Agrobacterium capable of resisting high glucose and producing curdlan at high yield and application of agrobacterium in production of curdlan

Abstract

The invention belongs to the technical field of biology, and relates to agrobacterium capable of resisting high glucose and producing curdlan at high yield and application of the agrobacterium in curdlan production. The Agrobacterium is radioactive Agrobacterium sp. LQC24G-08, and is preserved in the China Center for Type Culture Collection on February 24, 2025, the address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M 2025287. The Agrobacterium has the advantages that the Agrobacterium is used for culturing the Agrobacterium; when the agrobacterium provided by the invention is used for fermentation production of curdlan, the fermentation process is simple, the fermentation yield of the curdlan is higher, and the agrobacterium has high sugar tolerance and is beneficial to industrial application.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a high-sugar-tolerant and high-curdlan-producing Agrobacterium and application thereof in the production of curdlan. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Curdlan is a novel microbial extracellular polysaccharide with the unique property of forming a gel under heating conditions, hence its name, "thermogel." Curdlan possesses many unique physical and chemical properties and has broad application prospects in food processing, biomedicine, agriculture, tertiary oil recovery, and other fields. Curdlan production is primarily achieved through biosynthesis. Currently reported strains that synthesize curdlan include Agrobacterium and Agrobacterium. These strains are typically short, rod-shaped, aerobic, Gram-negative bacteria. Studies have reported that the biosynthesis of curdlan is typically achieved through the polymerization of glucose under nitrogen-limited conditions.

[0004] Currently reported research on the fermentation production of curdlan gum focuses on the optimization of fermentation medium, but it still has shortcomings such as long fermentation time, low yield, and high production cost. In addition, its process control is complex and its stability is insufficient. For example, the invention patent application with application number 202411732418.1 discloses a method for increasing the yield of curdlan gum. By adding aliphatic hydrocarbon alcohol or sorbitol to increase the yield of curdlan gum, the fermentation yield of curdlan gum is less than 30 g / L, which has the problem of low yield. The invention patent application with application number 201810017114.5 discloses a method for producing curdlan gum by fermentation. By adding a non-ionic surfactant, controlling the pH value of the fermentation process, and supplementing the carbon source, a series of complex process controls are performed. Although the yield of curdlan gum can reach 70.95 g / L, its process is complex, which limits its application in large-scale industrial production. Summary of the Invention

[0005] In order to overcome the deficiencies of the prior art, the present invention aims to provide a high-sugar-tolerant Agrobacterium strain and its application in the production of curdlan. When the Agrobacterium provided by the present invention is used to ferment curdlan, not only the fermentation process is simple, but also the yield of curdlan produced by fermentation is higher. It also has high sugar tolerance, which is conducive to industrial application.

[0006] In order to achieve the above object, the technical solution of the present invention is: In the first aspect, a strain of Agrobacterium that is tolerant to high sugar and produces high amounts of curdlan gum is provided, wherein the Agrobacterium is a radioactive Agrobacterium.Agrobacterium sp. LQC24G-08 was deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan, China on February 24, 2025, with the deposit number CCTCC NO: M 2025287.

[0007] Radioactive Agrobacterium provided by the present invention Agrobacterium sp. LQC24G-08 was produced by the strain producing curdlan gum during the screening of high-yielding strains of curdlan gum. Agrobacterium sp. LQC24-08 was obtained through multiple rounds of high sugar tolerance and high yield directional breeding. Agrobacterium sp. The advantages of LQC24G-08 include fast growth, high sugar tolerance, and high yield of curdlan.

[0008] In a second aspect, a method for culturing Agrobacterium is provided, comprising culturing the above-mentioned Agrobacterium.

[0009] In a third aspect, a bacterial agent comprises the above-mentioned Agrobacterium or its fermentation product, culture, or metabolite.

[0010] In a fourth aspect, a use of the above-mentioned Agrobacterium or bacterial agent in the fermentation production of curdlan.

