Method for improving curdlan production

Abstract

The invention belongs to the technical field of microbial fermentation production, and particularly relates to a method for improving curdlan production, which improves the fermentation yield and shortens the fermentation period by optimizing and adjusting a fermentation formula. The method disclosed by the invention is simple and easy to implement, low in cost and applicable to large-scale industrial production of the high-strength curdlan.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and in particular relates to a method for improving the yield of curdlan. Background Art

[0002] Curdlan is a new type of microbial extracellular polysaccharide. Due to its unique property of forming gel under heating conditions, it is also called thermogel.

[0003] Curdlan is produced by fermentation of bacteria such as Agrobacterium sp. or Rhizobium sp., and reproduces in the presence of a nitrogen source. When the nitrogen source is exhausted, curdlan is secreted and surrounds the bacteria. It is insoluble in water. Curdlan has the characteristics of forming a reversible gel under low temperature heating conditions and an irreversible gel under high temperature conditions. It is widely used in the food industry as a food additive. It can improve the water retention and stability of the product, and increase the hardness and taste of the product. In addition, it also has good applications in fields such as oil extraction.

[0004] However, the current method of producing curdlan by fermentation in my country requires the addition of multiple raw materials such as glucose, sodium nitrate, and dipotassium hydrogen phosphate during the fermentation process, which results in a complex feeding process, a prolonged fermentation cycle, a low gum production rate, and a low sugar conversion rate, thus leading to a high cost for producing curdlan by fermentation. Therefore, new technological breakthroughs and production processes are urgently needed to increase the gum production capacity and conversion rate of curdlan while reducing production costs.

[0005] CN2015102744542 discloses a method for increasing the production of bacterial curdlan using Acinetobacter lwoffii. The co-cultivation technology of Acinetobacter lwoffii and curdlan-producing bacteria is used to increase the production of curdlan by the curdlan-producing bacteria compared with using only rhizobia, thereby providing a new method for producing curdlan by microbial fermentation.

[0006] CN2017100776251 discloses a new process for producing curdlan by fermentation, which adopts the method of adding a soluble polysaccharide fermentation promoter and combining a stage control method to change the rotation speed for fermentation culture, thereby increasing the yield of curdlan and improving the quality of curdlan.

[0007] The fermentation conditions need to be further optimized to increase the yield of curdlan. Summary of the invention

[0008] The present invention provides a method for increasing the yield of curdlan gum, which increases the yield of curdlan gum by adjusting the fermentation formula. The process of the present invention has a high degree of controllability, low investment cost, and accurately controls the fermentation process to avoid fermentation out of control and cost increase, while reducing labor intensity, and the entire production process is simple and easy to operate.

[0009] The present invention is achieved through the following technical solutions.

[0010] A method for increasing the yield of curdlan, comprising the following steps: Step 1) primary seed culture, step 2) secondary seed culture, step 3) fermentation to produce curdlan.

[0011] Specifically, the method comprises the following steps: Step 1) Primary seed culture: Inoculate the curdlan-producing rhizobium into the primary seed culture medium and culture at 30±1℃ and 210rpm until the OD 600 =8, and the first-grade seed solution is obtained; Step 2) Secondary seed culture: The first-level seed solution obtained in step 1) was inoculated into the secondary seed culture medium at a rate of 5-10%, the temperature was controlled at 30°C, the stirring speed was 300 rpm, and the culture was continued until the OD 600 =15, and the secondary seed solution is obtained; Step 3) Fermentation to produce curdlan: 5-15% of the secondary seed liquid obtained in step 2) is added to the fermentation medium for fermentation, the temperature is controlled at 30°C, the stirring speed is 600 rpm, the dissolved oxygen is controlled at 30%, the pH is natural, and the culture is carried out for 50-60 hours to obtain curdlan fermentation liquid.

[0012] Preferably, the fermentation medium comprises sorbitol.

[0013] More preferably, the concentration of sorbitol in the fermentation medium is 1-8 g / L.

[0014] Most preferably, the components of the fermentation medium are: 46 g / L glucose, 4 g / L sorbitol, 2 g / L dipotassium hydrogen phosphate, 1.2 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate heptahydrate, 0.5 g / L light calcium carbonate, 2 g / L corn steep liquor powder, 0.1 g / L ferrous sulfate heptahydrate, and 0.05 g / L manganese sulfate monohydrate.

[0015] Preferably, the components of the primary seed culture medium are: 20 g / L glucose, 5 g / L dipotassium hydrogen phosphate, 1.5 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate heptahydrate, 3 g / L light calcium carbonate, and 1.5 g / L corn steep liquor powder.

