Xanthomonas campestris strain producing high yield, temperature-resistant and quick-dissolving xanthan gum and use thereof

Abstract

Disclosed in the present application are a Xanthomonas campestris strain and the use thereof. Xanthomonas campestris Y-11 provided by the present application has the deposit number of GDMCC No: 63460. Compared with an original strain, the xanthan gum yield of the Xanthomonas campestris Y-11 provided by the present application is increased by 10%-20%, and the xanthan gum obtained by means of fermentation has the characteristics of high dissolution speed, resistance to clumping, high-temperature resistance, etc. In addition, genetic stability analysis is carried out on the Xanthomonas campestris strain, which involves carrying out continuous subculturing, and measuring the xanthan gum yield every two passages. After 12 continuous passages, no significant changes are observed in the xanthan gum yield and gum quality.

Description

A strain of Xanthomonas campestris capable of producing high-yield, temperature-resistant and fast-dissolving xanthan gum and its application

[0001] This application claims priority to the Chinese patent application filed with the Patent Office of China on December 21, 2023, with application number CN202311763508.2 and invention name “A strain of Xanthomonas campestris producing high-yield, temperature-resistant and fast-soluble xanthan gum and its application”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present application belongs to the field of microbial technology, and specifically relates to a strain of Xanthomonas campestris that produces high-yield, temperature-resistant, and fast-dissolving xanthan gum and its application. Background Art

[0003] 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. Xanthan gum is an acidic exopolysaccharide produced by Xanthomonas campestris through aerobic fermentation of sugars, cleaving 1,6-glycosidic bonds, opening side chains, and synthesizing linear chains along 1,4-linkages. In 1963, the U.S. Department of Agriculture's Northern Regional Research Center discovered that xanthan gum was synthesized by Xanthomonas campestris NRRL B-1459, a polysaccharide. Large-scale commercial production began in early 1964. 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 has the general properties of long-chain polymers, but it contains more functional groups than ordinary polymers, which can lead to unique properties under specific conditions. The number of pyruvic acid groups at the end of the molecular side chains of xanthan gum significantly affects its performance. In industrial production, aerobic fermentation using Xanthomonas campestris is performed, and the fermentation broth is subjected to precipitation, centrifugation, washing, separation, drying, grinding, and packaging processes to ultimately produce xanthan gum.

[0004] The strain of bacteria is a very important issue in the fermentation industry. Currently, the most common industrial production strain is Xanthomonas aeruginosa, which ferments and secretes the product outside the cell. The fermentation broth is then processed and extracted to obtain the desired product. However, the degradation of the existing strains caused by the increasing number of strain passages directly affects the industrial production of xanthan gum. The xanthan gum products produced have a single performance, poor high temperature resistance and solubility, and do not meet some application scenarios. Therefore, seeking a good strain is extremely important for the xanthan gum industry to further increase the yield and improve the performance of the fermented xanthan gum to expand its application scenarios. Summary of the Invention

[0005] The purpose of the present application is to provide a strain of Xanthomonas campestris that produces high-yield, temperature-resistant, and fast-dissolving xanthan gum and its application, so as to solve the problems of low xanthan gum production rate in the existing industry and unsatisfactory high-temperature resistance and solubility of the produced xanthan gum.

[0006] To achieve the above objectives, the technical solutions adopted in this application are as follows:

[0007] The present application provides a strain of Xanthomonas campestris that produces high-yield, temperature-resistant, and fast-dissolving xanthan gum. The strain is Xanthomonas campestris Y-11, with a taxonomic name of Xanthomonas campestris. It was deposited in the Guangdong Provincial Microbiological Culture Collection on May 12, 2023, with a deposit number of GDMCC No: 63460, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0008] Preferably, the screening process is as follows:

[0009] (1) Thaw the preserved bacterial solution of the original strain (deposit number: CICC No: 10258), dip the preserved bacterial solution with an inoculating loop and streak it on a plate medium. After a single colony grows, pick a single colony and inoculate it into a liquid seed medium at a 1% inoculum. Incubate at 30°C, 180 rpm, and a constant temperature shaker for 24 h. Subculture twice to restore the original activity of the original bacteria.

