Xanthomonas campestris strain producing transparent xanthan gum by fermentation and use thereof

Abstract

A Xanthomonas campestris strain producing transparent xanthan gum by fermentation and the use thereof. The Xanthomonas campestris strain is Xanthomonas campestris F417-6 and deposited in the Guangdong Microbial Culture Collection Center on 12 May 2023 with the deposit number of GDMCC NO: 63459. According to the provided Xanthomonas campestris strain, the yield of xanthan gum is increased by 15%-30%, the light transmittance of the aqueous solution of xanthan gum obtained by means of the fermentation is improved by 30%-50% compared to that of the original strain. The genetic stability of the Xanthomonas campestris strain is analyzed and continuous subcultures are carried out, and the yield of xanthan gum is measured every two subcultures. The provided Xanthomonas campestris strain has no significant changes in yield and quality after 12 continuous subcultures, indicating that the Xanthomonas campestris strain has good stability and can be used as an industrial production strain for large-scale production of transparent xanthan gum.

Description

A strain of Xanthomonas campestris producing transparent xanthan gum through fermentation and its application Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a strain of Xanthomonas campestris capable of producing transparent xanthan gum through fermentation and application thereof. Background Art

[0002] Xanthan gum, also known as xanthan gum or xanthan gum, is a water-soluble microbial exopolysaccharide produced by Xanthomonas campestris using carbohydrates as the primary raw material through aerobic fermentation. It is currently the world's largest microbial polysaccharide. Due to its unique macromolecular structure and colloidal properties, xanthan gum possesses excellent physical and chemical properties, such as suspending, emulsifying, thickening, pseudoplasticity, and thermal stability. It is widely used in various fields as a thickener, emulsifier, stabilizer, gelling agent, wetting agent, and film-forming agent, making it one of the most superior bio-glue products. Xanthan gum possesses the general properties of long-chain polymers, but it contains more functional groups than ordinary polymers, resulting in unique properties under specific conditions. The number of pyruvic acid groups at the end of the side chains of xanthan gum molecules significantly influences its performance.

[0003] While China's xanthan gum industry boasts advantages in terms of total output value, its industrialized xanthan gum production technology still has shortcomings compared to international xanthan gum production technologies, such as low yields and suboptimal light transmittance of fermented xanthan gum solutions. Domestic and international research indicates that strain modification and breeding are the most effective methods for improving xanthan gum yield and quality, offering the greatest potential for industrial production. While the selection of culture medium components is crucial in the xanthan gum fermentation industry, the choice of strain also requires careful consideration. The strain is the "chip" of the biofermentation industry, a decisive factor in product yield and quality, significantly impacting economic, social, and ecological benefits. Therefore, identifying optimal strains is crucial for the xanthan gum industry to further improve yields and the light transmittance of fermented xanthan gum solutions. Summary of the Invention

[0004] The purpose of the present invention is to provide a strain of Xanthomonas campestris that produces transparent xanthan gum through fermentation and its application, so as to solve the problems of low xanthan gum production rate in the existing xanthan gum industry and unsatisfactory light transmittance of the xanthan gum aqueous solution obtained by fermentation.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] A strain of Xanthomonas campestris that ferments and produces transparent xanthan gum. The strain is Xanthomonas campestris F417-6, and was deposited in the Guangdong Provincial Microbiological Culture Collection on May 12, 2023. The deposit number is GDMCCNO: 63459, and the deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0007] More preferably, the screening process is as follows:

[0008] (1) Thaw the original wild Xanthomonas campestris bacterial solution, dip the bacterial solution with an inoculating loop and streak it on a plate culture medium. After a single colony grows, pick a single colony and inoculate it into a liquid seed culture medium at a 1% inoculum. Cultivate it at 30°C, 180 rpm, and a constant temperature shaker for 24 hours. Subculture twice to restore the original activity of the original wild Xanthomonas campestris.

[0009] (2) The activated original strain was inoculated into a 250 mL conical flask containing 100 mL of liquid seed culture medium at a 1% inoculum size and cultured in a constant temperature shaker at 30°C and 180 rpm for 24 h to complete the liquid seed culture;

[0010] (3) Take the bacterial suspension of the original wild Xanthomonas campestris grown to the logarithmic phase and mix it into a uniform bacterial suspension using a vortex instrument; take 1 mL of the prepared bacterial suspension in a 35 mm irradiated dish, seal it with a sealing film, and use 12 C 6+ Ion beam mutagenesis was performed with an extraction energy of 80 MeV / u and an LET of 35.5 keV / mm. Eleven mutagenic doses, including 0 Gy, 20 Gy, 40 Gy, 60 Gy, 80 Gy, 100 Gy, 120 Gy, 140 Gy, 160 Gy, 180 Gy, and 200 Gy, were selected for irradiation.