[0011] The beneficial effects of the present invention are: 1. The present invention discloses a strain of Agrobacterium that produces high amounts of curdlan gum, namely, Agrobacterium radioactive Agrobacterium sp. LQC24G-08 CCTCC NO: M 2025287.

[0012] 2. The Agrobacterium provided by the present invention is tolerant to high sugar, can grow in a 150 g / L sucrose medium and efficiently synthesize curdlan gum with a fermentation yield of up to 100 g / L, which can significantly enhance the market competitiveness of curdlan gum.

[0013] 3. The Agrobacterium provided by the present invention is a stable and high-yielding strain of curdlan gum-producing strain obtained through high-sugar tolerant directional breeding and evolution, and can be directly applied to industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0015] 图1 This is a picture of the aniline blue plate screening of the strain in Example 1 of the present invention.

[0016] 图2 For Example 2 of the present invention, radioactive Agrobacterium Agrobacterium sp. Typical curve of LQC24G-08 fermentation process.

[0017] 图3 This is a typical curve of the fermentation process of the starting strain LQC24-08 that was evolved through high-sugar tolerance directed breeding in Comparative Example 1 of the present invention.

[0018] 图4 For comparative example 2 of the present invention, a typical strain of Agrobacterium for curdlan biosynthesis was used. Agrobacterium sp. Typical curve of ATCC31749 fermentation process. DETAILED DESCRIPTION

[0019] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0020] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0021] In view of the problems of low yield and complex process in the existing fermentation production of curdlan, the present invention provides an Agrobacterium that is tolerant to high sugar and produces high curdlan and its application in the production of curdlan.

[0022] A typical embodiment of the present invention provides a high sugar tolerant and high curdlan gum producing Agrobacterium, wherein the Agrobacterium is a radioactive Agrobacterium. Agrobacterium sp. LQC24G-08 was deposited in the China Center for Type Culture Collection on February 24, 2025, with the deposit number CCTCC NO: M 2025287.

[0023] Another embodiment of the present invention provides a method for culturing Agrobacterium, which comprises culturing the above-mentioned Agrobacterium.

[0024] In an embodiment of the present invention, the culture includes LBS slant culture, seed culture and / or fermentation.

[0025] Specifically, the medium used for LBS slant culture is LBS medium. More specifically, the composition of LBS medium per liter of medium includes: 18-22 g sucrose, 9-11 g peptone, 4.5-5.5 g yeast powder, 9-11 g NaCl, and 16-20 g agar powder. The pH value is 7.0-7.1.

[0026] Specifically, the seed culture medium composition includes, per liter of culture medium: 18-22 g sucrose, 1.3-1.7 g corn steep liquor, 4.5-5.5 g diammonium hydrogen phosphate, 4.5-5.5 g potassium dihydrogen phosphate, and 0.9-1.1 g magnesium sulfate; pH 7.0-7.2.

[0027] Specifically, the fermentation medium used in the fermentation comprises, per liter of medium, 75-150 g of sucrose, 6-8 g of diammonium hydrogen phosphate, 1-2 g of corn steep liquor, 2-3 g of potassium dihydrogen phosphate, 2-4 g of magnesium sulfate, and a pH of 7.0-7.2.

[0028] Specifically, the LBS slant culture time is 46-50 h.

[0029] Specifically, the temperature of seed culture is 29~31°C, and the culture time is 15~17 h.

[0030] Specifically, during fermentation, the liquid seeds are inoculated into the fermentation medium at a volume percentage of 9-11%, and aeration fermentation culture is performed.

[0031] A third embodiment of the present invention provides a bacterial agent comprising the above-mentioned Agrobacterium or its fermentation product, culture, or metabolite.

[0032] A fourth embodiment of the present invention provides a use of the above-mentioned Agrobacterium or bacterial agent in the fermentation production of curdlan.

[0033] In an embodiment of the present invention, the Agrobacterium or bacterial agent is added to a culture medium for fermentation production.

