Composite leavening agent and application thereof in preparation of Ectoin

Abstract

The invention discloses a composite leavening agent and application thereof in preparation of Ectoine, and belongs to the technical field of microbial fermentation. The preparation method comprises the following steps: mixing halomonas elongata, halomonas canparia and halomonas fence according to a ratio to prepare a compound fermentation agent, adding the compound fermentation agent into a compound fermentation culture medium containing a water spinach extract, and adding supplementary materials in the fermentation process, so as to finally prepare and produce Ectodoine. According to the fermentation method disclosed by the invention, the compound leavening agent, the compound fermentation culture medium and the supplementary material are subjected to proper component proportioning, bacteria in the compound leavening agent grow fast and play a synergistic role, the Ectoine fermentation liquor with the concentration up to 26.4 g / L is prepared, and the yield and the fermentation rate of Ectoine are improved. A novel method is provided for microbial fermentation preparation of Ectoine, and industrial development of Ectoine is promoted.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, and in particular to a composite leavening agent and application thereof in the preparation of ectoine. Background Art

[0002] Ectoine (2-methyl-1,4,5,6,-tetrahydropyrimidine-4-carboxylic acid), also known as tetrahydropyrimidine, is a derivative of aspartic acid synthesized by halophilic bacteria in extreme environments. Ectoine can increase the hydration force of membrane lipid surfaces, maintain the cytoplasmic space and normal cell morphology required for cell metabolism, and also has good effects in protecting and stabilizing skin cells. At present, Ectoine is widely used in cosmetics, medicine, food and other fields as a high-value compound.

[0003] Ectoine can be obtained by chemical synthesis and biological fermentation, but because ectoine contains a chiral carbon atom, it is difficult to synthesize it by chemical methods; therefore, biological fermentation has become the main production method of ectoine. The paper "Growth characteristics of a strain of alkaliphilic halomonas ZB109 and optimization of tetrahydropyrimidine fermentation conditions" uses alkaliphilic halomonas ZB109 to conduct intracellular ectoine accumulation experiments, but the problem with the above methods is that they are too dependent on a single strain and have low potential for improvement. The patent "CN113481233B Method for Constructing Ectoine Production Bacteria" uses genetic engineering to transform lysine-producing Corynebacterium glutamicum as the starting strain to construct ectoine production bacteria. The early construction technology of this method is complex and has not achieved the original intention of reducing overall costs. Therefore, how to optimize the utilization of existing natural strains and fully tap their ability to synthesize ectoine is of practical significance for the development of the ectoine industry.

[0004] Ipomoea aquatica Forssk.in Forssk.&Niebuhr, also known as water spinach, is an annual herb of the genus Ipomoea of ​​the family Convolvulaceae. Ipomoea aquatica is native to China and is now widely distributed in Asia, Africa, Oceania and other regions. Ipomoea aquatica is rich in protein, soluble sugars, vitamins and minerals. It is now widely cultivated as a vegetable and can be eaten by humans or used as a palatable feed for poultry. In addition, the stems, leaves and roots of Ipomoea aquatica can be used as medicine. Its stems and leaves are sweet and cold in nature, and have the effects of cooling blood, clearing heat, promoting dampness and detoxifying. Although Ipomoea aquatica is relatively common as a food, there has been no report on its use in the production of ectoine. Summary of the invention

[0005] In view of the above-mentioned prior art, the purpose of the present invention is to provide a composite leavening agent and its application in the preparation of ectoine.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] In a first aspect of the present invention, a composite fermentation agent is provided, wherein the composite fermentation agent is mixed according to a ratio of live bacteria count of bacteria A: bacteria B: bacteria C = (0.1-5): (0.1-5): (0.1-5);

[0008] The bacterium A is Halomonas elongata, with a preservation number of GDMCC NO.: 1.3716; the bacterium B is Halomonas campaniensis, with a preservation number of GDMCC NO.: 802887; and the bacterium C is Halomonas campaniensis, with a preservation number of CICC 24079.

[0009] Furthermore, the ratio of the number of live bacteria of each bacterial species in the composite fermentation agent is bacteria A: bacteria B: bacteria C = 1:2:1.

