Synergist and application thereof in fermentation production of xanthan gum

Abstract

The invention relates to the technical field of microbial fermentation culture, and particularly discloses a synergist and application thereof in fermentation production of xanthan gum. According to the method for producing xanthan gum through fermentation, a fermentation culture medium comprises a synergist, and the synergist comprises betaine substances, phosphate or polyphosphate, zinc sulfate and manganese sulfate; the betaine substance comprises one or more of betaine, a betaine hydrate or a betaine inorganic salt. When the synergist is used for xanthan gum fermentation, the high-temperature tolerance of zymophyte and the fermentation strength and conversion rate of xanthan gum are improved, and the obtained product is high in pyruvic acid content and large in viscosity.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation and cultivation, in particular to a synergist and application thereof in the fermentation production of xanthan gum. Background Art

[0002] Xanthan gum is a microbial polysaccharide with important industrial application value. It is composed of a cellulose backbone of β-1,4-linked D-glucose units, which are replaced by trisaccharide side chains on alternating glucose residues. The trisaccharide side chains are composed of two mannose units separated by glucuronic acid. Xanthan gum is produced by fermentation of Xanthomonas campestris with carbohydrates such as starch or glucose as the main raw materials. It has the characteristics of viscosity and thickening, suspension, emulsification and stabilization, and is widely used in many industries such as food, daily chemicals, oil production, textiles, printing and dyeing, and medicine.

[0003] With the further expansion of xanthan gum application areas, despite the full efforts of various manufacturers, the existing xanthan gum yield still cannot fully meet the market demand, which also causes a certain degree of product price increase. At the same time, since xanthan gum belongs to typical aerobic fermentation, with the increase of production intensity, the existing equipment cannot fully meet the production of high-intensity and high-yield products, so some product indicators, such as viscosity, are reduced.

[0004] In view of the above problems, it is an urgent need for production companies and the market to further improve the fermentation intensity of existing xanthan gum, improve the fermentation yield, and improve performance indicators such as product viscosity in order to further release production capacity. Summary of the invention

[0005] One of the purposes of the present invention is to provide a fermentation enhancer which can improve the fermentation intensity of xanthan gum, the high temperature resistance of fermentation bacteria and the product quality, and a method for efficiently producing xanthan gum.

[0006] In order to achieve this object, the technical solution of the present invention is as follows:

[0007] A method for producing xanthan gum by fermentation, wherein the fermentation medium includes a synergist, the synergist includes: betaine substances, phosphates or polyphosphates, zinc sulfate and manganese sulfate; the betaine substances include one or more of betaine, betaine hydrates or betaine inorganic salts;

[0008] When intermittent single batch aerobic fermentation is used during fermentation, the concentration of the synergist in the fermentation medium is 2 to 6 g / L;

[0009] When the fermentation is carried out in fed-batch fermentation, the concentration of the synergist in the fermentation medium is 1-2 g / L.

[0010] The present invention has found that adding a specific fermentation synergist during the xanthan gum fermentation process can improve the stress capacity during the xanthan gum fermentation process, and provide specific nutritional requirements required for satisfying the xanthan gum fermentation, thereby improving the intensity and output of the xanthan gum fermentation. In particular, in the case of a high-concentration carbon source, the fermentation synergist plays a synergistic effect, can improve the tolerance of the strain to the high-concentration carbon source, and improve the output and conversion rate of xanthan gum. The synergist of the present invention can also further improve the viscosity and other properties of the xanthan gum product. Compared to the fermentation of ordinary xanthan gum, after adding the fermentation synergist of the present invention, the fermentation cycle is significantly shortened, the gum production rate is significantly improved, and the pyruvic acid content and viscosity of the final product are improved at the same time.

[0011] In the method of the present invention, when the synergist includes betaine substances, phosphate, zinc sulfate and manganese sulfate, the mass ratio of the betaine substances, phosphate, zinc sulfate and manganese sulfate is (1000-2000): (1000-2000): (1-2): (0.5-1);

[0012] When the synergist includes betaine, polyphosphate, zinc sulfate and manganese sulfate, the mass ratio of the betaine, polyphosphate, zinc sulfate and manganese sulfate is (1000-2000): (900-1100): (1.8-2.2): (1.8-2.2), preferably (1000-2000): 1000: 2: 2;

[0013] In the method of the present invention, the betaine substance includes one or more of anhydrous betaine, monohydrate betaine, betaine hydrochloride, betaine sulfate, betaine citrate, and betaine phosphate; the phosphate includes one or more of dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, potassium phosphate, sodium phosphate, and hydrates thereof;

[0014] The polyphosphate includes one or more of tripolyphosphate, hexametaphosphate and hydrates thereof.

