A method for the separation and purification of indigo from a microbial fermentation broth

Abstract

The present invention relates to a method for separating and purifying indigo from a microbial fermentation broth, which comprises the following steps: Step 1, pretreatment of the microbial cells: separating the solid and liquid of the fermentation broth of indigo-producing microorganisms to obtain microbial cells; Step 2, cell disruption: disrupting the microbial cells obtained in Step 1 by mechanical disruption, chemical disruption or a combination of mechanical and chemical disruption to allow the content to flow out, obtaining a disrupted sample; Step 3, washing and centrifugation: adding a washing solvent to the disrupted sample obtained in Step 2, washing the disrupted sample several times, centrifuging, pouring off the upper liquid, collecting and combining the bottom solids to obtain solids; Step 4, drying: drying the solids obtained in Step 3 to obtain an indigo product. The process flow of the present invention is simple, the obtained indigo has a high color value, good purity and high yield, and the equipment used in the separation and purification process are all mature industrialized equipment with large capacity, realizing the industrialization of the separation and purification of indigo from microbial fermentation broth.

Description

Technical Field

[0001] The present invention belongs to the technical field of separation and purification, and particularly relates to a method for separating and purifying indigo from microbial fermentation broth. Background Art

[0002] At present, there are mainly three methods for producing indigo: 1) the method of extracting from plant fermentation, which has low yield and complex extraction process; 2) the method of chemical synthesis, which can meet the needs of large-scale production, but will produce pollutants and toxic substances, and is difficult to treat; 3) the method of microbial fermentation synthesis, which is green and environmentally friendly and has high synthesis efficiency, and can meet the needs of large-scale production. However, in the extraction process, indigo is mixed with a large amount of impurities such as miscellaneous proteins, cell debris, and cell contents from the fermentation broth, and indigo itself is insoluble in water, which makes its separation and purification extremely difficult. Therefore, it has become the biggest obstacle restricting its replacement of chemical synthesis. For example, the method for extracting indigo from microbial fermentation broth disclosed in the prior art is to first use lysozyme and protease to break the cells, and then use redox method to separate indigo. Its disadvantages are: after treating the cells with lysozyme and protease, the cell contents flow out, and the liquid in the system is viscous, making it difficult to directly separate indigo from solid impurities. In addition, the enzyme itself, as a protein, further increases the difficulty of indigo separation and purification. Therefore, it is necessary to first reduce the insoluble indigo to soluble indigo white by a reducing agent for solid-liquid separation to remove solid impurities, and then oxidize indigo white to indigo by oxidation for solid-liquid separation to obtain indigo products. Its operation is cumbersome, and during the reduction and oxidation process, due to the influence of impurities, parameter control is extremely demanding, and in the amplification process, the redox reaction is unstable. In addition, it also has defects such as high cost of lysozyme and protease and limited by the equipment capacity. Therefore, it cannot be used for industrial production and can only be used for small-scale fine experiments in the laboratory. Summary of the Invention

[0003] The purpose of the present invention is to overcome the defects in the prior art and provide a method for separating and purifying indigo from microbial fermentation broth, which has a simple process flow, high indigo color value, good purity, high yield, and the equipment used in the separation and purification process are all mature industrial equipment with large capacity, realizing the industrial production of indigo separation and purification from microbial fermentation broth.

[0004] To achieve the above purpose, the technical solutions adopted by the present invention are as follows:

[0005] A method for separating and purifying indigo from microbial fermentation broth, comprising the following steps:

[0006] Step 1, pretreatment of the cells: separating the solid and liquid of the fermentation broth of indigo-producing microorganisms to obtain microbial cells;

[0007] Step 2, cell disruption: The microbial cells obtained in Step 1 are disrupted by mechanical disruption, chemical disruption, or a combination of mechanical and chemical disruption to allow the content to flow out, obtaining a disrupted sample.