[0011] (4) Observe the morphology and size of the colonies under different irradiation doses, pick the larger, smoother and more transparent colonies and inoculate them on the screening medium, number them, and culture them at 37°C for 72 hours; after the colonies grow, add Lugol's iodine solution to the screening medium and observe the transparent zone around the colonies; measure the colony diameter C and the transparent zone diameter H, and calculate the H / C value; strains with larger H / C values ​​are inoculated into liquid culture medium and cultured in a constant temperature shaker at 30°C and 180 rpm for 72 hours. These strains are the primary screening strains;

[0012] (5) The primary screened strains were numbered, cultured to the logarithmic phase, and inoculated into 75 mL of fermentation medium at a 10% inoculation rate. The strains were cultured in a constant temperature shaker at 37°C and 180 r / min for 96 h. After the fermentation, the gum production rate and the transmittance of the fermentation product xanthan gum aqueous solution were measured. The gum production rate and the transmittance of the aqueous solution of the primary screened strains were compared, and the optimal strain was selected as the re-screened strain. The re-screened strains were tested for genetic stability, and finally the strain with the best gum production rate, aqueous solution transmittance, and genetic stability test was selected as the target strain.

[0013] The present invention also provides the use of the above-mentioned Xanthomonas campestris as a fermentation strain in the fermentation preparation of xanthan gum.

[0014] Specifically, the above application includes the following steps:

[0015] (1) Activation of bacterial strains: Thaw the preserved bacterial solution containing Xanthomonas campestris with the deposit number of GDMCC NO: 63459, dip an inoculating loop into the preserved bacterial solution and streak it on a plate culture medium. After a single colony grows, pick a single colony and inoculate it into a liquid seed culture medium at a 1% inoculum. Ferment at 30°C and 180 rpm constant temperature shaker for 24 h. Subculture twice to restore the original activity of Xanthomonas campestris.

[0016] (2) Liquid seed culture: The activated Xanthomonas campestris was inoculated into the liquid seed culture medium at a 1% inoculum rate, and the fermentation temperature was 30°C and the temperature was shaken at 180 rpm for 24 h to complete the liquid seed culture;

[0017] (3) Fermentation culture: 5-20% of the seed liquid of Xanthomonas campestris grown to the logarithmic phase was inoculated into the fermentation medium, and the fermentation temperature was 30-40°C and the shaking was constant at 180 rpm for 96 h;

[0018] (1) Xanthan gum was extracted from the fermentation broth by ethanol precipitation.

[0019] Furthermore, the plate culture medium in step (1) includes the following components in weight percentage: 0.5% soluble starch, 1% peptone, 0.3% beef extract, 0.5% sodium chloride, and 2% agar. The pH of the plate culture medium is 6.5-7.0, the sterilization temperature is 115° C., the sterilization time is 30 min, and the plate culture time is 72 h.

[0020] Furthermore, in step (1) and step (2), the liquid seed culture medium includes the following components in weight percentage: 2.0% soluble starch, 0.5% peptone, 0.3% potassium dihydrogen phosphate, and 0.2% sodium chloride, and the pH value of the liquid seed culture medium is 7.0.

[0021] Furthermore, the inoculation amount in step (3) is 15%.

[0022] Furthermore, the fermentation temperature in step (3) is 40°C.

[0023] Furthermore, the fermentation medium in step (3) comprises the following components in weight percentage: 6.0% corn starch, 1.0% glucose, 2.0% soy protein, 0.1% magnesium sulfate heptahydrate, 0.1% dipotassium hydrogen phosphate, and 0.1% potassium dihydrogen phosphate, and the pH value of the fermentation medium is 7.0.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The Xanthomonas campestris provided by the present invention (deposit number: GDMCC NO: 63459) has a xanthan gum production rate increased by 15% to 30% compared with the original strain, and the transmittance of the xanthan gum aqueous solution obtained by fermentation is increased by 30% to 50% compared with the original strain.

