A method for producing erythritol by fermentation

Abstract

The invention relates to a fermentation method of erythritol, and belongs to the technical field of erythritol fermentation. The method comprises a fermentation strain shake flask culture stage, a primary seed tank expansion culture stage and a fermentation stage. The erythritol fermentation production method adopts a batch sugar supplementation mode to be put into a fermentation tank. The batch sugar supplementation fermentation is adopted, which is conducive to extending the fermentation cycle but not too long, and the time is controlled within the range of 70 to 100 hours, thereby improving the fermentation conversion rate, effectively improving the erythritol yield, and being suitable for large-scale industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of erythritol fermentation, and more specifically relates to a method for producing erythritol by fermentation. Background Art

[0002] The molecular formula of erythritol is C4H 10 O4, molecular weight 122.12. White crystals, slightly sweet, relative sweetness 0.65, refreshing, low calorific value, about one tenth of sucrose calorific value. Soluble in water (37%, 25℃). Easy to crystallize due to low solubility.

[0003] Erythritol is suitable for foods that need the taste of sucrose, such as chocolate and table sugar. It is not degraded by enzymes and can only be excreted from the blood to the urine through the kidneys (easily absorbed by the small intestine). It does not participate in sugar metabolism and blood sugar changes. It has been used as a low-calorie sugar alcohol for decades and is one of the ideal sweeteners for diabetic patients. It does not ferment in the colon, which can avoid gastrointestinal discomfort. It does not cause tooth decay. In addition, erythritol is very stable to heat and acid, and it will hardly brown or decompose under general food processing conditions.

[0004] At present, erythritol has strong competitiveness as a new functional food additive in the international sweetener market. In terms of food processing, erythritol has the following advantages: erythritol has a high sweetness and can be used as a sweetener for food and beverages; erythritol has a small molecular weight, and the solution of the same concentration has a higher osmotic pressure and a lower water activity, which can be used for the processing and preservation of low water activity foods; erythritol has a large heat of dissolution, so solid foods and candies made with it have a more obvious cool feeling; erythritol has a refreshing sweetness, and when it is compounded with other high-sweetness sweeteners such as protein sugar and stevia, it can effectively mask the bitter aftertaste of these sweeteners; erythritol has low calories, so it can be used to produce low-calorie foods and beverages; erythritol can also promote the combination of ethanol molecules and water molecules in the solution, thereby reducing the odor and sensory stimulation of alcohol in alcoholic beverages and improving the quality of distilled spirits and wines.

[0005] Erythritol is also widely used in the pharmaceutical and chemical industries. In the pharmaceutical industry, erythritol can be used as a flavoring agent for medicines and an excipient for tablets, which can effectively improve the taste of medicines. Erythritol can be used as a raw material to synthesize a variety of drugs. For example, dideoxyerythritol generated by deoxygenation of erythritol has anti-HIV activity, and nitroerythritol generated by nitration of erythritol can be used to treat cardiovascular and cerebrovascular diseases and asthma. In the chemical industry, erythritol, as an intermediate in organic synthesis, can be used in the synthesis of alkyd resins, polyesters, polyethers, as well as in the manufacture of paints and explosives. In cosmetics, erythritol can replace part of the glycerol to prevent cosmetics from deteriorating. With technological advances, new uses for erythritol will continue to be developed.

[0006] However, the current fermentation process for producing erythritol in my country has defects such as low acid production capacity and conversion rate, and high fermentation cost. Chinese patent CN200410035911 discloses a method for producing erythritol by fermentation of Candida lipolytica IFFI-Y3068, with a maximum yield of 160g / L. CN200510102929 discloses a method for producing erythritol by fermentation of Candida lipolytica, with a maximum yield of 162g / L and a sugar-acid conversion rate of 46.3%. CN201010611106 discloses a method for producing erythritol by fermentation of corn flour by Candida lipolytica, with a maximum yield of 165g / L for 120 hours of fermentation. CN110804632A continuously optimizes fermentation conditions and produces erythritol by fermentation of specific Candida lipolytica CGMCC NO.6364, with the content of erythritol in the fermentation broth reaching about 190g / L and a sugar-acid conversion rate of 61%. Summary of the invention

[0007] In order to solve the problems existing in the prior art, the present invention provides a method for producing erythritol by fermentation, which has high fermentation efficiency, simple and feasible production process, and is easy to promote and use.