[0008] Step 3, washing and centrifugation: Add a washing solvent to the disrupted sample obtained in Step 2, wash the disrupted sample several times, centrifuge, pour off the upper liquid, collect and combine the solids at the bottom layer to obtain solids.

[0009] Step 4, drying: Dry the solids obtained in Step 3 to obtain indigo products.

[0010] As a further technical solution, in Step 2, the combination of mechanical and chemical disruption is carried out in the order of first performing mechanical disruption and then chemical disruption.

[0011] As a further technical solution, in Step 2, before cell disruption, water needs to be added to the microbial cells, and the addition amount of water is 5 - 15 times the weight of the microbial cells, which can be 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, etc., preferably 10 times.

[0012] The mechanical disruption in Step 2 uses bead milling and / or high-pressure homogenization.

[0013] In Step 2, the disruption reagents used for chemical disruption include guanidine hydrochloride and / or urea.

[0014] As a further technical solution, in Step 2, the chemical reagents used for chemical disruption further include surfactants.

[0015] As a further technical solution, in the disruption system for chemical disruption in Step 2, the concentration of guanidine hydrochloride and / or urea is 1 - 5 mol / L; the weight percentage concentration of the surfactant is 0.5% - 5%.

[0016] The surfactant includes one or more of Triton X-100, SDS, Tween 20, and Tween 80.

[0017] As a further technical solution, in Step 3, the washing solvent uses water.

[0018] In Step 3, during the washing process, the usage amount of the washing solvent is 10 - 200 times the weight of the microbial cells (preferably 100 times).

[0019] As a further technical solution, in step 3, the number of washing times is 1 - 3 times, and the rotation speed of centrifugation is 3000 - 8000 rpm. Values such as 3000 rpm, 4000 rpm, 5000 rpm, 6000 rpm, 7000 rpm, 8000 rpm, etc. can be selected, and 4000 rpm is preferred; the duration of a single centrifugation is 5 - 20 min. Values such as 5 min, 10 min, 15 min, 20 min, etc. can be selected, and 10 min is preferred.

[0020] As a further technical solution, in step 1, centrifugation is used for solid-liquid separation.

[0021] In step 1, after solid-liquid separation, the bacterial cells need to be washed.

[0022] In step 1, the washing solvent for washing the bacterial cells is water, and the number of washing times is 1 - 3 times.

[0023] As a further technical solution, in step 4, before drying the solid matter collected in step 3, solid-liquid separation is required to remove excess water, and then drying treatment is carried out.

[0024] The drying methods in step 4 include at least one of drying, spray drying, and freeze drying.

[0025] As a further technical solution, in step 4, solid-liquid separation is carried out by centrifugation or microfiltration membrane filtration.

[0026] The rotation speed of the centrifugation is 3000 - 10000 rpm.

[0027] The pore size of the microfiltration membrane is 0.1 - 0.5 μm.

[0028] As a further technical solution, when bead milling is used for cell disruption in step 2, the parameters of the bead mill are as follows: the diameter of the beads is 0.2 mm, the added volume of the beads is 50 - 70% (60% is preferred), and the disruption time is 4 - 6 min (5 min is preferred).

[0029] As a further technical solution, when high-pressure homogenization is used for cell disruption in step 2, a high-pressure homogenizer is used for homogenization. The pressure of homogenization is 110 - 130 MPa. Values such as 110 MPa, 120 MPa, 130 MPa, etc. can be selected, and 120 MPa is preferred; the flow rate of a single homogenization is 10 - 20 L / h. Values such as 10 L / h, 11 L / h, 12 L / h, 13 L / h, 14 L / h, 15 L / h, 16 L / h, 17 L / h, 18 L / h, 19 L / h, 20 L / h, etc. can be selected, and 13 L / h is preferred.

