Purification method of biological indigo

Abstract

The invention belongs to the technical field of purification, and particularly provides a biological indigo purification method which comprises the following steps: culturing a biological indigo fermentation product based on an engineering strain for producing indigo modified by synthetic biology; hydrolyzing the biological indigo fermented product with a hydrolysis reagent to obtain a biological indigo precipitate; purifying the biological indigo precipitate in a solvent pulping manner to obtain a biological indigo refined substance; and drying the purified biological indigo refined substance to obtain a high-purity biological indigo and / or indirubin mixture. Based on the purification method provided by the invention, the content of the biological indigo can reach 95.1%, the purity of the indigo is greatly improved, and the method is more suitable for the biological indigo in the fields of foods, cosmetics, medicines and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of purification and relates to a method for purifying biological indigo. Background Art

[0002] Indigo is a natural blue dye with a long history, widely used in textiles, food, medicine and other fields. Traditional indigo preparation methods mainly include plant extraction and chemical synthesis, but the former is limited by the long planting cycle, low content (only 2%), and high extraction cost, while the latter relies on toxic chemicals such as aniline as raw materials, which poses environmental pollution and health risks. With the global annual demand for indigo exceeding 50,000 tons, the development of green and efficient biosynthesis technology and the provision of high-purity biological indigo have become urgent needs of the industry and the development trend of bio-pigments in the future. In recent years, breakthroughs in synthetic biology technology have provided a new path for indigo production, exploring key enzymes for high-yield and stable synthesis of indigo, using genetically engineered strains to construct microbial cell factories, and obtaining high-purity biological indigo through purification processes. This can retain the environmental characteristics of natural products, significantly reduce production costs, and achieve mass production of high-purity biological indigo.

[0003] Although there are many plants that can be used to extract indigo, such as indigo, isatis, indigo, and horsetail, their indigo content is low, the extraction process is complicated, and a large amount of solvent and alkaline solution is required; although chemical synthesis of indigo has high purity and conversion rate, it involves harmful chemicals such as aniline, and there are problems such as environmental pollution and safety hazards. The synthesis of biological indigo is always accompanied by the overexpression of protein and the growth of engineered bacteria. Indigo has very low solubility in water. Centrifugation can remove most of the fermentation broth impurities, but it is impossible to separate indigo from the bacteria. It is urgent to study the effective extraction and purification method of biological indigo to obtain high-purity biosynthetic indigo. Patent CN113698790A only removes the protein in the bacteria and then purifies the indigo by reduction. Although the protein removal rate is high, it does not mention whether other impurities (such as nucleic acids and sugars) have an impact on purification and the final indigo content. Patent CN117430970A uses protein hydrolysis, acid hydrolysis, and alkaline hydrolysis to purify bio-indigo dye. Studies have shown that high-temperature alkaline hydrolysis is the most effective in purifying indigo, followed by high-temperature acid hydrolysis, and proteinase hydrolysis is the least effective. The article mentions that when using an acid or base as a hydrolyzing agent, the optimal equivalent concentration should be between 1N and 18N for optimal hydrolysis. The optimal yield (78.8%) and purity (90.1%) were achieved using a high-concentration base (6N NaOH) combined with a high temperature (100°C) and hydrolysis for 15 hours. However, the high-temperature alkaline hydrolysis method of the patent CN117430970A method has shortcomings: (1) the amount of alkali used is too large and the high-temperature hydrolysis time is too long; (2) the overall purification test yield is low. This is because indigo will be destroyed under strong alkali and high-temperature conditions. The longer the hydrolysis time, the lower the indigo yield; (3) the purified indigo is filtered by suction. However, during the experimental process of this study, it was found that the suction filtration method takes a long time to filter indigo. Instead, centrifugation is more convenient and faster to collect indigo. During the indigo hydrolysis process, in the presence of alkaline conditions and various reducing substances, a small amount of indigo may be reduced to soluble leuco indigo. Therefore, this study increased ventilation after the hydrolysis is completed to allow oxygen to reduce the leuco indigo to indigo.

