A method for extracting erythritol through fermentation

Abstract

The invention belongs to the technical field of erythritol production, and discloses a method for extracting erythritol by fermentation, which mainly utilizes ceramic membrane filtration technology to remove bacterial protein, and uses decolorization membrane filtration technology to basically achieve complete separation between erythritol and miscellaneous acids and other impurities. The invention can more effectively remove impurities in erythritol fermentation liquid to obtain high-purity fine erythritol; and after membrane filtration and concentration, evaporation and crystallization can also reduce production costs, save energy and water, and meet the concept of environmental protection.

Description

Technical Field

[0001] The invention belongs to the technical field of erythritol production, and particularly relates to a method for extracting erythritol through fermentation. Background Art

[0002] Erythritol is a bulking sweetener with a molecular formula of C4H10O4. Erythritol is widely found in nature, such as fungi such as mushrooms and lichens, melons, grapes, and pears, and can also be detected in small amounts in animal eye lenses, plasma, fetal fluid, semen, and urine. It also exists in small amounts in fermented foods such as wine, beer, soy sauce, and Japanese sake. Erythritol is made by fermenting glucose and is a white crystalline powder with a refreshing sweet taste. It is not hygroscopic, stable at high temperatures, stable in a wide pH range, and has a mild cool feeling when dissolved in the mouth, making it suitable for a variety of foods.

[0003] In recent years, erythritol has been used in the development of new zero-calorie and low-calorie beverages. Erythritol can increase the sweetness, thickness and lubricity of beverages, reduce bitterness, cover up other odors and improve the flavor of beverages. Erythritol can also be used in refreshing solid beverages. Erythritol can promote the solution combination of ethanol molecules and water molecules. Alcoholic beverages can reduce the sensory stimulation of odor and alcohol, and can effectively improve the quality of liquor and wine. Erythritol can also significantly improve the unpleasant odor of plant extracts, collagen, peptides and other substances. With the improvement of people's living standards, the baking industry is developing towards nutrition, health, functionality, low calories and other aspects. Erythritol can not only replace sucrose to increase flavor, but also reduce food calories. At the same time, food has better structural tightness and softness, and has a smooth and round taste. In addition, in the pharmaceutical industry, the anti-caries, antioxidant, moisturizing and non-flammable properties of erythritol have continuously expanded its application in the fields of medicine and daily chemicals.

[0004] With the upgrading of consumption and the improvement of human health awareness, residents of various countries pay more attention to food health, the market demand for erythritol has increased, and the industry has developed rapidly. my country is a major producer of erythritol, and domestic production capacity is mainly used for export. In the future, erythritol has high development potential in both domestic and overseas markets, and there are great development opportunities in the erythritol industry. The domestic production method of erythritol is mainly based on fermentation, and the extraction and separation process can largely determine the quality and yield of erythritol. Therefore, studying methods to improve the extraction yield of erythritol has important theoretical significance and practical value. Summary of the invention

[0005] The invention provides a method for extracting erythritol with simple operation and high efficiency, which can be used for producing erythritol by fermentation method.

[0006] The present invention is achieved through the following technical solutions.

[0007] A method for extracting erythritol by fermentation comprises the following steps: step 1) fermentation, step 2) ceramic membrane filtration, step 3) decolorization, step 4) four-effect evaporation concentration, step 5) decolorization again, and step 6) concentration and crystallization.

[0008] Furthermore, the method comprises the following steps:

[0009] Step 1) fermentation: inoculating Candina lipolytica Ery-2020-12 seed liquid at an inoculum amount of 5-15% in a fermenter filled with fermentation medium, culturing at a temperature of 30° C., a ventilation ratio of 1:1.5, a tank pressure of 0.08 MPa, and a dissolved oxygen control of 35%, and a fermentation period of 80 h;

[0010] Step 2) Ceramic membrane filtration: The erythritol fermentation liquid is heated to 60° C. and poured into an extraction tank. After adjusting the pH value to 3-5 with hydrochloric acid, the fermentation liquid is filtered through a ceramic membrane to obtain a filtered filtrate;