[0010] Furthermore, the number of viable bacteria in the composite fermentation agent was 2×10 7 cfu / ml-9×10 9 cfu / ml.

[0011] The second aspect of the present invention provides the use of the composite fermentation agent in the preparation of ectoine. The application method is to inoculate the composite fermentation agent into the composite fermentation medium at an inoculation amount of 1-15% by volume according to the volume of the composite fermentation medium, and ferment by feeding to obtain ectoine.

[0012] Furthermore, the composition of the composite fermentation medium is: 10 g / L of L-sodium glutamate, 3 g / L of KH2PO4, 11.8 g / L of K2HPO4·3H2O, 0.4 g / L of MgSO4·7H2O, 0.01 g / L of MnSO4·H2O, 0.05 g / L of FeCl2, 2 g / L of ammonium chloride, 15 g / L of yeast extract, 40 g / L of glucose, 75 g / L of sodium chloride, pH=7.2, and 9 g / L of Yongcai extract.

[0013] Furthermore, the extract of Yongcai is prepared by the following method:

[0014] After washing the Yongcai, dry it in an oven at 75°C, crush it to 400 mesh and sieve it to obtain Yongcai powder; extract the Yongcai powder in 75% v / v ethanol at room temperature with a solid-liquid ratio of 1:2, each extraction time is 16 hours, and the extraction is performed 3 times in total; the extracts obtained each time are combined, concentrated under reduced pressure at 50°C, and then dried in an oven at 75°C to obtain a Yongcai extract.

[0015] Furthermore, when the fermentation time is 10-30 hours, 20-100 g / L of sodium chloride is added at one time, and from then on, 1-10 g / L of sodium L-glutamate and 10-50 g / L of glucose are added every hour according to the existing liquid volume in the fermenter; the total fermentation time is 30-70 hours.

[0016] Furthermore, during the fermentation process, the stirring speed is 100-500 r / m, the dissolved oxygen content is maintained at 10-50%, the temperature is maintained at 20-40° C., and the pH of the composite fermentation medium is maintained at 5-8.

[0017] Beneficial effects of the present invention:

[0018] The present invention changes the previous method of preparing ectoine by fermenting a single strain of bacteria. Elongated Halomonas, Campanian Halomonas and Fan's Halomonas are mixed in proportion to prepare a microbial fermentation liquid, the microbial fermentation liquid is added to a composite fermentation medium containing a Yongcai extract, and supplementary materials are added during the fermentation process to finally prepare and output ectoine. The fermentation method of the present invention prepares an ectoine fermentation liquid with a concentration of up to 26.4 g / L by making appropriate component ratios of the microbial fermentation liquid, the fermentation medium and the supplementary materials, and regulating the fermentation conditions. The average synthesis rate is 5.28 g / L·10h, which increases the yield and fermentation rate of ectoine, provides a new method for the microbial fermentation preparation of ectoine, and is conducive to promoting the industrialization development and popularization and application of ectoine. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a graph of OD values ​​in the fermentation broth of the present invention. DETAILED DESCRIPTION

[0020] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0021] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0022] The experimental materials used in the examples of the present invention but not specifically described are all conventional experimental materials in the art and can be purchased through commercial channels.

[0023] In the production of ectoine by microbial fermentation, most of the methods currently used are single strain fermentation. In terms of strain selection for the above fermentation method, strains that have undergone genetic engineering are still preferred, but this method still has the problem of high genetic modification costs and inability to fully utilize existing strains; and in the process of mixed fermentation using natural strains, it is not a random choice, but there are still many considerations, such as: different bacteria require different substrates, temperatures, culture media and other environments for the production of ectoine, different utilization efficiencies for the same substrate, and different tolerance levels for the substrate and product ectoine; therefore, it is challenging but still practical for manufacturers to rationally select strains and explore suitable culture conditions to further improve the yield and production efficiency of ectoine.