[0004] Although a certain amount of indigo can be produced through biosynthesis (30g / L, with a content of about 30% after drying), the impurities are numerous and complex, including a large amount of protein and a small amount of sugars, lipids, nucleic acids, and intermediates in the indigo synthesis pathway. These impurities can be hydrolyzed into small molecules soluble in water under appropriate conditions and can be removed by subsequent centrifugation. Therefore, research on the purification process of bio-indigo is of certain significance and can expand the application of bio-indigo in food, cosmetics, and medicine. Summary of the Invention

[0005] The present invention provides a method for purifying biological indigo, comprising the following steps:

[0006] Step 1: Cultivating a bio-indigo fermentation product using an engineered strain for producing indigo modified by synthetic biology;

[0007] Step 2: hydrolyzing the bio-indigo fermentation product with a hydrolysis reagent to obtain a bio-indigo precipitate;

[0008] Step 3: Purifying the bio-indigo precipitate by solvent beating to obtain a bio-indigo refined product;

[0009] Step 4: Drying the purified biological indigo product to obtain a high-purity biological indigo and / or indirubin mixture.

[0010] Furthermore, the specific process of cultivating the biological indigo fermentation product is as follows:

[0011] Cultivate the engineered strain for indigo production at 35°C-40°C;

[0012] The cultured engineered strain was cooled to 25°C-30°C for induction;

[0013] The induced engineered strain was fermented under the conditions of tryptophan as substrate, pH value of 7±0.2, and dissolved oxygen concentration of 30% to obtain a bio-indigo fermentation liquid;

[0014] Based on the bio-indigo fermentation broth, a bio-indigo fermentation product is obtained.

[0015] Furthermore, during the fermentation process of the induced engineered strain, the concentration of tryptophan is always controlled within 0.1-5 g / L.

[0016] Furthermore, the specific process of obtaining the biological indigo fermentation product is as follows:

[0017] The bio-indigo fermentation liquor is directly used as the bio-indigo fermentation product for hydrolysis.

[0018] Furthermore, the specific process of obtaining the biological indigo fermentation product is as follows:

[0019] The bio-indigo fermentation broth is centrifuged, the supernatant is discarded, and the wet cells are collected;

[0020] The wet cells are used as the bio-indigo fermentation product for hydrolysis.

[0021] Furthermore, the specific process of obtaining the biological indigo fermentation product is as follows:

[0022] The bio-indigo fermentation broth is centrifuged, the supernatant is discarded, and the wet cells are collected;

[0023] The wet bacterial cells are dried, and the dried bacterial cell powder is taken as the bio-indigo fermentation product for hydrolysis.

[0024] Furthermore, the specific process of hydrolyzing the biological indigo fermentation product is as follows:

[0025] S2.1. Add 2-5 times the mass of the bacterial cells in water and stir to obtain indigo hydrolyzate.

[0026] S2.2, adding sodium hydroxide to the indigo hydrolyzate to adjust its pH to pH 13, to obtain a pH-adjusted indigo hydrolyzate;

[0027] S2.3. Hydrolyzing the pH-adjusted indigo hydrolyzate at 85°C-95°C to remove impurities such as cellular components;

[0028] S2.4. The indigo hydrolyzate is naturally cooled to room temperature and then aerated for 1 hour. The supernatant is then discarded by centrifugation and the precipitate is collected to obtain a biological indigo precipitate.

[0029] Furthermore, the concentration of sodium hydroxide in the indigo hydrolyzate after pH adjustment is 0.4-1 mol / L.

[0030] Furthermore, the specific process of purifying the biological indigo precipitate is as follows:

[0031] The mixture is pulped with an organic solvent to further remove impurities and separate some indigo red. After centrifugation, the supernatant and precipitate are collected. The precipitate is the refined biological indigo.

[0032] Furthermore, the specific process for obtaining a high-purity biological indigo and / or indirubin mixture is as follows:

[0033] The bio-indigo precipitate after hydrolysis is dried to obtain a bio-indigo with a content of 91.8%;

[0034] The refined bio-indigo product is dried to obtain bio-indigo with a content of 95.1%;

[0035] The supernatant obtained during the purification is concentrated to obtain an indigo red mixture.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] Based on the purification method provided by the present invention, the content of biological indigo can reach 95.1%, greatly improving the purity of indigo, making it more suitable for biological indigo in the fields of food, cosmetics, and medicine.