[0010] (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 obtain liquid seeds that had grown to the logarithmic phase, completing the liquid seed culture;

[0011] (3) 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 irradiation dish, seal it with a sealing film, and use a 12C 6+ ion beam for irradiation mutagenesis. The extraction energy of the ion beam is 80 MeV / u and the LET is 35.5 keV / mm; the mutagenic dose is selected from 11 doses of 0 Gy, 20 Gy, 40 Gy, 60 Gy, 80 Gy, 100 Gy, 120 Gy, 140 Gy, 160 Gy, 180 Gy, and 200 Gy for irradiation;

[0012] (4) Observe the morphology and size of the colonies under different irradiation doses, pick the larger, smoother and more transparent colonies and spot 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;

[0013] (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.

[0014] The present application also provides the use of the above-mentioned Xanthomonas campestris as a fermentation strain in the fermentation preparation of xanthan gum.

[0015] Preferably, the application comprises the following steps:

[0016] (1) Activation of bacterial strains: Thaw the preserved bacterial solution containing Xanthomonas campestris Y-11 with the deposit number of GDMCC No: 63460, dip the preserved 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. Ferment at 30°C and 180 rpm constant temperature shaker for 24 h. Subculture twice to restore the original activity of Xanthomonas campestris Y-11.

[0017] (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;

[0018] (3) Fermentation culture: Liquid seeds grown to the logarithmic phase were inoculated into the fermentation medium at an inoculum size of 5% to 20%, and the fermentation temperature was 30 to 40°C and the shaking incubator was kept constant at 180 rpm for 96 h;

[0019] (4) Xanthan gum was extracted from the fermentation broth using ethanol precipitation method.

[0020] Preferably, 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.

[0021] Preferably, in step (1) and step (2), the liquid seed culture medium comprises 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.

[0022] Preferably, the inoculation amount in step (3) is 15%.

[0023] Preferably, the fermentation temperature in step (3) is 40°C.

[0024] Preferably, the fermentation medium in step (3) comprises the following components in weight percentage: 4.5% soluble starch, 0.5% glucose, 1.0% 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.

[0025] Preferably, the ethanol precipitation method for extracting xanthan gum from the fermentation broth in step (4) comprises: mixing the fermentation broth with anhydrous ethanol and centrifuging to obtain xanthan gum.

[0026] Preferably, before mixing with anhydrous ethanol, the fermentation broth is first mixed with deionized water for dilution to obtain a dilution liquid, and the volume ratio of the dilution liquid to deionized water is 1:3.

[0027] The present invention provides a method for preparing xanthan gum by fermenting Xanthomonas campestris Y-11 as a fermentation strain, comprising the following steps:

[0028] (1) Activation of bacterial strains: Thaw the preserved bacterial solution containing Xanthomonas campestris with the deposit number GDMCC No. 63460, dip the preserved 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. Ferment at 30°C, 180 rpm constant temperature shaker for 24 h, and subculture twice to restore the original activity of GDMCC63460.

[0029] (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;

[0030] (3) Fermentation culture: Liquid seeds grown to the logarithmic phase were inoculated into the fermentation medium at an inoculum size of 5% to 20%, and the fermentation temperature was 30 to 40°C and the shaking incubator was kept constant at 180 rpm for 96 h;

[0031] (4) Xanthan gum was extracted from the fermentation broth using ethanol precipitation method.

[0032] Preferably, 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.

[0033] Preferably, in step (1) and step (2), the liquid seed culture medium comprises 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.

[0034] Preferably, the inoculation amount in step (3) is 15%.

[0035] Preferably, the fermentation temperature in step (3) is 40°C.

[0036] Preferably, the fermentation medium in step (3) comprises the following components in weight percentage: 4.5% soluble starch, 0.5% glucose, 1.0% 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.

[0037] Preferably, the ethanol precipitation method for extracting xanthan gum from the fermentation broth in step (4) comprises: mixing the fermentation broth with anhydrous ethanol and centrifuging to obtain xanthan gum.

[0038] Preferably, before mixing with anhydrous ethanol, the fermentation broth is first mixed with deionized water for dilution to obtain a dilution liquid, and the volume ratio of the dilution liquid to deionized water is 1:3.

