A method for preparing xanthan gum with reduced pyruvic acid content and improved salt tolerance

Abstract

The present invention belongs to the technical field of microbial fermentation. Provided is a method for preparing xanthan gum with reduced pyruvic acid content and improved salt tolerance. The xanthan gum is prepared by means of a fermentation method using Xanthomonas campestris with the deposit number of GDMCC No: 63427. In the fermentation process, the fermentation medium used comprises the following components in percentage by weight: 4.0% to 8.0% of corn starch, 0.5% to 2.5% of industrial ammonium sulfate, 0.3% to 0.7% of ferrous sulfate, 0.1% to 0.2% of dipotassium hydrogen phosphate and 0.1% to 0.2% of potassium dihydrogen phosphate. The problem of undesirable pyruvic acid content and salt resistance of xanthan gum produced in the prior art is solved, the product performance of xanthan gum is improved, the yield of the gum is effectively increased, and the low-cost production of xanthan gum is achieved.

Description

A method for preparing xanthan gum with reduced pyruvic acid content and improved salt tolerance Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and particularly relates to a method for preparing xanthan gum with reduced pyruvic acid content and improved salt tolerance. 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. Xanthan gum is an acidic exopolysaccharide produced by Xanthomonas campestris through aerobic fermentation of sugars, which cleaves 1,6-glycosidic bonds, opens side chains, and synthesizes 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.

[0003] 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 exhibit unique properties under specific conditions. The number of pyruvic acid groups at the end of the side chains of xanthan gum molecules significantly affects its performance. In industrial production, aerobic fermentation is carried out using Xanthomonas campestris. The fermentation broth undergoes precipitation, centrifugation, washing, separation, drying, grinding, and packaging to ultimately produce xanthan gum. The strain of bacteria is a crucial issue in the fermentation industry. Currently, the most common industrial production strain is Xanthomonas aeruginosa, which ferments xanthan gum to produce the desired product. The fermentation strain secretes the product extracellularly, and the fermentation broth is then processed and extracted to obtain the desired product.

[0004] However, xanthan gum produced using existing fermentation technologies has suboptimal pyruvic acid content and salt tolerance (high pyruvic acid content and poor salt tolerance), two important indicators for evaluating xanthan gum product performance. Therefore, it is necessary to develop a new fermentation technology to improve the product performance of xanthan gum. Summary of the Invention

[0005] The object of the present invention is to provide a method for preparing xanthan gum with reduced pyruvic acid content and improved salt tolerance, so as to solve the problem that xanthan gum produced by the prior art has unsatisfactory pyruvic acid content and salt tolerance.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] A method for preparing xanthan gum with reduced pyruvate content and improved salt tolerance is disclosed. The xanthan gum is prepared by fermentation using Xanthomonas campestris F417, which has a taxonomic name of Xanthomonas campestris and was deposited in the Guangdong Provincial Microbial Culture Collection on May 8, 2023, with a deposit number of GDMCC No: 63427. The deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. During the fermentation process, the fermentation medium used comprises the following components in weight percentage: 4.0-8.0% corn starch, 0.5-2.5% industrial ammonium sulfate, 0.3-0.7% ferrous sulfate, 0.1-0.2% dipotassium hydrogen phosphate, and 0.1-0.2% potassium dihydrogen phosphate.

[0008] Furthermore, the preparation method comprises the following steps:

[0009] (1) Activation of bacterial strains: Thaw the preserved bacterial solution containing Xanthomonas campestris with the deposit number of GDMCC No: 63427, 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 Xanthomonas campestris.

[0010] (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;

[0011] (3) Fermentation culture: Liquid seeds grown to the logarithmic phase were inoculated into a fermentation medium at an inoculum size of 5-20%, and the fermentation temperature was 35-40° C. and the shaking incubator was kept constant at 180 rpm for 96 h, wherein the pH value of the fermentation medium was 7.0;

[0012] (4) Xanthan gum was extracted from the fermentation broth using ethanol precipitation method.

