CN110241145B

CN110241145B - Fermentation preparation method of mannitol

Info

Publication number: CN110241145B
Application number: CN201910570857.XA
Authority: CN
Inventors: 吴汉夔; 李刚; 孙凯; 邓德华; 段宁慧; 王宇航
Original assignee: Anyang Normal University
Current assignee: Anyang Normal University
Priority date: 2019-06-27
Filing date: 2019-06-27
Publication date: 2023-04-25
Anticipated expiration: 2039-06-27
Also published as: CN110241145A

Abstract

The invention belongs to the technical field of compound preparation, and particularly discloses a fermentation preparation method of mannitol, which comprises the following steps: (1) Inoculating the carbon horn fungus strain into a culture bottle, and culturing for 6-8 days; (2) Taking 50 conical flasks, filling rice and water into the conical flasks, sterilizing, and cooling to room temperature; inoculating the strain in the step (1) into a rice culture medium; (3) Adding ethanol solution into the culture flask in the step (2) to soak for 28-30 days to obtain a first mixture; (4) Filtering the first mixture in the step (3), putting the filtrate into a conical flask, adding an ethanol solution into the flask, and soaking for 6-8 days to obtain a second mixture; (5) Filtering the second mixture in the step (4), putting the filtrate into the conical flask in the step (4), removing the solvent, and performing extraction crystallization to obtain a white needle-like crystal product. The method is mainly used for preparing mannitol, and solves the problem of complex production process flow of mannitol synthesized by the prior art.

Description

Fermentation preparation method of mannitol

Technical Field

The invention belongs to the technical field of compound preparation, and particularly discloses a fermentation preparation method of mannitol.

Background

Mannitol has wide application (such as diuretic, anti-sticking of maltose, chewing gum and rice cake, plasticizer of polyvinyl chloride, etc.), and can be widely applied to medicine, food, industrial production, etc. At present, the world industrial production of mannitol mainly comprises two processes, namely, taking kelp as a raw material, producing alginate and simultaneously carrying out concentration, impurity removal, ion exchange, evaporation concentration and cooling crystallization on kelp soak solution subjected to iodine extraction for a plurality of times; one is obtained by taking sucrose and glucose as raw materials, hydrolyzing, epimerising and enzymatically isomerizing, and then hydrogenating.

The process for extracting mannitol from kelp has been used for decades, and is simple and feasible, but is limited by raw material resources, extraction yield, climatic conditions, energy consumption and the like, and the development of the process is limited for a long time. The synthesis process in China starts the test in the eighties of the last century and is available in the nineties, and has been developed to a great extent because the synthesis process is not limited by raw materials and is suitable for large-scale production. However, the method for synthesizing mannitol in the prior art has the defects of low purity of the obtained product, complex production process flow, low production yield and low production efficiency.

Disclosure of Invention

The invention aims to provide a fermentation preparation method of mannitol, which aims to solve the problem of complex production process flow of mannitol synthesis in the prior art.

In order to achieve the above purpose, the technical scheme of the invention is as follows: a fermentation preparation method of mannitol, comprising the following steps:

(1) Inoculating the strain of the carbon horn fungus into 5 culture bottles filled with culture medium, standing overnight, and culturing at the constant temperature of 23-25 ℃ for 6-8 days;

(2) Taking 50 500mL clean conical flasks, filling 70-80g of rice and 110-120mL of water into each flask, sterilizing, and cooling to room temperature; inoculating the strain in the step (1) into a rice culture medium, and standing and culturing in an air-conditioning room at 28 ℃ for 35-37 days;

(3) Adding 250-300mL of 80% ethanol solution into the culture flask in the step (2), and soaking for 28-30 days to obtain a first mixture;

(4) Filtering the first mixture in the step (3), putting the filtrate into a conical flask, adding 240-250ml of 95% ethanol solution into the flask, and soaking for 6-8 days to obtain a second mixture;

(5) Filtering the second mixture in the step (4), putting the filtrate into a conical flask in the step (4), removing the solvent to obtain a crude product, extracting the crude product with an organic solvent for 2-3 times to obtain a product solution, removing the organic solvent, and standing for 0.5-1 days to obtain a white needle-like crystal product.

The working principle and the beneficial effects of the technical scheme are as follows:

(1) Compared with the prior art, the preparation method has the advantages that the preparation process is simple, mannitol is prepared by colony fermentation, the number of used raw materials is reduced, the colony fermentation is adopted, the preparation condition is simple, the energy consumption is low compared with the synthesis process, and the production cost is low;

(2) The method has less toxic and harmful gas discharged in the process of preparing mannitol and less environmental pollution.

Further, inoculating the strain of the carbon-horn-shaped bacteria in the step (1), and simultaneously inoculating and culturing the strain in a flat dish at 25 ℃. Therefore, the strain can be further preserved for the subsequent experiment to be used again.

Further, in the step (2), the sterilization temperature is 120-122 ℃, and the sterilization time is 20-25min. The temperature and time in the above range can be used for fully sterilizing the conical flask, rice and water, and prevent pollution in the culture process.

Further, the organic solvent in the step (4) is ethyl acetate, n-butanol or petroleum ether. The ethyl acetate or petroleum ether is used for extraction, the solvent is easy to obtain and remove, and the used solvent has little pollution to the environment.

Further, the strain in the step (2) is inoculated into a rice culture medium and placed in an air-conditioning room at 28 ℃ for static culture for 36 days, and the fungus grows in the whole culture bottle within the time.

Further, in the step (3), the mixture is soaked in an ethanol solution with the mass fraction of 80% for 30 days. The extraction rate can be higher by adopting the time, and the extraction rate can not be improved by soaking for a longer time.