[0024] The bacterial species used in the present invention are: Bacteria A: Halomonas elongata, deposited in Guangdong Microbiological Culture Collection Center in Guangzhou, China, with a deposit number of GDMCC NO.: 1.3716; Bacteria B: Halomonas campaniensis, deposited in Guangdong Microbiological Culture Collection Center in Guangzhou, China, with a deposit number of GDMCC NO.: 802887; Bacteria C: Halomonas campaniensis, deposited in China Industrial Microbiological Culture Collection Management Center in Beijing, China, with a deposit number of CICC 24079. The above bacterial species were purchased through public channels.

[0025] Example 1

[0026] Resuscitation of fungi and cultivation of seeds

[0027] (1) Resuscitation of bacterial strains

[0028] The frozen Halomonas elongata, Halomonas campania and Halomonas van der Waals were thawed, and then streaked onto seed culture media respectively, and cultured at 30°C for 12 hours to obtain bacterial culture fluid.

[0029] Seed culture medium: L-glutamate 10 g / L, KH2PO4 3 g / L, K2HPO4·3H2O 11.8 g / L, MgSO4·7H2O0.4 g / L, MnSO4·H2O 0.01 g / L, FeCl2 0.05 g / L, yeast extract 5 g / L, glucose 10 g / L, sodium chloride 30 g / L, pH = 7.2.

[0030] (2) Seed cultivation

[0031] The bacterial culture liquid obtained in step (1) was inoculated into 500 mL of seed culture medium at a volume of 1% v / v, and the shaking speed was 150 rpm, and the culture was carried out at 30°C for 12 h to obtain the secondary seed liquid. The OD values ​​of the secondary seed liquids of the three bacteria were measured respectively. 600 The value is measured and used.

[0032] The composition of the liquid fermentation medium: L-glutamate 10 g / L, KH2PO4 3 g / L, K2HPO4·3H2O 11.8 g / L, MgSO4·7H2O 0.4 g / L, MnSO4·H2O 0.01 g / L, FeCl2 0.05 g / L, ammonium chloride 2 g / L, yeast extract 15 g / L, glucose 40 g / L, sodium chloride 75 g / L, pH = 7.2.

[0033] Preparation of extracts from Eupatorium odoratum

[0034] After washing the Yongcai, dry it in an oven at 75°C, crush it to 400 mesh and sieve it to obtain Yongcai powder; extract the Yongcai powder in 75% v / v ethanol at room temperature with a solid-liquid ratio of 1:2, each extraction time is 16 hours, and the extraction is performed 3 times in total; the extracts obtained each time are combined, concentrated under reduced pressure at 50°C, and then dried in an oven at 75°C to obtain a Yongcai extract.

[0035] Preparation of composite fermentation medium

[0036] The extract of Yongcai was added to the liquid fermentation medium to obtain a composite fermentation medium, the specific composition of which is as follows:

[0037] Sodium L-glutamate 10g / L, KH2PO4 3g / L, K2HPO4·3H2O 11.8g / L, MgSO4·7H2O 0.4g / L, MnSO4·H2O 0.01g / L, FeCl2 0.05g / L, ammonium chloride 2g / L, yeast extract 15g / L, glucose 40g / L, sodium chloride 75g / L, pH=7.2, Yongcai extract 9g / L.

[0038] Example 2

[0039] Preparation of compound fermentation agent

[0040] The bacterial culture solution after the secondary seed culture was taken and mixed according to the proportion of viable bacteria to form a mixed bacterial solution with a concentration of 3×10 9 cfu / ml, the mixed bacterial solution is the composite fermentation agent.

[0041] The ratio of the live counts of bacteria A, bacteria B and bacteria C in the composite leavening agent is 1:1:1, 2:1:1 and 1:2:1.

[0042] Example 3

[0043] The bacterial culture solution after the secondary seed culture is taken and mixed according to the live bacterial count ratio of bacteria A: bacteria B: bacteria C = 1:2:1 to form a mixed bacterial solution.

[0044] Take bacterial A solution, bacterial B solution, bacterial C solution and mixed bacterial solution, the concentration of each solution is 3×10 9 cfu / ml, 300 ml of the above four bacterial solutions were inoculated into a 5 L fermenter filled with 3 L of culture medium. The culture medium used was liquid fermentation medium or composite fermentation medium.