[0013] Furthermore, in step (1) and step (2), the liquid seed culture medium includes the following components in weight percentage: 2% glucose, 0.5% industrial ammonium sulfate, 0.3% potassium dihydrogen phosphate and 0.2% sodium chloride, and the pH value of the liquid seed culture medium is 7.0.

[0014] Furthermore, the inoculation amount in step (3) is 10%.

[0015] Furthermore, the fermentation temperature in step (3) is 40°C.

[0016] Furthermore, the weight percentage of corn starch in the fermentation medium is 6%.

[0017] Furthermore, the weight percentage of industrial ammonium sulfate in the fermentation medium is 1.5%.

[0018] Furthermore, the weight percentage of ferrous sulfate in the fermentation medium is 0.4%.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention has found through multiple studies and verifications that by using Xanthomonas campestris (deposit number: GDMCC No: 63427) in combination with a designed fermentation medium to prepare xanthan gum through a fermentation method, the xanthan gum can have the advantages of low pyruvate content, good salt tolerance, and unaffected structure. This not only improves the product performance of the xanthan gum, but also effectively increases the gum yield, thereby achieving low-cost production of xanthan gum. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG1 is a growth curve of Xanthomonas campestris with a deposit number of GDMCC No: 63427;

[0022] FIG2 is a physical diagram of xanthan gum extracted from fermentation broth by ethanol precipitation;

[0023] FIG3 is a diagram showing the optimization results of the inoculum size during the fermentation culture stage;

[0024] FIG4 is a diagram showing the optimization results of the liquid loading amount during the fermentation culture stage;

[0025] FIG5 is a diagram showing the fermentation temperature optimization results during the fermentation culture stage;

[0026] FIG6 is a diagram showing the pH optimization results during the fermentation culture stage;

[0027] FIG7 is a diagram showing the fermentation time optimization results during the fermentation culture stage;

[0028] FIG8 is a diagram showing the optimization results of corn starch content during the fermentation and cultivation stage;

[0029] FIG9 is a diagram showing the optimization results of industrial ammonium sulfate content during the fermentation and cultivation stage;

[0030] FIG10 is a diagram showing the optimization results of ferrous sulfate content during the fermentation culture stage;

[0031] Figure 11 is a SEM analysis of xanthan gum obtained under the optimal fermentation conditions. DETAILED DESCRIPTION

[0032] 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.

[0033] In this example, Xanthomonas campestris with a deposit number of GDMCC No: 63427 was used to produce xanthan gum according to the following steps:

[0034] 1. Production process

[0035] 1.1 Bacteria activation

[0036] After thawing, the preserved bacterial liquid containing Xanthomonas campestris with a deposit number of GDMCC No: 63427 (hereinafter referred to as the "preserved strain") was dipped in the preserved bacterial liquid with an inoculating loop and streaked on a plate culture medium. After a single colony grew, a single colony was picked and inoculated into a 250 mL conical flask containing 100 mL of liquid seed culture medium at a 1% inoculum size. The culture was incubated in a constant temperature shaking incubator at 30°C and 180 rpm for two passages to restore the original activity of the preserved strain.

[0037] In the above steps, the liquid seed culture medium includes the following components in weight percentage: 2% glucose, 0.5% industrial ammonium sulfate, 0.3% potassium dihydrogen phosphate and 0.2% sodium chloride, and the pH value of the liquid seed culture medium is 7.0.

[0038] 1.2 Liquid seed culture

[0039] The bacterial liquid of the preserved strain that has been passaged 2-3 times was inoculated into 100 ml of liquid seed culture medium (same as 1.1) at a 1% inoculum volume, and cultured in a shaking incubator at 30°C and 180 rpm. The absorbance of the seed culture medium at a wavelength of 600 nm was measured 0 h after inoculation. Samples were taken every 2 h thereafter to measure the OD values ​​of the preserved 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 The data were recorded and the growth curve was drawn, as shown in Figure 1: The fermentation cycle of the preserved strain is roughly 36-72 hours. Before 36 hours, the main period is the growth and enrichment of the bacteria. After 36 hours, it enters the gel production period. The growth curve of the preserved 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 0-8 hours. The strain grows rapidly in an exponential form within 8-24 hours. At 24 hours, 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 600The culture medium gradually decreases and the bacteria die, which reduces the number of mycelium in the culture medium. It can be seen that the liquid seed culture should be carried out for 24 hours.