Drawings

FIG. 1 is a diagram showing the structural formula of mannitol in an example of a method for producing mannitol by fermentation according to the present invention;

FIG. 2 is a DEPT135 spectrum of mannitol in an embodiment of the present invention;

FIG. 3 is a nuclear magnetic resonance chromatogram of mannitol in an embodiment of the present invention;

FIG. 4 is a hydrogen nuclear magnetic resonance spectrum of mannitol in an embodiment of the present invention;

FIG. 5 is a DEPT90 spectrum of mannitol in an example of the present invention.

Detailed Description

The following is a further detailed description of the embodiments:

the embodiment is basically as shown in fig. 1, and the fermentation preparation method of mannitol comprises the following steps:

(1) Inoculating the strain BNCC-119006 (inclined plane) of the genus Xylaria into a culture flask containing 5 culture medium in a conical flask with 100mL potato dextrose broth, standing overnight, and culturing at 23-25deg.C for 6-8 days; simultaneously inoculating and culturing in a flat dish at 25 ℃;

(2) Taking 50 500mL clean conical flasks, filling 70-80g of rice and 110-120mL of water into each conical flask, sterilizing, and cooling to room temperature, wherein the sterilization temperature is 120-122 ℃ and the sterilization time is 20-25min; inoculating the strain in the step (1) into a rice culture medium, and standing and culturing in an air-conditioning room at 28 ℃ for 35-37 days, wherein the preferred time is 36 days in the embodiment;

(3) Adding 250-300mL of 80% ethanol solution into the culture flask in the step (2) to soak for 28-30 days to obtain a first mixture, wherein the soaking time is preferably 30 days in the embodiment;

(4) Filtering the first mixture in the step (3), putting the filtrate into a 2000mL conical flask, adding 240-250mL of 95% ethanol solution into the flask, and soaking for 6-8 days to obtain a second mixture, wherein the soaking time is preferably 7 days in the embodiment;

(5) Filtering the second mixture in the step (4), putting the filtrate into a conical flask in the step (4), removing the solvent to obtain a crude product, extracting the crude product with ethyl acetate or petroleum ether for 2-3 times to obtain a product solution, removing the organic solvent, and standing for 0.5-1 days to obtain a white needle-like crystal product 1.

The compounds were tested using a nuclear magnetic resonance spectrometer from Avance-III 400MHz, bruker, switzerland, which shows that there is a pronounced polyhydroxy compound in the hydrogen spectrum between delta 3.30 and 3.65, as shown in FIG. 4. The nmr spectrum showed three sets of carbon signals, shown in fig. 2 as δ:71.9, 70.1, 64.4. According to DEPT135 spectrum 3, three groups of carbon have two upward peaks and one reverse peak respectively, so that the compound is judged to have two groups of oxygen-linked CH and one group of CH2. Further description of chemical shift δ71.9, 70.1 is methine of oxygen according to DEPT90 spectrum 5. These information are substantially consistent with the standard mannitol data in the AIST database, and their HMBC, HMQC spectra further illustrate that the compound is mannitol. From the literature database (https:// sdbs.db.air. Go. Jp/sdbs/cgi-bin/cre_index. Cgi) query, six carbon spectrum carbon signals of sorbitol in heavy water were 73.98, 72.32, 72.22, 70.75, 63.93, 63.58, respectively. The compound 1 has only three groups of carbon signals, so that the structure of the compound is better symmetrical, and the structure of the compound 1 can be further deduced as shown in figure 1.

The foregoing is merely exemplary embodiments of the present invention, and specific structures and features that are well known in the art are not described in detail herein. It should be noted that modifications and improvements can be made by those skilled in the art without departing from the structure of the present invention, and these should also be considered as the scope of the present invention, which does not affect the effect of the implementation of the present invention and the utility of the patent.

Claims

1. The fermentation preparation method of mannitol is characterized by comprising the following steps:

(1) Inoculating the carbon horn fungus strain BNCC-119006 into 5 culture bottles filled with culture medium, standing overnight, and culturing at 23-25deg.C for 6-8 days;

(2) Taking 50 500mL clean conical flasks, filling 70-80g of rice and 110-120mL of water into each conical flask, sterilizing, and cooling to room temperature; inoculating the strain in the step (1) into a rice culture medium, and standing and culturing in an air-conditioning room at 28 ℃ for 35-37 days;

(4) Filtering the first mixture in the step (3), putting the filtrate into a 2000mL conical flask, adding 240-250mL of 95% ethanol solution into the flask, and soaking for 6-8 days to obtain a second mixture;

(5) Filtering the second mixture in the step (4), putting the filtrate into a conical flask in the step (4), removing the solvent to obtain a crude product, extracting the crude product with an organic solvent for 2-3 times to obtain a product solution, removing the organic solvent, and standing for 0.5-1 day to obtain a white needle-like crystal product.

2. The method for producing mannitol according to claim 1, wherein the step (1) comprises inoculating a strain of carbon-horn-shaped bacteria and simultaneously inoculating and culturing the strain in a dish at 25 ℃.

3. The method for producing mannitol according to claim 1, wherein the sterilization temperature in step (2) is 120-122 ℃ and the sterilization time is 20-25min.

4. The method for producing mannitol according to claim 1, wherein the organic solvent in step (5) is ethyl acetate, n-butanol or petroleum ether.

5. The method for preparing mannitol by fermentation according to claim 1, wherein the strain in the step (2) is inoculated into a rice culture medium and placed in an air conditioning room at 28 ℃ for static culture for 36 days.

6. The method for fermentative preparation of mannitol according to claim 1, wherein in step (3), 80% by mass of ethanol solution is used for soaking for 30 days.