Separation method of neohesperidin finished product

Abstract

The invention provides a separation method of a neohesperidin finished product, and belongs to the technical field of separation and purification. In the implementation process, a whole set of separation treatment including alkali dissolution, membrane separation, acid precipitation, concentration, cooling crystallization and centrifugal separation is carried out on the neohesperidin obtained after fermentation is finished, the yield of the obtained neohesperidin finished product is about 85%, and industrialization of the neohesperidin fermentation process is achieved.

Description

Technical Field

[0001] This invention belongs to the field of separation and purification technology, specifically relating to a method for separating a new hesperidin product. Background Technology

[0002] Neohesperidin is a flavonoid glycoside sweetener and a type of vitamin P drug. Its hydroxylation yields neohesperidin dihydrochalcone (NHDC), which is 1500-1800 times sweeter than sucrose. NHDC has extremely low caloric value (8.36 J / g) and high safety. Furthermore, NHDC possesses strong antioxidant properties and is already used as a cosmetic additive. NHDC also exhibits anti-inflammatory, hypoglycemic, and hepatoprotective activities.

[0003] Currently, the main focus is on constructing a Yersinia lipolytica chassis for the synthesis of neohesperidin and screening for highly efficient endogenous methyltransferases. The synthesis of neohesperidin from naringenin, starting with tyrosine or phenylalanine, requires approximately nine steps. The synthesis of neohesperidin from naringenin requires multi-step catalysis by p450 enzymes, oxygen methyltransferases, and glycosyltransferases. The reaction mechanism is as follows:

[0004]

[0005] Existing neohesperidin products are basically extracted directly from plants using plant extraction methods. While the separation process for neohesperidin produced by fermentation is mature, it cannot be scaled up. To address the shortcomings of existing processes, it is necessary to develop a method for preparing neohesperidin products that can achieve continuous industrial production. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the purpose of this invention is to extract neohesperidin from fermentation broth. Utilizing the characteristic that neohesperidin is readily soluble in alkaline water but poorly soluble in acidic water, the pH of the fermentation broth is adjusted, impurities in the fermentation broth are removed using membrane separation, and then the product is obtained through concentration, cooling, and crystallization.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] On the one hand, the present invention provides a method for separating a novel hesperidin product, comprising the following steps:

[0009] (1) Add alkali to the new hesperidin fermentation tank after fermentation is completed, stir, adjust the pH of the fermentation liquid to alkaline, and then separate the fermentation liquid after adding alkali through a membrane to obtain clear liquid and concentrated liquid. Discharge the concentrated liquid into the sewage tank and pump the clear liquid into the concentration tank.

[0010] (2) Add acid to the clear liquid in the concentration vessel, stir, adjust the pH of the clear liquid to acidic, then turn on the system vacuum pump to perform negative pressure treatment on the concentration vessel, turn on the steam in the reaction vessel jacket to heat up the concentration vessel, concentrate the clear liquid, and pump the obtained concentrated liquid into the crystallization vessel.

[0011] (3) Start the crystallization kettle to stir and cool the crystallization kettle jacket cooling water, stir the concentrated liquid to cool down and crystallize, and obtain the crystallized liquid. Then, centrifuge the crystallized liquid to obtain the new hesperidin product.

[0012] As a preferred embodiment, the pH of the fermentation broth in step (1) is adjusted to an alkaline pH of 10-12.

[0013] As a preferred embodiment, the alkali mentioned in step (1) above is a sodium hydroxide solution, and the concentration of the sodium hydroxide solution is 32%.

[0014] As a preferred embodiment, the membrane separation in step (1) above is performed using a ceramic membrane with a porosity of 50 μm.

[0015] As a preferred embodiment, the pH of the clear liquid in step (2) above is adjusted to an acidic pH of 5-7.

[0016] As a preferred embodiment, the acid mentioned in step (2) above is hydrochloric acid, and the concentration of the hydrochloric acid is 31%.

[0017] As a preferred embodiment, the volume ratio of concentrated liquid to clear liquid in step (1) above is 1:10.

[0018] As a preferred embodiment, the crystallization temperature in step (3) above is 10-30°C.

[0019] As a preferred embodiment, the crystallization time in step (3) above is 20-24 hours.

[0020] On the other hand, the present invention also provides a new hesperidin product obtained by the above separation method.

[0021] Furthermore, this invention also provides the application of the novel hesperidin product obtained by the above separation method in the preparation of food additives.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] (1) This invention utilizes the solubility of neohesperidin to separate neohesperidin produced by fermentation of Yeast lipolyticis into a finished product.

[0024] (2) By separating the new hesperidin, the entire production process is industrialized, which significantly increases the economic benefits of enterprises. Attached Figure Description

[0025] Figure 1 This is a simplified process flow diagram of the present invention. Specific implementation methods

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the tables in the embodiments of the present invention. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0027] The following examples and comparative examples of the present invention are shown to provide a more detailed description of the compositions of the present invention, but the present invention is not limited thereto.