[0016] Preferably, the components of the secondary seed culture medium are: 20 g / L glucose, 5 g / L dipotassium hydrogen phosphate, 1.5 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate heptahydrate, 3 g / L light calcium carbonate, and 1.5 g / L corn steep liquor powder.

[0017] Preferably, the fermentation period is 54 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 :The effects of fatty alcohols on the synthesis and metabolism of curdlan; Figure 2 :Effect of sorbitol on the fermentation cycle of curdlan produced by rhizobium. DETAILED DESCRIPTION

[0019] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The products and methods of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the products and methods described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention. In order to further understand the present invention, the present invention is described in detail below in conjunction with embodiments. Example

[0020] A method for increasing the yield of curdlan, the specific operation steps are as follows: Step 1) Take out the rhizobium ATCC 31750 and activate it, then inoculate it to the LB slant plate and culture it in the dark for 48 hours. Inoculate the slant bacteria into the primary seed culture medium and culture it at 30±1℃ and 210rpm until the OD 600 =8, and the first-grade seed solution is obtained.

[0021] Step 2) The first-level seed solution obtained in step 1) was inoculated into the second-level seed tank at a 5% inoculation rate, the temperature was controlled at 30°C, the stirring speed was 300 rpm, and the OD was 0. 600 =15, and the secondary seed solution is obtained.

[0022] Among them, the first and second level seed culture media are the same, and the components are: glucose 20g / L, dipotassium hydrogen phosphate 5g / L, potassium dihydrogen phosphate 1.5g / L, magnesium sulfate heptahydrate 1g / L, light calcium carbonate 3g / L, corn steep liquor powder 1.5g / L.

[0023] Step 3) The secondary seed liquid obtained in step 2) is inoculated at 10% into a fermentation medium containing 50 g / L glucose, 2 g / L dipotassium hydrogen phosphate, 1.2 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate heptahydrate, 0.5 g / L light calcium carbonate, 2 g / L corn syrup dry powder, 0.1 g / L ferrous sulfate heptahydrate, and 0.05 g / L manganese sulfate monohydrate for fermentation. The temperature is controlled at 30°C, the stirring speed is 600 rpm, the dissolved oxygen is controlled at 30%, the pH is natural, and the culture is carried out for 60 hours to obtain a curdlan fermentation liquid. The curdlan content is 23.7 g / L after testing. Example

[0024] The synthetic pathway of curdlan is mainly divided into three steps: absorption of reaction substrates, bio-enzyme-catalyzed polymerization of monosaccharides into polysaccharides, and secretion of polysaccharide products outside the cell. Curdlan is an extracellular polysaccharide that requires a phosphate carrier to transport it outside the cell. The phosphate carrier plays a key role in the synthetic metabolic pathway of curdlan. This study chose to experiment with fatty alcohol substances that may have positive regulation on phosphate synthesis to explore the effects of fatty alcohol substances on the synthetic metabolism of curdlan.

[0025] 1. This study selected four fatty alcohols, glycerol, erythritol, xylitol and sorbitol, in the culture medium for the experiment.

[0026] The above study was conducted on the basis of Example 1. Four substances, glycerol, erythritol, xylitol and sorbitol, were added to the fermentation medium to replace part of the glucose, and the addition amounts were 0, 1, 2, 4, 8, and 16, respectively, in g / L. Accordingly, each group removed the same weight of glucose, and the rest was the same as Example 1; the control group was Example 1 (no glucose replacement). Figure 1 As shown in the figure, the addition of glycerol, erythritol and xylitol had a certain adverse effect on the fermentation of curdlan gum. With the increase of the addition amount, the yield of curdlan gum decreased. The most serious decrease was in the glycerol group. Taking 8g / L as an example, it decreased by 15% compared with the control group (Example 1 group); while the sorbitol group had a positive effect on the fermentation of curdlan gum. With the increase of the addition amount of sorbitol, the yield of curdlan gum also increased. When the addition amount of sorbitol reached 4g / L, the yield of curdlan gum was close to the peak value. The yield of curdlan gum did not change much when the addition amount of sorbitol continued to increase. The possible reason is that rhizobia can effectively use sorbitol as a substrate source of phosphate ester, rather than just using sorbitol as a carbon source, so that the intracellular concentration of phosphate ester increases, and more curdlan gum can be transported out of the cell.

