Method for preparing vitexin through solid-state fermentation of hawthorn leaves
By using Bacillus pumilus solid-state fermentation of hawthorn leaves and enhancing the enzymatic hydrolysis process with a compound inducer, the problems of cumbersome processes, environmental pollution, and low extraction efficiency in traditional methods have been solved, achieving high-yield and high-purity preparation of vitexin, which is suitable for industrial application.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-08
- Publication Date
- 2026-04-07
AI Technical Summary
Existing methods for preparing vitexin from hawthorn leaves suffer from problems such as cumbersome processes, high energy consumption, high costs, serious environmental pollution, and low extraction efficiency. In particular, the traditional chemical extraction-chemical hydrolysis process requires a large amount of organic solvents and has harsh reaction conditions, resulting in low yield and purity of vitexin.
After activation with Bacillus pumilus and the compound inducers phenylalanine and Tween 80, hawthorn leaves were fermented in a solid state. Cellulase and pectinase destroyed the cell wall, and enhanced rhamnosidase hydrolyzed glycosidic bonds to generate free vitexin. Antimicrobial substances were used to inhibit miscellaneous bacteria, simplifying the process and reducing solvent use.
It achieves efficient and simple extraction of vitexin, improves yield and purity, reduces solvent consumption and environmental pollution, is suitable for industrial production, has high resource utilization, and low equipment requirements.
Smart Images
Figure CN121801984A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of plant extraction technology, specifically relating to a method for preparing vitexin by solid-state fermentation of hawthorn leaves. Background Technology
[0002] Vitexin is one of the important active ingredients in hawthorn leaves, but it mostly exists in the form of glycosides such as vitexin rhamnoside and vitexin glucoside. Vitexin can only be obtained by hydrolyzing and breaking the glycoside bonds.
[0003] Existing processes for preparing vitexin from hawthorn leaves mainly rely on chemical extraction followed by chemical hydrolysis. The main drawbacks are threefold: First, the extraction process requires large amounts of organic solvents (such as ethanol), leading to high costs and environmental pollution. Second, the hydrolysis step often employs strong acid conditions, hydrolyzing the crude extract of hawthorn leaves in a strongly acidic aqueous solution, precipitating crystals, and then recrystallizing to obtain the target product, vitexin. These harsh reaction conditions easily degrade the vitexin flavonoid nucleus, reducing product yield and purity. Third, the process is a step-by-step process (raw material pretreatment → solvent extraction → concentration → chemical hydrolysis → purification), which is cumbersome, energy-intensive, and results in low raw material utilization and significant waste.
[0004] While there have been attempts to apply fermentation technology to hawthorn leaf processing in the existing technology, most of them are liquid fermentation. Generally, the effective components in hawthorn leaves need to be extracted into liquid systems such as ethanol before fermentation and conversion. This still requires the use of a large amount of organic solvents for extraction, resulting in low conversion efficiency, complex process, and potential problems such as excessive alcohol content or solution residue in the later stages.
[0005] Therefore, developing a method for preparing vitexin from hawthorn leaves that has high extraction efficiency, requires no large amount of organic solvents, has a simple process, high conversion efficiency, and is environmentally friendly has become an urgent technical problem to be solved in this field. Summary of the Invention
[0006] To address the aforementioned deficiencies in existing technologies, this invention provides a method for preparing vitexin through solid-state fermentation of hawthorn leaves, comprising the following steps.
[0007] 1) Activate Bacillus pumilus using a compound inducer to obtain an activated bacterial solution; 2) Solid-state fermentation culture medium was prepared using hawthorn leaves as raw material and adding compound inducers; 3) The activated bacterial solution is inoculated into the solid fermentation medium, mixed evenly, and then fermented. After fermentation is completed, the fermentation product is obtained. 4) The fermentation products are eluted with an organic solvent to obtain the eluent; 5) The eluent was concentrated under reduced pressure, purified, and dried to obtain vitexin; The complex inducer in steps 1) and 2) includes phenylalanine and Tween 80.
[0008] In this scheme, hawthorn leaves were treated through preliminary experiments, and the content of vitexin rhamnoside in the hawthorn leaves and the extracted vitexin were detected. The experimental results showed that the content of vitexin rhamnoside in hawthorn leaves was positively correlated with the content of the final extracted vitexin. Subsequently, hawthorn leaves with a vitexin rhamnoside content of not less than 2.0 mg / g were selected as raw materials to improve the extraction efficiency of vitexin.
