Method for extracting cereal blue from cereal blue fermentation liquor

Abstract

The invention discloses a method for extracting cereal blue from cereal blue fermentation liquor, which is characterized in that lauryl sodium sulfate and ammonia water are used as extraction solvents, cereal blue with better quality can be extracted from the fermentation liquor, and the product purity of the cereal blue is improved; by utilizing the extraction method, the use of organic reagents in the extraction process can be reduced, the whole purification process is simplified, the process difficulty can be reduced, and large-scale industrial application is facilitated; meanwhile, an extracting agent lauryl sodium sulfate used in the method is a substance with extremely high biodegradability, so that pollution and damage to the environment are avoided; ammonia water is a cheap raw material, and the extraction cost can be reduced by using the ammonia water as the extraction agent.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology. Specifically, it relates to a method for extracting indigoidine from indigoidine fermentation broth. Background Art

[0002] Indigoidine (CAS#2435598) is a non-toxic natural blue substance of microorganisms. Due to its natural occurrence and being obtained based on glutamic acid, it can be used as a coloring agent in food, beverages, and cosmetics; the excellent color display effect and natural environmental degradability of indigoidine have attracted more and more attention in its application in the textile industry; indigoidine can also be used as a screening marker in molecular biology, such as the blue-white screening system for gene expression, etc.; the molecular structure of indigoidine has a bipyridine ring structure, forming a double large π bond in the molecular space, making it have the potential to be used as an organic semiconductor material, and has broad development and application prospects in solar energy, information recording, liquid crystal materials, etc.

[0003] Indigoidine exists in nature, but its content is scarce. Some studies have used Escherichia coli, Pseudomonas, Corynebacterium glutamicum, etc. as expression systems for heterologous expression of indigoidine. However, since the indigoidine produced by fermentation contains a large amount of impurities such as bacteria and proteins, its purity is affected, which also limits its application in the fields of food, cosmetics, medicine, printing and dyeing, new materials, etc.

[0004] At present, centrifugation is the main means to separate bacteria and products, which can remove most of the impurities in the fermentation broth, but it cannot separate indigoidine from bacteria. At the same time, due to the non-water-soluble nature of indigoidine itself, further extraction and purification are required to obtain indigoidine products. The existing methods usually reduce indigoidine to a leuco form, dissolve and remove impurities with reagents multiple times, centrifuge, and then oxidize it to the original pigment. This process not only has a complicated process flow, but also easily causes losses during the extraction of indigoidine; the existing methods also use methanol, ethyl acetate, and n-hexane to extract indigoidine from the fermentation broth, but methanol, ethyl acetate, and n-hexane in this method are all toxic reagents. Due to these problems such as complicated processes, excessive use of chemical reagents, low product purity, and poor safety, it restricts the large-scale industrial application of indigoidine.

[0005] In view of this, the present invention is specifically proposed. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for extracting indigoidine from indigoidine fermentation broth. Through the method of the present invention, indigoidine products with better quality can be obtained, and the operation method is simplified, and the process difficulty of purification is reduced.

[0007] The present invention is implemented as follows:

[0008] A method for extracting gulain from gulain fermentation broth, which comprises: adding sodium dodecyl sulfate (SDS) and ammonia water to the gulain fermentation broth, and then obtaining pure gulain through separation and purification.

[0009] In some embodiments, the above method comprises: adding a solution containing sodium dodecyl sulfate to the gulain fermentation broth, adjusting the pH to weak acidity or neutrality and then stirring the mixed solution; separating the crude gulain from the mixed solution, adding ammonia water and stirring, and separating to obtain a precipitate; washing the obtained precipitate multiple times with water and drying to obtain pure gulain.

[0010] In order to simplify the gulain extraction process, the present invention first proposes using SDS and ammonia water as its extractants, and a gulain product with relatively high purity can be obtained through water washing. Using the extraction method of the present invention can overcome the problems of complex existing processes, excessive use of chemical reagents, and low product purity, which is beneficial to the large-scale industrial application of gulain.