[0040] 1.3 Fermentation culture

[0041] When the OD of liquid seeds 600 When the value was 0.8, the liquid seeds grown to the logarithmic phase were inoculated into a 250 mL conical flask containing 100 mL of fermentation medium and cultured at 30 °C with a shaking incubator at 180 rpm for 72 h.

[0042] The fermentation medium includes the following components in weight percentage: 6% corn starch, 1.5% industrial ammonium sulfate, 0.4% ferrous sulfate, 0.1% dipotassium hydrogen phosphate, and 0.1% potassium dihydrogen phosphate, and the pH value is 7.0.

[0043] 1.4 Extraction of xanthan gum

[0044] Using the ethanol precipitation method, 3 times the volume of deionized water was added to the fermentation broth to dilute the fermentation broth, and then 3 times the volume of anhydrous ethanol was added to precipitate xanthan gum. The mixture was centrifuged at 8000 r / min for 15 minutes (as shown in Figure 2), the supernatant was discarded, and the precipitate was retained. The precipitate was washed 1-2 times with anhydrous ethanol, dried in an oven at 60°C, and ground to obtain the fermentation product xanthan gum, which was weighed.

[0045] 1.5 Calculation of rubber yield

[0046] The xanthan gum yield is obtained by dividing the mass of the extracted xanthan gum by the mass of the fermentation liquid.

[0047]

[0048] 2. Optimize production conditions

[0049] 2.1 Optimization of inoculum size

[0050] The inoculum size of the preserved strain was optimized. Liquid seeds cultured to the logarithmic growth phase were inoculated into the initial fermentation medium at inoculum ratios of 5%, 10%, 15%, 20%, and 25%. The culture temperature was 30°C, the shaker speed was 180 rpm, and the fermentation time was 72 hours. After fermentation, the fermentation broth was diluted with 3 volumes of deionized water. Then, 3 volumes of anhydrous ethanol were added to precipitate xanthan gum. The mixture was centrifuged at 8000 rpm for 15 minutes, the supernatant was discarded, and the precipitate was washed one to two times with anhydrous ethanol, dried in a 60°C oven, ground, and weighed. The xanthan gum yield was calculated. Three replicates were performed in each group.

[0051] As shown in Figure 3, as the inoculum size continues to increase, the xanthan gum production rate of the preserved strain shows a trend of first increasing and then decreasing. When the inoculum size is less than 10.0%, the xanthan gum production rate gradually increases, reaching a peak of 4.892% at an inoculum size of 10.0%. When the inoculum size is greater than 10.0%, the xanthan gum production rate shows a downward trend, the fermentation process is slow, and the production rate within the specified fermentation time is low. Excessive inoculum size can shorten the strain growth cycle, but it will cause the nutrients in the culture medium to be utilized prematurely, resulting in insufficient substrate required for the later strain to synthesize xanthan gum, reducing xanthan gum synthesis. Therefore, choosing an inoculum size of 10.0% is most appropriate.

[0052] 2.2 Liquid volume optimization

[0053] The inoculum volume of the deposited strain was optimized. Seed broth cultured to the logarithmic growth phase was inoculated into 250 mL Erlenmeyer flasks containing 25 mL, 50 mL, 75 mL, 100 mL, and 125 mL of initial fermentation medium, respectively, using the optimal inoculum volume optimized in 2.1. The incubation temperature was 30°C, the shaker speed was 180 rpm, and the fermentation time was 72 h. After fermentation, the fermentation broth was diluted with 3 volumes of deionized water and then precipitated with 3 volumes of anhydrous ethanol. The xanthan gum was then centrifuged at 8000 rpm for 15 min, the supernatant discarded, and the precipitate retained. The precipitate was washed one to two times with anhydrous ethanol, dried in a 60°C oven, ground, and weighed. The xanthan gum yield was calculated. Three replicates were performed in each group.

