Method for solid-state fermentation of sealed fresh strobilanthes cusia and water extraction and separation of indigo blue
By using a method of sealed solid-state fermentation of fresh Indigofera tinctoria and water extraction to separate indigo, the problem of efficient targeted transformation and enrichment of indigo has been solved, resulting in high-purity indigo extract suitable for the development of high-grade dyes and pharmaceuticals, while reducing the risk of environmental pollution.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-25
- Publication Date
- 2026-03-31
AI Technical Summary
Existing fermentation methods for preparing indigo cannot effectively solve the problems of efficient and targeted conversion and enrichment of indigo, and chemical synthesis preparation carries pollution risks.
A method for separating indigo from fresh senna indigo using sealed solid-state fermentation and water extraction was developed. This method includes sealed solid-state fermentation of fresh senna indigo, preparation of crude indigo extract, indigo membrane separation and solvent extraction, purification using ceramic membranes and organic solvents, and optimization of fermentation conditions and extraction parameters to improve the conversion rate and purity of indigo.
This method achieves efficient targeted conversion and enrichment of indigo, yielding high-purity indigo extract suitable for the development of high-grade dyes and pharmaceuticals, while reducing the risk of environmental pollution.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of fermentation refining development, and specifically relates to a method for sealed solid-state fermentation of fresh strychnine and water extraction to separate indigo. Background Technology
[0002] Indigofera tinctoria, also known as southern indigo root or blue indigo grass, is a shrubby perennial herb. Because it is tolerant of different climatic conditions and soil pH values, it is mainly distributed in the humid areas on the edge of mountain forests in southwestern and southern my country.
[0003] Indigo, a major component of the traditional Chinese medicine Qingdai, possesses anti-inflammatory and hepatoprotective properties. Indigo pigments are among the oldest known pigments, initially extracted from indigo plants. They are water-soluble, non-azo colorants, widely used in the food, pharmaceutical, and dyeing industries in their sodium sulfonate salt or aluminum-containing forms as food colorings. Initially extracted from plants, indigo was later synthesized chemically. The plant extraction method involves piling indigo leaves, frequently watering them, and allowing them to ferment for 2-3 months, resulting in black clods. After being pounded in a mortar, these clods are called "ball indigo," containing 2%-10% indigo pigment. When ball indigo is mixed with wood ash, lime, and wheat bran, and then water is added, the mixture is heated to 30-40°C, and exposed to air to produce insoluble blue indigo, resulting in a low yield. The chemical synthesis of indigo pollutes the environment and poses potential carcinogenic risks; therefore, the plant-based preparation of indigo has a promising future.
[0004] While existing fermentation methods for producing indigo can improve its bioavailability to some extent, they still cannot solve the problems of efficient targeted conversion and enrichment. Therefore, finding an effective strategy that can simultaneously address both the efficient targeted conversion and enrichment of indigo is urgently needed. This study proposes a method involving sealed solid-state fermentation of *Indigofera tinctoria* leaves to rapidly and directionally convert indole glycosides or free indolephenol into indigo, followed by purification using a combination of ceramic and organic membranes and macroporous resins. Currently, research on the development of indigo through sealed solid-state fermentation of *Indigofera tinctoria* leaves is rarely reported both domestically and internationally. Summary of the Invention
[0005] In view of this, the present invention provides a method for sealing fresh strychnine solid-state fermentation and water extraction to separate indigo.
[0006] To achieve the above objectives, the present invention adopts the following technical solution: A method for sealing fresh strychnos nux-vomica through solid-state fermentation and water extraction to separate indigo, comprising the following steps: S1. Sealed Fresh Indigofera tinctoria Solid Fermentation Take fresh woad and store it in a sealed bag at 10℃~30℃ for 5~30 days; S2. Preparation of crude indigo extract After solid-state fermentation, strychnos nux-vomica was extracted with water at a solid-liquid ratio (g / mL) of 1:5~30. 0.1%~1% enzyme was added, and the extraction was carried out at 10℃~30℃ for 0.5~2 hours. The crude indigo extract was obtained by filtration.
[0007] S3, indigo membrane separation and solvent extraction Preferably, in the above-mentioned method for sealing fresh strychnine solid-state fermentation and water extraction to separate indigo, the step S1 described above... Fresh sage includes sage leaves, sage stems, and the whole sage plant.
[0008] Preferably, in the above-mentioned method for sealing fresh strychnos nux-vomica solid-state fermentation and water extraction to separate indigo, the sealing in step S1 includes either vacuum or nitrogen filling, with nitrogen filling being preferred.