[0008] The present invention is achieved through the following technical solutions:

[0009] Candina lipolytica Ery-2020-12, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.23582.

[0010] Application of Candina lipolytica Ery-2020-12 in fermentation production of erythritol.

[0011] A microbial preparation for fermentation production of erythritol, characterized in that the microbial preparation comprises Candida lipolytica.

[0012] A method for producing erythritol by fermenting Candina lipolytica Ery-2020-12, comprising:

[0013] Step 1) activation, step 2) primary seed culture, step 3) secondary seed culture, 4) fermentation.

[0014] Specifically, the method comprises the following steps:

[0015] Step 1) Activation: Activating the low-temperature stored Candina lipolytica Ery-2020-12;

[0016] Step 2) primary seed culture: inoculate the bacterial solution obtained in step 1) into a shake flask containing a primary seed culture medium at an inoculum size of 0.1% to 1%, and culture until the OD value of the bacterial solution is in the range of 0.38 to 0.42, and then terminate the culture;

[0017] Step 3) secondary seed culture: the primary shake flask seed liquid obtained in step 2) is inoculated into a seed tank of the secondary seed culture medium at an inoculum amount of 0.8% to 1.2%, the liquid volume is 40% to 45%, the temperature is 28 to 32° C., the ventilation ratio is 1:0.5 to 1:1.2, the tank pressure is 0.05 to 0.1 MPa, the dissolved oxygen is controlled at 30% to 40%, and the culture is performed until the OD value of the bacterial liquid is in the range of 0.42 to 0.51, and the culture is terminated;

[0018] Step 4) Fermentation: The secondary seed liquid obtained in step 3) is inoculated into a fermentation tank filled with fermentation medium at an inoculation rate of 10% to 13% for fermentation culture, and the fermentation culture cycle is 70 to 100 hours.

[0019] Preferably, the components of the primary culture medium are: 40-50 g / L glucose and 10-12 g / L peptone.

[0020] Preferably, the components of the secondary seed culture medium are: 150-250 g / L glucose, 8-12 g / L yeast extract, 0.5-1.0 g / L potassium dihydrogen phosphate, 0.2-0.6 g / L magnesium sulfate, 3.0-5.0 g / L citric acid, and 0.02-0.04 g / L defoaming agent.

[0021] Preferably, the components of the fermentation medium are: 330-336 g / L glucose, 6-10 g / L yeast extract, 0.3-0.5 g / L potassium dihydrogen phosphate, 5-10 g / L citric acid, 0.08-1.2 g / L magnesium sulfate, 0.003-0.006 g / L copper chloride, and 0.12-0.18 g / L defoaming agent.

[0022] The technical solution of the present invention has the following outstanding advantages and uniqueness:

[0023] The invention obtains the Candida lipolytica with high erythritol production by mutagenesis, has high fermentation efficiency, simple and feasible production process and great application value.

[0024] The present invention designs a sugar distribution component to add sugar so that the added sugar can be quickly and evenly distributed in the fermentation liquid, effectively solving the problem of excessive osmotic pressure of the fermentation liquid caused by excessive local sugar concentration; it also solves the problem of substrate limitation caused by excessive local glucose concentration in the fermentation liquid, causing the residual sugar in the fermentation liquid to be quickly exhausted, resulting in the strain production capacity not being able to be maximized and entering a decline period.

[0025] By controlling the fermentation culture dissolved oxygen content, ventilation volume, fermentation formula and other conditions during the fermentation process, the fermentation conditions are optimized and the residual sugar content is monitored to achieve the purpose of increasing the erythritol yield and conversion rate.

[0026] Compared with the existing production process, the present invention stabilizes the growth cycle of the seed tank and prolongs the fermentation cycle of the fermentation tank without adding any additional equipment and manpower investment. The fermentation cycle is controlled within the range of 70 to 100 hours, and the stable output of erythritol can be maintained, the output of erythritol is effectively increased, the labor intensity is reduced, the production cost is reduced, the entire process is simplified, and it is suitable for industrial production. DETAILED DESCRIPTION

[0027] 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.