[0045] During the fermentation process, the fermentation temperature was maintained at 30° C., the rotation speed was 150 rpm, and the relative concentration of dissolved oxygen (DO) in the fermentation liquid was maintained at 30%. The fermentation was terminated after 20 hours.

[0046] Example 4

[0047] The bacterial culture solution after the secondary seed culture was mixed, and the mixed bacterial solution was the composite fermentation agent. The number of viable bacteria in the composite fermentation agent was 3×10 9 cfu / ml, where the ratio of the viable counts of bacteria A, bacteria B and bacteria C is 1:1:1.

[0048] 6 L of composite fermentation medium and 200 mL of composite fermentation agent were added into a 10 L fermenter for fermentation.

[0049] During the fermentation process, the fermentation temperature was maintained at 30°C, the rotation speed was 150rpm, the relative concentration of dissolved oxygen in the fermentation liquid was maintained at 30%, and the pH of the fermentation liquid was maintained at 6.5 with ammonia water; after 20 hours of fermentation, 60g / L of sodium chloride was added at one time, and from then on, 6g / L of sodium L-glutamate and 20g / L of glucose were added every hour according to the existing liquid volume in the fermenter; the total fermentation time was 50 hours.

[0050] Example 5

[0051] The bacterial culture solution after the secondary seed culture was mixed, and the mixed bacterial solution was the composite fermentation agent. The number of viable bacteria in the composite fermentation agent was 3×10 9 cfu / ml, where the ratio of the viable counts of bacteria A, bacteria B and bacteria C is 2:1:1.

[0052] 6 L of composite fermentation medium and 200 mL of composite fermentation agent were added into a 10 L fermenter for fermentation.

[0053] During the fermentation process, the fermentation temperature was maintained at 30°C, the rotation speed was 150rpm, the relative concentration of dissolved oxygen in the fermentation liquid was maintained at 30%, and the pH of the fermentation liquid was maintained at 6.5 with ammonia water; after 20 hours of fermentation, 60g / L of sodium chloride was added at one time, and from then on, 6g / L of sodium L-glutamate and 20g / L of glucose were added every hour according to the existing liquid volume in the fermenter; the total fermentation time was 50 hours.

[0054] Example 6

[0055] The bacterial culture solution after the secondary seed culture was mixed, and the mixed bacterial solution was the composite fermentation agent. The number of viable bacteria in the composite fermentation agent was 3×10 9 cfu / ml, where the ratio of the viable counts of bacteria A, bacteria B and bacteria C is 1:2:1.

[0056] 6 L of composite fermentation medium and 200 mL of composite fermentation agent were added into a 10 L fermenter for fermentation.

[0057] During the fermentation process, the fermentation temperature was maintained at 30°C, the rotation speed was 150rpm, the relative concentration of dissolved oxygen in the fermentation liquid was maintained at 30%, and the pH of the fermentation liquid was maintained at 6.5 with ammonia water; after 20 hours of fermentation, 60g / L of sodium chloride was added at one time, and from then on, 6g / L of sodium L-glutamate and 20g / L of glucose were added every hour according to the existing liquid volume in the fermenter; the total fermentation time was 50 hours.

[0058] Comparative Example 1

[0059] Take the culture solution of bacteria A after the secondary seed culture. The number of viable bacteria in the culture solution of bacteria A is 3×10 9 cfu / ml.

[0060] 6 L of composite fermentation medium and 200 mL of bacterial A culture solution were added to a 10 L fermenter for fermentation.

[0061] During the fermentation process, the fermentation temperature was maintained at 30°C, the rotation speed was 150rpm, the relative concentration of dissolved oxygen in the fermentation liquid was maintained at 30%, and the pH of the fermentation liquid was maintained at 6.5 with ammonia water; after 20 hours of fermentation, 60g / L of sodium chloride was added at one time, and from then on, 6g / L of sodium L-glutamate and 20g / L of glucose were added every hour according to the existing liquid volume in the fermenter; the total fermentation time was 50 hours.

[0062] Comparative Example 2

[0063] Take the culture solution of bacteria B after the secondary seed culture. The number of viable bacteria in the culture solution of bacteria B is 3×10 9 cfu / ml.