[0011] In some embodiments, the mass concentration of sodium dodecyl sulfate in the mixed solution is 1.0% - 2.0%. When the addition concentration of sodium dodecyl sulfate is within the above range, the purification efficiency of the present invention is higher.

[0012] In some embodiments, the mass concentration of sodium dodecyl sulfate in the mixed solution is 1.0%.

[0013] In some embodiments, the solution containing sodium dodecyl sulfate also contains citric acid, and its mass concentration in the above mixed solution is 0.1% - 0.2%. Among them, the role of citric acid is to protect the gulain structure.

[0014] In some embodiments, the pH of the mixed solution is adjusted to 6 - 7.

[0015] The purpose of adjusting the pH is to keep the solution neutral or weakly acidic to protect the gulain structure from being damaged.

[0016] In some embodiments, the conditions for separating the crude gulain are: centrifuging the mixed solution at 4000 rpm - 6000 rpm for 10 min - 30 min.

[0017] In some embodiments, the conditions for adding ammonia water to the crude gulain are: the mass concentration of the added ammonia water is 5.0% - 15.0%. When the addition concentration of ammonia water is within the above range, the purification efficiency of the present invention is higher.

[0018] In some embodiments, the mass concentration of the added ammonia water is 5.0%.

[0019] In some embodiments, the separation conditions for the precipitate are: centrifuging at 4000 rpm - 6000 rpm for 10 min - 30 min.

[0020] In some embodiments, ammonia extraction is repeated multiple times before the precipitated material is washed with water; preferably, the number of repetitions is 2 to 3 times.

[0021] In some embodiments, the water washing includes: adding distilled water to the precipitate, stirring for 10 min to 20 min, and centrifuging at 4000 rpm to 6000 rpm for 10 min to 30 min; preferably, the number of water washing times is 5 to 10 times.

[0022] In some embodiments, the precipitated material needs to be dried after water washing, and the drying temperature is 55°C - 65°C.

[0023] In some embodiments, the drying temperature is 60°C.

[0024] The present invention has the following beneficial effects:

[0025] (1) The present invention uses sodium dodecyl sulfate and ammonia water as extraction solvents, and can extract good-quality glaucocalyxin from the fermentation broth, improving the product purity of glaucocalyxin.

[0026] (2) Using the extraction method of the present invention can reduce the use of organic reagents in the extraction process, and the whole purification process is also simplified, which can reduce the process difficulty and is beneficial to its large-scale industrial application.

[0027] (3) The extraction agent sodium dodecyl sulfate used in the present invention is a substance with extremely high biodegradability and will not cause pollution and damage to the environment; while ammonia water is a cheap raw material, and using it as an extraction agent can reduce the extraction cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can also be obtained based on these drawings without creative efforts.

[0029] Figure 1 It is the standard curve made in Experimental Example 1. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0030] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments not specified by the manufacturer are all conventional products that can be obtained through commercial purchase.

[0031] The features and properties of the present invention will be further described in detail below in conjunction with embodiments.

[0032] Embodiment 1

[0033] a. Take a certain amount of Gulan fermentation broth, prepare a corresponding amount of a mixed solution of 1.0% SDS and 0.1% citric acid, add it to the fermentation broth, adjust the pH to 7.0 with NaOH solution, and then stir the mixture for 60 min;

[0034] b. Centrifuge the mixture at 4000 rpm for 10 min, and take the precipitated crude Gulan;

[0035] c. Add a corresponding amount of 5.0% ammonia water according to the weight of the crude Gulan, and stir for 60 min;

[0036] d. Centrifuge the mixture of crude Gulan and ammonia water again at 4000 rpm for 10 min, and take the precipitated substance;

[0037] e. Repeat steps c and d twice;

[0038] f. Take the precipitate, add it to distilled water, stir for 10 min, and centrifuge at 4000 rpm for 10 min;

[0039] g. Repeat step f five times;

[0040] h. Dry the precipitated substance at 60 °C to finally obtain pure Gulan.

