Construction method of high / low pyruvate xanthan gum production strain
By using ambient temperature and pressure plasma mutagenesis and high-throughput screening technology, efficient and stable strains for producing high or low pyruvate xanthan gum were constructed, solving the problems of low screening efficiency and poor stability in existing technologies and meeting the needs of different application scenarios.
Patent Information
- Application Number
- CN202511217841.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-28
- Publication Date
- 2025-12-02
AI Technical Summary
In existing technologies, the pyruvate content of xanthan gum producing strains is concentrated at around 2.5%, which is difficult to meet the differentiated needs of niche markets, and the screening efficiency is low and the stability of mutant strains is poor.
A combination of ambient temperature and pressure plasma mutagenesis and high-throughput screening was used to induce targeted mutagenesis of Xanthomonas brassicae. Stable mutant strains with high or low pyruvate content were screened through high-throughput primary screening and multiple rounds of secondary screening. The pyruvate content was quantified using the 2,4-dinitrophenylhydrazine colorimetric method combined with a standard curve. Single colonies were selected using a high-throughput colony selection workstation, shortening the screening cycle.
The mutant strains with pyruvate content significantly deviating from the original strain were efficiently screened. The high pyruvate content of the strains can reach more than 5%, while the low pyruvate content is as low as 1.0% to 1.2%. The xanthan gum production capacity is on par with the original strains, which solves the problem of poor stability of mutant strains and ensures the continuity and economy of industrial production.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, specifically to a method for constructing a high / low pyruvate xanthan gum production strain. Background Technology
[0002] Xanthan gum, an important microbial extracellular polysaccharide, has its rheological properties, temperature resistance, and synergistic effects directly influenced by the pyruvate content in its molecular structure, thus determining its application scenarios in food, oil extraction, and pharmaceutical fields. Xanthan gum with high pyruvate content is suitable for high-temperature, high-shear environments, while xanthan gum with low pyruvate content is suitable for low-viscosity, low-shear-sensitivity environments.
[0003] Currently, the pyruvate content of xanthan gum industrial production strains is mostly concentrated around 2.5%, which is insufficient to meet the needs of niche markets. Existing technologies for improving xanthan gum production strains primarily focus on increasing yield or optimizing solubility. There is a lack of systematic research on methods for constructing xanthan gum production strains with extremely high or low pyruvate content, and these methods suffer from low screening efficiency and poor stability of mutant strains. Therefore, developing an efficient and stable method for constructing xanthan gum production strains with high / low pyruvate content is of great significance. Summary of the Invention
[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for constructing high / low pyruvate xanthan gum production strains, which can efficiently obtain mutant strains with stable pyruvate content and excellent gum production capacity, thus meeting the differentiated needs of xanthan gum in different application scenarios.
[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:
[0006] A method for constructing a high / low pyruvate xanthan gum production strain, comprising:
[0007] S1, prepare the original strain Xanthomonas campestris;
[0008] S2, determine the growth curve of the original strain to identify the logarithmic growth phase;
[0009] S3, using ARTP mutagenesis of the original strain in the logarithmic growth phase, with a mutagenesis time of 160-200s;
[0010] S4, High-throughput initial screening: The mutagenized strains are cultured to obtain fermentation broth, the pyruvate content in the fermentation broth is measured, and multiple strains with high or low pyruvate content are screened out.
[0011] S5, Secondary screening: The strains selected in step S4 are screened again to select strains with stable pyruvate content and xanthan gum production that are the same as the original strains.
[0012] Furthermore, the original strain Xanthomonas campestris was purchased from the China General Microbiological Culture Collection Center, with accession number CGMCC1.1781.
[0013] Furthermore, in step S3, the original bacterial culture in the logarithmic growth phase is taken and diluted to a bacterial concentration of 10. 5 CFU / mL, take 10 μL of bacterial suspension and spread it evenly on a sterile stainless steel slide, then place it in the ARTP mutagenesis system for mutagenesis.
[0014] Furthermore, in step S3, the parameters for ARTP mutagenesis are: RF power 100W, distance between plasma torch nozzle and substrate 2mm, air flow rate 10mL / min, and plasma flow temperature 25℃~35℃.