[0064] 6 L of composite fermentation medium and 200 mL of bacterial B culture solution were added to a 10 L fermenter for fermentation.

[0065] During the fermentation process, the fermentation temperature was maintained at 30°C, the rotation speed was 150rpm, the relative concentration of dissolved oxygen in the fermentation liquid was maintained at 30%, and the pH of the fermentation liquid was maintained at 6.5 with ammonia water; after 20 hours of fermentation, 60g / L of sodium chloride was added at one time, and from then on, 6g / L of sodium L-glutamate and 20g / L of glucose were added every hour according to the existing liquid volume in the fermenter; the total fermentation time was 50 hours.

[0066] Comparative Example 3

[0067] The bacterial C culture solution after secondary seed culture was taken. The number of viable bacteria in the bacterial C culture solution was 3×10 9 cfu / ml.

[0068] 6 L of composite fermentation medium and 200 mL of bacterial C culture solution were added to a 10 L fermenter for fermentation.

[0069] During the fermentation process, the fermentation temperature was maintained at 30°C, the rotation speed was 150rpm, the relative concentration of dissolved oxygen in the fermentation liquid was maintained at 30%, and the pH of the fermentation liquid was maintained at 6.5 with ammonia water; after 20 hours of fermentation, 60g / L of sodium chloride was added at one time, and from then on, 6g / L of sodium L-glutamate and 20g / L of glucose were added every hour according to the existing liquid volume in the fermenter; the total fermentation time was 50 hours.

[0070] Test Example 1:

[0071] The fermentation process of Example 3 was monitored, and the fermentation liquid in the fermenter was taken and the OD 600 The OD value of the fermentation liquid after fermentation is completed 600 The results of the values ​​(unit: 1) are shown in Table 1; the OD values ​​of the mixed bacterial solution in the liquid fermentation medium and the composite fermentation medium 600 The value (unit is 1) is measured, and the OD 600 The results of the value determination are shown in Figure 1 .

[0072] Table 1 Example 3 Fermentation broth OD determination

[0073]

[0074] According to the results in Table 1, compared with the use of a single strain, after the bacterial solution of bacteria A, bacteria B, and bacteria C were mixed in proportion to form a composite fermentation agent, the OD value in the fermentation broth using the composite fermentation agent was larger. This shows that the various strains not only did not inhibit each other, but also achieved faster growth in the culture medium, which is advantageous for the accumulation of later products. At the same time, when the composite fermentation medium was used, the OD value of each strain in the fermentation broth was higher, which was better than the OD value when the liquid fermentation medium was used.

[0075] according to Figure 1 The results showed that compared with the liquid fermentation medium, when the composite starter made of mixed bacterial liquid was cultured in the composite fermentation medium, the OD value in the fermentation liquid increased faster. After 8 hours, the OD value was at a high level, which indicated that bacteria A, bacteria B and bacteria C grew and multiplied rapidly, reflecting that the bacteria had a strong demand for the utilization of the substrate and could quickly convert the substrate into products after rapid reproduction.

[0076] Therefore, in terms of strain selection, bacterial liquid of bacteria A, bacterial liquid of bacteria B, and bacterial liquid of bacteria C are used together, and the three bacteria are mixed as a composite fermentation agent; in terms of culture medium selection, a composite fermentation medium added with Yongcai extract is used. The combination of these two methods will be beneficial to the growth of bacteria and the preparation of the product ectoine.

[0077] Test Example 2:

[0078] According to the method of Examples 4-6 and Comparative Examples 1-3, the fermentation was carried out for 50 hours. After the fermentation was completed, the fermentation liquid formed by the fermentation of the composite fermentation medium in the fermenter was taken, diluted 3 times with ultrapure water, and then glass grinding beads were added to the frozen grinder at 65Hz for 30 minutes. After taking it out, it was centrifuged at 12000r / min for 5min to remove the precipitate, and the supernatant was filtered through a 0.22μm water system membrane and stored in a liquid phase vial. The ectoine content was detected by HPLC, and the results are shown in Table 2.