[0034] In an embodiment of the present invention, the process of producing curdlan by fermentation sequentially comprises LBS slant culture, seed culture and fermentation.

[0035] Specifically, the medium used for LBS slant culture is LBS medium. More specifically, LBS medium comprises, per liter of medium, 18-22 g of sucrose, 9-11 g of peptone, 4.5-5.5 g of yeast powder, 9-11 g of NaCl, and 16-20 g of agar powder. The pH is 7.0-7.1.

[0036] Specifically, the seed culture medium used for seed culture includes, per liter of culture medium: 18-22 g of sucrose, 1.3-1.7 g of corn steep liquor, 4.5-5.5 g of diammonium hydrogen phosphate, 4.5-5.5 g of potassium dihydrogen phosphate, and 0.9-1.1 g of magnesium sulfate; pH 7.0-7.2.

[0037] Specifically, the fermentation medium used in the fermentation comprises, per liter of medium, 75-150 g of sucrose, 6-8 g of diammonium hydrogen phosphate, 1-2 g of corn steep liquor, 2-3 g of potassium dihydrogen phosphate, 2-4 g of magnesium sulfate, and a pH of 7.0-7.2.

[0038] Specifically, the LBS slant culture time is 46-50 h.

[0039] Specifically, the temperature of seed culture is 29~31°C, and the culture time is 15~17 h.

[0040] Specifically, during fermentation, the liquid seeds are inoculated into the fermentation medium at a volume percentage of 9-11%, and aeration fermentation culture is performed.

[0041] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples.

[0042] Example 1: Directed breeding of high sugar tolerant and high yield strains Curdlan gum producing strains Agrobacterium sp. LQC24-08 was used as the starting strain.

[0043] (1) After 48 h of LBS slant culture, a ring of bacterial moss was added to liquid culture medium (100 mL / 500 mL) and cultured at 30°C and 200 rpm for 16 h to obtain liquid seeds.

[0044] (2) The liquid seeds were first inoculated into a fermentation medium (100 mL / 500 mL) containing 75 g / L sucrose at a 10% inoculum volume (v / v) and cultured for 24-36 h. Then, the liquid seeds were used as seeds and inoculated into a fermentation medium (100 mL / 500 mL) containing 75 g / L sucrose at a 10% inoculum volume (v / v). After further culture for 24-36 h, 1 mL was taken and appropriately diluted with 0.85% sterile saline. 100 μL of the dilution was spread on an LBS-aniline blue solid (sucrose 75 g / L) plate. After inverted culture at 30°C for 3-5 days, single colonies with larger blue circles were picked and transferred to an LBS slant culture medium and cultured at 30°C for 24-48 h.

[0045] (3) Inoculate a ring of the LBS slant culture in step (2) into the liquid culture medium (100 mL / 500 mL), and culture at 30°C and 200 r / min for 16 h to obtain liquid seeds.

[0046] (4) The liquid seeds obtained in step (3) were first inoculated into a fermentation medium containing 100 g / L sucrose at a 10% inoculum volume (v / v) and cultured for 24 to 36 h. Then, the liquid seeds were used as seeds and inoculated into a fermentation medium containing 100 g / L sucrose at a 10% inoculum volume (v / v). After further culture for 24 to 36 h, 1 mL was taken and appropriately diluted with 0.85% sterile saline. 100 μL was spread on an LBS-aniline blue solid (sucrose 100 g / L) plate. After inverted culture at 30°C for 3-5 days, single colonies with larger blue circles were picked and transferred to an LBS slant culture medium and cultured at 30°C for 24 to 48 h.

[0047] (5) Inoculate a ringlet of the bacterial moss from the LBS slant culture in step (4) into the liquid culture medium (100 mL / 500 mL), and culture in a shaking incubator at 30°C and 200 rpm for 16 h to obtain liquid seeds.