[0028] Combination Figure 1 The present invention will be further described below.

[0029] Basic Example: A Method for Separating a Novel Hesperidin Product

[0030] Includes the following steps:

[0031] (1) Start stirring and slowly add 32% liquid alkali to the fermentation broth in the 5000L fermenter (new hesperidin concentration of about 20g / L) to adjust the pH of the fermentation broth to 10-12. After stirring for half an hour, the pH of the fermentation broth stabilizes between 10-12. Start the ceramic membrane equipment and pump the fermentation broth into the ceramic membrane feed settling tank. Separate the fermentation broth through the ceramic membrane and obtain the ceramic membrane clear liquid and ceramic membrane concentrated liquid by membrane separation. Discharge the ceramic membrane concentrated liquid into the sewage tank and pump the ceramic membrane clear liquid into the concentration kettle.

[0032] (2) Open the 5000L concentration vessel and stir. Slowly add 31% hydrochloric acid to the concentration vessel to adjust the pH of the ceramic membrane solution to 5-7. Stir for half an hour to stabilize the pH of the ceramic membrane solution between 5-7.

[0033] (3) Turn on the system vacuum pump and the steam in the jacket of the concentrator to concentrate the ceramic membrane solution in the concentrator until it is concentrated to about 10% of the ceramic membrane solution volume. Then turn off the steam, turn off the system vacuum pump, and stop the concentration. Pump the concentrated solution into the crystallization tank.

[0034] (4) Turn on the crystallization vessel and stir. Turn on the 1000L crystallization vessel jacket cooling water and control the temperature of the crystallization vessel between 10-30 degrees. After stirring and crystallizing for 24 hours, centrifuge the crystallized liquid through a centrifuge to obtain the new hesperidin product.

[0035] Example 1: A method for separating a new hesperidin product

[0036] Includes the following steps:

[0037] (1) Start stirring and slowly add 300L of 32% liquid alkali to 3000L of fermentation liquid (neohesperidin concentration 20g / L) in a 5000L fermenter. Adjust the pH of the fermentation liquid to 10-12. After stirring for half an hour, the pH of the fermentation liquid stabilizes at 11.2. Start the ceramic membrane equipment and pump the fermentation liquid into the ceramic membrane feed settling tank. Separate the 3 cubic meters of fermentation liquid through the ceramic membrane to obtain 3000L of clear ceramic membrane liquid and 1000L of concentrated ceramic membrane liquid. Pump the clear liquid into the concentration kettle and the concentrated liquid into the sewage tank.

[0038] (2) Slowly add 300L of 31% hydrochloric acid to the concentration vessel to adjust the pH of the ceramic membrane solution to 5-7. After stirring for half an hour, the pH of the ceramic membrane solution stabilizes at 6.1.

[0039] (3) Start the system vacuum pump, turn on the steam in the jacket of the concentrator, and concentrate the ceramic membrane clear liquid in the concentrator. When the volume of the distillate reaches 2900L, turn off the steam, turn off the system vacuum pump, stop the concentration, and pump the concentrated liquid into the crystallization vessel.

[0040] (4) Turn on the crystallizer and stir. Turn on the 1000L crystallizer jacket cooling water and control the temperature of the crystallizer at 20 degrees. After stirring and crystallizing for 24 hours, put the crystallized liquid into the centrifuge to centrifuge and obtain 280L of centrifuged mother liquor, 100kg of neohesperidin product, 45% moisture, and 95% neohesperidin content.

[0041] Example 2: A method for separating a new hesperidin product

[0042] Includes the following steps:

[0043] (1) Start stirring and slowly add 310L of 32% liquid alkali to 3000L of fermentation liquid (neohesperidin concentration 20.2g / L) in a 5000L fermenter. Adjust the pH of the fermentation liquid to 10-12. After stirring for half an hour, the pH of the fermentation liquid stabilizes at 11. Start the ceramic membrane equipment and pump the fermentation liquid into the ceramic membrane feed settling tank. Separate the 3 cubic meters of fermentation liquid through the ceramic membrane to obtain 3000L of clear ceramic membrane liquid and 1000L of concentrated ceramic membrane liquid. Pump the clear liquid into the concentration kettle and the concentrated liquid into the sewage tank.

[0044] (2) Slowly add 31% hydrochloric acid 312L to the concentration vessel to adjust the pH of the ceramic membrane clear solution to 5-7. After stirring for half an hour, the pH of the ceramic membrane clear solution stabilizes at 6.3.

[0045] (3) Start the system vacuum pump, turn on the steam in the jacket of the concentrator, and concentrate the ceramic membrane clear liquid in the concentrator. When the volume of the distillate reaches 2900L, turn off the steam, turn off the system vacuum pump, stop the concentration, and pump the concentrated liquid into the crystallization vessel.