[0015] Furthermore, in step S4, the mutagenized strain is diluted and spread onto a plate. Single colonies are picked using a high-throughput colony selection workstation and inoculated into seed culture medium. The single colonies are then inoculated into fermentation medium and incubated at 30°C and 200 rpm for 16 h. Finally, the culture is inoculated into fermentation medium and incubated at 30°C and 200 rpm for 96 h to obtain fermentation broth.
[0016] Furthermore, the seed culture medium comprises: 20 g / L sucrose, 5 g / L tryptone, 3 g / L beef extract, 1 g / L yeast extract, and pH adjusted to 7.0–7.2.
[0017] Furthermore, the fermentation medium comprises: 50 g / L sucrose, 0.08 g / L yeast extract, 15 g / L potassium chloride, 0.6 g / L potassium dihydrogen phosphate, 0.08 g / L magnesium sulfate, 5 g / L calcium carbonate, and 0.015 g / L citric acid, with the pH adjusted to 7.0–7.2.
[0018] Furthermore, in step S4, the method for determining the pyruvate content in the fermentation broth is as follows: take 1 mL of fermentation broth, dilute it 50 times, add 1.5 mol / L HCl to hydrolyze for 10 min, then add 1 mL of 0.5% 2,4-dinitrophenylhydrazine hydrochloric acid solution and react for 5 min. Add 5 mL of 1.5 mol / L NaOH solution, shake well and develop color. Record the absorbance at 520 nm and calculate by comparing with the pyruvate standard curve.
[0019] Furthermore, in step S5, the method for determining the xanthan gum yield during re-screening includes: taking the fermentation broth, centrifuging at 8000 rpm for 15 min at 4°C, taking the supernatant, adding 3 times the volume of anhydrous ethanol for precipitation, letting it stand at 4°C for 2 h, filtering under reduced pressure, drying the precipitate under vacuum at 55°C for 24 h to constant weight, weighing and calculating the yield.
[0020] The present invention also provides the application of the strains constructed by the above method in the preparation of high-pyruvate xanthan gum or low-pyruvate xanthan gum.
[0021] Compared with the prior art, the present invention has the following beneficial effects:
[0022] (1) This invention uses ARTP mutagenesis to induce targeted mutagenesis of Xanthomonas brassicae, and combines high-throughput primary screening with multiple rounds of secondary screening to efficiently screen mutant strains with pyruvate content significantly deviating from the original strain. Among them, the high-pyruvate strain has a pyruvate content of more than 5%, which is suitable for high temperature and strong shear scenarios; the low-pyruvate strain has a pyruvate content as low as 1.0% to 1.2%, which is suitable for low viscosity and weak shear sensitivity scenarios, filling the gap in the existing industrial strains in specific fields.
[0023] (2) Using the mutagenesis technology provided by this invention, while the pyruvate content deviates significantly from the original strain, its xanthan gum production capacity remains the same as the original strain. Through three repeated fermentation verifications, the pyruvate content of the low-pyruvate strain Z3 and the high-pyruvate strain 2H7 fluctuates very little, and the xanthan gum yield remains at a relatively high level of 16-18 g / L. This solves the problems of poor stability and decreased xanthan gum production capacity of mutant strains in the prior art, ensuring the continuity and economy of industrial production.
[0024] (3) This invention employs a high-throughput colony selection workstation and standardized detection methods (such as quantifying pyruvate content using the 2,4-dinitrophenylhydrazine colorimetric method combined with a standard curve, and determining xanthan gum yield using the ethanol precipitation-vacuum drying method), significantly shortening the screening cycle. Compared to traditional methods, the initial screening of this invention can process a large number of single colonies at once, and the secondary screening eliminates accidental variations through multiple rounds of verification, significantly improving screening efficiency and result reliability. Attached Figure Description
[0025] Figure 1 The growth curve of Xanthomonas aeruginosa in rapeseed;
[0026] Figure 2 The lethality curve of Xanthomonas campestris mutagenesis;
[0027] Figure 3 This is the standard curve for pyruvate.
[0028] Figure 4 The results of the first secondary screening of Xanthomonas spp. that produces low-pyruvate xanthan gum;
[0029] Figure 5 The results show the xanthan gum production capacity of strains after the first secondary screening of strains with low pyruvate xanthan gum content.