[0009] Preferably, in the above-mentioned method for sealing fresh strychnine solid-state fermentation and water extraction to separate indigo, the solid-state fermentation in step S1 includes solid-state fermentation at different times and solid-state fermentation at different temperatures, with the fermentation temperature preferably being 25℃~30℃ and the fermentation time preferably being 20~30 days.
[0010] Preferably, in the above-mentioned method for sealing fresh strychnos nux-vomica solid-state fermentation and water extraction to separate indigo, the extraction and preparation of crude indigo extract in step S2 includes water extraction and extraction with different solvents, with methanol, ethanol and ethyl acetate being preferred solvents.
[0011] Preferably, in the above-mentioned method for sealing fresh strychnos nux-vomica solid-state fermentation and water extraction to separate indigo, the extraction and preparation of crude indigo extract in step S2 includes different times, different temperatures, and different solid-liquid ratios. Preferably, the time is 1-2 hours, the temperature is 25℃-30℃, and the solid-liquid ratio (g / mL) is 1:20-30.
[0012] Preferably, in the above-mentioned method for sealing fresh strychnos nux-vomica solid-state fermentation and water extraction to separate indigo, the enzyme added in step S2 for preparing the crude indigo extract includes any one of glycosidase, cellulase, amylase, or a self-made enzyme. The self-made enzyme is prepared using a low-temperature water extraction membrane separation freeze-drying method.
[0013] Preferably, in the above-mentioned method for sealing fresh strychnos nux-vomica solid-state fermentation and water extraction to separate indigo, the membrane separation in step S3 includes ceramic membranes with different molecular weight cutoffs and ultrafiltration hollow fiber membranes. Preferably, the ceramic membrane is 0.5µm to 50µm, and the ultrafiltration hollow fiber membrane preferably has a molecular weight of 10,000 to 30,000 D. Preferably, in the above-mentioned method for sealing fresh strychnos nux-vomica solid-state fermentation and water extraction to separate indigo, the solvent extraction in step S3 includes organic solvents such as ethyl acetate, chloroform, n-butanol, and acetone.
[0014] Preferably, in the above-mentioned method for sealing fresh strychnos nux-vomica solid-state fermentation and water extraction to separate indigo, the purity of indigo in the indigo extract in step S3 is higher than 85%.
[0015] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a method for sealing and solid-state fermentation of fresh strychnos nux-vomica and water extraction to separate indigo. This method uses sealed and efficient fermentation to transform indigo, and then uses membrane-combined solvent extraction to prepare high-purity indigo extract, which can provide theoretical reference and technical support for the development and application of high-grade dyes and pharmaceuticals. Attached Figure Description
[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings and tables can be obtained based on the provided drawings without creative effort.
[0017] Figure 1 HPLC chromatogram of indigo in crude indigo extract Figure 2 HPLC chromatogram of indigo in indigo extract Detailed Implementation The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.
[0018] Example 1: Sealed solid-state fermentation of fresh strychnine Take 100g of Indigofera tinctoria leaves, seal them in a vacuum bag, ferment them in solid state at 30℃ for 30 days, and extract them twice with water at 30℃, with a solid-liquid ratio (g / mL) of 1:30 each time, and extract for 2 hours each time to obtain crude indigo extract, which contains 42% indigo.
[0019] Take 100g of senna stems, seal them in a vacuum bag, ferment them in solid state at 30℃ for 30 days, and extract them twice with water at 30℃, with a solid-liquid ratio (g / mL) of 1:30 each time, and extract for 2 hours each time to obtain crude indigo extract, which contains 24% indigo.
[0020] Take 100g of whole Strobilanthes cusia plant, vacuum seal it in a bag, ferment it in solid state at 30℃ for 30 days, and extract it twice with water at 30℃, with a solid-liquid ratio (g / mL) of 1:30 each time, and extract for 2 hours each time to obtain crude indigo extract, which contains 35% indigo.
[0021] Take 100g of Indigofera tinctoria leaves, seal them in a nitrogen-filled bag, ferment them in a solid state at 30℃ for 30 days, and extract them twice with water at 30℃, with a solid-liquid ratio (g / mL) of 1:30 each time, and extract for 2 hours each time to obtain crude indigo extract, which contains 37% indigo.
[0022] Comparison of vacuum fermentation and nitrogen-filled solid-state fermentation of leaves, stems, and whole plant of *Indigofera tinctoria*. Fresh scotch Sealing process Fermentation temperature (°C) Fermentation time (days) Indigo content (%) in crude indigo extract Indigofera tinctoria Vacuuming 30 30 42 Indigo stem Vacuuming 30 30 24 whole plant of Strobilanthes cusia Vacuuming 30 30 35 Indigofera tinctoria Vacuuming 30 30 37 Example 2 Preparation of crude indigo extract Take 100g of woad leaves, vacuum seal them in a bag, and ferment them at 30℃ for 30 days. Take the sealed solid fermented leaves of Indigofera tinctoria, extract them twice with water at 30℃, each time with a solid-liquid ratio (g / mL) of 1:30, and extract for 2 hours to obtain crude indigo extract with an indigo content of 42%.