[0038] In addition to the above-described objects, features and advantages, the present invention has other objects, features and advantages. The present invention will be further described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0040] Figure 1 1 is a schematic flow chart of a method for purifying biological indigo according to an embodiment of the present invention;

[0041] Figure 2 This is a chromatogram of the purified biological indigo powder in Experimental Example 1 of the present invention;

[0042] Figure 3 is a chromatogram of the fermentation liquid in Experimental Example 2 of the present invention;

[0043] Figure 4 This is the centrifuged wet bacterial cell chromatogram in Experimental Example 2 of the present invention;

[0044] Figure 5 is a chromatogram of the unpurified biological indigo powder in Experimental Example 2 of the present invention;

[0045] Figure 6 This is a chromatogram of the purified biological indigo powder in Experimental Example 4 of the present invention (wherein the indigo content is 95.1% and the indirubin content is 1.48%);

[0046] Figure 7 This is a chromatogram of the crude indirubin powder in Experimental Example 4 of the present invention (wherein the indigo content is 4.9% and the indirubin content is 11.5%). DETAILED DESCRIPTION

[0047] In order to make the above-mentioned objects, features and advantages of the present invention more clear and easy to understand, the specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0048] Example:

[0049] See also Figure 1 As shown, the present invention provides a method for purifying biological indigo, comprising the following steps:

[0050] Step 1: Cultivating a bio-indigo fermentation product using an engineered strain for producing indigo modified by synthetic biology;

[0051] The specific process is as follows:

[0052] S1.1. Select an engineered indigo-producing strain modified using synthetic biology or other technologies, and culture the engineered indigo-producing strain in liquid culture at 37°C until the strain reaches a certain weight;

[0053] S1.2. Cool the cultured engineered strain to 25-30°C for induction;

[0054] S1.3. Fermenting the induced engineered strain using tryptophan as a substrate at a pH of 7±0.2 and a dissolved oxygen concentration of 30% to obtain a bio-indigo fermentation broth; wherein, during the fermentation of the induced engineered strain, the tryptophan concentration is always controlled within a range of 0.1-5 g / L;

[0055] S1.4. The bio-indigo fermentation broth is directly used as the bio-indigo fermentation product for hydrolysis; alternatively, the indigo fermentation broth after fermentation is centrifuged, the supernatant is discarded, and the wet cells are collected, and the wet cells are used as the bio-indigo fermentation product for hydrolysis; alternatively, the indigo fermentation broth after fermentation is centrifuged, the supernatant is discarded, and the wet cells are collected, and the wet cells are then dried, and the dried powder is used as the bio-indigo fermentation product for hydrolysis.

[0056] Step 2: hydrolyzing the bio-indigo fermentation product with a hydrolysis reagent to obtain a bio-indigo precipitate;

[0057] The specific process is as follows:

[0058] S2.1. Add 2-5 times the mass of the bacterial cells in water and stir to obtain indigo hydrolyzate.

[0059] S2.2. Adding sodium hydroxide to the indigo hydrolyzate to adjust its pH to 13; wherein the concentration of sodium hydroxide in the indigo hydrolyzate after pH adjustment is 0.4-1 mol / L;

[0060] S2.3, hydrolyzing the indigo hydrolyzate after adjusting the pH value at a high temperature of 85°C to 95°C to remove impurities such as cellular components;

[0061] S2.4. Cool the indigo hydrolyzate to room temperature and ventilate for 1 hour. Centrifuge and discard the supernatant to collect the precipitate to obtain the biological indigo precipitate.

[0062] Step 3: Purifying the biological indigo precipitate by beating;

[0063] The specific process is:

[0064] The biological indigo precipitate is pulped with an organic solvent to further remove impurities and separate some indirubin. After centrifugation, the supernatant and precipitate are collected. The precipitate is the refined biological indigo.