[0015] By adopting the specific proportions of the substances in the present invention, the fermentation effect can be better achieved.

[0016] The raw materials used in the present invention, such as betaine substances, phosphate substances, zinc sulfate, manganese sulfate, etc., are all commercially available products.

[0017] In the method of the present invention, intermittent single batch aerobic fermentation is adopted during fermentation, and the fermentation medium comprises: glucose, 90-100 g / L; fish peptone, 8-10 g / L; corn steep liquor, 8-10 g / L; MgSO4·7H2O, 3-4 g / L; calcium carbonate, 3-4 g / L; the synergist 2-6 g / L; pH is 7.2±0.1;

[0018] Alternatively, the fermentation is carried out by fed-batch fermentation, wherein the fermentation medium comprises: glucose, 10-20 g / L; fish peptone, 8-10 g / L; corn steep liquor, 8-10 g / L; MgSO4·7H2O, 3-4 g / L; calcium carbonate, 3-4 g / L; fermentation enhancer, 1-2 g / L; pH is 7.2±0.1;

[0019] When the residual sugar content of the fermentation broth is less than 0.3%, feed medium (sterilized in advance and cooled to room temperature) is used for feeding at a rate of 600-800 mL / h, and the total volume of the feed is 9-11% of the volume of the initial fermentation broth;

[0020] The feed medium comprises:

[0021] The synergist is 10-30 g / L; glucose is 500-800 g / L; and the pH is 7.1±0.1.

[0022] The synergist of the present invention can be used as a fermentation medium base component in single batch aerobic fermentation, and can also be further used in fed batch fermentation, and can be used as a fermentation medium base component and a feed medium component for fed addition at the same time.

[0023] In the method of the present invention, the fermentation conditions are:

[0024] Within 0-20h, the air volume is 0.4-0.6vvm, the temperature is 29.5±0.5℃, and the stirring speed is 200-400rpm; the fermentation liquid can be fully stirred and turned;

[0025] Within 20-24h, the air volume is 0.8-1.0vvm, the temperature is 30.0±0.5℃~31.0±0.5℃, and the stirring speed is 400-500rpm; the fermentation liquid can be fully stirred and turned;

[0026] Within 24-48h, the air volume is 1.0-1.1vvm, the temperature is 30±0.5℃~31.5±0.5℃, and the stirring speed is 500-650rpm; the fermentation liquid can be fully stirred and turned;

[0027] After 48 hours, the air volume is 1.1-0.8vvm, the temperature is 30±0.5℃~32.0±0.5℃, and the stirring speed is 650-700rpm; the fermentation liquid can be fully stirred and turned;

[0028] During the fermentation, the pH of the fermentation liquid is controlled at 6.5-7.2 (preferably with a 30-40% sodium hydroxide solution sterilized at high temperature), and the fermentation is terminated when the residual sugar content in the fermentation liquid no longer changes or is less than 0.1%.

[0029] The use of the synergist of the present invention can improve the tolerance of fermentation bacteria to high temperature, thereby allowing fermentation to be carried out at a higher temperature, which is beneficial to improving the fermentation conversion rate.

[0030] The method of the present invention further comprises the step of obtaining xanthan gum from the fermentation liquid after the fermentation is completed; the step of obtaining xanthan gum comprises:

[0031] (1) mixing the fermentation liquid after fermentation with a soluble inorganic salt and letting it stand to obtain an extract; preferably, the soluble inorganic salt is dissolved in a small amount of water in advance, then mixed with the fermentation liquid, stirred evenly and let stand for 15 to 45 minutes;

[0032] (2) extracting the extract with ethanol, wherein the final ethanol concentration of the system during the extraction is 50-55%, to obtain a first material containing xanthan gum fibers;

[0033] (3) separating the first material to obtain a crude wet xanthan gum product;

[0034] (4) extracting the wet xanthan gum crude product with ethanol, wherein the final ethanol concentration of the system during the extraction is 70-80%, to obtain a second material;

[0035] (5) separating the second material to obtain a wet xanthan gum product;

[0036] (6) drying and crushing the wet xanthan gum product.