[0023] Take the sealed solid fermented leaves of Indigofera tinctoria and extract them twice with ethanol at 30℃, each time with a solid-liquid ratio (g / mL) (1:30) for 2 hours to obtain crude indigo extract with an indigo content of 45%.
[0024] Take the sealed solid fermented leaves of Strobilanthes cusia and extract them twice with methanol at 30℃, with a solid-liquid ratio (g / mL) of 1:30 each time, for 2 hours each time to obtain crude indigo extract, which contains 44% indigo.
[0025] Take the sealed solid fermented leaves of Strobilanthes cusia and extract them twice with ethyl acetate at 30℃, with a solid-liquid ratio (g / mL) of 1:30 each time, for 2 hours each time to obtain crude indigo extract with an indigo content of 40%.
[0026] Comparison table of preparation of crude indigo extract using water, ethanol, methanol, and ethyl acetate. Extraction reagents Solid-liquid ratio (g:mL) Extraction temperature (°C) Extraction time (h) Indigo content (%) in crude indigo extract water 1:30 30 2 42 ethanol 1:30 30 2 45 methanol 1:30 30 2 44 Ethyl acetate 1:30 30 2 40 Example 3 Preparation of crude indigo extract Take the sealed solid fermented leaves of Strobilanthes cusia, add 1% glycosidase, extract twice with water at 30℃, each time with a solid-liquid ratio (g / mL) (1:30), extract for 2 hours to obtain crude indigo extract, in which the indigo content is 47%.
[0027] Take the sealed solid fermented leaves of Strobilanthes cusia, add 1% cellulase, extract twice with water at 30℃, each time with a solid-liquid ratio (g / mL) (1:30), extract for 2 hours to obtain crude indigo extract, in which the indigo content is 42%.
[0028] Take the sealed solid fermented leaves of Strobilanthes cusia, add 1% amylase, extract twice with water at 30℃, each time with a solid-liquid ratio (g / mL) (1:30), extract for 2 hours to obtain crude indigo extract, in which the indigo content is 38%.
[0029] Take the sealed solid fermented leaves of Strobilanthes cusia, add 1% of self-made enzyme (prepared by low temperature water extraction membrane separation freeze drying method), extract twice with water at 30℃, each time with a solid-liquid ratio (g / mL) (1:30), extract for 2 hours to obtain crude indigo extract, in which the indigo content is 49.5%.
[0030] Comparison table of preparation of crude indigo extract by adding different enzymes to aqueous extracts Enzyme (1% addition) Solid-liquid ratio (g:mL) Extraction temperature (°C) Extraction time (h) Indigo content (%) in crude indigo extract glycosidase 1:30 30 2 47 Cellulase 1:30 30 2 42 amylase 1:30 30 2 38 Homemade enzymes 1:30 30 2 49.5
[0031] Example 4: Indigo membrane separation and solvent extraction The crude indigo extract was separated and purified using a ceramic membrane (50 μm) to obtain the ceramic membrane permeate, which was then extracted with acetone. The extract was concentrated and dried to obtain the indigo extract, which contained 65% indigo.
[0032] The crude indigo extract was separated and purified using a ceramic membrane (0.5 μm) to obtain the ceramic membrane permeate, which was then extracted with acetone. The extract was concentrated and dried to obtain the indigo extract, which contained 72% indigo.
[0033] The crude indigo extract was purified using a ceramic membrane (0.5 μm) to obtain the ceramic membrane permeate; this permeate was then purified using an ultrafiltration hollow fiber membrane (30,000 molecular weight cutoff) to obtain the ultrafiltration permeate. The permeate was extracted with acetone, concentrated, and dried to obtain the indigo extract, which contained 78% indigo.
[0034] The crude indigo extract was purified using a ceramic membrane (0.5 μm) to obtain the ceramic membrane permeate, which was then purified using an ultrafiltration hollow fiber membrane (20,000 molecular weight cutoff) to obtain the ultrafiltration permeate. The permeate was extracted with acetone, concentrated, and dried to obtain the indigo extract, which contained 82% indigo.
[0035] The crude indigo extract was purified using a ceramic membrane (0.5 μm) to obtain the ceramic membrane permeate, which was then purified using an ultrafiltration hollow fiber membrane (10,000 molecular weight cutoff) to obtain the ultrafiltration permeate. The permeate was extracted with acetone, concentrated, and dried to obtain the indigo extract, which contained 86% indigo.