[0030] Figure 6 The results of the second screening of Xanthomonas spp. that produces low-pyruvate xanthan gum;
[0031] Figure 7 The results of the third screening of Xanthomonas spp. that produces low-pyruvate xanthan gum;
[0032] Figure 8 For the first secondary screening of Xanthomonas spp. that produces high levels of xanthan gum pyruvate;
[0033] Figure 9 Second screening of Xanthomonas spp. that produces high levels of xanthan gum pyruvate;
[0034] Figure 10 This is the third rescreening of Xanthomonas oryzae, a bacterium that produces high levels of xanthan gum pyruvate. Detailed Implementation
[0035] The present invention will be further described below with reference to the accompanying drawings and embodiments. The embodiments of the present invention include, but are not limited to, the following embodiments. All experiments related to these embodiments were completed on the experimental platform of the Key Laboratory of Microbial Metabolism and Green Fermentation Engineering in Inner Mongolia Autonomous Region.
[0036] This embodiment provides a method for constructing high / low pyruvate xanthan gum production strains. The method uses *Xanthomonas campestris* as the original strain and obtains the strain through ambient temperature and pressure plasma mutagenesis combined with high-throughput screening technology. The specific steps are as follows:
[0037] I. Materials and Instruments
[0038] (1) Original strain: Xanthomonas campestris, purchased from China General Microbiological Culture Collection Center, accession number: CGMCC1.1781;
[0039] (2) Other materials and reagents: Beef extract, yeast extract and potassium dihydrogen phosphate were purchased from Sinopharm Chemical Reagent Co., Ltd., and sucrose, tryptone and other reagents were purchased from Sangon Biotech Co., Ltd.
[0040] (3) Experimental instruments: atmospheric pressure room temperature plasma mutagenesis breeding system (Beijing Siqingyuan Biotechnology Co., Ltd.); high-throughput fully automated microbial colony selection workstation (Molecular Instruments, Inc., USA);
[0041] (4) The culture media used in this embodiment are (all in 1L, diluted with deionized water):
[0042] Seed culture medium: 20 g / L sucrose, 5 g / L tryptone, 3 g / L beef extract, 1 g / L yeast extract, pH adjusted to 7.0–7.2;
[0043] Slant culture medium: Add 20 g / L agar to the seed culture medium and adjust the pH to 7.0–7.2;
[0044] Shake-flask fermentation medium: sucrose 50 g / L, yeast extract 0.08 g / L, potassium chloride 15 g / L, potassium dihydrogen phosphate 0.6 g / L, magnesium sulfate 0.08 g / L, calcium carbonate 5 g / L, citric acid 0.015 g / L, pH adjusted to 7.0–7.2.
[0045] II. Construction Method
[0046] Step 1: Construct a growth curve for Xanthomonas brassicae (to determine the logarithmic growth phase):
[0047] The original strain *Xanthomonas campestris* was inoculated from glycerol tubes onto agar slant culture medium and cultured at 30°C for 24 h. It was then transferred to liquid seed culture medium and cultured at 30°C with shaking at 200 rpm for 12 h. Finally, it was transferred at a 10% inoculum to a fresh seed culture medium (30°C, 200 rpm). Samples were taken every 2 h, and absorbance (OD) was measured at 600 nm. 600 ), plot the growth curve, such as Figure 1 As shown, this is to determine the logarithmic growth phase range of the growing strain.
[0048] according to Figure 1 It is known that the logarithmic growth phase of Xanthomonas brassicae is between 7 and 18 hours. During the logarithmic growth phase, the bacteria are more susceptible to mutagenesis. Therefore, bacterial culture of 12 hours was selected for subsequent ARTP mutagenesis.