[0026] 2. The present invention conducted a genetic stability analysis on Xanthomonas campestris with a deposit number of GDMCC NO: 63459, carried out continuous subculture, and measured the xanthan gum yield every two subcultures. It can be seen that the xanthan gum yield and gum quality of the Xanthomonas campestris provided by the present invention did not change significantly after 12 continuous subcultures, demonstrating that it has good stability and can be used as an industrial production strain for scaled production of highly transparent xanthan gum. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a growth curve diagram of the original strain;

[0028] FIG2 is a line graph showing the relationship between the lethality of the original strain and the irradiation dose;

[0029] FIG3 is a line graph showing the relationship between the positive mutation rate of the original strain and the irradiation dose;

[0030] FIG4 is a bar chart showing the gel yield of the rescreened strains;

[0031] FIG5 is a histogram showing the transmittance of xanthan gum aqueous solution obtained from fermentation of the rescreened strains;

[0032] FIG6 is a bar chart showing the genetic stability of the rubber yield of Xanthomonas campestris (deposit number: GDMCC NO: 63459) obtained in the present invention. DETAILED DESCRIPTION

[0033] The present invention will be further described below in conjunction with various embodiments. The embodiments of the present invention include but are not limited to the following embodiments.

[0034] Example

[0035] The Xanthomonas campestris obtained in the present invention (deposit number: GDMCC NO: 63459) was obtained by heavy ion beam irradiation mutagenesis technology, and the starting strain Xanthomonas campestris was irradiated and mutated, and then screened and bred. The experimental materials involved in this example were all commercially available, for example, soluble starch, glucose, corn starch, and sodium chloride were purchased from Tianjin Hongyan Chemical Reagent Factory; peptone and soy protein were purchased from Guangdong Huankai Microbiological Technology Co., Ltd.; potassium dihydrogen phosphate and dipotassium hydrogen phosphate were purchased from Tianjin Yongsheng Fine Chemical Co., Ltd.; magnesium sulfate heptahydrate was purchased from Tianjin Baishi Chemical Co., Ltd.; and beef extract was purchased from Beijing Bioxing Biotechnology Co., Ltd.

[0036] The specific mutagenesis technology screening process is as follows:

[0037] 1. Bacteria culture

[0038] Thaw the original Xanthomonas campestris bacterial suspension, dip a small amount of the suspension with an inoculating loop, and streak it onto a plate. Once a single colony grows, pick it and inoculate it into a liquid seed medium. Incubate it in a constant temperature shaker at 30°C, 180 rpm, for 24 hours. Then, inoculate it into a 250ml conical flask containing 100ml of liquid seed medium at a 1% inoculum rate and incubate it in a constant temperature shaker at 30°C, 180 rpm, for 24 hours. Passage the culture twice to restore its original activity. This original strain was isolated from the leaves of the cruciferous plant Brassica oleracea.

[0039] The plate culture medium is 0.5% soluble starch, 1% peptone, 0.3% beef extract, 0.5% sodium chloride, and 2% agar. The pH of the plate culture medium is 6.57.0, the sterilization temperature is 115°C, and the sterilization time is 30 minutes.

[0040] 2. Liquid seed culture

[0041] The activated original strain was inoculated into a 250 mL conical flask containing 100 mL of liquid seed culture medium at a 1% inoculation rate, and cultured in a constant temperature shaker at 30°C and 180 r / min for 24 h to complete the liquid seed culture.

[0042] The liquid seed culture medium consists of 2.0% soluble starch, 0.5% peptone, 0.3% potassium dihydrogen phosphate, and 0.2% sodium chloride. The pH of the seed culture medium is 7.0. The volume is 100 mL / 250 mL.

[0043] 3. Growth curve determination

[0044] The bacterial liquid of Xanthomonas campestris that had been passaged 2-3 times was inoculated into 100 ml of liquid seed culture medium at a 1% inoculum volume, and cultured at 30°C and 180 r / min in a shaking incubator. The absorbance of the seed culture medium at a wavelength of 600 nm was measured at 0 h after inoculation. Samples were then taken every 2 h to measure the OD of the original strain at 0 h, 2 h, 4 h, 8 h, 12 h, 18 h, 24 h, 30 h, 36 h, 48 h, 54 h, 60 h, and 72 h. 600 Values, record data and draw growth curves.