[0065] Step 4: drying the purified biological indigo product and concentrating the supernatant to obtain a high-purity biological indigo and / or indirubin mixture;

[0066] The specific process is:

[0067] The bio-indigo refined product is dried to obtain high-purity bio-indigo, and the supernatant is concentrated to obtain the indigo red mixture.

[0068] Furthermore, the specific process for obtaining a high-purity biological indigo and / or indirubin mixture is as follows:

[0069] The bio-indigo precipitate after hydrolysis is dried to obtain a bio-indigo with a content of 91.8%;

[0070] The refined bio-indigo product is dried to obtain bio-indigo with a content of 95.1%;

[0071] The supernatant obtained during the purification is concentrated to obtain an indigo red mixture.

[0072] Experimental Example 1: Effects of pH, time, and aeration during hydrolysis on indigo purification

[0073] The fermentation broth had a bio-indigo concentration of 30 g / L. The same volume of fermentation broth was centrifuged, and 100 g of cells were collected. Purified water (3 times the cell mass) was added, and the bio-indigo was hydrolyzed at different pH values ​​(adjusted with sodium hydroxide) and hydrolysis times (maintaining a constant temperature of 90°C). The broth was naturally cooled to room temperature (approximately 2 hours), aerated (aeration time), and centrifuged to separate the indigo. After drying at 80°C, the indigo content and yield (mass of indigo after purification / mass of indigo before purification) were calculated. The indigo content was determined using high-performance liquid chromatography (HPLC) using an external standard method by comparing the peak areas of the sample and the standard. The sample was dissolved by ultrasonication in DMF and fixed to volume. A C18 column (150 mm × 4.6 mm, 2.7 μm) was used, the column temperature was 30 °C, the mobile phase was methanol: water (7:3, V / V), the flow rate was 0.5 mL / min, the detection wavelength was 287 nm, the injection volume was 5 μL, and three parallel samples were taken for each group; the purified biological indigo was detected as follows; Figure 2 shown.

[0074] Table 1 Effects of pH, time and ventilation on indigo purification

[0075]

[0076]

[0077] The results showed that when the pH was between 13.0 and 13.6, the hydrolysis time was 6 to 8 hours, and the aeration was 1 hour, the indigo content could be increased to over 90%, and the yield to over 88%. Experiments 1-3 were the best, with an indigo content of 91.8% and an indirubin content of 1.88%.

[0078] Experimental Example 2: Indigo state before hydrolysis (wet cells, fermentation liquid, dry powder)

[0079] Fermentation broth is a liquid mixture formed during microbial metabolism. It is a multiphase system containing unconsumed culture medium components (amino acids, sugars, lipids, inorganic salts, etc.), microbial metabolites, and living microorganisms. It is characterized by its liquid state and high viscosity. Various impurities are slow to dissolve during alkaline hydrolysis, significantly reducing the indigo purification effect. Wet cells are cell aggregates separated from the fermentation broth by centrifugation. They retain some water and contain very small amounts of incompletely removed culture medium components. They appear as moist clumps or slurries and are living microorganisms. Proteins, nucleic acids, sugars, lipids, etc. can be completely hydrolyzed, resulting in optimal purification. Dry powder is a powdered product formed after high-temperature drying of wet cells. It has an extremely low moisture content (<5%). High-temperature denaturation of proteins hinders the breakdown of protein structures during subsequent alkaline hydrolysis, slightly reducing the indigo purification effect.

[0080] The fermentation broth has a bio-indigo concentration of 30 g / L. Equal volumes of fermentation broth were centrifuged. Wet cells: After centrifugation, 100 g of centrifuged cells were collected and purified water (3 times the cell mass) was added. Fermentation broth: Three parallel 30 ml aliquots of fermentation broth were centrifuged and the supernatant removed. The cell concentration (g / L) was calculated using the formula (precipitate mass g / 30 ml) × 1000. The fermentation broth volume required to collect 100 g of wet cells was calculated (required fermentation broth volume L = 100 g / cell concentration g / L), and purified water was added to the same volume. Dry powder: 100 g of centrifuged wet cells were dried at 80°C, ground into powder, and the same volume was added with purified water. The pH was adjusted to 13.0 using sodium hydroxide, and the cells were hydrolyzed at 90°C for 8 hours. The cells were cooled to room temperature, aerated for 1 hour, and the indigo was separated by centrifugation. The cells were then dried at 80°C.