[0049] Step 2, ARTP mutagenesis treatment (determining optimal mutagenesis conditions):
[0050] Take the bacterial culture in the logarithmic growth phase and serially dilute it to a bacterial concentration of 10⁻⁶. 5 CFU / mL, 10 μL of bacterial suspension was evenly spread onto a sterile stainless steel slide and placed in an ARTP mutagenesis system. The mutagenesis parameters were set as follows: radio frequency power 100 W, plasma torch nozzle-slide distance 2 mm, air flow rate 10 mL / min, plasma temperature 25℃~35℃. Mutagenesis was performed for 40 s, 80 s, 120 s, 160 s, 200 s, and 240 s. After mutagenesis, the slide was immersed in an EP tube containing 500 μL of liquid seed culture medium and vortexed for 30 s to detach the bacteria, ensuring all bacteria entered the buffer solution. The bacterial suspension was then diluted 10... 4 After doubling the induction time, 100 μL was evenly spread onto solid selection medium and incubated at 30°C for 2–3 days. Three replicates were prepared for each mutagenesis time, and the number of colonies on the plates was recorded. The optimal mutagenesis time was determined based on the lethality rate, calculated using the following formula:
[0051] Lethality rate = (Number of colonies in control group - Number of colonies in experimental group) / Number of colonies in control group × 100%
[0052] The curves of lethality and mutagenesis time are as follows: Figure 2 As shown, according to Figure 2 It can be seen that the mortality rate of Xanthomonas brassicae increases with time, and when the mutagenesis time is 160s, the mortality rate is about 95%, which is the optimal mutagenesis condition.
[0053] Step 3, High-throughput primary screening:
[0054] The mutagenized strain was diluted and spread onto plates. Single colonies were picked using a high-throughput colony selection workstation and inoculated into 96-well plates containing 1 mL of seed culture medium. The plates were incubated at 30°C and 200 rpm for 16 h. The inoculum was then transferred to 96-well plates with 10% inoculum (1 mL per well) and incubated at 30°C and 200 rpm for 96 h.
[0055] After fermentation, take 1 mL of fermentation broth, dilute it 50 times, add 1.5 mol / L HCl for 10 min of hydrolysis, then add 1 mL of 0.5% 2,4-dinitrophenylhydrazine hydrochloric acid solution and react for 5 min, then add 5 mL of 1.5 mol / L HCl solution. NaOH solution was shaken and the color was developed. The absorbance was recorded at 520 nm and compared with the pyruvate standard curve to calculate the pyruvate concentration of the fermentation broth. In this example, 20 strains with low pyruvate concentrations were initially screened and numbered sequentially as Z1-Z20. 23 strains with high pyruvate concentrations were selected and numbered sequentially as follows: 2H1, 2H10, 10A1, 2F12, 2H4, 8H5, 8H1, 7A1, 8H3, 2F2, 10C12*, 8H8, 4A9, 10C6, 2H7, 9A1, 6H11, 8H3, 2F2, 10C12, 7H12, 6D1, 2H6.
[0056] In this step, the pyruvate standard curve was obtained as follows: A 1 mg / mL pyruvate standard solution was prepared and diluted 10 times to obtain a 0.1 mg / mL pyruvate standard solution. Six 10 mL stoppered test tubes were taken, and 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of pyruvate standard solution and 1 mL of distilled water were added respectively. The volume was then adjusted to 1 mL with distilled water to obtain solutions with pyruvate contents of 20 μg, 40 μg, 60 μg, 80 μg, 100 μg, and 0 μg, respectively. Each tube was placed in a metal bath and heated at 110 °C for 4 h. After cooling to room temperature, 1 mL of 0.5% 2,4-dinitrophenylhydrazine solution was added, and the mixture was shaken well and reacted for 5 min. Then, 5 mL of 1.5 mol / L NaOH solution was added, and the mixture was shaken well for color development. The absorbance of each tube was measured at 520 nm using a spectrophotometer. A standard curve was plotted with pyruvate content (μg) on the x-axis and the corresponding absorbance on the y-axis, as shown below. Figure 3As shown, its regression equation is: y = 0.012x + 0.03(R²). 2 =0.998).