[0009] In this scheme, *Bacillus pumilus* is activated using a composite inducer. *Bacillus pumilus* can secrete basal amounts of cellulase, pectinase, and glycoside hydrolysis-related enzymes. Adding the composite inducer significantly enhances its enzyme secretion capacity, eliminating the need for cumbersome screening and domestication steps. Phenylalanine promotes preferential synthesis of the target enzyme, while Tween 80 improves enzyme secretion efficiency and substrate contact efficiency. Cellulase and pectinase disrupt the cell wall structure of hawthorn leaves, releasing bound vitexin glycosides. The enhanced rhamnosidase can directionally hydrolyze the glycosidic bonds of vitexin rhamnosides, generating free vitexin. Simultaneously, *Bacillus pumilus* secretes antimicrobial peptides and other antimicrobial substances, inhibiting contamination by other microorganisms during fermentation and preventing the metabolic destruction of vitexin by these microorganisms.
[0010] Among them, using LB medium containing compound inducers can simultaneously enhance enzyme activity during the strain activation stage, making the process simpler; LB medium is widely available and inexpensive, enabling rapid strain activation.
[0011] Preferably, the composite inducer in step 1) includes phenylalanine at a mass fraction of 0.5-1.0% and Tween 80 at a volume fraction of 0.1-0.3%.
[0012] Preferably, the composite inducer in step 2) includes phenylalanine at a mass fraction of 0.3-0.8% and Tween 80 at a volume fraction of 0.1-0.2%.
[0013] Preferably, the bacterial concentration of the activated bacterial solution in step 1) is controlled to be 10. 7 -10 8 CFU / mL.
[0014] Preferably, in step 2), the hawthorn leaf raw material is pretreated by drying and crushing the hawthorn leaves into powder.
[0015] Preferably, the inoculation amount of the activated bacterial solution in step 3) is 5-10 wt%.
[0016] Preferably, in step 4), the organic solvent comprises an ethanol solution with a volume fraction of 50-60%.
[0017] Preferably, in step 4), the mass ratio of fermentation product to organic solvent is 1:8-12.
[0018] Preferably, in step 5), the concentration is carried out under reduced pressure at a temperature of 50-60℃ and a pressure of -0.08 to -0.06 MPa, and the ethanol is recovered.
[0019] In this scheme, Bacillus pumilus activated by the compound inducer secretes a large amount of cellulase and pectinase, which destroys the cell wall of hawthorn leaf in situ and releases vitexin glycosides. The enhanced rhamnosidase simultaneously hydrolyzes the glycosidic bonds of vitexin rhamnoside in situ to generate free vitexin. At the same time, the antibacterial substances secreted by the strain can inhibit the growth of miscellaneous bacteria, and the mild environment created by its metabolic activities can reduce the oxidative loss of vitexin, thus completing a highly efficient integrated conversion.
[0020] Compared with the prior art, the present invention has the following innovations: 1. Achieves integrated transformation and simplified process: Bacillus pumilus combined with compound inducers eliminates the need for cumbersome screening and domestication steps, shortening the process cycle by more than 30%; in the solid-state fermentation system, the in-situ transformation of "hawthorn leaf cell wall destruction - vitexin rhamnoside hydrolysis - vitexin enrichment" is achieved simultaneously, replacing the multi-step operation of traditional processes; at the same time, the strain can secrete antimicrobial peptides and other antimicrobial substances to inhibit contamination by miscellaneous bacteria, solving the problem of vitexin being destroyed by miscellaneous bacteria during fermentation, eliminating the need to add preservatives, simplifying the process and improving product safety.
[0021] 2. Green and environmentally friendly: Using hawthorn leaves as a solid substrate, there is no need for a large amount of organic solvents for preliminary extraction; ethanol can be recovered and reused during the elution process, greatly reducing solvent consumption; the fermentation process is mild, requiring no strong acids or alkalis, and there are no serious pollutant emissions, meeting the requirements of green production.
[0022] 3. High yield and purity: The compound inducer can significantly enhance the enzyme secretion ability of Bacillus pumilus, realize the targeted and efficient hydrolysis of vitexin glycosides, and avoid the degradation of vitexin caused by traditional strong acid hydrolysis; the antibacterial substances secreted by it can reduce the consumption of vitexin by miscellaneous bacteria, and the mild environment created by metabolism reduces the oxidative loss of vitexin, significantly improving the yield and purity of vitexin.
[0023] 4. High resource utilization rate: Hawthorn leaves do not require defatting or dephenolization pretreatment and can be used directly for fermentation; the residue after fermentation can be further developed and utilized, realizing the full utilization of hawthorn leaf resources and reducing the production cost per unit of vitexin.