[0045] Xanthomonas campestris is a Gram-negative pathogen that is obligately aerobic, usually rod-shaped, with a polar flagellum, and has a suitable growth temperature range of 25-30°C. The extracellular polysaccharide secreted by Xanthomonas campestris is called xanthan gum, which is a biological glue with excellent performance and can be widely used in multiple industries with huge market prospects. The fermentation cycle of the original strain is roughly 36-72h. Before 36h, it is mainly the growth and enrichment of the bacteria, and after 36h, it enters the gum production period. The growth curve of the original strain in liquid culture medium is shown in Figure 1. As can be seen from the figure, the growth curve of the original strain conforms to the "S" curve, which is consistent with the characteristics of normal growth and reproduction of microorganisms. The growth delay period of the strain is 0-8h, and the strain grows rapidly in an exponential form within 8-24h. At 24h, the OD 600 It reaches 0.813, which is the logarithmic growth period of the strain. It then continues to grow but the growth rate decreases, reaching a peak between 36-48h, which is the stable growth period of the strain. It enters the decay period in the subsequent culture, and its OD 600 The bacteria slowly decrease and die, which reduces the number of mycelium in the culture medium.

[0046] 4. Heavy ion irradiation mutagenesis

[0047] Take the bacterial suspension of the original strain that has grown to the logarithmic phase and mix it with a vortexer to form a uniform bacterial suspension. Take 1 mL of the prepared bacterial suspension in a 35 mm irradiated dish, seal it with a sealing film, and use the heavy ion mutagenesis research facility (HIRFL) in Lanzhou to generate 12 C 6+ Ion beam mutagenesis was performed with an extraction energy of 80 MeV / u and an LET of 35.5 keV / mm. Eleven doses of mutagenesis were selected: 0 Gy, 20 Gy, 40 Gy, 60 Gy, 80 Gy, 100 Gy, 120 Gy, 110 Gy, 160 Gy, 180 Gy, and 200 Gy.

[0048] 5. Calculate the fatality rate

[0049] After gradient dilution of bacterial suspension with irradiation doses of 0Gy, 20Gy, 40Gy, 60Gy, 80Gy, 100Gy, 120Gy, 140Gy, 160Gy, 180Gy, and 200Gy, 40μL of the bacterial suspension was spread on solid culture medium and cultured in a constant temperature shaker at 37°C and 180r / min for 96h. Three parallel experiments were performed in each group. The number of live bacteria on the plate at different irradiation doses was recorded. The number of colonies after irradiation was divided by the number of colonies in the blank control to calculate the lethality. The lethality curve was drawn with the irradiation dose as the horizontal axis and the lethality as the vertical axis.

[0050]

[0051] In this experiment, the heavy ion beam was controlled within a stable range, and the relative irradiation dose was used as the horizontal axis and the lethality was used as the vertical axis to calculate the lethality and make a lethality curve. The lethality of the original wild Xanthomonas campestris under different irradiation doses is shown in Table 1 and Figure 2. As can be seen from Figure 2, 12 C 6+ For heavy ion irradiation doses between 0 and 200 Gy, the lethality curve exhibits an initial increase, then a decrease, and then a gradual increase again, forming a "saddle"-like curve. At a dose of 120 Gy, the lethality rate briefly dips to 83.89%, then rises again at 140 Gy, reaching a peak of 98.90% at 200 Gy. With increasing irradiation dose, the lethality rate again shows a significant downward trend, forming a concave shape. This characteristic "saddle"-shaped curve is believed to result from the combined effects of damage from energy and momentum and protection and stimulation from mass and charge. As the heavy ion irradiation dose increases, repair pathways within the strain cells activate, protecting DNA from damage, leading to a gradual decrease in lethality. However, when the irradiation dose reaches a certain level, the heavy ion irradiation inactivates repair enzymes within the cells, preventing proper cell repair, resulting in cell damage or death, and a gradual increase in lethality.