[0048] (6) The liquid seeds obtained in step (5) were first inoculated into a fermentation medium containing 125 g / L sucrose at a 10% inoculum volume (v / v) and cultured for 24-36 h. Then, the liquid seeds were used as seeds and again inoculated into a fermentation medium containing 125 g / L sucrose at a 10% inoculum volume (v / v). After further culture for 24-36 h, 1 mL was taken and appropriately diluted with 0.85% sterile saline. 100 μL was spread on an LBS-aniline blue solid (sucrose 125 g / L) plate. After inverted culture at 30°C for 3-5 days, single colonies with larger blue circles were picked and transferred to an LBS slant culture medium and cultured at 30°C for 24-48 h.

[0049] (7) Inoculate a ringlet of the bacterial moss from the LBS slant culture in step (6) into the liquid culture medium (100 mL / 500 mL) and culture in a shaking incubator at 30°C and 200 rpm for 16 h to obtain liquid seeds.

[0050] (8) The liquid seeds obtained in step (7) were first inoculated into a fermentation medium containing 150 g / L sucrose at a 10% inoculum volume (v / v) and cultured for 24 to 36 h. The liquid seeds were then used as seeds and again inoculated into a fermentation medium containing 150 g / L sucrose at a 10% inoculum volume (v / v). After further culture for 24 to 36 h, 1 mL was taken and appropriately diluted with 0.85% sterile saline. 100 μL was spread on an LBS-aniline blue solid (sucrose 150 g / L) plate. After inverted culture at 30°C for 3-5 days, single colonies with larger blue circles were picked and transferred to an LBS slant culture medium and cultured at 30°C for 24 to 48 h.

[0051] After several rounds of directed breeding and evolution in high-sugar medium, a strain with high sugar tolerance and high curdlan gum production was obtained and named Agrobacterium radiobacterium. Agrobacterium sp. LQC24G-08, and was deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan, China on February 24, 2025, with the deposit number CCTCC NO: M2025287.

[0052] The LBS medium in this example (g / L) contains: 20 g sucrose, 10 g peptone, 5 g yeast powder, 10 g NaCl, 18 g agar powder, pH 7.0, and sterilized at 121°C for 20 min.

[0053] The LBS-aniline blue medium in this example (g / L) contains the following: actual amount of sucrose, 10 g of peptone, 5 g of yeast powder, 10 g of NaCl, 1 g of aniline blue, 18 g of agar powder, pH 7.0, and sterilization at 121°C for 20 min.

[0054] The liquid seed culture medium in this example (g / L) includes: 20 g sucrose, 1.5 g corn steep liquor, 5 g diammonium hydrogen phosphate, 5 g potassium dihydrogen phosphate, 1 g magnesium sulfate, pH 7.0-7.2, and sterilization at 121°C for 20 min.

[0055] The above fermentation medium (g / L): sucrose 75-150 g, diammonium phosphate 6-8 g, corn steep liquor 1-2 g, potassium dihydrogen phosphate 2-3 g, magnesium sulfate 2-4 g, pH 7.0-7.2, sterilized at 121°C for 20 min.

[0056] Example 2: Radioactive Agrobacterium Agrobacterium sp. Fermentation production of curdlan by strain LQC24G-08 (1) After 48 h of LBS slant culture, a ring of bacterial moss was added to the liquid seed culture medium (100 mL / 500 mL). After shaking culture at 30°C and 200 rpm for 16 h, liquid seeds were obtained.

[0057] (2) The liquid seeds were inoculated into a fermentation medium (6L / 10L fermenter) at a 10% inoculum volume (v / v). The pH of the initial fermentation medium was controlled at approximately 7.0-7.2, and aeration and stirring were performed to produce curdlan.

[0058] (3) Typical curves of the fermentation process are shown in the attached 图2 The content of curdlan in the fermentation broth was obtained by drying and weighing the precipitated broth with alcohol. The yield of curdlan in this example was 100.2±1.9 g / L, and the gel strength of the fermentation broth was 3320.1±64 g / cm 2 .