[0011] Step 3) decolorization: the erythritol filtrate obtained after filtering in step 2) is passed through a decolorization membrane for decolorization and filtration to obtain a high-purity erythritol filtrate;

[0012] Step 4) Four-effect evaporation concentration: the high-purity erythritol filtrate obtained in step 3) is subjected to four-effect evaporation concentration at a temperature of 80-120° C. and a steam pressure of 0.2-0.4 MPa, and crystallized after cooling, and centrifuged to obtain crude erythritol;

[0013] Step 5) decolorizing again: dissolving the crude erythritol obtained in step 4) in water, controlling the pH to 5.5-6.0 and the liquid temperature to 70-80° C., decolorizing and filtering again using a decolorizing membrane to obtain an erythritol solution with higher purity;

[0014] Step 6) Concentration and crystallization: The erythritol solution obtained in step 5) is concentrated twice, crystallized, and centrifuged to obtain fine erythritol.

[0015] Preferably,

[0016] The fermentation medium comprises the following components: 330 g / L glucose, 8 g / L yeast extract, 5 g / L ammonium chloride, 0.3 g / L potassium dihydrogen phosphate, 5 g / L citric acid, 0.5 g / L magnesium sulfate, 0.005 g / L copper chloride and 0.16 g / L defoamer.

[0017] Preferably,

[0018] The molecular weight cut-off of the ceramic membrane is 2000Da.

[0019] Preferably,

[0020] The parameters of the decolorization and filtration in step 3) are: temperature of 38±5° C. and pressure of 0.3±0.05 MPa.

[0021] The technical solution of the present invention has the following outstanding advantages and uniqueness:

[0022] 1. The present invention adopts multi-stage membrane to extract erythritol, and its production process is simple, easy to operate and maintain, small in footprint, reduces material utilization, saves costs, conforms to environmental protection concepts, and can be continuously produced industrially.

[0023] 2. The erythritol solution extracted by the present invention is concentrated and crystallized, and the total extraction yield is greater than 92%, and the product purity is greater than 99%.

[0024] 3. The production process of the present invention is fully automated, has low labor intensity, and can effectively save costs.

[0025] 4. In the process of extracting erythritol, the present invention recycles bacterial protein and the like, which greatly reduces the discharge of wastewater and avoids the pollution of the environment caused by waste liquid, which is green and environmentally friendly; and nutrient-rich bacterial protein feed is obtained, which effectively recycles resources and realizes the resource utilization of waste. DETAILED DESCRIPTION

[0026] In order to enable those skilled in the art to better understand the technical solution in the present application, the technical solution will be fully described below in conjunction with the specific embodiments of the present application.

[0027] Example 1

[0028] Candina lipolytica Ery-2020-12 was deposited on October 12, 2021 in the General Microbiology Center of China Microorganism Culture Collection (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101), with the deposit number CGMCC No. 23582.

[0029] The suspension of Candida lipolytica Ery-2020 retained in the laboratory for the production of erythritol was taken and poured into a sterilized plate for ultraviolet irradiation mutagenesis. Different ultraviolet irradiation mutagenesis times were set, namely 10, 20, 40, and 80 min, and the time dose was gradually increased from low to high. After each mutagenesis, enriched culture medium was added for culturing for 24 hours, and then the next mutagenesis was continued. Finally, a strain of Candida lipolytica Ery-2020-12 with high erythritol production was obtained through conventional separation and purification methods.

[0030] Example 2

[0031] A method for producing erythritol by fermentation using erythritol-producing bacteria, comprising a fermentation strain shake flask culture stage, a primary seed tank expansion culture stage and a fermentation stage, and a production process in which an erythritol fermentation sugar-supplementing device is used to evenly distribute the added sugar and perform efficient fermentation culture, comprising the following steps:

[0032] Step 1): Activate the low-temperature stored Candina lipolytica Ery-2020-12.

[0033] Step 2): The bacterial solution obtained in step 1) was inoculated into a 2L shake flask containing a primary seed culture medium at an inoculum size of 0.5%. After the culture medium was sterilized, the pH value was controlled at 7.0 by ammonia water before inoculation. The temperature was 30°C, and the culture was cultured at 100 rpm for 24 hours until the OD value of the bacterial solution reached 0.4, and the culture was terminated.