[0046] (2) Turn on the crystallizer and stir. Turn on the 1000L crystallizer jacket cooling water and control the temperature of the crystallizer at 20 degrees. After stirring and crystallizing for 24 hours, put the crystallized liquid into the centrifuge to centrifuge and obtain 270L of centrifuged mother liquor, 102kg of neohesperidin product, 48% moisture, and 97.5% neohesperidin content.

[0047] Example 3: A method for separating a new hesperidin product

[0048] Includes the following steps:

[0049] (1) Start stirring and slowly add 304L of 32% liquid alkali to 3000L of fermentation liquid (neohesperidin concentration 19.5g / L) in a 5000L fermenter. Adjust the pH of the fermentation liquid to 10-12. After stirring for half an hour, the pH of the fermentation liquid stabilizes at 10.95. Start the ceramic membrane equipment and pump the fermentation liquid into the ceramic membrane feed settling tank. Separate the 3 cubic meters of fermentation liquid through the ceramic membrane to obtain 3000L of clear ceramic membrane liquid and 1000L of concentrated ceramic membrane liquid. Pump the clear liquid into the concentration kettle and the concentrated liquid into the sewage tank.

[0050] (2) Slowly add 305L of 31% hydrochloric acid into the concentration vessel to adjust the pH of the ceramic membrane solution to 5-7. After stirring for half an hour, the pH of the ceramic membrane solution stabilizes at 6.0.

[0051] (3) Start the system vacuum pump, turn on the steam in the jacket of the concentrator, and concentrate the ceramic membrane clear liquid in the concentrator. When the volume of the distillate reaches 2900L, turn off the steam, turn off the system vacuum pump, stop the concentration, and pump the concentrated liquid into the crystallization vessel.

[0052] (4) Turn on the crystallizer and stir. Turn on the 1000L crystallizer jacket cooling water and control the temperature of the crystallizer at 20 degrees. After stirring and crystallizing for 24 hours, put the crystallized liquid into the centrifuge to centrifuge and obtain 273L of centrifuged mother liquor, 103kg of neohesperidin product, 47% moisture, and 97% neohesperidin content.

[0053] The specific separation yields are shown in Table 1 below.

[0054] Table 1. Yield of neohesperidin product in different examples

[0055] Weight of neohesperidin before separation Weight of neohesperidin after separation Separation yield Example 1 3000 * 20 = 60 kg 100 * 0.55 * 0.97 = 52.25 kg 87％ Example 2 3000 * 20.2 = 60.6 kg 102 * 0.52 * 0.975 = 51.71 kg 85.3％ Example 3 3000 * 19.5 = 58.5 kg 103 * 0.53 * 0.97 = 52.95 kg 90.5％

[0056] As shown in Table 1, after a complete set of separation processes including alkaline dissolution, membrane separation, acid precipitation, concentration, cooling crystallization, and centrifugal separation, the yield of neohesperidin product is about 87%, thus realizing the industrialization of neohesperidin fermentation process.

[0057] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the scope of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the scope of the present invention, shall still fall within the protection scope of the present invention.

Claims

1. A method for isolating a new hesperidin product, characterized by: It comprises the following steps: (1) adding alkali to the new hesperidin fermentation tank at the end of fermentation, stirring, adjusting the pH of the fermentation liquor to alkaline, then separating the alkali-added fermentation liquor by membrane to obtain clear liquid and thick liquid, discharging the thick liquid into a sewage tank, and pumping the clear liquid into a concentration kettle; (2) adding acid to the clear liquid in the concentration kettle, stirring, adjusting the pH of the clear liquid to acidic, then opening the system vacuum pump to perform negative pressure treatment on the concentration kettle, opening the reaction kettle jacket steam to heat the concentration kettle, concentrating the clear liquid, and pumping the obtained concentrated liquid into a crystallization kettle; (3) opening the crystallization kettle stirring, opening the crystallization kettle jacket cooling water, stirring and cooling the concentrated liquid to crystallize, obtaining crystallization liquid, and obtaining new hesperidin finished product by centrifugal separation of the crystallization liquid.

2. The separation method of claim 1, wherein: The pH of the fermentation liquor adjusted to alkaline in step (1) is 10-12.

3. The separation method of claim 1, wherein: The alkali in step (1) is sodium hydroxide solution, and the concentration of the sodium hydroxide solution is 32%.

4. The separation method of claim 1, wherein: The membrane separation in step (1) is carried out by using a ceramic membrane with a porosity of 50 μm.

5. The separation method of claim 1, wherein: The pH of the clear liquid adjusted to acidic in step (2) is 5-7.

6. The separation method of claim 1, wherein: The acid in step (2) is hydrochloric acid, and the concentration of the hydrochloric acid is 31%.

7. The separation method of claim 1, wherein: The temperature of the crystallization in step (3) is 10-30℃.

8. The separation method of claim 1, wherein: The time of the crystallization in step (3) is 20-24 hours.

9. A new hesperidin finished product obtained by the separation method of any one of claims 1-8.

10. Use of the new hesperidin finished product obtained by the separation method of any one of claims 1-8 in the preparation of food additives.

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