[0059] The LBS medium in this example (g / L) contains: 20 g sucrose, 10 g peptone, 5 g yeast powder, 10 g NaCl, 18 g agar powder, pH 7.0, and sterilized at 121°C for 20 min.

[0060] The liquid seed culture medium in this example (g / L) includes: 20 g sucrose, 1.5 g corn steep liquor, 5 g diammonium hydrogen phosphate, 5 g potassium dihydrogen phosphate, 1 g magnesium sulfate, pH 7.0-7.2, and sterilization at 121°C for 20 min.

[0061] The fermentation medium in this example (g / L) includes: 150 g sucrose, 6 g diammonium hydrogen phosphate, 1 g corn steep liquor, 2 g potassium dihydrogen phosphate, 2 g magnesium sulfate, pH 7.0-7.2, and sterilization at 121°C for 20 min.

[0062] Comparative Example 1: Fermentation of curdlan by the starting strain LQC24-08 (1) After 48 h of LBS slant culture, a ring of bacterial moss was added to the liquid seed culture medium (100 mL / 500 mL). After shaking culture at 30°C and 200 rpm for 16 h, liquid seeds were obtained.

[0063] (2) The liquid seeds were inoculated into a fermentation medium (6L / 10L fermenter) at a 10% inoculum volume (v / v). The pH of the initial fermentation medium was controlled at approximately 7.0-7.2, and aeration and stirring were performed to produce curdlan.

[0064] (3) Typical curves of the fermentation process are shown in the attached 图3 The content of curdlan in the fermentation broth was obtained by drying and weighing the precipitated broth with alcohol. The yield of curdlan in this comparative example was 32.2±1.1 g / L, and the gel strength of the fermentation broth was 1221±30 g / cm 2 .

[0065] The LBS medium in this comparative example (g / L): 20 g sucrose, 10 g peptone, 5 g yeast powder, 10 g NaCl, 18 g agar powder, pH 7.0, sterilized at 121°C for 20 min.

[0066] The liquid seed culture medium of this comparative example (g / L): 20 g sucrose, 1.5 g corn steep liquor, 5 g diammonium hydrogen phosphate, 5 g potassium dihydrogen phosphate, 1 g magnesium sulfate, pH 7.0-7.2, sterilized at 121°C for 20 min.

[0067] The fermentation medium of this comparative example (g / L): 150 g sucrose, 6 g diammonium hydrogen phosphate, 1 g corn steep liquor, 2 g potassium dihydrogen phosphate, 2 g magnesium sulfate, pH 7.0-7.2, sterilized at 121°C for 20 min.

[0068] Comparative Example 2: Agrobacterium Agrobacterium sp. Curdlan produced by fermentation of ATCC31749 (1) After 48 h of LBS slant culture, a ring of bacterial moss was added to the liquid culture medium (100 mL / 500 mL). The culture was shaken at 30°C and 200 r / min for 20 h to obtain liquid seeds.

[0069] (2) The liquid seeds are inoculated into the fermentation medium at a 10% inoculum volume (v / v). The pH of the initial fermentation medium is controlled at approximately 7.0-7.2, and aeration and stirring are performed to ferment and produce curdlan.

[0070] (3) Typical curves of the fermentation process are shown in the attached 图4 The content of curdlan in the fermentation broth was obtained by drying and weighing the precipitated broth with alcohol. The yield of curdlan in this comparative example was 28.2±1.9 g / L, and the gel strength of the fermentation broth was 978±35 g / cm 2 .

[0071] The LBS medium in this comparative example (g / L): 20 g sucrose, 10 g peptone, 5 g yeast powder, 10 g NaCl, 18 g agar powder, pH 7.0, sterilized at 121°C for 20 min.

[0072] The liquid seed culture medium of this comparative example (g / L): 20 g sucrose, 1.5 g corn steep liquor, 5 g diammonium hydrogen phosphate, 5 g potassium dihydrogen phosphate, 1 g magnesium sulfate, pH 7.0-7.2, sterilized at 121°C for 20 min.