[0039] Compared with the prior art, this application has the following beneficial effects:

[0040] 1. The Xanthomonas campestris provided in this application (deposit number: GDMCC No: 63460) has a xanthan gum yield that is 10% to 20% higher than that of the original strain. The xanthan gum obtained by fermentation has the characteristics of fast dissolution speed, non-agglomeration and high temperature resistance, and is suitable for the fermentation production of temperature-resistant instant xanthan gum.

[0041] 2. This application conducted a genetic stability analysis on Xanthomonas campestris with a deposit number of GDMCC No: 63460, 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 Xanthomonas campestris after 12 continuous subcultures did not change significantly, proving that it has good stability and can be used as an industrial production strain for large-scale production of temperature-resistant and instant-soluble xanthan gum. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments.

[0043] FIG1 is a growth curve of the original strain of the present application (deposit number: CICC No: 10258);

[0044] FIG2 is a line graph showing the relationship between the lethality and irradiation dose of the original strain of the present application (deposit number: CICC No: 10258);

[0045] FIG3 is a line graph showing the relationship between the positive mutation rate and the irradiation dose of the original strain of the present application (deposit number: CICC No: 10258);

[0046] FIG4 is a bar chart showing the gel yield of the primary screening strains of the present application;

[0047] FIG5 is a bar chart showing the gel yield of the rescreened strains of the present application;

[0048] FIG6 is a bar chart showing the genetic stability of the rubber yield of Xanthomonas campestris (GDMCC No: 63460) and the original strain (CICC No: 10258) of the present application;

[0049] FIG7 is a bar chart showing the complete dissolution time of xanthan gum, a fermentation product of Xanthomonas campestris (GDMCC No: 63460) and the original strain (CICC No: 10258) of the present application, at a concentration of 1% by mass;

[0050] FIG8 is a line graph showing the temperature resistance of xanthan gum fermented by the present application's Xanthomonas campestris (deposit number: GDMCC No: 63460) and the original strain (deposit number: CICC No: 10258).

[0051] Biological Deposit Description

[0052] Xanthomonas campestris Y-11 was deposited in Guangdong Provincial Microbiological Culture Collection on May 12, 2023, with the deposit number GDMCC No: 63460, and the deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. DETAILED DESCRIPTION

[0053] The present application is further described below in conjunction with various embodiments. The methods of the present application include but are not limited to the following embodiments.

[0054] The Xanthomonas campestris obtained in the present application (with a deposit number of GDMCC No: 63460) was obtained by subjecting the original strain Xanthomonas campestris (with a deposit number of CICC No: 10258, hereinafter referred to as the "original strain") to irradiation mutagenesis using heavy ion beam irradiation mutagenesis technology, and then screening and breeding. The screening process of the mutagenesis technology is as follows:

[0055] 1. Bacteria culture

[0056] Thaw the bacterial liquid of the original strain, dip a small amount of bacterial liquid with an inoculating loop and streak on the flat culture medium. After a single colony grows, pick a single colony and inoculate it into a liquid seed culture medium. Culture it in a constant temperature shaker at 30°C and 180 r / min for 24 hours. Inoculate it into a 250 mL conical flask containing 100 mL of liquid seed culture medium at a 1% inoculum size and culture it in a constant temperature shaker at 30°C and 180 r / min for 24 hours. Subculture it twice to restore its original activity.

[0057] The plate culture medium comprises 0.5% soluble starch, 1% peptone, 0.3% beef extract, 0.5% sodium chloride, and 2% agar, has a pH of 6.5-7.0, a sterilization temperature of 115° C., and a sterilization time of 30 minutes.

[0058] 2. Liquid seed culture

[0059] 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.

[0060] 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 liquid seed culture medium is 7.0. The volume is 100 mL / 250 mL.

[0061] 3. Growth curve determination

[0062] The bacterial liquid of the original strain that was repeatedly passaged 2-3 times was inoculated into 100 ml of liquid seed culture medium at a 1% inoculum volume, and the temperature was 30°C and the shaking was constant temperature cultured at 180 r / min. The absorbance of the seed culture medium at a wavelength of 600 nm was measured at 0 h of inoculation. Samples were taken every 2 h thereafter 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.