[0024] 5. Good industrialization prospects: Solid-state fermentation process is simple, requires low equipment, consumes little energy, and is easy to scale up, making it suitable for large-scale industrial preparation of vitexin. Attached Figure Description
[0025] Figure 1This is the QDa mass spectrometry molecular map of vitexin.
[0026] Figure 2 This is a liquid chromatography peak diagram of hawthorn leaf standard.
[0027] Figure 3 The image shows the liquid phase peaks of hawthorn leaf solid-state fermentation for 12 hours in Example 1.
[0028] Figure 4 The image shows the liquid phase peaks of hawthorn leaf solid-state fermentation for 24 hours in Example 1.
[0029] Figure 5 The image shows the liquid phase peaks of hawthorn leaf solid-state fermentation for 36 hours in Example 1.
[0030] Figure 6 The image shows the liquid phase peaks of hawthorn leaf solid-state fermentation for 48 hours in Example 1. Detailed Implementation
[0031] The embodiments of the present invention are described in detail below. These embodiments are only used to explain the present invention and are not intended to limit the scope of the present invention.
[0032] Pre-experiment Hawthorn leaves from four production areas—Huixian and Fengqiu in Henan Province, Yuncheng in Shanxi Province, and Feixian in Shandong Province—were sampled in early September. The leaves were required to be free of rotten material. After drying and pulverizing, they underwent the same pretreatment. The content of precursor substances was determined by HPLC, and three groups of raw materials were ultimately selected. ① High precursor group: vitexin rhamnoside 2.79 mg / g, vitexin content 0.72 mg / g; ②Mid-progenitor group: vitexin rhamnoside 1.81 mg / g, vitexin content 0.69 mg / g; ③ Low precursor group: Vitexin rhamnoside 1.10 mg / g, vitexin content 0.65 mg / g.
[0033] Example 1 Step 1: Activation of Bacillus pumilus. Bacillus pumilus was selected and inoculated into LB medium containing a compound inducer (0.5% phenylalanine and 0.1% Tween 80). The medium was incubated at 30°C with shaking at 220 rpm for 18 hours, yielding a bacterial concentration of 5 × 10⁻⁶. 7 CFU / mL activated bacterial solution.
[0034] Step 2: Pre-treatment of hawthorn leaf raw materials. Wash the hawthorn leaves thoroughly, dry them at 50℃ to constant weight, pulverize them, and pass them through a 40-mesh sieve to obtain hawthorn leaf powder, which is then directly used for solid-state fermentation.
[0035] Step 3: Solid-state fermentation reaction. Take 100g of hawthorn leaf powder as the solid substrate, add deionized water to adjust the material-to-water ratio to 1:1.4, add 1.5g of sucrose and 0.8g of yeast extract, and simultaneously add 0.3% phenylalanine (by mass) and 0.1% Tween 80 (by volume). After mixing evenly, put it into a fermentation tank and sterilize at 121℃ and 0.1MPa for 25min. Cool to room temperature. Inoculate with activated bacterial solution at an inoculum rate of 5wt% and mix evenly. Ferment for 48h at a temperature of 35℃, humidity of 65%, and aeration rate of 0.4vvm, stirring once every 12h.
[0036] Step 4: Elution and purification of vitexin. After fermentation, 1200 mL of 50% ethanol solution (solid-to-liquid ratio 1:12) was added to the fermentation product, and ultrasonic elution was performed at 55℃ and 180W for 25 min. The mixture was filtered, and the eluent was collected. The filter residue was then eluted again with 800 mL of 50% ethanol solution (solid-to-liquid ratio 1:8), and the two eluents were combined. The eluent was concentrated under reduced pressure at 50℃ and -0.08 MPa to recover the ethanol. The concentrated solution was passed through a macroporous adsorption resin column (column diameter ratio 1:8, flow rate 2 BV / h), first eluted with deionized water until the effluent was colorless (elution volume 3 BV), and then eluted with a gradient of 75% ethanol solution (elution volume 5 BV) to collect the vitexin fraction. The collected fraction was dried under reduced pressure at 50℃ and -0.08 MPa to obtain the vitexin product.
[0037] According to HPLC analysis, the yield of vitexin in this example was 3.76‰, and the purity was 88.6%.
[0038] Example 2 Step 1: Activation of Bacillus pumilus. Bacillus pumilus was selected and inoculated into LB medium containing a compound inducer (1.0% phenylalanine and 0.3% Tween 80). The medium was incubated at 37°C with shaking at 180 rpm for 12 hours to obtain a bacterial concentration of 5 × 10⁻⁶. 8 CFU / mL activated bacterial solution.