[0034] Step 3): The first-level shake flask seeds obtained in step 2) were inoculated at an inoculum of 1.2% into 25 m of the second-level seed culture medium. 3 In the seed tank, the liquid volume is 40%, the temperature is 30℃, the ventilation ratio is 1:1.2, the tank pressure is 0.08MPa, the dissolved oxygen is controlled at 30%, and the pH value of the culture medium is controlled at 7.0 by ammonia water before inoculation after sterilization. The culture is cultured at 200rpm for 12 hours until the OD value of the bacterial liquid is in the range of 0.42~0.51, and the culture is terminated.

[0035] Step 4): The secondary fermentation tank seeds obtained in step 3) are inoculated with 10% inoculation amount in a fermentation tank filled with fermentation medium for cultivation, with a liquid volume of 50%, a temperature of 30°C, a ventilation ratio of 1:1.5, a rotation speed of 300rpm, a tank pressure of 0.08MPa, and a dissolved oxygen control of 35%. The pH value of the culture medium is adjusted to 5.0 by ammonia water before sterilization, and the pH value is controlled at 7.0 before inoculation after sterilization. During the fermentation process, the residual sugar content is detected every 4 hours. When the residual sugar content is detected to be 30 g / L, glucose with a volume of 10% of the liquid volume and a mass fraction of 75% is added. Monitoring and addition are performed twice until the sugar content is exhausted and the fermentation ends. The fermentation cycle is 80 hours.

[0036] The components of the primary culture medium are: glucose 40 g / L, peptone 10 g / L.

[0037] The components of the secondary seed culture medium are: glucose 150 g / L, yeast extract powder 8 g / L, potassium dihydrogen phosphate 0.5 g / L, magnesium sulfate 0.2 g / L, citric acid 3.0 g / L, and defoaming agent 0.02 g / L.

[0038] The components of the fermentation medium are: 330 g / L glucose, 8 g / L yeast extract powder, 0.3 g / L potassium dihydrogen phosphate, 5 g / L citric acid, 0.5 g / L magnesium sulfate, 0.005 g / L copper chloride, and 0.16 g / L defoaming agent.

[0039] Example 3

[0040] The fermentation production of erythritol by different strains was compared, and the fermentation process was referred to Example 2.

[0041] Table 1

[0042] strain Erythritol yield g / L Sugar-acid conversion rate% Ery-2020 197.6 53.7 Ery-2020-12 261.3 61.4

[0043] As shown in Table 1 above, compared with the starting strain, after mutagenesis screening, the fermentation production of erythritol by strain Ery-2020-12 was significantly increased, which was more than 30% higher than that of the starting strain. Correspondingly, the sugar-acid conversion rate also increased by 7.7 percentage points.

[0044] Example 4

[0045] The inorganic nitrogen source ammonium chloride was increased by 5 g / L in the fermentation medium formula, while the other components and dosages remained unchanged, and the fermentation method remained unchanged.

[0046] Ery-2020-12 strain: Different from Example 2, it consumes sugar slightly slower. It takes a longer time to consume the same amount of sugar. The final erythritol production is 287.3 g / L.

[0047] Starting strain Ery-2020: There was no significant change in the yield of erythritol and sugar consumption time during fermentation.

[0048] Example 5

[0049] Effect of fermentation temperature on erythritol production of different strains.

[0050] The fermentation process is described in Example 2, as shown in Table 2:

[0051] Table 2

[0052] strain 29℃ 31℃ Ery-2020 195.8 194.4 Ery-2020-12 245.9 251.7

[0053] As shown in Table 2 above, Ery-2020-12 is more sensitive to temperature, and changes in temperature can significantly affect the erythritol production; while the starting strain Ery-2020 is not sensitive to temperature changes and has no significant effect on the fermentation erythritol production.