[0063] 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 many industries with huge market prospects. The growth curve of the original strain in liquid culture medium is shown in Figure 1. As can be seen from Figure 1, 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 12-30h. At 24h, the OD 600 When the growth rate reaches 1.175, it is the logarithmic growth period of the bacteria. During this period, the bacteria grow fastest and metabolize most vigorously. Then it continues to grow but the growth rate decreases, reaching a peak between 30-36 hours, which is the stable growth period of the bacteria. In the subsequent culture, it enters the decline period, and its OD 600 The bacteria slowly decrease and die, which reduces the number of mycelium in the culture medium.

[0064] 4. Heavy ion irradiation mutagenesis

[0065] 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, 140 Gy, 160 Gy, 180 Gy, and 200 Gy.

[0066] 5. Calculate the fatality rate

[0067] The bacterial suspension with irradiation doses of 0Gy, 20Gy, 40Gy, 60Gy, 80Gy, 100Gy, 120Gy, 140Gy, 160Gy, 180Gy, and 200Gy was gradiently diluted and 40 μL of the bacterial suspension was spread on the solid culture medium.

[0068] Here we need to determine the optimal dilution gradient. The purpose of the optimal dilution gradient experiment is to observe the single colonies of Xanthomonas campestris and prevent the bacterial growth density from being too high. Cultivating the colonies under the optimal dilution gradient can disperse the cells. The number of colonies on the solid plate is between 50 and 200, which is conducive to better observation of single bacterial colonies. As shown in Table 1, when the dilution gradient is 10 -5 When the gradient is applied, the number of colonies on the plate is less than 200, i.e. 10 -5 The gradient is the optimal dilution gradient.

[0069] Table 1 Dilution gradient

[0070] The culture was carried out in a constant temperature shaker at 37°C and 180 rpm for 96 h, with three parallel experiments in each group. The number of viable bacteria on the plate at different irradiation doses was recorded, and 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.

[0071] Lethality rate (%) = [1-(number of colonies grown in the irradiated group / number of colonies grown in the control group)] × 100%.

[0072] In this experiment, the heavy ion beam current was controlled within a stable range. The relative irradiation dose was used as the horizontal axis and the lethality was used as the vertical axis to calculate the lethality and create a lethality curve. The lethality of the original strain under different irradiation doses is shown in Figure 2. As can be seen from Figure 2, with the increase of irradiation dose, the lethality of the original strain generally increases first and then decreases, which is a typical "saddle-shaped" trend. The lethality of the strain at 160Gy is the highest, at 86.67%. Within the 0-60Gy mutagenic dose, the lethality of bacteria gradually increases. Within the 60-120Gy mutagenic dose, the lethality tends to be stable. Within the 120-160Gy mutagenic dose, the lethality shows an upward trend. The lethality at 180Gy decreases slightly. At the 200Gy irradiation dose, the lethality of bacteria increases again. When cells are irradiated, the cell structure will be destroyed, the number of bacterial deaths will increase, and the mortality rate will increase. Later, the bacteria will repair some damaged cells, and the mortality rate will increase slightly. Therefore, the mortality rate curve shows a trend of increasing, decreasing, and then increasing again.

[0073] 6. Calculate the positive mutation rate

[0074] The bacterial suspension of the original strain 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 circle around the colonies was observed. The colony diameter (C) and the transparent circle 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 circle 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.

[0075] Positive mutation rate (%) = (the number of colonies whose H / C value of the mutant strain is more than 20% higher than that of the original strain / the total number of test colonies) × 100%.

[0076] The positive mutation rate of the original strain was calculated based on the ratio of the clear zone diameter to the colony diameter. The positive mutation rate was calculated using the relative irradiation dose as the horizontal axis and the positive mutation rate as the vertical axis. A positive mutation rate curve was constructed. Figure 3 shows the positive mutation rate of Xanthomonas campestris under different irradiation doses. As shown in Figure 3, the positive mutation rate of the original strain increases with increasing irradiation dose, then decreases, then increases, then decreases, then increases again. The positive mutation rate reaches a maximum of 44.00% at an irradiation dose of 80 Gy, indicating that 80 Gy irradiation is beneficial for selecting strains with positive mutations. At high irradiation doses, internal cellular structures, such as DNA and proteins, are disrupted, genetic information mutates, and bacteria are more susceptible to mutations. Consequently, positive mutations are also more likely to occur, which facilitates the selection of higher-yielding strains among the induced strains.