[0041] Embodiment 2

[0042] a. Take a certain amount of Gulan fermentation broth, prepare a corresponding amount of a mixed solution of 2.0% SDS and 0.2% citric acid, add it to the fermentation broth, adjust the pH to 6.0 with NaOH solution, and then stir the mixture for 120 min;

[0043] b. Centrifuge the mixture at 6000 rpm for 30 min, and take the precipitated crude Gulan;

[0044] c. Add a corresponding amount of 15.0% ammonia water according to the weight of the crude Gulan, and stir for 90 min;

[0045] d. Centrifuge the mixture of crude Gulan and ammonia water again at 6000 rpm for 30 min, and take the precipitated substance;

[0046] e. Repeat steps c and d three times;

[0047] f. Add the precipitate to distilled water, stir for 10 min, and centrifuge at 6000 rpm for 30 min;

[0048] g. Repeat step f 10 times;

[0049] h. Dry the precipitate at 60 °C to finally obtain pure gulain.

[0050] Example 3

[0051] a. Take a certain amount of gulain fermentation broth, prepare a corresponding amount of a mixed solution of 1.5% SDS and 0.1% citric acid and add it to the fermentation broth, adjust the pH to 7.0 with NaOH solution, and then stir the mixture for 90 min;

[0052] b. Centrifuge the mixture at 5000 rpm for 20 min and take the precipitated crude gulain;

[0053] c. Add a corresponding amount of 10.0% ammonia water according to the weight of the crude gulain and stir for 80 min;

[0054] d. Centrifuge the mixture of crude gulain and ammonia water again at 5000 rpm for 20 min and take the precipitate;

[0055] e. Repeat steps c and d 2 times;

[0056] f. Add the precipitate to distilled water, stir for 15 min, and centrifuge at 5000 rpm for 20 min;

[0057] g. Repeat step f 8 times;

[0058] h. Dry the precipitate at 60 °C to finally obtain pure gulain.

[0059] Comparative Example 1

[0060] This comparative example is a conventional method for purifying pigments in the industry, namely the leuco body oxidation-reduction method.

[0061] a. Take a certain amount of gulain fermentation broth and adjust the pH to 7.0 with NaOH solution;

[0062] b. Add a 2.0% thiourea dioxide solution to the above fermentation broth and stir for 60 min to fully reduce gulain to the leuco body;

[0063] c. After the above steps are completed, centrifuge the gulain leuco body at 4000 rpm for 10 min and take the precipitate;

[0064] d. Add the precipitate obtained in the above steps to a 0.1% citric acid solution to promote its contact oxidation with air and oxidize it back to the pigment precipitate;

[0065] e. Centrifuge the glutamine blue pigment precipitate again at 4000 rpm for 10 min;

[0066] f. After the above steps are completed, dry the centrifuged precipitate at 60 °C to finally obtain the pure glutamine blue product.

[0067] Comparative Example 2

[0068] The difference between this comparative example and Example 1 lies in the too high SDS concentration, and the rest of the operation steps are exactly the same. Specifically:

[0069] a. Take a certain amount of glutamine blue fermentation broth, prepare a corresponding amount of a mixed solution of 5.0% SDS and 0.1% citric acid and add it to the fermentation broth, adjust the pH to 7.0 with NaOH solution, and then stir the mixed solution for 60 min;

[0070] b. Centrifuge the mixed solution at 4000 rpm for 10 min to obtain the crude glutamine blue product as the precipitate;

[0071] c. Add a corresponding amount of 5.0% ammonia water according to the weight of the crude glutamine blue product and stir for 60 min;

[0072] d. Centrifuge the mixture of the crude glutamine blue product and ammonia water again at 4000 rpm for 10 min to obtain the precipitate;

[0073] e. Repeat steps c and d twice;

[0074] f. Take the precipitate and add it to distilled water, stir for 10 min, and centrifuge at 4000 rpm for 10 min;

[0075] g. Repeat step f five times;

[0076] h. Dry the precipitate at 60 °C to finally obtain the pure glutamine blue product.