[0054] Example 6

[0055] A method for extracting erythritol using a multi-stage membrane, the specific implementation steps are as follows:

[0056] Step 1) The erythritol fermentation liquid is heated to 60° C. and pumped into an extraction tank. After adjusting the pH value to 4 with hydrochloric acid, the fermentation liquid is filtered through a ceramic membrane. The pore size of the ceramic membrane is 50-70 nm and the molecular weight cutoff is 2000 Da. The ceramic membrane filtration can remove macromolecular bacterial proteins and residues to obtain a filtered filtrate.

[0057] Step 2) The erythritol filtrate obtained after filtering in step 1) is passed through a decolorizing membrane, the temperature is controlled at 38° C., and the pressure is 0.3 MPa, and decolorization and filtration are performed to basically achieve complete separation of erythritol from miscellaneous acids and other impurities, thereby obtaining a high-purity erythritol filtrate.

[0058] Step 3) The high-purity erythritol filtrate obtained in step 2) is subjected to four-effect evaporation concentration at a temperature of 100° C. and a steam pressure of 0.3 MPa, cooled and crystallized, and centrifuged to obtain crude erythritol.

[0059] Step 4) The crude erythritol obtained in step 3) is dissolved in water, the pH is controlled to be 5.5, the liquid temperature is controlled to be 70° C., and the erythritol is decolorized and filtered again using a decolorizing membrane to obtain an erythritol solution with higher purity.

[0060] Step 5) The erythritol solution obtained in step 4) is concentrated twice, crystallized, and centrifuged to obtain fine erythritol.

[0061] After testing: the total extraction yield is greater than 92%, and the product purity is greater than 99%.

[0062] Although the above describes the specific implementation of the present invention in combination with the embodiments, it is not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that it should be clear to those skilled in the art that these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection of the present invention.

Claims

1. A method for extracting erythritol by fermentation, comprising the following steps: step 1) fermentation, step 2) ceramic membrane filtration, step 3) decolorization, step 4) four-effect evaporation concentration, step 5) decolorization again, and step 6) concentration and crystallization; Step 1) Fermentation: Candida lipolytica (CGMCC No. 23582) Candina lipolytica ) Ery-2020-12 seed liquid was inoculated in a fermentation tank filled with fermentation medium at an inoculum of 5-15%, the temperature was 30°C, the ventilation ratio was 1:1.5, the tank pressure was 0.08MPa, the dissolved oxygen was controlled at 35%, and the fermentation period was 80h; Step 2) Ceramic membrane filtration: The erythritol fermentation liquid is heated to 60° C. and poured into an extraction tank. After adjusting the pH value to 3-5 with hydrochloric acid, the erythritol fermentation liquid is poured into a ceramic membrane for filtration to obtain a filtered filtrate. Step 3) Decolorization: The filtrate obtained in step 2) is passed through a decolorization membrane, decolorized and filtered to obtain a high-purity erythritol filtrate; Step 4) Four-effect evaporation concentration: the high-purity erythritol filtrate obtained in step 3) is subjected to four-effect evaporation concentration at a temperature of 80-120° C. and a steam pressure of 0.2-0.4 MPa, and crystallized after cooling, and centrifuged to obtain crude erythritol; Step 5) decolorizing again: dissolving the crude erythritol obtained in step 4) in water, controlling the pH to 5.5-6.0 and the liquid temperature to 70-80° C., decolorizing and filtering again using a decolorizing membrane to obtain an erythritol solution with higher purity; Step 6) Concentration and crystallization: The erythritol solution obtained in step 5) is concentrated twice, crystallized, and centrifuged to obtain fine erythritol.

2. The method according to claim 1, characterized in that The fermentation medium comprises the following components: 330 g / L glucose, 8 g / L yeast extract, 5 g / L ammonium chloride, 0.3 g / L potassium dihydrogen phosphate, 5 g / L citric acid, 0.5 g / L magnesium sulfate, 0.005 g / L copper chloride and 0.16 g / L defoamer.

3. The method according to claim 1, characterized in that The molecular weight cut-off of the ceramic membrane is 2000Da.

4. The method according to claim 1, characterized in that The parameters of the decolorization and filtration in step 3) are: temperature of 38±5° C. and pressure of 0.3±0.05 MPa.