[0054] As shown in Figure 4, the xanthan gum yield of the deposited strains first increased and then decreased with increasing liquid volume. Lower liquid volumes reduce the nutrients available to the strain, resulting in lower xanthan gum yields. Higher liquid volumes, on the other hand, reduce the dissolved oxygen content in the fermentation environment, hindering air circulation and hindering bacterial growth, leading to lower xanthan gum yields. When the liquid volume was below 75 mL, the xanthan gum yield gradually increased, reaching a maximum of 4.976% at 75 mL. When the liquid volume exceeded 75 mL, the yield gradually decreased. Therefore, the optimal liquid volume should be 75 mL.

[0055] 2.3 Fermentation temperature optimization

[0056] Fermentation temperature optimization was performed for the preserved strain. Seed broth cultured to the logarithmic growth phase was inoculated into the initial fermentation medium at the optimal inoculum size optimized in 2.1. Shake flask fermentations were performed at temperatures of 25, 30, 35, 40, 45, and 50°C for the optimal fermentation time optimized in 2.5, with a shaker speed of 180 rpm. After fermentation, the fermentation broth was diluted with 3 volumes of deionized water and then precipitated with 3 volumes of anhydrous ethanol. The xanthan gum was then centrifuged at 8000 rpm for 15 minutes, the supernatant discarded, and the precipitate retained. The precipitate was washed one to two times with anhydrous ethanol, dried in a 60°C oven, ground, and weighed. The xanthan gum yield was calculated. Three replicates were performed for each group.

[0057] For the preserved strain, its optimal growth temperature is different from the optimal temperature for xanthan gum biosynthesis. The optimal growth temperature is 30°C, and the bacterial concentration increases rapidly, while the optimal fermentation temperature for xanthan gum biosynthesis is 35-40°C. As shown in Figure 5, with the increase in temperature, the gum production rate of the preserved strain shows a trend of first increasing and then decreasing, reaching a peak of 4.862% at a fermentation temperature of 40°C. As the fermentation temperature continues to rise, the gum production rate gradually decreases. This may be because the fermentation temperature is too high, and the internal temperature of the culture medium cannot be transmitted, resulting in the normal growth and metabolism of the strain being affected, thereby reducing the production of xanthan gum. Therefore, the optimal fermentation temperature for the preserved strain is determined to be 40°C.

[0058] 2.4 Optimization of initial pH conditions

[0059] The initial pH of the preserved strain was optimized. The pH of the fermentation medium was adjusted to 6.0, 6.5, 7.0, 7.5, and 8.0. Seed broth cultured to the logarithmic growth phase was inoculated into the initial fermentation medium at different pH values ​​using the optimal inoculum size optimized in 2.1. The fermentation time was the optimal fermentation time optimized in 2.5, the fermentation temperature was the optimal fermentation temperature optimized in 2.3, and the shaker speed was 180 rpm. After fermentation, the fermentation broth was diluted with 3 volumes of deionized water and then precipitated with 3 volumes of anhydrous ethanol. The xanthan gum was then centrifuged at 8000 rpm for 15 minutes, the supernatant discarded, and the precipitate retained. The precipitate was washed one to two times with anhydrous ethanol, dried in a 60°C oven, ground, and weighed. The xanthan gum yield was calculated. Three replicates were performed in each group.