[0079] Table 2. Comparison of edodene fermentation results

[0080]

[0081] The results are shown in Table 2. Using the fermentation method of this embodiment, the maximum ectoine production after 50 hours of fermentation was 26.4 g / L (i.e., each L of fermentation liquid contained 26.4 g of ectoine), and the average synthesis rate was 5.28 g / L·10h (i.e., the increase in ectoine product per liter of fermentation liquid every 10 hours).

[0082] The present invention selects elongated salt mononas, campanian salt mononas and fan's salt mononas to form a composite fermentation agent, which is used for a composite fermentation medium added with a Yongcai extract. In the composite fermentation medium, the three bacteria can fully utilize the substrate, the bacterial body grows fast, and has good tolerance to high-concentration substrates and products, which is conducive to the production of ectoine. The present invention simultaneously meets the different growth requirements of multiple bacteria in the fermenter, and the mixed bacteria have the characteristics of fast growth and can play a synergistic role. Finally, a co-culture fermentation method of different microbial strains producing ectoine is obtained. Compared with genetic engineering of the strain, the method of the present invention has simple procedures and high production efficiency, and has a demonstration effect on the high yield and promotion and application of ectoine.

[0083] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A composite leavening agent, characterized in that: The composite fermentation agent is mixed according to the ratio of live bacteria count of bacteria A: bacteria B: bacteria C = (0.1-5): (0.1-5): (0.1-5); The bacterium A is Halomonas elongata, with a preservation number of GDMCC NO.: 1.3716; the bacterium B is Halomonas campaniensis, with a preservation number of GDMCC NO.: 802887; and the bacterium C is Halomonas campaniensis, with a preservation number of CICC 24079.

2. The composite leavening agent according to claim 1, characterized in that The ratio of the number of live bacteria of each bacterial species in the composite fermentation agent is bacteria A: bacteria B: bacteria C = 1:2:

1.

3. The composite leavening agent according to claim 1, characterized in that The number of viable bacteria in the composite fermentation agent is 2×10 7 cfu / ml-9×10 9 cfu / ml.

4. The use of the composite leavening agent according to any one of claims 1 to 3 in the preparation of ekdoin, characterized in that: The application method is to inoculate the composite fermentation agent into the composite fermentation medium at an inoculation amount of 1-15% by volume according to the volume of the composite fermentation medium, and perform feed addition to obtain ectoine.

5. The use of the composite leavening agent according to claim 4 in the preparation of ekdoin, characterized in that: The composition of the composite fermentation medium is: L-glutamate 10 g / L, KH2PO4 3 g / L, K2HPO4·3H2O 11.8 g / L, MgSO4·7H2O0.4 g / L, MnSO4·H2O 0.01 g / L, FeCl2 0.05 g / L, ammonium chloride 2 g / L, yeast extract 15 g / L, glucose 40 g / L, sodium chloride 75 g / L, pH=7.2, Yongcai extract 9 g / L.

6. The use of the composite leavening agent according to claim 5 in the preparation of ekdoin, characterized in that: The Yongcai extract is prepared by the following method: After washing the Yongcai, dry it in an oven at 75°C, crush it to 400 mesh and sieve it to obtain Yongcai powder; extract the Yongcai powder in 75% v / v ethanol at room temperature with a solid-liquid ratio of 1:2, each extraction time is 16 hours, and the extraction is performed 3 times in total; the extracts obtained each time are combined, concentrated under reduced pressure at 50°C, and then dried in an oven at 75°C to obtain a Yongcai extract.

7. The use of the composite leavening agent according to claim 5 in the preparation of ekdoin, characterized in that: When the fermentation time is 10-30 hours, add 20-100g / L of sodium chloride at one time, and then add 1-10g / L of sodium L-glutamate and 10-50g / L of glucose every hour according to the existing liquid volume in the fermenter; the total fermentation time is 30-70 hours.

8. The use of the composite leavening agent according to claim 7 in the preparation of ekdoin, characterized in that: During the fermentation process, the stirring speed is 100-500r / m, the dissolved oxygen content is maintained at 10-50%, the temperature is maintained at 20-40°C, and the pH of the composite fermentation medium is maintained at 5-8.

Images