[0030] As a further technical solution, when the cell disruption in Step 2 is carried out by chemical disruption, the concentration of guanidine hydrochloride in the disruption system is 1 - 5 mol / L, and values such as 1 mol / L, 2 mol / L, 3 mol / L, 4 mol / L, 5 mol / L, etc. can be selected. When only chemical disruption is used without combining with mechanical disruption, it is preferably 5 mol / L; the weight percentage concentration of the surfactant is 0 - 5%, and values such as 0 mol / L, 1 mol / L, 2 mol / L, 3 mol / L, 4 mol / L, 5 mol / L, etc. can be selected, and it is preferably 2%.

[0031] As a further technical solution, when the cell disruption in Step 2 is carried out by combining bead milling disruption and chemical disruption, the parameters of the bead mill are: the diameter of the beads is 0.2 mm, the added volume of the beads is 50 - 70% (preferably 60%), and the disruption time is 4 - 6 min (preferably 5 min); the concentration of guanidine hydrochloride in the disruption system is 1 - 5 mol / L, and values such as 1 mol / L, 2 mol / L, 3 mol / L, 4 mol / L, 5 mol / L, etc. can be selected. When only chemical disruption is combined with mechanical disruption, it is preferably 2 mol / L; the weight percentage concentration of the surfactant is 0 - 5%, and values such as 0%, 1%, 2%, 3%, 4%, 5% etc. can be selected, and it is preferably 2%.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0033] 1. By studying the cell disruption process, the present invention discovers that when mechanical disruption and / or chemical disruption of cells is carried out under the condition of adding water, the state of the solid impurities obtained after disruption can be controlled by limiting the parameters in the disruption process, so that they can be suspended in the liquid phase under centrifugation conditions and separated from indigo, which is also a solid, by centrifugation. The present invention can effectively achieve the separation and purification of indigo by using two key steps of cell disruption and centrifugation. Compared with the traditional lysozyme disruption technology, it does not require an oxidation-reduction reaction with strictly controlled parameters, nor does it need to use expensive lysozyme and protease, and can further avoid the defects of sample viscosity caused by the introduction of lysozyme and protease and the deterioration of the indigo centrifugation separation effect caused by the additional introduction of solid protein.

[0034] 2. The present invention washes the collected microbial cells for solid-liquid separation multiple times, which can remove the water-soluble impurities in the culture medium. On the one hand, it can reduce the impurity residue in the indigo product and improve the purity of the indigo product. On the other hand, it can reduce the adverse effects on the subsequent chemical disruption process.

[0035] In summary, the present invention uses the indigo-producing microbial fermentation broth as a raw material, and separates and purifies indigo in the microbial fermentation broth through solid-liquid separation, washing the bacterial cells, mechanically and / or chemically breaking the bacterial cells, washing, centrifugal separation, and drying, to prepare an indigo product with a purity greater than 30%, a recovery rate greater than 45%, and a chromaticity greater than 150. The process is simple and convenient to operate. The obtained indigo has a high color value, good purity, and high yield. Moreover, the equipment used in the separation and purification process are all mature industrialized equipment with large capacities, realizing the industrial production of indigo separation and purification from microbial fermentation broth. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is a flow chart for the separation and purification of indigo in the microbial fermentation broth of the present invention; DETAILED DESCRIPTION OF THE EMBODIMENTS

[0037] The technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0038] In addition, it should be noted that the term "comprising", "including" or any other variant thereof is intended to cover a non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device.

[0039] The fermentation broth of indigo-producing microorganisms is obtained by fermenting and culturing the indigo-producing engineering strain BLT3 (this engineering strain is disclosed in the patent with the application number ZL202410351281.9).

[0040] In the present invention, unless otherwise specified, the raw materials used are commercially available raw materials.