[0060] As shown in Figure 6, the initial pH value of the culture medium has a great influence on the fermentation process. The pH affects the metabolic pathways during fermentation and also affects the activity of xanthan gum. The appropriate pH will promote fermentation, so it is extremely important to choose the right pH value during the fermentation process. The effect of the initial pH value on the gum production rate of the preserved strain is shown in Figure 6. As can be seen from Figure 6, when the initial pH value of the fermentation medium is 7.0, the gum production rate of the preserved strain reaches the highest, which is 4.919%. As the pH value gradually increases, the gum production rate gradually decreases. Therefore, the optimal fermentation pH value for the preserved strain is determined to be 7.0.

[0061] 2.5 Fermentation time optimization

[0062] Fermentation time was optimized for the preserved strain. Seed broth cultured to the logarithmic growth phase was inoculated into the initial fermentation medium at the optimal inoculum size optimized in 2.1. Shake flask fermentations were performed for 48, 72, 96, 120, and 144 hours, respectively. The culture temperature was 30°C and the shaker speed was 180 rpm. After fermentation, the fermentation broth was diluted with 3 volumes of deionized water. Three volumes of anhydrous ethanol were then added to precipitate xanthan gum. The mixture was centrifuged at 8000 rpm for 15 minutes, the supernatant discarded, and the precipitate retained. The precipitate was washed one to two times with anhydrous ethanol, dried in a 60°C oven, ground, and weighed. The xanthan gum yield was calculated. Three replicates were performed for each group.

[0063] Fermentation time is also a significant factor influencing xanthan gum production by Xanthomonas campestris. In the early stages of fermentation, the preserved strain primarily focuses on bacterial growth and accumulation. Once the growth curve reaches a plateau, it begins to synthesize and secrete the exopolysaccharide xanthan gum. The effect of fermentation time on the xanthan gum production rate of the preserved strain is shown in Figure 7. As shown in Figure 7, when the fermentation time is less than 96 hours, the xanthan gum production rate of the preserved strain gradually increases with increasing fermentation time, reaching a peak of 4.938% at 96 hours. When the fermentation time exceeds 96 hours, the xanthan gum production rate shows a downward trend. In the early stages of fermentation, the preserved strain of Xanthomonas campestris accumulates and synthesizes xanthan gum, with production increasing with increasing fermentation time. However, when the fermentation time is too long, the carbon source in the culture medium is fully utilized, and the preserved strain utilizes xanthan gum as a new carbon source to survive, resulting in a downward trend in xanthan gum production at 96 hours. Therefore, 96 hours was selected as the optimal fermentation time for xanthan gum production.

[0064] 3. Optimization of fermentation medium composition

[0065] 3.1 Corn starch concentration optimization

[0066] The amount of corn starch added was optimized. 4.0%, 5.0%, 6.0%, 7.0%, and 8.0% corn starch were added to the initial fermentation medium, respectively. Industrial ammonium sulfate was added at 1%, and ferrous sulfate was added at 0.5%. All other ingredients remained unchanged. Shake flask fermentation was performed under the optimal fermentation conditions determined in 2. After fermentation, the fermentation broth was diluted with 3 volumes of deionized water. Then, 3 volumes of anhydrous ethanol were added to precipitate xanthan gum. Centrifuge at 8000 rpm for 15 minutes, discard the supernatant, and retain the precipitate. The precipitate was washed 1-2 times with anhydrous ethanol, dried in a 60°C oven, ground, and weighed. The xanthan gum yield was calculated. Three replicates were performed in each group.

[0067] The results of the gelatin production rate of the preserved strain at different corn starch concentrations are shown in Figure 8. Xanthomonas campestris requires a high C / N ratio during xanthan gum production. As shown in Figure 8, the gelatin production rate of the preserved strain continues to increase with increasing soluble starch content, reaching a maximum of 5.065% at a content of 6.0%. Thereafter, the gelatin production rate begins to decline. When the soluble starch content is too high, the dissolved oxygen content in the fermentation environment is insufficient, and the osmotic pressure within the culture medium is too high. The preserved strain loses water under osmotic pressure, causing plasmolysis, which affects the normal growth process of the strain and leads to a decrease in gelatin production. During the fermentation process, insufficient soluble starch content will also affect the normal growth of the preserved strain, thereby affecting the gelatin production rate. Therefore, in actual production, in order to maximize the gelatin production rate while controlling costs, the optimal concentration of soluble starch is determined to be 6.0%.