[0052] Table 1 Calculation of mortality rate

[0053]

[0054] 6. Calculate the positive mutation rate

[0055] The bacterial suspension of the original wild Xanthomonas campestris induced by different irradiation doses was appropriately diluted, 0.1 mL was aspirated and spread on the solid culture medium, and cultured at 37 ° C for 48 hours. The morphology and size of the colonies under different irradiation doses were observed, and the larger colonies were picked and inoculated on the screening culture medium, and cultured at 37 ° C for 72 hours. After the colonies grew, Lugol's iodine solution was added to the screening culture medium, and the transparent zone around the colonies was observed. The colony diameter (C) and the transparent zone diameter (H) were measured, and the positive mutation rate was calculated according to the formula below. The positive mutation rate curve was drawn with the irradiation time as the horizontal axis and the positive mutation rate as the vertical axis. The strain with the larger transparent zone around the colony was selected and inoculated into the liquid culture medium. After cultured in a constant temperature shaker at 37 ° C and 180 r / min for 24 hours, it was preserved for the next fermentation screening.

[0056]

[0057] The positive mutation rate of the original wild campestris induced by the ratio of the transparent zone diameter to the colony diameter was calculated. The positive mutation rate was calculated with the relative irradiation dose as the horizontal axis and the positive mutation rate as the vertical axis. The positive mutation rate curve was made. The positive mutation rates of wild campestris under different irradiation doses are shown in Table 2 and Figure 3. As can be seen from Figure 3, 12 C 6+ For heavy ion irradiation doses between 0 and 200 Gy, the positive mutation rate curve shows a pattern of first decreasing, then increasing, then decreasing again, and then increasing with increasing irradiation dose. The positive mutation rate reached its lowest point, 8.33%, at 40 Gy. Within the 40-200 Gy range, the positive mutation rate exhibited an "increase-decline-increase" trend, reaching a low point at 140 Gy, reaching 33.33%. The positive mutation rate reached its highest point, 83.33%, at 200 Gy, where the H / C ratio was maximized. This indicates that higher irradiation doses increase the positive mutation rate, the more severe the DNA damage within the bacteria, and the higher the potential mutation rate of the strain. High irradiation doses disrupt internal cellular structures, such as DNA and proteins, leading to genetic variations. Bacteria are more susceptible to mutation, particularly positive mutations, which facilitates the selection of more productive strains from induced strains.

[0058] Table 2 Calculation of positive mutation rate

[0059]

[0060] 7. Initial screening of mutagenic strains

[0061] Observe the morphology and size of colonies at different irradiation doses. Select the larger, smoother, and more translucent colonies and inoculate them onto screening medium. These colonies are numbered and incubated at 37°C for 72 hours. Once colonies have grown, add Lugol's iodine solution to the screening medium and observe the clear zone surrounding the colonies. Measure the colony diameter (C) and the clear zone diameter (H), and calculate the H / C ratio. Strains with higher H / C ratios are inoculated into liquid culture medium and incubated at 30°C, 180 rpm, and a constant temperature shaker for 72 hours. These strains are considered for initial screening.

[0062] The H / C values ​​of the original wild Xanthomonas campestris colonies picked at different irradiation doses after inoculation are shown in Table 3.

[0063] Table 3 H / C value results of original wild Xanthomonas campestris

[0064]

[0065]

[0066]

[0067]

[0068] The H / C values ​​of strains F-0-1 to F-0-12 are the same as those of the original wild Xanthomonas campestris. As shown in Table 3, after the original wild Xanthomonas campestris was induced by heavy ion irradiation, the transparent zone of most colonies became larger and the H / C value increased, indicating that its ability to utilize starch was improved. The H / C value of some colonies decreased, which was a negative mutation. By comparing the size of the H / C value, a total of 23 strains were obtained in the initial screening and named according to the irradiation dose of the strains, namely F-20-1, F-20-7, F-20-8, F-20-9, F-20-10, F-20-11, F-20-12, F-20-13, F-20-14, F-20-15, F-20-16, F-20-17, F-20-18, F-20-19, F-20-21, F-20-23, F-20-27, F-20-28, F-20-29, F-20-21 0-12, F-40-4, F-60-5, F-60-11, F-60-12, F-80-2, F-100-11, F-120-1, F-120-4, F-140-3, F-140-12 , F-160-9, F-160-11, F-180-1, F-180-5, F-180-11, F-200-3, F-200-4, F-200-5, F-200-8, F-200-12. The 23 mutant strains were cultured to the logarithmic phase and inoculated into the fermentation medium at an inoculum size of 5%. The culture was carried out at 37°C and 180 rpm in a shaking incubator for 96 h. The xanthan gum yield was measured. The fermentation product xanthan gum was dissolved in deionized water to prepare a solution with a concentration of 2.0 g / L. The transmittance of the aqueous solution was measured. The measured xanthan gum yield and transmittance of the aqueous solution are shown in Table 4.

