27.3%.

[0016] Total polyphenol content: Folin-Ciocalteu method (control: gallic acid, Sigma-Aldrich), content 186mg GAE / g; DPPH free radical scavenging rate: spectrophotometry (517nm), scavenging rate 91.5% at a concentration of 0.5mg / mL. Example

[0017] The present application provides a kind of active dandelion extract fermentation preparation method with dispelling inflammation function, steps are identical with example one, only the activity unit ratio of cellulase, pectinase, β-glucanase in composite enzyme is adjusted to 2:3:1 (cellulase 4000U, pectinase 6000U, β-glucanase 2000U, total enzyme amount is still 1.0% of dandelion powder mass), unchanged the rest conditions.

[0018] Detection results: chicoric acid content 8.7% (w / w), small molecule glycopeptide (<10 kDa) content 26.1%, cell wall breakage rate 89%, indicating that the composite enzyme ratio in the range of (2-4): (1-3):1 can realize efficient extraction, and there is no significant difference.

[0019] Comparative example one The present application compares the dandelion with traditional water extraction method, and the operation steps are as follows: Take dandelion powder 1kg, add 10L deionized water, 90℃ water bath extraction 2 hours, 0.45 μm filter membrane filtration, vacuum concentration (60℃, -0.08MPa) of filtrate to solid content 35%, spray drying (air inlet 180℃, air outlet 85℃), get extract 140g (moisture content 5.1%).

[0020] Detection results: chicoric acid content 2.1% (w / w), small molecule glycopeptide (<10kDa) content 7.8%, total polyphenol content 92mg GAE / g, DPPH free radical scavenging rate 45.3% (0.5mg / mL), significantly lower than example one.

[0021] Comparative example two The present application compares the dandelion with single lactic acid bacteria fermentation method, and the operation steps are as follows: The steps S1-S3 (complex enzymolysis + Lactobacillus plantarum anaerobic fermentation) of Example One were performed, step S4 (yeast fermentation) was omitted, and steps S5-S6 (post-treatment and drying) were directly performed to obtain the extract 132 g.

[0022] The detection results were as follows: the content of chicoric acid was 8.9% (w / w), but the content of small molecule sugar peptides (<10 kDa) was only 12.4%; the abnormal cell proliferation inhibition rate in vitro was 42.5% (100 μg / mL), and the rat mammary nodule reduction rate was 31.2% (1.0 g / kg·d), and the resolving activity was weaker than that of Example One.

[0023] Comparative Example Three In the present application, dandelion was extracted by single yeast fermentation method, and the operation steps were as follows: The steps S1-S2 (raw material pretreatment + complex enzymolysis) of Example One were performed, step S3 (lactic acid bacteria fermentation) was skipped, and the subsequent steps were the same as Example One to obtain the extract 128 g.

[0024] The detection results were as follows: the content of small molecule sugar peptides (<10 kDa) was 23.1%, but the content of chicoric acid was reduced to 4.3% (because the esterase secreted by yeast degraded chicoric acid); the abnormal cell inhibition rate in vitro was 38.7% (100 μg / mL), and the rat mammary nodule reduction rate was 29.5% (1.0 g / kg·d), and the synergistic effect was absent, and the resolving activity was poor.

[0025] Functional verification test: Experiment 1: In vitro anti-abnormal proliferation activity (MTT method) Experimental materials: human breast epithelial cells MCF-10A (purchased from Shanghai Valley Industry Co., Ltd.); TGF-β1 (purchased from PeproTech, item number 100-21); MTT reagent (purchased from Sigma-Aldrich, item number M5655).

[0026] Experimental method: Cell culture: DMEM culture medium (containing 10% fetal bovine serum), 37°C, 5% CO2 incubator culture; Model establishment: 10 ng / mL TGF-β1 was added to induce abnormal cell proliferation, and incubated for 24 hours; Drug treatment: Example One extract, Comparative Example One extract (concentration 100 μg / mL), positive drug tamoxifen (concentration 10 μg / mL) were added respectively, and the blank group was added with the same amount of culture medium, and incubated for 48 hours; Detection: MTT solution (5 mg / mL) was added, incubated for 4 hours, the supernatant was discarded, dissolved with DMSO, and the absorbance was measured by enzyme marker (490 nm), and the inhibition rate was calculated.

[0027] The experimental results are shown in the table below:

[0028] Conclusion: The extract of this invention can significantly inhibit the abnormal proliferation of mammary epithelial cells at low concentrations, with activity close to that of positive control drugs, and is superior to traditional water extracts.

[0029] Experiment 2: Effect of nodule dispersal on animal model (rat mammary hyperplasia model) Experimental materials: SPF-grade female SD rats (6–8 weeks old, weighing 180–220 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.); testosterone propionate (purchased from Sigma-Aldrich, catalog number T1500); IL-6 ELISA kit (purchased from R&D Systems, catalog number DY526).

[0030] Experimental methods: Model establishment: Rats were subcutaneously injected with testosterone propionate (0.5 mg / 100 g body weight) once a day for 21 consecutive days, while the control group was injected with an equal volume of physiological saline. Grouped administration: The model rats were randomly divided into 3 groups (n=6): model group (no administration), extract group of Example 1 (1.0 g / kg·d, by gavage), extract group of Comparative Example 1 (1.0 g / kg·d, by gavage), and blank group by gavage with an equal volume of physiological saline. The administration was carried out continuously for 4 weeks. Detection: Nodule volume: Measure the length, width, and height of the breast nodule with calipers, and calculate the volume using the formula "volume = length × width × height × 0.5236". Test once a week. Inflammatory factors: After the administration of the drug, the rats were sacrificed, and the serum was collected. The IL-6 content was detected by ELISA. Pathological sections: Breast tissue was taken, stained with hematoxylin and eosin (HE), and the tissue morphology was observed under an optical microscope.