[0081] Furthermore, the wet cells, fermentation broth, and dry powder were first enzymatically hydrolyzed using neutral protease (0.5 g) at pH 7 and 45°C for 4 h, then sodium hydroxide was added to adjust the pH to 13.0, and hydrolyzed at 90°C for 8 h. The mixture was cooled to room temperature, ventilated for 1 h, and indigo was separated by centrifugation and dried at 80°C.

[0082] The indigo was quantitatively determined by the high performance liquid chromatography method of Example 1. The fermentation broth test results were as follows: Figure 3 As shown, the centrifugal wet cell test is as follows Figure 4 As shown, the unpurified biological indigo powder was detected as Figure 5 shown.

[0083] Table 2 Different indigo states (wet bacteria, fermentation liquid, dry powder)

[0084]

[0085]

[0086] The results showed that the overall indigo purification efficiency ranked in the order of wet cells > dry powder > fermentation broth. Only in the wet cell state could the indigo content be increased to over 90%, and protease hydrolysis failed to improve the indigo content at this stage. Adding protease to the fermentation broth for enzymatic hydrolysis followed by alkaline hydrolysis increased the indigo content. Under the same purification conditions, the hydrolysis efficiency of dry powder indigo was intermediate between that of wet cells and fermentation broth.

[0087] Experimental Example 3: Alkaline Water Liberation Experiment

[0088] Wet cells: Collect 20 kg of centrifuged wet cells after centrifugation and add purified water 3 times the mass of the cells. Dry powder: Collect 20 kg of dried indigo powder, add purified water 9 times the mass of the powder, and stir at room temperature for 12 hours to fully soak it. Then, adjust the pH to 13.2 with sodium hydroxide (measure the pH after 6 hours and adjust it to 13.2), hydrolyze at high temperature for 8-12 hours, cool to room temperature, ventilate for 1 hour, separate the indigo by centrifugation, and dry at 80°C.

[0089] Table 3 Alkaline hydrolysis experiment

[0090] serial number Indigo State Temperature / ℃ Hydrolysis time / h Indigo content / % Indigo yield / % 3-1 Wet bacteria 85 8 81.6 90.3 3-2 Wet bacteria 90 8 85.1 91.7 3-3 Wet bacteria 90 12 90.0 89.4 3-4 Wet bacteria 95 12 87.3 85.6 3-5 dry powder 85 8 73.2 89.2 3-6 dry powder 90 8 84.6 91.3 3-7 dry powder 90 12 88.6 88.6 3-8 dry powder 95 12 81.9 83.8

[0091] The results showed that when the sample mass increased, increasing the hydrolysis temperature and time could increase the indigo content, but reduce the indigo yield.

[0092] Experimental Example 4: Bio-indigo Refining

[0093] The solubility of indigo and indirubin in various organic solvents is relatively low. Beating at the reflux temperature of the solvent and centrifuging while hot can increase the solubility of impurities at this temperature, which is beneficial to the separation of impurities from indigo and indirubin. Take 25g of the biological indigo purified in the above experiment, take 500ml of different organic solvents (or use solvents alternately), beat for 2 hours at the reflux temperature of the solvent (ethanol 78°C, ethyl acetate 77°C, methanol 65°C, dichloromethane 40°C, chloroform 61°C, acetone 56°C, 75% ethanol 78°C, 95% ethanol 78°C), centrifuge while hot, and precipitate and dry at 80°C. The supernatant is concentrated using a rotary evaporator, replaced with water after ethanol replacement, and dried at 80°C. The high performance liquid chromatography method of Example 1 is used to quantitatively determine indigo and indirubin at the same time. The detection results of the biological indigo refinement are as follows: Figure 6 As shown, the crude indirubin test results are as follows Figure 7 shown.