[0041] Example 1

[0042] A method for separating and purifying indigo in microbial fermentation broth, comprising the following steps:

[0043] S1. Pretreatment; 50 L of the fermentation broth of indigo-producing microorganisms is centrifuged at 5000 rpm for 5 min for solid-liquid separation, and the bacterial cells are washed thoroughly 3 times with deionized water to remove soluble impurities in the fermentation broth, obtaining microbial bacterial cells;

[0044] S2. Cell disruption: Add water with a weight 10 times that of the microbial cells obtained in S1, mix well, and then use a high-pressure homogenizer to homogenize 5 times at a pressure of 120 MPa for cell disruption to obtain a disrupted sample. Among them, the flow rate for single homogenization is 13 L / h.

[0045] S3. Washing: Using water as the solvent, wash the disrupted sample obtained in S2 3 times. The amount of solvent used each time is 100 times the weight of the microbial cells. Centrifuge at 4000 rpm for 10 min, pour off the upper suspension, leave the bottom solid, and combine the bottom solids to obtain a solid matter.

[0046] S3. Solid-liquid separation and drying: Use a 0.22-μm microporous membrane to perform solid-liquid separation on the solid matter obtained in S3. After filtering off the excess water, dry it to obtain an indigo product.

[0047] Example 2

[0048] A method for separating and purifying indigo from a microbial fermentation broth, comprising the following steps:

[0049] S1. Pretreatment: Centrifuge 50 L of the fermentation broth of indigo-producing microorganisms at 5000 rpm for 5 min for solid-liquid separation. Wash the cells thoroughly 3 times with deionized water to remove soluble impurities in the fermentation broth to obtain microbial cells.

[0050] S2. Cell disruption: Add water with a weight 10 times that of the microbial cells obtained in S1, mix well, and then use a bead mill to disrupt the cells to obtain a disrupted sample. Among them, the parameters of the bead mill are: the diameter of the beads is 0.2 mm, the added volume of the beads is 60%, and the disruption time is 5 min.

[0051] S3. Washing: Using water as the solvent, wash the disrupted sample obtained in S2 3 times. The amount of solvent used each time is 100 times the weight of the microbial cells. Centrifuge at 4000 rpm for 10 min, pour off the upper suspension, leave the bottom solid, and combine the bottom solids to obtain a solid matter.

[0052] S4. Solid-liquid separation and drying: Use a 0.22-μm microporous membrane to perform solid-liquid separation on the solid matter obtained in S3. After filtering off the excess water, dry it to obtain an indigo product.

[0053] Example 3

[0054] A method for separating and purifying indigo from a microbial fermentation broth, comprising the following steps:

[0055] S1. Pretreatment: Centrifuge 50 L of the fermentation broth of indigo-producing microorganisms at 5000 rpm for 5 min for solid-liquid separation. Wash the cells thoroughly 3 times with deionized water to remove soluble impurities in the fermentation broth to obtain microbial cells.

[0056] S2, Cell disruption: Add 10 times the weight of water to the microbial cells obtained in S1. After thorough mixing, add guanidine hydrochloride and Triton X-100 until the concentration of guanidine hydrochloride in the system is 5 mol / L and the weight percentage concentration of Triton X-100 is 0.2%. React at 40 °C for 30 min to disrupt the cells and obtain the disrupted sample. Among them, the parameters of the bead mill are: the bead diameter is 0.2 mm, the added volume of beads is 60%, and the disruption time is 5 min.

[0057] S3, Washing: Wash the disrupted sample obtained in S2 3 times with water as the solvent. The amount of solvent used each time is 100 times the weight of the microbial cells. Centrifuge at 4000 rpm for 10 min, pour off the upper suspension, and keep the bottom solid. Combine the bottom solids to obtain the solid matter.

[0058] S4, Solid-liquid separation and drying: Use a 0.22 μm microporous filter membrane to perform solid-liquid separation on the solid matter obtained in S3. After filtering off the excess water, dry it to obtain the indigo product.