[0068] 3.2 Optimization of industrial ammonium sulfate concentration

[0069] Optimize the amount of industrial ammonium sulfate added. Add 0.5%, 1.0%, 1.5%, 2.0%, and 2.5% industrial ammonium sulfate to the initial fermentation medium, respectively. Corn starch was added according to the optimal concentration determined in 3.1, and ferrous sulfate was added at 0.5%. All other ingredients remained unchanged. Shake flask fermentation was carried out under the optimal fermentation conditions determined in 2. After fermentation, the fermentation broth was diluted with 3 volumes of deionized water. Then, 3 volumes of anhydrous ethanol were added to precipitate xanthan gum. Centrifuge at 8000 rpm for 15 minutes, discard the supernatant, and retain the precipitate. Wash the precipitate 1-2 times with anhydrous ethanol, dry it in a 60°C oven, grind it, weigh it, and calculate the xanthan gum yield. Three parallel experiments were performed in each group.

[0070] Figure 9 shows the results of the gelatin production rate of the preserved strain at different industrial ammonium sulfate concentrations. Nitrogen is an essential element for microbial growth. Excessive nitrogen content in the culture medium affects the C / N ratio during fermentation and inhibits bacterial growth. As shown in Figure 9, the gelatin production rate of the preserved strain increases when the industrial ammonium sulfate addition level is within the range of 0.5-1.5%. When the industrial ammonium sulfate concentration reaches 1.5%, the gelatin production rate reaches a peak of 5.172%. As the industrial ammonium sulfate concentration continues to increase, the gelatin production rate decreases. This may be because the nitrogen content in the culture medium exceeds the amount required for normal growth of the preserved strain, resulting in an imbalance in the C / N ratio and increased osmotic pressure, further affecting the gelatin production rate of the preserved strain. Therefore, the optimal addition level of industrial ammonium sulfate was determined to be 1.5%.

[0071] 3.3 Optimization of ferrous sulfate concentration

[0072] Optimize the amount of ferrous sulfate added. Add 0.3%, 0.4%, 0.5%, 0.6%, and 0.7% ferrous sulfate to the initial fermentation medium, respectively. Add corn starch according to the optimal concentration determined in 3.1, and industrial ammonium sulfate according to the optimal concentration determined in 3.2. Keep other ingredients unchanged, and perform shake flask fermentation under the optimal fermentation conditions determined in 2. After fermentation, dilute the fermentation broth with 3 times the volume of deionized water, then add 3 times the volume of anhydrous ethanol to precipitate xanthan gum. Centrifuge at 8000 rpm for 15 minutes, discard the supernatant, retain the precipitate, and wash the precipitate 1-2 times with anhydrous ethanol. Dry in a 60°C oven, grind, weigh, and calculate the xanthan gum yield. Perform three parallel experiments in each group.

[0073] Figure 10 shows the results of the gel production rate of the preserved strain at different ferrous sulfate concentrations. Inorganic salts are essential nutrients for microorganisms during the fermentation process. Their main functions include forming cell components, regulating the pH and osmotic pressure of the culture medium, and serving as important components of enzymes. Although the content of inorganic salts in the culture medium is very small, they do affect the growth of the fermenting bacteria and the synthesis of metabolites to a certain extent. As shown in Figure 10, the gel production rate of the preserved strain gradually increases with increasing ferrous sulfate concentration, reaching a peak of 5.227% at an addition level of 0.4%. As the ferrous sulfate concentration continues to increase, the gel production rate shows a downward trend. This may be due to the increased osmotic pressure in the culture medium, which makes the internal environment less suitable for bacterial growth and reproduction, resulting in a decrease in gel production. Therefore, the optimal addition level of ferrous sulfate is determined to be 0.4%.