[0077] 7. Initial screening of mutagenic strains

[0078] 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.

[0079] Sixteen strains with high H / C values ​​were selected and cultured in the above steps and designated as Y-1, Y-2, Y-3, Y-4, Y-5, Y-6, Y-7, Y-8, Y-9, Y-10, Y-11, Y-12, Y-13, Y-14, Y-15, and Y-16. The 16 mutagenic strains selected from the initial screening were cultured to the logarithmic phase and inoculated into a fermentation medium. After a period of time, xanthan gum was extracted and its yield was measured to identify the high-yielding strain.

[0080] The extraction process of strain xanthan gum and calculation of the yield rate are as follows:

[0081] 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.

[0082] The fermentation medium is composed of 4.5% soluble starch, 0.5% glucose, 1.0% soy protein, 0.1% magnesium sulfate heptahydrate, 0.1% dipotassium hydrogen phosphate, and 0.1% potassium dihydrogen phosphate; the pH of the fermentation medium is 7.0. The liquid volume is 75mL / 250mL.

[0083] 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.

[0084] Xanthan gum yield (%) = [xanthan gum sample mass (g) / xanthan gum fermentation broth mass (g)] × 100%.

[0085] Figure 4 shows that six relatively high-yielding mutagenized strains, Y-1, Y-4, Y-8, Y-9, Y-11, and Y-14, were selected. Among these, Y-11 had the highest xanthan gum yield, at 4.11%. This indicates that heavy ion beam mutagenesis of the original strain can yield xanthan gum-producing strains with even higher yields, with the induced strains undergoing positive mutations. These six high-yielding strains exhibited strong xanthan gum production, surpassing the original strain in production efficiency. Individual colonies of these six mutagenized strains were stored in a strain collection to facilitate further screening and other property determinations.

[0086] 8. Rescreening of mutagenic strains

[0087] Initially screened strains were numbered and, after reaching the logarithmic phase, inoculated at 10% into 75 mL of fermentation medium. The culture was 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.

[0088] The six mutagenized strains initially screened in the above steps were inoculated into fermentation medium for culture, and xanthan gum was extracted and measured for gum production. As shown in Figure 5, the gum production rate of Y-11 was significantly higher than that of the other strains irradiated with the mutagenizing dose, reaching 4.22%. This confirmed that Y-11 was the optimal strain relative to the other mutagenized strains.

[0089] 9. Genetic stability determination

[0090] 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.

[0091] The high-yield strain Y-11 and the original strain were continuously subcultured 12 times under the same conditions, and inoculated into the fermentation medium every two generations to determine the gum production rate. The results are shown in Figure 6. Under the analysis of standard deviation, the difference in the gum production rate is not large. This shows that the genetic performance of this high-yield strain Y-11 is stable. This experiment uses physical mutagenesis of heavy ion mutagenesis for artificial breeding, which can obtain a Y-11 mutant strain with stable performance, not prone to recovery and high yield. The mutant strain Y-11 is preserved with the preservation number GDMCC No: 63460 (hereinafter referred to as "mutagenic strain"), the taxonomic name is: Xanthomonas campestris, and it was preserved in the Guangdong Provincial Microbial Culture Collection Center on May 12, 2023.

[0092] 10. Product solubility determination

[0093] The screened mutagenic strain and the fermentation product xanthan gum of the original strain were prepared into a 1% mass concentration aqueous solution and stirred at 600 r / min until completely dissolved. The time for the xanthan gum to be completely dissolved at a 1% mass concentration was measured. Three parallel experiments were performed in each group and the data were recorded.

[0094] As shown in Figure 7, the complete dissolution time of the xanthan gum produced by the original strain at a 1% mass concentration was 13.95 minutes. The xanthan gum produced by the mutant strain was prepared into a 1% mass concentration aqueous solution and stirred at 600 rpm until completely dissolved. The complete dissolution time of the xanthan gum at 1% mass concentration was 8.67 minutes, which was 60.90% shorter than that of the original strain. This indicates that the xanthan gum produced by the mutant strain can be completely dissolved within 10 minutes when added to a deionized water solution at a 1% mass concentration.