[0059] Example 4

[0060] A method for separating and purifying indigo from a microbial fermentation broth, comprising the following steps:

[0061] S1, Pretreatment; Centrifuge 50 L of the fermentation broth of indigo-producing microorganisms at 5000 rpm for 5 min for solid-liquid separation. Wash the cells thoroughly 3 times with deionized water to remove soluble impurities in the fermentation broth and obtain the microbial cells.

[0062] S2, Cell disruption: Add 10 times the weight of water to the microbial cells obtained in S1. After thorough mixing, use a bead mill to disrupt the cells. The parameters of the bead mill are: the bead diameter is 0.2 mm, the added volume of beads is 60%, and the disruption time is 5 min. After the bead milling is completed, add guanidine hydrochloride until the concentration of guanidine hydrochloride in the system is 2 mol / L. React at 40 °C for 30 min to disrupt the cells and obtain the disrupted sample.

[0063] S3, Washing: Wash the disrupted sample obtained in S2 3 times with water as the solvent. The amount of solvent used each time is 100 times the weight of the microbial cells. Centrifuge at 4000 rpm for 10 min, pour off the upper suspension, and keep the bottom solid. Combine the bottom solids to obtain the solid matter.

[0064] S4, Solid-liquid separation and drying: Use a 0.22 μm microporous filter membrane to perform solid-liquid separation on the solid matter obtained in S3. After filtering off the excess water, dry it to obtain the indigo product.

[0065] Example 5

[0066] A method for separating and purifying indigo from a microbial fermentation broth, comprising the following steps:

[0067] S1. Pretreatment: 50 L of the fermentation broth of indigo-producing microorganisms is centrifuged at 5000 rpm for 5 min for solid-liquid separation. The microbial cells are thoroughly washed 3 times with deionized water to remove soluble impurities in the fermentation broth, obtaining microbial cells;

[0068] S2. Cell disruption: 10 times the weight of water is added to the microbial cells obtained in S1. After thorough mixing, the cells are disrupted by homogenizing 3 times with a high-pressure homogenizer at a pressure of 120 MPa, with a single-pass flow rate of 13 L / h. After homogenization, guanidine hydrochloride is added thereto until the concentration of guanidine hydrochloride in the system is 2 mol / L, and the reaction is carried out at a reaction temperature of 40 °C for 30 min to disrupt the cells, obtaining a disrupted sample;

[0069] S3. Washing: Using water as a solvent, the disrupted sample obtained in S2 is washed 3 times, with the amount of solvent used each time being 100 times the weight of the microbial cells. Centrifugation is carried out at 4000 rpm for 10 min, and the upper suspension is poured off, leaving the bottom solid. The bottom solids are combined to obtain a solid;

[0070] S4. Solid-liquid separation and drying: The solid obtained in S3 is subjected to solid-liquid separation using a 0.22-μm microporous membrane. After removing the excess water, it is dried to obtain an indigo product.

[0071] Example 6

[0072] A method for separating and purifying indigo from a microbial fermentation broth, comprising the following steps:

[0073] S1. Pretreatment: 50 L of the fermentation broth of indigo-producing microorganisms is centrifuged at 5000 rpm for 5 min for solid-liquid separation. The microbial cells are thoroughly washed 3 times with deionized water to remove soluble impurities in the fermentation broth, obtaining microbial cells;

[0074] S2. Cell disruption: 10 times the weight of water is added to the microbial cells obtained in S1. After thorough mixing, the cells are disrupted by homogenizing 3 times with a high-pressure homogenizer at a pressure of 120 MPa, with a single-pass flow rate of 13 L / h. After homogenization, guanidine hydrochloride and X-triton-100 are added thereto until the concentration of guanidine hydrochloride in the system is 2 mol / L and the percentage weight concentration of X-triton-100 is 0.2%, and the reaction is carried out at a reaction temperature of 40 °C for 30 min to disrupt the cells, obtaining a disrupted sample;

[0075] S3. Washing: Using water as the solvent, wash the crushed sample obtained in S2 three times. The amount of solvent used each time is 100 times the weight of the microbial cells. Centrifuge at 4000 rpm for 10 minutes, pour off the upper suspension, and keep the bottom solid. Combine the bottom solids to obtain the solid matter.