[0074] 4. Study on the pyruvic acid content of xanthan gum

[0075] According to the above-mentioned optimized fermentation medium and culture conditions, the xanthan gum (gum yield 5.227%) prepared was used as the experimental group, and the xanthan gum obtained by replacing the industrial ammonium sulfate in the optimal fermentation conditions of this embodiment with the same amount of soy protein was used as the control group 1; and according to the same fermentation conditions and the same fermentation medium, the wild campestris 1 (Latin name Xanthomonas campestris Y-11, taxonomic name Xanthomonas campestris, and deposited in 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) and the wild campestris 2 (Latin name Xanthomonas campestris F417-6, taxonomic name Xanthomonas campestris, and deposited in Guangdong Provincial Microbiological Culture Collection on May 12, 2023, with a deposit number of GDMCC No.: 63459, the deposit address is: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou), and the obtained xanthan gum was used as control group 2 and control group 3.

[0076] To determine the pyruvic acid content of xanthan gum: Accurately weigh 62.5 mg of sodium pyruvate and dissolve it in 100 mL of 0.1 mol / L H₂SO₄ solution. Weigh 100 mg of 2,4-dinitrophenylhydrazine and dissolve it in 100 mL of 2 mol / L hydrochloric acid solution. Weigh 50 mg of the fermentation product xanthan gum and add 25 mL of 0.1 mol / L H₂SO₄ solution. Hydrolyze for 3 hours to release the pyruvic acid from the xanthan gum side chains. Add 100 μL of the hydrolyzate to 900 μL of deionized water and 1 mL of 0.1% 2,4-dinitrophenylhydrazine solution. Shake well and let stand at room temperature for 10 minutes. Using deionized water as a blank control, measure the absorbance at 520 nm. Construct a standard curve using the absorbance as the ordinate and the pyruvic acid solution as the standard.

[0077] The pyruvic acid content in xanthan gum affects its viscosity. The lower the pyruvic acid content, the lower the viscosity. Higher pyruvic acid content improves xanthan gum viscosity at the same concentration, enhances acid resistance and compatibility, and increases hydration rate. As shown in Table 1, the yield of the deposited strain after determining the optimal fermentation conditions was 5.227%. The viscosity of the xanthan gum obtained by fermentation at a mass concentration of 5.0 g / L was only 97.10 mPa·s, and the pyruvic acid content was 1.43%. The experimental results of Xanthomonas campestris 1 and Xanthomonas campestris 2 were relatively poor, indicating that the optimal conditions explored in this example are only applicable to the deposited strains provided in this example. The pyruvic acid content produced by soy protein fermentation was slightly higher than that of the experimental group, but the yield was lower than that of the experimental group.

[0078] Table 1 Rubber production parameters

[0079]

[0080] 5. Study on the salt tolerance of xanthan gum

[0081] According to the above optimized fermentation medium and culture conditions, xanthan gum was prepared (gum yield 5.227%).

[0082] Xanthan gum salt tolerance test: To 1000 mL of a 5.0 g / L xanthan gum solution, add 0.1, 0.2, 0.3, 0.4, and 0.5 g of NaCl, respectively. Stir until completely dissolved and stabilize at 25°C for 1 hour. Measure the viscosity of the solution at different sodium chloride concentrations. Turn on the NDJ-5S viscometer, select rotor 1, set the speed to 6 r / min, and press OK to begin the measurement. The measurement is completed when the reading appears on the screen when the rotor stops rotating. Perform three replicates per group. Record the viscosity of the xanthan gum solution at different sodium chloride concentrations in mPa·s.

[0083] The xanthan gum obtained above (gum yield 5.227%) was used as the experimental group, and the xanthan gum obtained by fermenting Xanthomonas campestris 1 (deposit number GDMCC No: 63460) using the same fermentation medium and the same fermentation conditions was used as the control group.