[0037] Preferably, the final ethanol concentration of the system during the extraction in step (2) is less than the final ethanol concentration of the system during the extraction in step (4). The final ethanol concentration setting in the two extraction systems of the present invention is conducive to improving the extraction yield. Both extractions can be carried out using high-concentration ethanol. During the second extraction, the ethanol concentration can be adjusted by adding additional water for ease of operation. The ethanol-containing mother liquor obtained after each extraction and separation can be recycled and reused by a distillation device.

[0038] In the method of the present invention, the mass volume ratio of the soluble inorganic salt to the fermentation liquid is (0.2-1): 100 g / ml;

[0039] And / or, the soluble inorganic salt is one or more of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, and ferrous chloride;

[0040] And / or, in steps (2) and (4), the concentration of ethanol is 92-95%;

[0041] And / or, in step (6), the drying temperature is 60-105° C. and the drying time is 3-10 h.

[0042] In the method of the present invention, hydrochloric acid or sodium hydroxide solution can be used to adjust the pH value of each culture medium.

[0043] In the method of the present invention, the seed culture medium of the fermentation bacteria comprises: sucrose, 15-20 g / L; peptone, 3-4 g / L; yeast powder, 1-2 g / L; beef extract, 4-5 g / L; pH is 7.0±0.1;

[0044] And / or, the fermentation bacteria is Xanthomonas campestris.

[0045] The present invention also provides a synergist, and the synergist is as described above.

[0046] The present invention further provides an application of the above method or synergist in any of the following aspects:

[0047] (1) Improve the conversion rate of xanthan gum production by fermentation;

[0048] (2) Shorten the fermentation cycle of xanthan gum;

[0049] (3) Improve the tolerance of fermentation bacteria to high concentrations of carbon sources;

[0050] (4) increasing the pyruvic acid content in xanthan gum produced by fermentation;

[0051] (5) Improve the viscosity of xanthan gum produced by fermentation;

[0052] (6) Improve the tolerance of fermentation bacteria to fermentation temperature.

[0053] The present invention further provides a xanthan gum prepared by the above method; preferably, the content of pyruvic acid in the xanthan gum is ≥4.7%.

[0054] The beneficial effects of the present invention are at least:

[0055] According to the in-depth study of the fermentation characteristics of xanthan gum, the invention improves the high temperature tolerance of the fermentation bacteria, the fermentation intensity and the conversion rate of xanthan gum by improving the fermentation medium and preferably further improving the culture method. The obtained product has high pyruvic acid content and high viscosity. At the same time, the product meets the standard of "GB1886.41-2015 Food Additive Xanthan Gum", has good technical feasibility and operational feasibility, and can provide technical guidance for production. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 The results are a comparison of the bacterial growth curves of Examples 1-4 of the present invention and Comparative Examples 1-3 before fermentation for 24 hours.

[0057] Figure 2 The results are a comparison of the bacterial growth curves of Examples 1 and 7 of the present invention and Comparative Examples 5, 7, 9, and 10 before fermentation for 24 hours. DETAILED DESCRIPTION

[0058] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0059] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available or prepared according to conventional methods in the art unless otherwise specified.

[0060] The fermentation bacteria used in the specific embodiments of the present invention is Xanthomonas campestris unless otherwise specified; its deposit number is CGMCC No. 14771, which is disclosed in Chinese patent CN110229771B.

[0061] The seed culture medium used was as follows (g / L): sucrose, 15; peptone, 4; yeast powder, 2; beef extract, 4; pH 7.0±0.1.

[0062] The seed culture method is: inoculation amount 1%, air volume 1.0 vvm, stirring 200-400 rpm, culture temperature 28.5±0.5°C, and culture for 16 hours.

[0063] The inoculation amount of the fermentation culture is: 10% by volume (V / V).

[0064] The residual sugar detection method is: use SBA-40C biosensor analyzer to detect the glucose in the fermentation process, dilute it by a reasonable multiple and shake it well, so that the detection result falls within the detection range of the instrument, and finally obtain the residual sugar detection result in the fermentation process according to the dilution multiple.