[0076] S4. Solid-liquid separation and drying: Use a 0.22 μm microporous filter membrane to perform solid-liquid separation on the solid matter obtained in S3. After filtering out the excess water, dry it to obtain the indigo product.

[0077] Effect examples: Weigh and detect the purity (i.e., color value) of the indigo products prepared in each example, and calculate the recovery rate of indigo. The results are shown in Table 1.

[0078] 1) Purity detection:

[0079] Perform a full-wavelength scan on the indigo standard sample to measure the maximum absorption wavelength of indigo at 620 nm and prepare a standard curve. Weigh 1 g of the sample to be tested (the indigo product prepared in each example), dissolve and dilute the sample to an appropriate concentration with DMSO, and then use a microplate reader to measure the absorbance at 620 nm. Calculate the purity of the indigo product through the standard curve.

[0080] Indigo product purity = indigo concentration in the sample × sample dilution volume / 1 × 100%;

[0081] 2) Recovery rate calculation

[0082] Take 1 mL of indigo-producing microbial fermentation broth, centrifuge at 10,000 rpm for 10 minutes, discard the supernatant, wash the precipitate thoroughly with 100% methanol, centrifuge at 10,000 rpm for 5 minutes. After the methanol has fully evaporated, add 2 mL of DMSO and use ultrasonic disruption to dissolve the precipitate. Centrifuge the resulting liquid at 10,000 rpm for 10 minutes, take the supernatant, detect it with a microplate reader, and calculate the indigo concentration in the microbial fermentation broth.

[0083] Total amount of indigo in the fermentation broth = total volume of the fermentation broth × indigo concentration in the fermentation broth;

[0084] Indigo recovery rate = (indigo product purity × indigo product weight) / total weight of indigo in the fermentation broth × 100%;

[0085] The measured purity of the purified indigo is 67.75%, the recovery rate is 70.22%, and the color value E 1cm 1％ (620 nm) = 264. 3) Color value: The color value is expressed as E 1cm 1％ (620 nm).

[0086] Table 1

[0087] Purity Recovery rate Color value (E) Example 1 67.75％ 70.22％ 264 Example 2 33.75％ 63.48％ 166 Example 3 30.97％ 58.35％ 154 Example 4 42.14％ 46.78％ 205 Example 5 42.14％ 46.78％ 222 Example 6 50.44％ 62.38％ 243

[0088] The following conclusions can be drawn from the data in Table 1:

[0089] 1. Among the three cell disruption processes, homogenization disruption is the most beneficial for the separation and extraction of indigo in microbial fermentation broth. Followed by bead milling disruption and chemical disruption;

[0090] 2. When using the chemical disruption method alone to disrupt cells, the best effect is achieved when the dosage of guanidine hydrochloride is 5 mol / L. When chemical disruption is combined with mechanical disruption (bead milling disruption or homogenization disruption), the dosage of guanidine hydrochloride can be reduced to 2 mol / L. Therefore, the combination of chemical disruption and mechanical disruption can reduce the dosage of disruption reagents in chemical disruption compared to pure chemical disruption;

[0091] 3. The energy consumption of the homogenization process is relatively large. When chemical disruption is combined with homogenization disruption, compared to pure homogenization disruption, the number of homogenization times can be reduced from 5 times to 3 times. Therefore, it can reduce the number of homogenization disruptions, thereby reducing the energy consumption cost of indigo separation and extraction. Compared to pure chemical disruption, it can reduce the dosage of disruption reagents.

[0092] 4. When homogenization disruption and chemical disruption are combined, and a certain amount of surfactant is added during chemical disruption, compared to the technology of combining homogenization disruption and chemical disruption without adding surfactant, it can improve the purity and recovery rate of indigo products.