[0077] Comparative Example 3

[0078] The difference between this comparative example and Example 1 lies in the too high pH, and the rest of the operation steps are exactly the same. Specifically:

[0079] a. Take a certain amount of glutamine blue fermentation broth, prepare a corresponding amount of a mixed solution of 1.0% SDS and 0.1% citric acid and add it to the fermentation broth, adjust the pH to 10.0 with NaOH solution, and then stir the mixed solution for 60 min;

[0080] b. Centrifuge the mixed solution at 4000 rpm for 10 min, and take the crude product of gulain in the precipitate.

[0081] c. Add the corresponding amount of 5.0% ammonia water according to the weight of the crude gulain product, and stir for 60 min.

[0082] d. Centrifuge the mixture of the crude gulain product and ammonia water again at 4000 rpm for 10 min, and take the precipitated substance.

[0083] e. Repeat steps c and d twice.

[0084] f. Take the precipitate, add it to distilled water, stir for 10 min, and centrifuge at 4000 rpm for 10 min.

[0085] g. Repeat step f five times.

[0086] h. Dry the precipitated substance at 60 °C to finally obtain the pure gulain product.

[0087] Comparative Example 4

[0088] The difference between this comparative example and Example 1 is that the concentration of ammonia water is too high, and the rest of the operation steps are exactly the same. Specifically:

[0089] a. Take a certain amount of gulain fermentation broth, prepare a mixed solution of the corresponding amount of 1.0% SDS and 0.1% citric acid and add it to the fermentation broth, adjust the pH to 7.0 with NaOH solution, and then stir the mixed solution for 60 min.

[0090] b. Centrifuge the mixed solution at 4000 rpm for 10 min, and take the crude gulain product in the precipitate.

[0091] c. Add the corresponding amount of 25.0% ammonia water according to the weight of the crude gulain product, and stir for 60 min.

[0092] d. Centrifuge the mixture of the crude gulain product and ammonia water again at 4000 rpm for 10 min, and take the precipitated substance.

[0093] e. Repeat steps c and d twice.

[0094] f. Take the precipitate, add it to distilled water, stir for 10 min, and centrifuge at 4000 rpm for 10 min.

[0095] g. Repeat step f five times.

[0096] h. Dry the precipitated substance at 60 °C to finally obtain the pure gulain product.

[0097] Comparative Example 5

[0098] The difference between this comparative example and Example 1 is that citric acid is not added, and the remaining operation steps are exactly the same. Specifically:

[0099] a. Take a certain amount of Gulan fermentation broth, add the corresponding amount of 1.0% SDS solution to the fermentation broth, adjust the pH to 7.0 with NaOH solution, and then stir the mixture for 60 min;

[0100] b. Centrifuge the mixture at 4000 rpm for 10 min, and take the precipitated crude Gulan;

[0101] c. Add the corresponding amount of 5.0% ammonia water according to the weight of the crude Gulan, and stir for 60 min;

[0102] d. Centrifuge the mixture of crude Gulan and ammonia water again at 4000 rpm for 10 min, and take the precipitated substance;

[0103] e. Repeat steps c and d twice;

[0104] f. Take the precipitate and add it to distilled water, stir for 10 min, and centrifuge at 4000 rpm for 10 min;

[0105] g. Repeat step f five times;

[0106] h. Dry the precipitated substance at 60 °C to finally obtain pure Gulan.

[0107] Experimental Example 1

[0108] At present, there is no standardized standard for measuring the various indicators of Gulan, a natural blue pigment, in the industry, and there is no standard product of this product on the market. At the same time, considering that the product and its related solutions are all colored substances / liquids, the standard curve of absorbance corresponding to purity can be used to measure the product concentration. The standard curve is formulated with pure Gulan with a currently measured purity of 99.0%:

[0109] (1) Weigh 1.01 mg of 99.0% Gulan sample, dissolve it in 10 mL of DMSO and sonicate for 60 min;

[0110] (2) The actual concentration of the Gulan sample solution is 0.101 g / L, and it is diluted successively to 0.0505 g / L, 0.02525 g / L, 0.012625 g / L, and 0.063125 g / L;

[0111] (3) Use a UV-visible spectrophotometer to measure the absorbance of each gradient sample, and make a standard curve of Gulan concentration based on the linear relationship between concentration and absorbance, as Figure 1 shown.