[0084] Table 2 shows the viscosity changes of xanthan gum solutions with a mass concentration of 5.0 g / L at sodium chloride concentrations of 0.1-0.5 g / L. After the addition of sodium chloride, the viscosity values ​​of the xanthan gum solutions in the experimental group ranged from 97.10 mPa·s to 75.15 mPa·s, while the viscosity values ​​of the xanthan gum solutions in the control group ranged from 179.54 mPa·s to 44.61 mPa·s. As the sodium chloride concentration increased, the viscosity of the xanthan gum solutions in the control group decreased more significantly than in the experimental group, indicating that the xanthan gum fermented in the experimental group possessed good salt tolerance. Salt concentration has a certain effect on the viscosity of xanthan gum solutions. At low concentrations, the addition of a small amount of sodium chloride can slightly decrease the viscosity of xanthan gum. This is primarily due to a reduction in intermolecular charge forces. The addition of sodium chloride reduces the degree of bonding between xanthan gum molecules. After the sodium chloride attacks and severs the xanthan gum backbone, it loses its thickening ability, resulting in a decrease in the viscosity of the xanthan gum solution.

[0085] Table 2 Salt tolerance test results

[0086]

[0087] 6. Xanthan gum microstructure analysis

[0088] According to the above optimized fermentation medium and culture conditions, xanthan gum was prepared (gum yield 5.227%).

[0089] Microscopic morphology observation: Fermented xanthan gum was ground into powder, and the surface morphology of the xanthan gum sample was observed using a field emission scanning electron microscope (S-3400N, HITACHI, Japan). A small amount of the fermented xanthan gum sample was placed on a conductive adhesive, evenly sprayed with gold, and then scanned using the instrument. The test voltage was 20 kV.

[0090] The secondary structure of xanthan gum consists of side chains coiled in opposite directions around the main chain, held together by hydrogen bonds to form a rod-like double helix. The tertiary structure of xanthan gum consists of a helical complex formed by weak nonpolar covalent bonds between the rod-like double helices. As shown in Figure 11, the xanthan gum fermented with the preserved strain exhibits wrinkled and nodular morphologies, with fibrous characteristics, indicating that its structure is not disrupted during the production process.

[0091] 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 method for preparing xanthan gum with reduced pyruvic acid content and improved salt tolerance, characterized in that: Xanthomonas campestris F417 was deposited in Guangdong Microbial Culture Collection Center on May 8, 2023, with a deposit number of GDMCC No: 63427, and a deposit address of 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou. The fermentation medium used in the fermentation process includes the following components in weight percentage: 4.0-8.0% corn starch, 0.5-2.5% industrial ammonium sulfate, 0.3-0.7% ferrous sulfate, 0.1-0.2% dipotassium hydrogen phosphate, and 0.1-0.2% potassium dihydrogen phosphate.

2. The preparation method according to claim 1, characterized in that The steps include: (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: inoculating the liquid seeds grown to the logarithmic phase into the fermentation medium at an inoculum amount of 5-20%, fermenting at a temperature of 35-40° C. and 180 rpm in a shaking incubator for 96 h, wherein the pH value of the fermentation medium is 7.0; (4) Xanthan gum was extracted from the fermentation broth by ethanol precipitation method.

3. The preparation method according to claim 2, characterized in that: In step (1) and step (2), the liquid seed culture medium includes the following components in weight percentage: 2% glucose, 0.5% industrial ammonium sulfate, 0.3% potassium dihydrogen phosphate and 0.2% sodium chloride, and the pH value of the liquid seed culture medium is 7.

0.

4. The preparation method according to claim 2, characterized in that: The inoculation amount in step (3) is 10%.

5. The preparation method according to claim 2, characterized in that: The fermentation temperature in step (3) is 40°C.

6. The preparation method according to claim 2, characterized in that: The weight percentage of corn starch in the fermentation medium is 6%.

7. The preparation method according to claim 2, characterized in that: The weight percentage of industrial ammonium sulfate in the fermentation medium is 1.5%.

8. The preparation method according to claim 2, characterized in that: The weight percentage of ferrous sulfate in the fermentation medium is 0.4%.

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