[0065] Example 1

[0066] This embodiment provides a fermentation enhancer for improving fermentation intensity and a method for producing xanthan gum using the same, comprising the following steps:

[0067] 1. Weigh anhydrous betaine: anhydrous dipotassium hydrogen phosphate: zinc sulfate: manganese sulfate in a mass ratio of 2000:4000:2:2, and mix them thoroughly to obtain a xanthan gum fermentation enhancer.

[0068] 2. Weigh the fermentation enhancer according to the formula amount and add it to the xanthan gum fermentation medium base material, sterilize and cool it after it is fully dissolved, and use it as the fermentation medium to directly carry out intermittent single batch aerobic fermentation of xanthan gum;

[0069] The fermentation medium of this embodiment is specifically as follows (g / L): glucose, 100; fish peptone, 10; corn steep liquor, 8; MgSO4·7H2O, 4; calcium carbonate, 3; the above-mentioned fermentation enhancer, 2; and the pH of the medium is adjusted to 7.28 with hydrochloric acid or sodium hydroxide solution.

[0070] 3. Fermentation conditions:

[0071] Within 0-20h, the air volume is 0.4-0.6vvm, the temperature is 29.5±0.5℃, and the stirring speed is 200-400rpm; the fermentation liquid should be fully stirred and turned.

[0072] Within 20-24 hours, the air volume is 0.8-1.0vvm, the temperature is 30.0±0.5℃, and the stirring speed is 400-500rpm; the fermentation liquid should be fully stirred and turned.

[0073] Within 24-48 hours, the air volume is 1.0-1.1 vvm, the temperature is 30.0±0.5℃, and the stirring speed is 500-650rpm; the fermentation liquid should be fully stirred and turned.

[0074] After 48 hours, the air volume is 1.1-0.8vvm, the temperature is 30.0±0.5℃, and the stirring speed is 650-700rpm. The fermentation liquid can be fully stirred and turned.

[0075] During the fermentation process, the pH of the fermentation liquid was controlled at 6.5-7.2 with 35% sodium hydroxide solution.

[0076] The fermentation was continued until the residual sugar content reached 0.05%, and the fermentation was terminated. The fermentation cycle was 62.5 hours.

[0077] 4. Based on the volume of the fermentation liquid, add 1.0g of sodium chloride (sodium chloride is prepared with water in advance to a solution with a mass volume ratio of 20%) for every 100ml of fermentation liquid, stir evenly and let stand for 40min;

[0078] 5. Add 92% ethanol solution and stir to extract until the final ethanol concentration of the system is 55%, to obtain a material containing xanthan gum fiber;

[0079] 6. Separate the material in step 5 to obtain a wet xanthan gum crude product and a mother liquor containing ethanol; the mother liquor can be used to recover ethanol through a distillation device, and the wet xanthan gum crude product is reserved for later use;

[0080] 7. The wet xanthan gum crude product of step 6 is extracted again with 92% ethanol solution, and the ethanol concentration is adjusted with water to a final ethanol concentration of 71%.

[0081] 8. Separate the material from step 7 to obtain a wet finished product and a mother liquor containing ethanol. The mother liquor can be distilled to recover ethanol. The wet finished product is dried in a 75°C forced air drying oven for 5 hours and then crushed and passed through an 80-mesh (0.175 mm) sieve to obtain the sieved material as a dry finished product.

[0082] Example 2

[0083] This embodiment provides a fermentation enhancer for improving fermentation intensity and a method for producing xanthan gum using the same, comprising the following steps:

[0084] 1. Weigh anhydrous betaine: anhydrous disodium hydrogen phosphate: zinc sulfate: manganese sulfate in a mass ratio of 4000:2000:4:1, and mix them thoroughly to obtain a xanthan gum fermentation enhancer.

[0085] 2. Weigh the fermentation enhancer according to the formula amount and add it to the xanthan gum fermentation medium base material, sterilize and cool it after it is fully dissolved, and use it as the fermentation medium to directly carry out intermittent single batch aerobic fermentation of xanthan gum;

[0086] The fermentation medium of this embodiment is specifically as follows (g / L): glucose, 90; fish peptone, 8; corn steep liquor, 8; MgSO4·7H2O, 4; calcium carbonate, 4; the above-mentioned fermentation enhancer, 6, and the pH of the medium is adjusted to 7.19 with dilute hydrochloric acid and sodium hydroxide solution.