[0069] Table 4 Preliminary screening results

[0070]

[0071] 8. Rescreening of mutagenic strains

[0072] Initially screened strains were numbered and, after reaching the logarithmic phase, inoculated at 10% into 75 mL of fermentation medium. The culture was then shaken at 37°C and 180 rpm for 96 hours. After fermentation, the gum production rate and the light transmittance of the xanthan gum aqueous solution were measured. The optimal strains were selected for rescreening by comparing the gum production rates and light transmittance of the aqueous solutions of the initial screened strains. The rescreened strains were then tested for genetic stability.

[0073] The extraction of strain xanthan gum, the calculation process of the yield and transmittance of the aqueous solution are as follows:

[0074] Extraction of xanthan gum: When the OD of liquid seeds 600 When the value is 0.8, the seed liquid of the target strain grown to the logarithmic phase is inoculated into a 250mL conical flask containing 100mL fermentation medium at an inoculum size of 15%, and cultured in a shaker at 40°C and 180r / min for 96h. The ethanol precipitation method is used to add 3 times the volume of deionized water to the fermentation liquid to dilute the fermentation liquid, and then add 3 times the volume of anhydrous ethanol to precipitate xanthan gum. Centrifuge at 8000r / min for 15min, discard the supernatant, retain the precipitate, and then wash the precipitate twice with anhydrous ethanol, dry it in a 60°C oven, grind it, and obtain the fermentation product xanthan gum, which is weighed.

[0075] The liquid fermentation medium is 6.0% corn starch, 1.0% glucose, 2.0% soy protein, 0.1% magnesium sulfate heptahydrate, 0.1% dipotassium hydrogen phosphate, and 0.1% potassium dihydrogen phosphate. The pH of the liquid fermentation medium is 7.0, and the liquid volume is 75mL / 250mL.

[0076] Calculate the xanthan gum yield: Divide the mass of the extracted xanthan gum by the mass of the fermentation liquid to get the xanthan gum yield.

[0077]

[0078] Calculate light transmittance: Dissolve the fermentation product xanthan gum in deionized water to a concentration of 2.0 g / L. Centrifuge at 8000 rpm for 10 minutes to remove bubbles. Measure the absorbance of the aqueous solution at 600 nm using a UV-visible spectrophotometer, using deionized water as a blank control. Perform three replicates per group. Record the data and calculate the light transmittance of the fermentation product xanthan gum aqueous solution according to the following formula:

[0079]

[0080] A——absorbance of the sample;

[0081] T——transmittance of the sample.

[0082] After preliminary screening and comparison of the rubber production rate and light transmittance of the primary screened strains, the six rescreened mutant strains were determined to be F-20-12, F-40-4, F-140-3, F-160-9, F-160-11, and F-180-1. The rubber production rate and light transmittance were determined by fermentation, and the results are shown in Table 5 and Figures 4 and 5 . As shown in Figures 4 and 5, the original wild Xanthomonas campestris had a gum production rate of 3.31% and an aqueous solution transmittance of 65.87%. The gum production rate of F-160-9 was 4.20%, a 26.89% increase compared to the original wild Xanthomonas campestris; the aqueous solution transmittance was 98.41%, a 49.40% increase compared to the original wild Xanthomonas campestris. After heavy ion irradiation, the xanthan gum production rate and aqueous solution transmittance of wild Xanthomonas campestris were significantly improved. Compared with the initial screening results, the H / C value had a certain positive correlation with the xanthan gum production rate, indicating that the initial screening results were reliable. By comparing the gum production rate and transmittance of the six re-screened strains, F-160-9 was finally determined to be the mutant strain obtained in the final screening. The results indicate that heavy ion beam mutagenesis of a native wild Xanthomonas campestris strain can produce a xanthan gum-producing strain with higher gum production and greater xanthan gum aqueous solution transmittance. The strain undergoing heavy ion irradiation and subsequent cultivation exhibited a positive mutation. The mutant strain F-160-9 was deposited with the Guangdong Provincial Microbial Culture Collection, whose biological material name and identification characteristics are Xanthomonas campestris F417-6. The deposit is with the Guangdong Provincial Microbiological Culture Collection, located at 5th Floor, Building 59, 100 Xianlie Middle Road, Guangzhou. The deposit number is GDMCC NO: 63459, and the date of deposit is May 12, 2023.