[0039] Step 2: Pre-treatment of hawthorn leaf raw materials. Wash the hawthorn leaves thoroughly, dry them at 60℃ to constant weight, pulverize them, and pass them through a 60-mesh sieve to obtain hawthorn leaf powder, which is then directly used for solid-state fermentation.
[0040] Step 3: Solid-state fermentation reaction. Take 100g of hawthorn leaf powder as the solid substrate, add deionized water to adjust the material-to-water ratio to 1:1.4, add 1.5g of sucrose and 0.8g of yeast extract, and simultaneously add 0.8% phenylalanine (by mass) and 0.2% Tween 80 (by volume). After mixing evenly, put it into a fermentation tank and sterilize at 121℃ and 0.1MPa for 25min. Cool to room temperature. Inoculate with activated bacterial solution at an inoculum rate of 10wt% and mix evenly. Ferment for 48h at a temperature of 35℃, humidity of 65%, and aeration rate of 0.4vvm, stirring once every 12h.
[0041] Step 4: Elution and purification of vitexin. After fermentation, 1200 mL of 60% ethanol solution (solid-to-liquid ratio 1:12) was added to the fermentation product, and the mixture was ultrasonically eluted at 55℃ and 180W for 25 min. The mixture was filtered, and the eluent was collected. The filter residue was then eluted again with 800 mL of 60% ethanol solution (solid-to-liquid ratio 1:8), and the two eluents were combined. The eluent was concentrated under reduced pressure at 60℃ and -0.06 MPa to recover the ethanol. The concentrated solution was passed through a macroporous adsorption resin column (column diameter ratio 1:8, flow rate 2 BV / h), first eluted with deionized water until the effluent was colorless (elution volume 3 BV), and then eluted with a gradient of 75% ethanol solution (elution volume 5 BV) to collect the vitexin fraction. The collected fraction was dried under reduced pressure at 60℃ and -0.06 MPa to obtain the vitexin product.
[0042] According to HPLC analysis, the yield of vitexin in this example was 4.02‰, and the purity was 89.8%.
[0043] Comparative Example 1 (Traditional Process) Take 100g of hawthorn leaf powder (the same as in Example 1), add 1000mL of 70% ethanol solution (solid-liquid ratio 1:10), reflux at 80℃ for 2h, filter, and collect the extract; add 800mL of 70% ethanol solution (solid-liquid ratio 1:8) to the filter residue and repeat the extraction once, combine the extracts; concentrate the extract under reduced pressure (temperature 60℃, pressure -0.08MPa) until there is no alcohol odor, add hydrochloric acid to adjust the pH to 0.8, and hydrolyze at 80℃ for 4h; defatt the hydrolysate three times with an equal volume of petroleum ether, take the lower aqueous phase, and then purify it through an HPD100 macroporous adsorption resin column to obtain vitexin product.
[0044] HPLC analysis showed that the yield of vitexin in this comparative example was 1.2‰, and the purity was 66.3%.
[0045] Comparative Example 2 (Solid-state fermentation of uninduced strains) Step 1: Select a commercially available Bacillus pumilus strain, inoculate it into LB medium, and culture at 35℃ with shaking at 200 rpm for 14 hours to obtain a bacterial concentration of 5 × 10⁻⁶. 7 CFU / mL activated bacterial solution.
[0046] Step 2: Same as in Example 1.
[0047] Step 3: Take 100g of hawthorn leaf powder, add deionized water to adjust the material-to-water ratio to 1:1.4, add 1.5g of sucrose and 0.8g of yeast extract, mix well and put into a fermentation tank, sterilize at 121℃ and 0.1MPa for 25min, and cool to room temperature; inoculate with 6% of the activated bacterial solution and mix well; ferment at 35℃, 65% humidity and 0.4vvm for 48h, stirring once every 12h.
[0048] Step 4: Same as in Example 1, elute and purify the fermentation product.
[0049] HPLC analysis showed that the yield of vitexin in this comparative example was 1.89‰, and the purity was 71.8%.
[0050] Examples 1 and 2 and Comparative Examples 1 and 2 used hawthorn leaves from the high-body group as raw materials.
[0051] Comparative Example 3 Hawthorn leaves (from the precursor group) were used as raw material, and the remaining process steps were the same as in Example 1. HPLC analysis showed that the yield of vitexin in this comparative example was 2.79‰, and the purity was 88.3%.
[0052] Comparative Example 4 Hawthorn leaves (low precursor group) were used as raw material, and the remaining process steps were the same as in Example 1. HPLC analysis showed that the yield of vitexin in this comparative example was 2.31‰, and the purity was 87.7%.