[0073] The fermentation medium of this comparative example (g / L): 150 g sucrose, 8 g diammonium hydrogen phosphate, 2 g corn steep liquor, 3 g potassium dihydrogen phosphate, 4 g magnesium sulfate, pH 7.0-7.2, sterilized at 121°C for 20 min.

[0074] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A high-sugar-tolerant and high-yielding Agrobacterium strain, characterized by: The Agrobacterium is radioactive Agrobacterium Agrobacterium sp. LQC24G-08 was deposited in the China Center for Type Culture Collection on February 24, 2025, with the deposit number CCTCC NO: M 2025287.

2. A method for culturing Agrobacterium, characterized in that: The Agrobacterium according to claim 1 is cultured.

3. The culture method according to claim 2, wherein The culture includes LBS slant culture, seed culture and / or fermentation.

4. The culture method according to claim 3, wherein The medium used for LBS slant culture is LBS medium; preferably, the LBS medium comprises, per liter of medium, 18-22 g of sucrose, 9-11 g of peptone, 4.5-5.5 g of yeast powder, 9-11 g of NaCl, and 16-20 g of agar powder; Alternatively, the LBS slant culture time is 46-50 h.

5. The culture method according to claim 3, wherein: The seed culture medium used for seed culture includes, per liter of culture medium, 18-22 g of sucrose, 1.3-1.7 g of corn steep liquor, 4.5-5.5 g of diammonium hydrogen phosphate, 4.5-5.5 g of potassium dihydrogen phosphate, and 0.9-1.1 g of magnesium sulfate; pH 7.0-7.2; Alternatively, the seed culture temperature is 29-31°C and the culture time is 15-17 h.

6. The culture method according to claim 3, wherein: The fermentation medium used in the fermentation comprises, per liter of medium, 75-150 g of sucrose, 6-8 g of diammonium hydrogen phosphate, 1-2 g of corn steep liquor, 2-3 g of potassium dihydrogen phosphate, 2-4 g of magnesium sulfate, and a pH of 7.0-7.2; Alternatively, during fermentation, the liquid seeds are inoculated into the fermentation medium at a volume percentage of 9-11% to carry out aerated fermentation culture.

7. A bacterial agent characterized by: The method comprises the Agrobacterium according to claim 1 or its fermentation product, culture, or metabolite.

8. Use of the Agrobacterium according to claim 1 or the bacterial agent according to claim 7 in the fermentation production of curdlan.

9. The use according to claim 8, characterized in that: The Agrobacterium or bacterial agent is added to the fermentation medium for fermentation production.

10. The use according to claim 8, characterized in that: The process of producing curdlan by fermentation includes LBS slant culture, seed culture and fermentation. Preferably, the medium used for LBS slant culture is LBS medium; more preferably, the LBS medium comprises, per liter of medium: 18-22 g sucrose, 9-11 g peptone, 4.5-5.5 g yeast powder, 9-11 g NaCl, and 16-20 g agar powder; the pH value is 7.0-7.1; Preferably, the seed culture medium used for seed culture comprises, per liter of culture medium: 18-22 g sucrose, 1.3-1.7 g corn steep liquor, 4.5-5.5 g diammonium hydrogen phosphate, 4.5-5.5 g potassium dihydrogen phosphate, 0.9-1.1 g magnesium sulfate; pH 7.0-7.2; Preferably, the fermentation medium used in the fermentation comprises, per liter of medium, 75-150 g of sucrose, 6-8 g of diammonium hydrogen phosphate, 1-2 g of corn steep liquor, 2-3 g of potassium dihydrogen phosphate, 2-4 g of magnesium sulfate, and a pH of 7.0-7.2; Preferably, the LBS slant culture time is 46-50 h; Preferably, the seed culture temperature is 29-31°C and the culture time is 15-17 hours; Preferably, during fermentation, the liquid seeds are inoculated into the fermentation medium at a volume percentage of 9-11% for aeration fermentation culture.

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