[0057] Step 4: Secondary screening of strains with low pyruvate content:
[0058] Twenty low-pyruvate bacterial strains obtained after high-throughput screening were inoculated into 500 mL shake flasks (containing 100 mL of seed culture medium) and cultured at 30 °C and 200 rpm for 18 h. Then, at a 10% inoculum size, they were transferred to 500 mL shake flasks containing 100 mL of fermentation medium and cultured at 30 °C and 200 rpm for 96 h. The fermentation broth was centrifuged at 4 °C and 8000 rpm for 15 min. Three times the volume of the supernatant was used for alcohol precipitation, and the mixture was allowed to stand at 4 °C for 2 h. The precipitate was collected by vacuum filtration and dried at 55 °C for 24 h to constant weight. After grinding into powder, the dried xanthan gum was obtained, and the xanthan gum yield was calculated. Weigh 0.0100 g of dried xanthan gum and redissolve it in 10 mL of distilled water. Pipette 1 mL of the aqueous solution into a stoppered test tube and heat at 110 °C for 4 h in a metal bath. After cooling to room temperature, add 1 mL of 0.5% 2,4-dinitrophenylhydrazine solution, shake well, and react for 5 min. Then add 5 mL of 1.5 mol / L NaOH solution, shake well, and develop color. Record the absorbance at 520 nm. Calculate the pyruvate content based on the standard curve (y = 0.012x + 0.03). The pyruvate content of these 20 strains (Z1-Z20) is as follows: Figure 4 As shown, six strains with relatively low pyruvate content were screened, numbered Z1, Z3, Z6, Z10, Z15, and Z18, with pyruvate content ranging from 1.0% to 1.2%. The xanthan gum yield of these six strains is as follows: Figure 5 As shown, from Figure 5 It can be seen that after room temperature and pressure plasma mutagenesis and high-throughput screening, the pyruvate content of the strain decreased during the fermentation production of xanthan gum, but the ability to produce xanthan gum remained at a high level (16-18 g / L).
[0059] The six selected strains underwent a second screening, using the same method as the shake-flask fermentation method described above. The results of pyruvate production for each strain are as follows: Figure 6 As shown, these six strains underwent a third screening, using the same method as the shake-flask fermentation method described above. The results are as follows. Figure 7 As shown.
[0060] In this embodiment, after mutagenesis of the original strain, a relatively stable xanthan gum-producing strain Z3 with low pyruvate content was obtained through one primary screening and three secondary screenings. The pyruvate content of the strain was between 1.0% and 1.2% in the three secondary screenings, indicating that the pyruvate content of the strain was stable and did not fluctuate significantly during the production of xanthan gum. Therefore, this strain was selected as the strain for subsequent fermentation. The selected strain Z3 was preserved for subsequent fermentation experiments.
[0061] The selected strain was named *Xanthomonas campestris* L1 and deposited on February 20, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC NO: 65931), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. This strain can be used as a fermentation strain for xanthan gum production, with an optimal fermentation temperature of 30℃ and a fermentation time of 96 hours.
[0062] Step 5: Secondary screening of strains with high pyruvate content:
[0063] The secondary screening method for strains with high pyruvate content was the same as that for strains with low pyruvate content. The xanthan gum yield (g / L) and pyruvate content (%) of the 23 strains obtained from the initial screening were as follows: Figure 8 As shown, six bacterial strains with relatively high pyruvate content (>5%) were screened: 2H4, 2H7, 8H8, 10A1, 6A11, and 8H5. A second screening was performed on the selected strains, and the results of pyruvate production for each strain are shown below. Figure 9 As shown, these 6 strains underwent a third screening, and the results are as follows. Figure 10 As shown.
[0064] Three rounds of strain screening were conducted. The first screening was conducted to select strains with high pyruvate content in xanthan gum. The second and third screenings were then conducted. After analysis, strain 2H7 produced xanthan gum with the highest pyruvate content. Furthermore, by comparing the results of the three screenings, the ability of this strain to produce pyruvate was relatively stable without significant fluctuations. Therefore, this strain was selected as the subsequent strain for fermentation. The selected strain 2H7 was preserved for use in subsequent fermentation experiments.
[0065] The selected strain 2H7 was named *Xanthomonas campestris* H1 and deposited on February 20, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC NO: 65930), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. This strain can be inoculated into fermentation medium to produce pyruvate. The optimal fermentation temperature is 30℃, and the fermentation time is 96 hours.
[0066] The above embodiments are merely one of the preferred embodiments of the present invention and should not be used to limit the scope of protection of the present invention. Any modifications or refinements made to the main design concept and spirit of the present invention that are not of substantial significance, but solve the same technical problem as the present invention, should be included within the scope of protection of the present invention.