[0112] On this basis, the calculation method of the purity of gulain is as follows:

[0113] Purity = (Absorbance test value × Dilution factor × 0.0132 - 0.0001) × Dissolution volume / Actual mass

[0114] According to the standard curve for the determination of gulain concentration and the calculation method of purity, the purity of gulain obtained in each example can be determined and calculated. Specifically, as shown in Table 1 of the results:

[0115] Table 1 Purity data

[0116] Group Purity Example 1 119.37％ Example 2 116.58％ Example 3 115.34％ Comparative Example 1 89.31％ Comparative Example 2 95.17％ Comparative Example 3 76.59％ Comparative Example 4 87.64％ Comparative Example 5 96.51％

[0117] From the results of Comparative Example 1 and each example, it can be seen that the gulain purification method provided by the present invention is superior to the conventional pigment purification methods in the industry.

[0118] From the results of Comparative Example 2 and Example 1, it can be seen that when the SDS concentration is too high, the purification efficiency of the method of the present invention is instead reduced. From the results of Comparative Example 3, when the pH is too high, to a certain extent, the structure of gulain itself is damaged, resulting in a significant reduction in the purification efficiency; from the results of Comparative Example 4, when the ammonia water concentration is too high, the purification efficiency of the method of the present invention is instead reduced; from the results of Comparative Example 5, when citric acid is not added, to a certain extent, the double bond of the structure of gulain itself is damaged during the purification process, thereby reducing the purification efficiency.

[0119] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for extracting indigofera from indigofera fermentation broth, characterized in that: include: Sodium dodecyl sulfate and ammonia water are added to the indigofermentation liquid, and then the indigofermentation pure product is obtained through separation and purification.

2. The method according to claim 1, characterized in that The method comprises: adding a solution containing sodium dodecyl sulfate to the indigofermented rice fermentation liquid, adjusting the pH to weak acidity or neutrality, and then stirring the mixed solution; Separate the crude indigo plant product from the mixed solution, add ammonia water, stir, and separate to obtain the precipitate; The obtained precipitate was washed with water for several times and dried to obtain pure indigo plant product.

3. The method according to claim 2, characterized in that The mass concentration of sodium dodecyl sulfate in the mixed solution is 1.0% to 2.0%; Preferably, the mass concentration of sodium dodecyl sulfate in the mixed solution is 1.0%.

4. The method according to claim 2, characterized in that: The solution containing sodium dodecyl sulfate also contains citric acid, and the mass concentration of citric acid in the mixed solution is 0.1% to 0.2%.

5. The method according to claim 2, characterized in that: Adjust the pH of the mixed solution to 6-7; Preferably, the pH of the mixed solution is adjusted to 7.

6. The method according to claim 2, characterized in that The conditions for separating the crude indigo plant product are as follows: centrifuging the mixed solution at 4000 rpm to 6000 rpm for 10 min to 30 min.

7. The method according to claim 2, characterized in that The mass concentration of ammonia water added to the crude indigo plant is 5.0% to 15.0%; Preferably, the mass concentration of the added ammonia water is 5.0%.

8. The method according to claim 7, characterized in that The separation conditions of the precipitated material are: centrifugation at 4000 rpm to 6000 rpm for 10 min to 30 min.

9. The method according to claim 8, characterized in that The ammonia extraction is repeated several times before the precipitated material is washed with water; Preferably, the repetition number is 2 to 3 times.

10. The method according to claim 2, characterized in that The obtained precipitate is washed and dried, wherein the washing comprises: adding distilled water to the precipitate, stirring for 10 to 20 minutes, centrifuging at 4000 to 6000 rpm for 10 to 30 minutes; and drying at a temperature of 55 to 65°C; Preferably, the number of water washings is 5 to 10 times; Preferably, the drying temperature is 60°C.

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