[0093] In the present invention, by controlling the parameters during the mechanical disruption and / or chemical disruption of cells under the condition of adding water, the state of the solid impurities obtained after disruption is controlled, so that they can be suspended in the liquid phase under centrifugation conditions and separated from indigo, which is also a solid, by centrifugation. Therefore, by using the disruption method and conditions defined in the present invention, the separation and purification of indigo can be achieved by a simple centrifugation method. Compared to the traditional lysozyme disruption technology, it does not require an oxidation-reduction reaction with strictly controlled parameters, nor does it need to use expensive lysozyme and protease. Moreover, it can avoid the defects of sample viscosity caused by the introduction of lysozyme and protease, and the deterioration of indigo centrifugation separation effect caused by the additional introduction of solid protein.

[0094] The above-described embodiments are only the preferred embodiments of the present invention, and not an exhaustive list of the feasible embodiments of the present invention. For those of ordinary skill in the art, any obvious modifications made without departing from the principles and spirit of the present invention should be considered to be included within the protection scope of the claims of the present invention.

Claims

1. A method for separating and purifying indigo from a microbial fermentation broth, characterized in that: The steps include: Step 1, microbial cell pretreatment: solid-liquid separation of the fermentation liquid of the indigo-producing microorganism to obtain microbial cells; Step 2, cell crushing: adding water to the microbial cells obtained in step 1, wherein the amount of water added is 5-15 times the weight of the microbial cells; then crushing the cells mechanically or by combining mechanical and chemical crushing to make the contents flow out, thereby obtaining a crushed sample; In step 2, mechanical crushing is performed by high-pressure homogenization; when only mechanical crushing is performed, the number of high-pressure homogenization is 6 times; when mechanical crushing and chemical crushing are used together, mechanical crushing is performed first, and then chemical crushing is performed, and the number of high-pressure homogenization is 3 times; In step 2, when high-pressure homogenization is used to break the bacteria, a high-pressure homogenizer is used for homogenization, the homogenization pressure is 110-130MPa, and the flow rate of a single homogenization is 10-20 L / h; In step 2, the chemical fragmentation reagents used include guanidine hydrochloride and / or urea, and also include a surfactant; the concentration of guanidine hydrochloride and / or urea is 1-5 mol / L; the percentage weight concentration of the surfactant is 0.5-5%; The surfactant includes one or more of Triton X-100, SDS, Tween 20, and Tween 80; Step 3, washing and centrifugation: adding a washing solvent to the crushed sample obtained in step 2, washing the crushed sample several times, centrifuging, pouring off the upper suspension, collecting and combining the bottom solids, and obtaining a solid; In step 3, the washing solvent is water; In step 3, during the washing process, the amount of washing solvent used is 10-200 times the weight of the microorganisms; In step 3, the washing times are 1-3 times, the centrifugal speed is 3000-8000 rpm, and the single centrifugation time is 5-20 min; Step 4, drying: drying the solid obtained in step 3 to obtain an indigo product.

2. The method for separating and purifying indigo in a microbial fermentation broth according to claim 1, characterized in that: In step 1, solid-liquid separation is carried out by centrifugation. In step 1, after solid-liquid separation, the bacteria need to be washed; In step 1, the washing solvent used for washing the bacteria is water, and the washing times are 1-3 times.

3. The method for separating and purifying indigo in a microbial fermentation broth according to claim 1, characterized in that: In step 4, before drying the solid collected in step 3, solid-liquid separation is required to remove excess water, and then drying is performed; The drying method in step 4 includes at least one of drying, spray drying and freeze drying.

4. The method for separating and purifying indigo in a microbial fermentation broth according to claim 3, characterized in that: In step 4, solid-liquid separation is performed by centrifugation or microporous membrane filtration; The centrifugal speed is 3000-10000rpm; The pore size of the microporous filter membrane is 0.1-0.5 μm.

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