[0027] 2. Based on the above research results, it can be determined that the fermentation yield of curdlan gum can be increased by adding 4g / L sorbitol to the fermentation medium to replace the same amount of glucose. On this basis, the effect of adding sorbitol on the fermentation cycle is further studied. Figure 2 As shown, the sorbitol group (4 g / L sorbitol to replace an equal amount of glucose) was close to the peak at 54 h of fermentation, while the glucose group (Example 1) was close to the peak at 58 h of fermentation. It can be seen that using sorbitol to replace part of the glucose in the fermentation medium can not only increase the yield of curdlan, but also shorten the fermentation cycle. Example

[0028] The optimized fermentation process of curdlan is as follows: Step 1) Take out the rhizobium ATCC 31750 and activate it, then inoculate it to the LB slant plate and culture it in the dark for 48 hours. Inoculate the slant bacteria into the primary seed culture medium and culture it at 30±1℃ and 210rpm until the OD 600 =8, and the first-grade seed solution is obtained.

[0029] Step 2) The first-level seed solution obtained in step 1) was inoculated into the second-level seed tank at a 5% inoculation rate, the temperature was controlled at 30°C, the stirring speed was 300 rpm, and the OD was 0. 600 =15, and the secondary seed solution is obtained.

[0030] Among them, the first and second level seed culture media are the same, and the components are: glucose 20g / L, dipotassium hydrogen phosphate 5g / L, potassium dihydrogen phosphate 1.5g / L, magnesium sulfate heptahydrate 1g / L, light calcium carbonate 3g / L, corn steep liquor powder 1.5g / L.

[0031] Step 3) The secondary seed liquid obtained in step 2) is inoculated at 10% into a fermentation medium containing 46 g / L glucose, 4 g / L sorbitol, 2 g / L dipotassium hydrogen phosphate, 1.2 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate heptahydrate, 0.5 g / L light calcium carbonate, 2 g / L corn syrup dry powder, 0.1 g / L ferrous sulfate heptahydrate, and 0.05 g / L manganese sulfate monohydrate for fermentation, the temperature is controlled at 30°C, the stirring speed is 600 rpm, the dissolved oxygen is controlled at 30%, the pH is natural, and the culture is carried out for 54 hours to obtain a curdlan fermentation liquid.

[0032] 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 increasing the yield of curdlan, comprising the following steps: Step 1) primary seed culture, step 2) secondary seed culture, step 3) fermentation to produce curdlan.

2. The method according to claim 1, characterized in that The method comprises the following steps: Step 1) Primary seed culture: Inoculate the curdlan-producing rhizobium into the primary seed culture medium and culture until OD 600 =8, and the first-grade seed solution is obtained; Step 2) Secondary seed culture: The primary seed solution obtained in step 1) was inoculated into the secondary seed culture medium at a rate of 5-10%, and cultured until OD 600 =15, and the secondary seed solution is obtained; Step 3) Fermentation to produce curdlan: 5-15% of the secondary seed liquid obtained in step 2) is added to the fermentation medium for fermentation, the temperature is controlled at 30°C, the stirring speed is 600 rpm, the dissolved oxygen is controlled at 30%, the pH is natural, and the culture is carried out for 50-60 hours to obtain curdlan fermentation liquid.

3. The method according to claim 1, characterized in that The fermentation medium contains sorbitol.

4. The method according to claim 3, characterized in that The concentration of sorbitol in the fermentation medium is 1-8 g / L.

5. The method according to any one of claims 1 to 4, characterized in that: The fermentation medium comprises the following components: 46 g / L glucose, 4 g / L sorbitol, 2 g / L dipotassium hydrogen phosphate, 1.2 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate heptahydrate, 0.5 g / L light calcium carbonate, 2 g / L corn steep liquor powder, 0.1 g / L ferrous sulfate heptahydrate, and 0.05 g / L manganese sulfate monohydrate.

6. The method according to any one of claims 1 to 4, characterized in that: The components of the primary seed culture medium are: 20 g / L glucose, 5 g / L dipotassium hydrogen phosphate, 1.5 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate heptahydrate, 3 g / L light calcium carbonate, and 1.5 g / L corn steep liquor powder.

7. The method according to any one of claims 1 to 4, characterized in that: The components of the secondary seed culture medium are: 20 g / L glucose, 5 g / L dipotassium hydrogen phosphate, 1.5 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate heptahydrate, 3 g / L light calcium carbonate, and 1.5 g / L corn steep liquor powder.

8. The method according to any one of claims 1 to 4, characterized in that: The fermentation period is 54 hours.

9. Curdlan prepared according to the method of claims 1-8.

10. Use of the curdlan according to claim 9 in the preparation of food or medicine.

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