[0036] Comparison table of indigo separated by different membranes Ceramic membrane (μm) Ultrafiltration hollow fiber membrane (10,000 units) Extraction solvent Indigo content (%) in crude indigo extract 50 - acetone 65 0.5 - acetone 72 0.5 3 acetone 78 0.5 2 acetone 82 0.5 1 acetone 86 Example 5: Indigo membrane separation and solvent extraction The crude indigo extract was purified using a ceramic membrane (0.5 μm) to obtain the ceramic membrane permeate, which was then purified using an ultrafiltration membrane (10,000 molecular weight cutoff) to obtain the ultrafiltration permeate. The permeate was extracted with acetone, concentrated, and dried to obtain the indigo extract, which contained 86% indigo.
[0037] The crude indigo extract was purified using a ceramic membrane (0.5 μm) to obtain the ceramic membrane permeate, which was then purified using an ultrafiltration hollow fiber membrane (10,000 molecular weight cutoff) to obtain the ultrafiltration permeate. The permeate was extracted with chloroform, concentrated, and dried to obtain the indigo extract, which contained 79% indigo.
[0038] The crude indigo extract was purified using a ceramic membrane (0.5 μm) to obtain the ceramic membrane permeate, which was then purified using an ultrafiltration hollow fiber membrane (10,000 molecular weight cutoff) to obtain the ultrafiltration permeate. The permeate was extracted with n-butanol, concentrated, and dried to obtain the indigo extract, which contained 71% indigo.
[0039] The crude indigo extract was purified using a ceramic membrane (0.5 μm) to obtain the ceramic membrane permeate, which was then purified using an ultrafiltration hollow fiber membrane (10,000 molecular weight cutoff) to obtain the ultrafiltration permeate. The permeate was extracted with ethyl acetate, concentrated, and dried to obtain the indigo extract, which contained 82% indigo.
[0040] Comparison table of refined indigo extracted with different solvents Ceramic membrane (μm) Ultrafiltration hollow fiber membrane (10,000 units) Extraction solvent Indigo content (%) in crude indigo extract 0.5 1 acetone 86 0.5 1 chloroform 79 0.5 1 n-Butanol 71 0.5 1 Ethyl acetate 82 The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. The solutions disclosed in the embodiments are described simply because they correspond to the methods disclosed in the embodiments; relevant parts can be found in the method section.
[0041] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. A method for separating indigo by sealing fresh indigofera tinctoria for solid state fermentation and water extraction, characterized in that The method comprises the following steps: S1, sealing fresh indigo solid fermentation Take fresh indigo, put it in a sealed bag and store it at 10-30℃ for 5-30 days; S2, preparation of indigo crude extract After solid-state fermentation, the indigo is extracted with water at a solid-liquid ratio (g / mL) of 1:5-30, 0.1%-1% enzyme is added, and the mixture is extracted at 10-30℃ for 0.5-2 hours. The filtrate is the indigo crude extract. 2.S3, indigo membrane separation and solvent extraction The indigo crude extract is used as raw material to prepare high-purity indigo extract by different membrane separation and solvent extraction.
3. The method for sealed fresh indigofera tinctoria solid state fermentation and water extraction separation of indigo according to claim 1, characterized in that, The fresh indigo in step S1 includes indigo leaves, indigo stems, and whole indigo plants.
4. The method for sealed fresh indigofera tinctoria solid state fermentation and water extraction separation of indigo according to claim 1, characterized in that, The sealing in step S1 includes any one of vacuum and nitrogen filling.
5. The method for sealed fresh indigofera tinctoria solid state fermentation and water extraction separation of indigo according to claim 1, characterized in that, The solid-state fermentation in step S1 has a fermentation temperature of 25-30℃ and a fermentation time of 20-30 days.
6. The method for sealed fresh indigofera tinctoria solid state fermentation and water extraction separation of indigo according to claim 1, characterized in that, The enzyme added in step S2 for preparing the indigo crude extract includes glycosidase, cellulase, amylase, self-made enzyme, etc.
7. The method for sealed fresh indigofera tinctoria solid state fermentation and water extraction separation of indigo according to claim 1, characterized in that, The membrane separation in step S3 includes ceramic membranes with different molecular weight cut-offs and ultrafiltration hollow fiber membranes.
8. The method for sealed fresh indigofera tinctoria solid state fermentation and water extraction separation of indigo according to claim 1, characterized in that, The solvent extraction in step S3 includes organic solvents such as ethyl acetate, chloroform, n-butanol, and acetone.
9. The method for sealing fresh indigofera tinctoria solid state fermentation and water extraction separation of indigo according to claim 1, characterized in that, The purity of indigo in the indigo extract in step S3 is higher than 85%.