[0031] The experimental results are shown in the table below:

[0032] Comparative data results: In Example 1, the nodule shrinkage rate was 67.4%, while in Comparative Example 1 it was 28.6%. The decrease in inflammatory factor IL-6 was 52.3% in Example 1 and 19.8% in Comparative Example 1; the pathological improvement in the Comparative Example group was weaker than that in Example 1.

[0033] Experiment 3: Core test data (all results were obtained at a concentration of 0.5 mg / ml) Using 0.5 mg / mL as the detection concentration, the absorbance was measured at a wavelength of 517 nm. The scavenging ability of the sample for DPPH free radicals was calculated, and the results are shown in the table below:

[0034] Figure 2 The HPLC chromatogram is shown in Example 1. Figure 3 This is the HPLC chromatogram for Comparative Example 1. The horizontal axis represents retention time (min), with the retention time of the characteristic peak of chicoric acid (t=33 min) marked; the vertical axis represents peak area (mV·s). Figure 2 and Figure 3 As shown, the peak area of ​​chicoric acid in Example 1 (12568 mV·s) is significantly larger than that in Comparative Example 1 (2845 mV·s), which directly reflects the difference in chicoric acid content. There are no impurity peaks interfering in the spectrum, indicating that the extract has good purity.

[0035] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A fermentation preparation method for an active dandelion extract with anti-inflammatory and nodule-dispersing functions, characterized in that: Includes the following steps: S1. Raw material pretreatment The dried whole dandelion was pulverized using a pulverizer and passed through a 40-60 mesh Tyler sieve to obtain dandelion powder. Deionized water was added at a solid-liquid ratio of 1:10 to 1:15 (w / v) and stirred to form a uniform suspension. S2. Synergistic enzymatic hydrolysis by complex enzymes Adjust the pH of the suspension to 4.5–5.5, add the compound enzyme preparation, and place it in a constant temperature water bath shaker at 45–55°C and 50 rpm for 2–4 hours for enzymatic hydrolysis; the compound enzyme preparation is composed of cellulase, pectinase, and β-glucanase in an activity unit ratio of (2–4):(1–3):1, and the total amount of enzyme added is 0.5%–1.5% of the mass of dandelion powder; S3. Primary anaerobic fermentation The enzymatic hydrolysate was transferred to a fermenter and sterilized at 120℃ and 0.1MPa for 15 min. After sterilization, it was cooled to 35-37℃ and inoculated with 5%-10% (v / v) of activated Lactobacillus plantarum seed culture. Fermentation was carried out at 37±1℃ under anaerobic conditions, either by standing or by stirring at 50 rpm. S4. Secondary aerobic fermentation After the primary fermentation is completed, no sterilization treatment is performed. Instead, 3% to 5% (v / v) of activated Saccharomyces cerevisiae seed culture is directly inoculated and aerobic fermentation is carried out for 60 to 75 hours at 28 to 32°C, an aeration rate of 1:0.8 to 1:1.2 vvm, and a stirring speed of 150 to 250 rpm. S5. Termination of Fermentation and Post-Processing After fermentation, the fermentation broth was transferred to a water bath and heated at 85°C for 10 minutes to terminate the reaction. After cooling to room temperature, it was centrifuged at 4°C and 8000 rpm for 20 minutes using a high-speed centrifuge. The supernatant was then filtered through a 0.45 μm microporous membrane to obtain a clear fermentation broth. S6. Concentration and Drying The clarified fermentation broth was placed in a rotary evaporator and vacuum concentrated to a solid content of 30%–40% under conditions of ≤50℃ and ≤-0.08MPa. The concentrated broth was then spray-dried in a spray dryer with the inlet air temperature set at 170–190℃, the outlet air temperature at 80–90℃, and the atomization pressure at 0.2MPa. After drying, dandelion fermentation extract was obtained.

2. The fermentation preparation method of an active dandelion extract with anti-inflammatory and nodule-dispersing functions according to claim 1, characterized in that: The activation medium for the Lactobacillus plantarum seed liquid in step S3 is MRS medium, and the activation conditions are static incubation at 37°C for 24 hours.

3. The fermentation preparation method of an active dandelion extract with anti-inflammatory and nodule-dispersing functions according to claim 1, characterized in that: The activation medium for the *Issa mesasura* seed culture in step S4 is YPD medium, and the activation conditions are 30°C and 200 rpm shaking culture for 18 hours.

4. The dandelion fermentation extract prepared according to any one of claims 1-3, characterized in that: The extract is a brownish-yellow powder with a water content of ≤5%, containing chicoric acid content of ≥8.5% and small molecule glycopeptides with a molecular weight of less than 10kDa content of ≥25%.

5. The application of the dandelion fermentation extract according to claim 4 in the preparation of drugs, health foods or cosmetics with nodule-dispersing function.

6. The application according to claim 5, characterized in that: The nodule-dispersing function includes inhibiting abnormal proliferation of mammary epithelial cells, promoting the resolution of inflammatory factors, shrinking mammary hyperplasia nodules, relieving lymph node enlargement, improving acne, or inhibiting the enlargement of thyroid nodules; in the application, the effective dosage of the extract is: 0.5-1.0g / person per day for pharmaceuticals, 0.3-0.5g / person per day for health foods, and 0.5%-2.0% (mass fraction) for cosmetics.

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