[0028] Example 1

[0029] 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.

[0030] 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.

[0031] Example 2

[0032] 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:

[0033] Step 1): Activate the low-temperature stored Candina lipolytica Ery-2020-12.

[0034] 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.

[0035] 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.

[0036] 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 300 rpm, 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.

[0037] The components of the primary culture medium are: glucose 40 g / L, peptone 10 g / L.

[0038] 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.

[0039] 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.

[0040] Example 3

[0041] The fermentation production of erythritol by different strains was compared, and the fermentation process was referred to Example 2.

[0042] Table 1

[0043] strain Erythritol yield g / L Sugar-acid conversion rate% Ery-2020 197.6 53.7 Ery-2020-12 261.3 61.4

[0044] 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 63.7 g / L higher than that of the starting strain. Correspondingly, the sugar-acid conversion rate also increased by 7.7 percentage points.

[0045] Example 4

[0046] 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.

[0047] Ery-2020-12 strain: Different from Example 2, it consumes sugar slightly slower. It takes a longer time to deplete the same amount of sugar. The final erythritol production is 287.3 g / L.

[0048] Starting strain Ery-2020: There was no significant change in the yield of erythritol and sugar consumption time during fermentation.

[0049] Example 5

[0050] Effect of fermentation temperature on erythritol production of different strains.

[0051] The fermentation process is described in Example 2, as shown in Table 2:

[0052] Table 2

[0053] strain 29℃ 31℃ Ery-2020 195.8 194.4 Ery-2020-12 245.9 251.7

[0054] 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.

[0055] 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. Candida lipolytica ( Candina lipolytica )Ery-2020-12, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number CGMCC No.23582.

2. The Candida lipolytica according to claim 1 ( Candina lipolytica )Application of Ery-2020-12 in fermentation production of erythritol.

3. A method of using the Candida lipolytica according to claim 1 ( Candina lipolytica )Ery-2020-12 A method for producing erythritol by fermentation, comprising: Step 1) activation, step 2) primary seed culture, step 3) secondary seed culture, 4) fermentation.

4. The method according to claim 3, characterized in that The method comprises the following steps: Step 1) Activation: The cryopreserved Candida lipolytica ( Candina lipolytica )Ery-2020-12 was activated; Step 2) primary seed culture: inoculate the bacterial solution obtained in step 1) into a shake flask containing a primary seed culture medium at an inoculum size of 0.1% to 1%, and culture until the OD value of the bacterial solution is in the range of 0.38 to 0.42, and then terminate the culture; Step 3) secondary seed culture: the primary shake flask seed liquid obtained in step 2) is inoculated into a seed tank of the secondary seed culture medium at an inoculum amount of 0.8% to 1.2%, the liquid volume is 40% to 45%, the temperature is 28 to 32° C., the ventilation ratio is 1:0.5 to 1:1.2, the tank pressure is 0.05 to 0.1 MPa, the dissolved oxygen is controlled at 30% to 40%, and the culture is terminated until the OD value of the bacterial liquid is in the range of 0.42 to 0.51; Step 4) Fermentation: The secondary seed liquid obtained in step 3) is inoculated into a fermentation tank filled with fermentation medium at an inoculation rate of 10% to 13% for fermentation culture, and the fermentation culture cycle is 70 to 100 hours.

5. The method according to claim 4, characterized in that The components of the primary seed culture medium are: 40-50 g / L glucose and 10-12 g / L peptone.

6. The method according to claim 4, characterized in that The components of the secondary seed culture medium are: 150-250 g / L of glucose, 8-12 g / L of yeast extract powder, 0.5-1.0 g / L of potassium dihydrogen phosphate, 0.2-0.6 g / L of magnesium sulfate, 3.0-5.0 g / L of citric acid, and 0.02-0.04 g / L of defoaming agent.

7. The method according to claim 4, characterized in that The components of the fermentation medium are: 330-336 g / L of glucose, 6-10 g / L of yeast extract powder, 0.3-0.5 g / L of potassium dihydrogen phosphate, 5-10 g / L of citric acid, 0.08-1.2 g / L of magnesium sulfate, 0.003-0.006 g / L of copper chloride, and 0.12-0.18 g / L of defoaming agent.