Claims
1. A method for constructing a high / low pyruvate xanthan gum production strain, characterized in that, include: S1, prepare the original strain Xanthomonas campestris; S2, determine the growth curve of the original strain to identify the logarithmic growth phase; S3, using ARTP mutagenesis of the original strain in the logarithmic growth phase, with a mutagenesis time of 160-200s; S4, High-throughput initial screening: The mutagenized strains are cultured to obtain fermentation broth, the pyruvate content in the fermentation broth is measured, and multiple strains with high or low pyruvate content are screened out. S5, Secondary screening: The strains selected in step S4 are screened again to select strains with stable pyruvate content and xanthan gum production that are the same as the original strains.
2. The method for constructing a high / low pyruvate xanthan gum production strain according to claim 1, characterized in that, The original strain, Xanthomonas campestris, was purchased from the China General Microbiological Culture Collection Center, with accession number CGMCC1.1781.
3. The method for constructing a high / low pyruvate xanthan gum production strain according to claim 2, characterized in that, In step S3, the original bacterial culture of the strain in the logarithmic growth phase is taken and diluted to a bacterial concentration of 10. 5 CFU / mL, take 10 μL of bacterial suspension and spread it evenly on a sterile stainless steel slide, then place it in the ARTP mutagenesis system for mutagenesis.
4. The method for constructing a high / low pyruvate xanthan gum production strain according to claim 3, characterized in that, In step S3, the parameters for ARTP mutagenesis are: RF power 100W, distance between plasma torch nozzle and substrate 2mm, air flow rate 10mL / min, and plasma flow temperature 25℃~35℃.
5. The method for constructing a high / low pyruvate xanthan gum production strain according to claim 4, characterized in that, In step S4, the mutagenized strain is diluted and spread onto a plate. Single colonies are picked using a high-throughput colony selection workstation and inoculated into seed culture medium. The single colonies are then inoculated into fermentation medium and incubated at 30°C and 200 rpm for 16 h. The inoculated culture medium is then inoculated into fermentation medium and incubated at 30°C and 200 rpm for 96 h to obtain the fermentation broth.
6. The method for constructing a high / low pyruvate xanthan gum production strain according to claim 5, characterized in that, The seed culture medium comprises: 20 g / L sucrose, 5 g / L tryptone, 3 g / L beef extract, 1 g / L yeast extract, and pH adjusted to 7.0–7.
2.
7. The method for constructing a high / low pyruvate xanthan gum production strain according to claim 6, characterized in that, The fermentation medium comprises: 50 g / L sucrose, 0.08 g / L yeast extract, 15 g / L potassium chloride, 0.6 g / L potassium dihydrogen phosphate, 0.08 g / L magnesium sulfate, 5 g / L calcium carbonate, and 0.015 g / L citric acid, with the pH adjusted to 7.0–7.
2.
8. The method for constructing a high / low pyruvate xanthan gum production strain according to claim 7, characterized in that, In step S4, the method for determining the pyruvate content in the fermentation broth is as follows: Take 1 mL of fermentation broth, dilute it 50 times, add 1.5 mol / L HCl to hydrolyze for 10 min, then add 1 mL of 0.5% 2,4-dinitrophenylhydrazine hydrochloric acid solution and react for 5 min. Add 5 mL of 1.5 mol / L NaOH solution, shake well and develop color. Record the absorbance at 520 nm and compare it with the pyruvate standard curve for calculation.
9. The method for constructing a high / low pyruvate xanthan gum production strain according to claim 8, characterized in that, In step S5, the method for determining the xanthan gum yield during re-screening includes: taking the fermentation broth, centrifuging at 8000 rpm for 15 min at 4℃, taking the supernatant, adding 3 times the volume of anhydrous ethanol for precipitation, letting it stand at 4℃ for 2 h, filtering under reduced pressure, drying the precipitate under vacuum at 55℃ for 24 h to constant weight, weighing and calculating the yield.
10. The use of the strain constructed by the method according to any one of claims 1-9 in the preparation of high-pyruvate xanthan gum or low-pyruvate xanthan gum.