[0083] Table 5 Rescreening results

[0084]

[0085] 9. Genetic stability determination

[0086] The genetic stability of the screened mutagenic strains was tested and they were subcultured 12 times under the same culture conditions. The xanthan gum yield and the transmittance of the aqueous solution after subculture were observed to determine whether they were genetically stable.

[0087] The mutant strain Xanthomonas campestris (GDMCC No. 63459) was serially passaged 12 times under the same conditions. Every two generations, the strain was inoculated into a liquid fermentation medium. The gum production rate (Figure 6) and the transmittance of the xanthan gum aqueous solution (Table 6) were measured. Standard deviation analysis revealed minimal differences in the yield and transmittance. This suggests that this high-yielding strain of Xanthomonas campestris is genetically stable. This experiment, using heavy ion mutagenesis as a physical mutagenesis technique, yielded a mutant strain with stable properties that is less susceptible to recovery. This strain can be considered for industrial production of highly transparent xanthan gum.

[0088] Table 6 Transmittance genetic stability

[0089]

[0090] The above embodiment is only one of the preferred implementation methods of the present invention and should not be used to limit the scope of protection of the present invention. Any changes or modifications that have no substantive meaning in the main design concept and spirit of the present invention, as long as the technical problems they solve are still consistent with the present invention, should be included in the scope of protection of the present invention.

Claims

1. A strain of Xanthomonas campestris that produces transparent xanthan gum by fermentation, characterized in that: The wild rapeseed Xanthomonas is Xanthomonas campestris F417-6, and it was deposited in the Guangdong Provincial Microbiological Culture Collection Center on May 12, 2023, with the collection number GDMCC NO: 63459, and the collection address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. Use of the Xanthomonas campestris as claimed in claim 1 as a fermentation strain in the fermentation preparation of xanthan gum.

3. The use according to claim 2, characterized in that The following steps are involved: (1) Activation of bacterial strains: After thawing the preserved bacterial liquid containing the Xanthomonas campestris described in claim 1, use an inoculation loop to dip the preserved bacterial liquid and streak it on a plate culture medium. After a single colony grows, pick a single colony and inoculate it into a liquid seed culture medium at a 1% inoculum amount. Ferment at 30°C and 180 rpm constant temperature shaking incubator for 24 hours, perform subculture twice, and restore the original activity of the Xanthomonas campestris. (2) Liquid seed culture: The activated Xanthomonas campestris was inoculated into the liquid seed culture medium at a rate of 1%, and the fermentation temperature was 30° C. and the temperature was shaken at 180 rpm for 24 h to complete the liquid seed culture; (3) Fermentation culture: inoculate the seed liquid of Xanthomonas campestris grown to the logarithmic phase into the fermentation medium at an inoculum amount of 5-20%, ferment at a temperature of 30-40° C., and culture at a constant temperature of 180 rpm in a shaking incubator for 96 h; (4) Xanthan gum was extracted from the fermentation broth by ethanol precipitation method.

4. The use according to claim 3, characterized in that The plate culture medium in step (1) comprises the following components in weight percentage: 0.5% soluble starch, 1% peptone, 0.3% beef extract, 0.5% sodium chloride, and 2% agar. The pH of the plate culture medium is 6.5-7.0, the sterilization temperature is 115° C., the sterilization time is 30 min, and the plate culture time is 72 h.

5. The use according to claim 3, characterized in that In step (1) and step (2), the liquid seed culture medium includes the following components in weight percentage: 2.0% soluble starch, 0.5% peptone, 0.3% potassium dihydrogen phosphate, and 0.2% sodium chloride. The pH value of the liquid seed culture medium is 7.

0.

6. The use according to claim 3, characterized in that The inoculation amount in step (3) is 15%.

7. The use according to claim 3, characterized in that The fermentation temperature in step (3) is 40°C.

8. The use according to claim 3, characterized in that The fermentation medium in step (3) comprises the following components in weight percentage: 6.0% corn starch, 1.0% glucose, 2.0% soy protein, 0.1% magnesium sulfate heptahydrate, 0.1% dipotassium hydrogen phosphate, 0.1% potassium dihydrogen phosphate, and the pH value of the fermentation medium is 7.0.

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