[0094] Table 4 Organic solvent refining experiment

[0095]

[0096] Table 5 Indigo red content after supernatant concentration

[0097]

[0098]

[0099] The results showed that organic solvents can remove some impurities remaining after hydrolysis and effectively increase the indigo content, but at the same time they can take away a small amount of indigo and indirubin, which will reduce the indigo yield.

[0100] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for purifying biological indigo, characterized in that: The following steps are involved: Step 1: Cultivating a bio-indigo fermentation product using an engineered strain for producing indigo modified by synthetic biology; Step 2: hydrolyzing the bio-indigo fermentation product with a hydrolysis reagent to obtain a bio-indigo precipitate; Step 3: Purifying the bio-indigo precipitate by solvent beating to obtain a bio-indigo refined product; Step 4: Drying the purified biological indigo product to obtain a high-purity biological indigo and / or indirubin mixture.

2. The method for purifying biological indigo according to claim 1, characterized in that: The specific process of cultivating biological indigo fermentation is: Cultivate the engineered strain for indigo production at 35°C-40°C; The cultured engineered strain was cooled to 25°C-30°C for induction; The induced engineered strain was fermented under the conditions of tryptophan as substrate, pH value of 7±0.2, and dissolved oxygen concentration of 30% to obtain a bio-indigo fermentation liquid; Based on the bio-indigo fermentation broth, a bio-indigo fermentation product is obtained.

3. The method for purifying biological indigo according to claim 2, characterized in that: During the fermentation process of the induced engineered strain, the concentration of tryptophan is always controlled within 0.1-5 g / L.

4. The method for purifying biological indigo according to claim 3, characterized in that: The specific process of obtaining biological indigo fermentation is as follows: The bio-indigo fermentation liquor is directly used as the bio-indigo fermentation product for hydrolysis.

5. The method for purifying biological indigo according to claim 3, characterized in that: The specific process of obtaining biological indigo fermentation is as follows: The bio-indigo fermentation broth is centrifuged, the supernatant is discarded, and the wet cells are collected; The wet cells are used as the bio-indigo fermentation product for hydrolysis.

6. The method for purifying biological indigo according to claim 3, characterized in that: The specific process of obtaining biological indigo fermentation is as follows: The bio-indigo fermentation broth is centrifuged, the supernatant is discarded, and the wet cells are collected; The wet bacterial cells are dried, and the dried bacterial cell powder is taken as the bio-indigo fermentation product for hydrolysis.

7. The method for purifying bio-indigo according to any one of claims 4 to 6, characterized in that: The specific process of hydrolyzing the biological indigo fermentation product is as follows: S2.

1. Add 2-5 times the mass of the bacterial cells in water and stir to obtain indigo hydrolyzate. S2.2, adding sodium hydroxide to the indigo hydrolyzate to adjust its pH to pH 13, to obtain a pH-adjusted indigo hydrolyzate; S2.

3. Hydrolyzing the pH-adjusted indigo hydrolyzate at 85°C-95°C to remove impurities such as cellular components; S2.

4. The indigo hydrolyzate is naturally cooled to room temperature and then aerated for 1 hour. The supernatant is then discarded by centrifugation and the precipitate is collected to obtain a biological indigo precipitate.

8. The method for purifying biological indigo according to claim 7, characterized in that: The concentration of sodium hydroxide in the indigo hydrolyzate after pH adjustment is 0.4-1 mol / L.

9. The method for purifying biological indigo according to claim 8, characterized in that: The specific process of purifying biological indigo precipitate is as follows: The mixture is pulped with an organic solvent to further remove impurities and separate some indigo red. After centrifugation, the supernatant and precipitate are collected. The precipitate is the refined biological indigo.

10. The method for purifying biological indigo according to claim 9, characterized in that: The specific process for obtaining a high-purity biological indigo and / or indirubin mixture is as follows: The bio-indigo precipitate after hydrolysis is dried to obtain a bio-indigo with a content of 91.8%; The refined bio-indigo product is dried to obtain bio-indigo with a content of 95.1%; The supernatant obtained during the purification is concentrated to obtain an indigo red mixture.

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