[0053] The vitexin products obtained in the above examples and comparative examples were analyzed by HPLC under the following conditions. QDa conditions Electrospray ionization source (ESI source) Positive ion detection mode; Capillary voltage: 0.8kV; Ion source temperature: 600℃; Selective ion detection (SIR) mode was used, and the cone voltage for each component was 15V.
[0054] Liquid phase conditions: The chromatographic column is C10. 18 4.6×250mm, 5μm Detection wavelength: 280nm Column temperature: 25℃ Flow rate 0.8 ml / min Gradient elution procedure: 0-12 min Acetonitrile 10%-21% 12-16 min Acetonitrile 21% 16-21 min Acetonitrile 21%-30% 21-50 min Acetonitrile 30%-60% 50-60 min Acetonitrile 60%-10% And compared with the standard (Vitexin QDa mass spectrometry molecular map as shown) Figure 1 As shown, the liquid chromatography peak chromatogram of hawthorn leaf standard is as follows: Figure 2 As shown, the liquid phase peak diagrams of hawthorn leaf solid-state fermentation in Example 1 at 12h, 24h, 36h, and 48h are as follows. Figure 3 , Figure 4 , Figure 5 , Figure 6 (As shown), calculate the yield and purity of vitexin.
[0055] The comparison shows that the yield and purity of vitexin in Example 1 are significantly higher than those in Comparative Examples 1 and 2, indicating that the solid-state fermentation method using induced strains can significantly improve the yield and purity of vitexin.
[0056] In addition, the yield and purity of vitexin in Example 1 were higher than those in Comparative Examples 3 and 4, indicating that selecting hawthorn leaf raw materials with high precursor content can also improve the yield of vitexin.
Claims
1. A method for preparing vitexin by solid-state fermentation of hawthorn leaves, characterized in that, Includes the following steps: 1) Activate Bacillus pumilus using a compound inducer to obtain an activated bacterial solution; 2) Solid-state fermentation culture medium was prepared using hawthorn leaves as raw material and adding compound inducers; 3) The activated bacterial solution is inoculated into the solid fermentation medium, mixed evenly, and then fermented. After fermentation is completed, the fermentation product is obtained. 4) The fermentation products are eluted with an organic solvent to obtain the eluent; 5) The eluent was concentrated under reduced pressure, purified, and dried to obtain vitexin; The complex inducer in steps 1) and 2) consists of phenylalanine and Tween 80.
2. The method for preparing vitexin by solid-state fermentation of hawthorn leaves according to claim 1, characterized in that, In step 2), the content of vitexin rhamnoside in hawthorn leaves is first tested, and hawthorn leaves with a vitexin rhamnoside content of not less than 2.0 mg / g are selected as raw materials.
3. The method for preparing vitexin by solid-state fermentation of hawthorn leaves according to claim 1, characterized in that, The complex inducer in step 1) includes phenylalanine at a mass fraction of 0.5-1.0% and Tween 80 at a volume fraction of 0.1-0.3%.
4. The method for preparing vitexin by solid-state fermentation of hawthorn leaves according to claim 1, characterized in that, In step 2), the complex inducer includes phenylalanine at a mass fraction of 0.3-0.8% and Tween 80 at a volume fraction of 0.1-0.2%.
5. The method for preparing vitexin by solid-state fermentation of hawthorn leaves according to claim 1, characterized in that, In step 1), the bacterial concentration of the activated bacterial solution is controlled to be 10. 7 -10 8 CFU / mL.
6. The method for preparing vitexin by solid-state fermentation of hawthorn leaves according to claim 1, characterized in that, In step 2), the hawthorn leaf raw material is pre-treated by drying and crushing the hawthorn leaves into powder.
7. The method for preparing vitexin by solid-state fermentation of hawthorn leaves according to claim 1, characterized in that, In step 3), the inoculation amount of the activated bacterial solution is 5-10 wt%.
8. The method for preparing vitexin by solid-state fermentation of hawthorn leaves according to claim 1, characterized in that, In step 4), the organic solvent contains an ethanol solution with a volume fraction of 50-60%.
9. The method for preparing vitexin by solid-state fermentation of hawthorn leaves according to claim 1, characterized in that, In step 4), the mass ratio of fermentation product to organic solvent is 1:8-12.
10. The method for preparing vitexin by solid-state fermentation of hawthorn leaves according to claim 8, characterized in that, In step 5), the concentration is carried out under reduced pressure at a temperature of 50-60℃ and a pressure of -0.08 to -0.06 MPa, and the ethanol is recovered.