[0095] 11. Determination of product temperature resistance

[0096] The xanthan gum obtained by fermentation of the screened mutagenic strain and the original strain was ground into powder. 1000 mL of the product xanthan gum solution with a mass concentration of 1% was placed in a water bath and heated at 10, 20, 30, 40, 50, 60, 70, and 80°C. The product xanthan gum aqueous solution was stabilized at each temperature for 1 h. The NDJ-5S rotational viscometer was turned on and the speed was set to 6 r / min. The viscosity values ​​of the product xanthan gum solution at different temperatures were recorded. Three parallel experiments were performed in each group, and the unit was mPa·s.

[0097] As can be seen from Figure 8, the viscosity of the xanthan gum solution obtained by the mutant strain fermentation showed a trend of first increasing and then decreasing in the temperature range of 10-80°C. The viscosity of the xanthan gum solution was the highest at 20°C, which was 180.34 mPa·s. As the temperature gradually increased, the viscosity of the xanthan gum solution gradually decreased, and the viscosity at 80°C was 117.06 mPa·s. The viscosity change in the entire temperature range was not obvious, the viscosity was moderate, and no agglomeration was generated. The viscosity change of the xanthan gum solution in the temperature range of 10-80°C was not significant compared with the xanthan gum solution produced by the original strain fermentation. The viscosity of the xanthan gum solution obtained by the original strain fermentation showed a trend of gradually decreasing in the temperature range of 10-80°C, with a viscosity of 151.43 mPa·s at 10°C and a minimum value of 27.66 mPa·s when the temperature was raised to 80°C. The viscosity change was more obvious.

[0098] Although the above embodiment provides a detailed description of the present application, it is only a part of the embodiments of the present application, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present application.

Claims

1. A strain of Xanthomonas campestris Y-11 which produces high-yield temperature-resistant and fast-soluble xanthan gum, with a deposit number of GDMCC No: 63460.

2. Use of the Xanthomonas campestris Y-11 described in claim 1 as a fermentation strain in the fermentation preparation of xanthan gum.

3. A method for preparing xanthan gum by fermenting Xanthomonas campestris Y-11 as a fermentation strain, characterized in that: The following steps are involved: (1) Activation of bacterial strains: After thawing the preserved bacterial liquid containing the wild Xanthomonas campestris Y-11 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 wild Xanthomonas campestris Y-11. (2) Liquid seed culture: The activated Xanthomonas campestris Y-11 was inoculated into a liquid seed culture medium at a 1% inoculation rate for culture at a temperature of 30° C. and a constant temperature shaker at 180 rpm for 24 h to obtain liquid seeds grown to the logarithmic phase, thereby completing the liquid seed culture; (3) Fermentation culture: Inoculate the liquid seeds grown to the logarithmic phase into the fermentation medium at an inoculum rate of 5% to 20%, ferment at a temperature of 30 to 40° C., and culture at a constant temperature of 180 rpm for 96 h in a shaking incubator; (4) Xanthan gum was extracted from the fermentation broth by ethanol precipitation method.

4. The method 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 value of the plate culture medium is 6.5-7.0, the sterilization temperature is 115° C., the sterilization time is 30 min, and the culture time on the plate culture medium is 72 h.

5. The method 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 method according to claim 3, characterized in that: The inoculation amount in step (3) is 15%.

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

8. The method according to claim 3, characterized in that The fermentation medium in step (3) comprises the following components in weight percentage: 4.5% soluble starch, 0.5% glucose, 1.0% 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.

9. The method according to claim 3, characterized in that: The ethanol precipitation method for extracting xanthan gum from the fermentation broth in step (4) comprises: mixing the fermentation broth with anhydrous ethanol, and centrifuging to obtain xanthan gum.

10. The method according to claim 9, characterized in that Before mixing with anhydrous ethanol, the fermentation liquid is first mixed with deionized water for dilution to obtain a diluent, and the volume ratio of the diluent to deionized water is 1:3.

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