[0087] 3. The fermentation conditions were the same as those in Example 1. The fermentation was continued until the residual sugar content reached 0.02%, and the fermentation was terminated. The fermentation period was 64.1 h.

[0088] 4. Add 0.6g of potassium chloride (prepare a 15% solution by mass to volume ratio with water) for every 100ml of fermentation liquid, stir evenly and let stand for 30min;

[0089] 5. Add 93.2% ethanol solution and stir to extract until the final ethanol concentration of the system is 50.8%, to obtain a material containing xanthan gum fiber;

[0090] 6. Separate the material in step 5 to obtain a wet xanthan gum crude product and a mother liquor containing ethanol; the mother liquor can be used to recover ethanol through a distillation device, and the wet xanthan gum crude product is used for later use;

[0091] 7. The wet xanthan gum crude product of step 6 is extracted again with 93.2% ethanol solution, and the ethanol concentration is adjusted with water to a final ethanol concentration of 76.2%.

[0092] 8. Separate the material from step 7 to obtain a wet finished product and a mother liquor containing ethanol. The mother liquor can be distilled to recover ethanol. The wet finished product is dried in a 75°C forced air drying oven for 5 hours and then crushed and passed through an 80-mesh (0.175 mm) sieve to obtain the sieved material as a dry finished product.

[0093] Example 3

[0094] This embodiment provides a fermentation enhancer for improving fermentation intensity and a method for producing xanthan gum using the same, comprising the following steps:

[0095] 1. Weigh betaine hydrochloride: disodium hydrogen phosphate dodecahydrate: zinc sulfate: manganese sulfate in a mass ratio of 4000:4000:2:2, and mix them thoroughly to obtain a xanthan gum fermentation enhancer.

[0096] 2. Weigh the above-mentioned fermentation enhancer according to the formula amount and add it to the xanthan gum fermentation medium base material. After fully dissolving, sterilize and cool it down, and use it as the fermentation medium to directly carry out the xanthan gum fed batch fermentation; in addition, weigh the above-mentioned fermentation enhancer according to the formula amount to prepare the feed medium.

[0097] The fermentation medium of this embodiment is specifically as follows (g / L): glucose, 10; fish peptone, 8; corn steep liquor, 8; MgSO4·7H2O, 4; calcium carbonate, 4; the above-mentioned fermentation enhancer, 1, and the pH of the medium is adjusted to 7.24 with dilute hydrochloric acid and sodium hydroxide solution.

[0098] The feed medium is specifically as follows (g / L): glucose, 800; the above-mentioned fermentation enhancer, 30; a small amount of sodium hydroxide solution is used to adjust the pH to 7.11, and sterilize for later use.

[0099] 3. The fermentation conditions were the same as those in Example 1, except that the residual sugar concentration was detected every 2-4 h according to the sugar consumption rate of the system. After 12 h of fermentation, the residual sugar concentration of the fermentation liquid was detected to be 0.19%. Feeding was started at a flow rate of 700±50 mL / h through the feed pump, and the cumulative feed volume was 1 / 10 of the initial volume.

[0100] The fermentation was run until the residual sugar reached 0.00%, and the fermentation was terminated. The fermentation cycle was 61.8 hours.

[0101] 4. Add 0.3g of anhydrous calcium chloride (calcium chloride is dissolved in water in advance to a 10% mass volume ratio solution) for every 100ml of fermentation liquid, stir evenly and let stand for 30 minutes;

[0102] 5. Add 92.7% ethanol solution and stir to extract until the final ethanol concentration of the system is 53.9% to obtain a material containing xanthan gum fiber;

[0103] 6. Separate the material in step 5 to obtain a wet xanthan gum crude product and a mother liquor containing ethanol; the mother liquor can be used to recover ethanol through a distillation device, and the wet xanthan gum crude product is used for later use;

[0104] 7. The wet xanthan gum crude product of step 6 is extracted again with 92.7% ethanol solution, and the ethanol concentration is adjusted with water to a final ethanol concentration of 73.1%.

[0105] 8. Separate the material from step 7 to obtain a wet finished product and a mother liquor containing ethanol. The mother liquor can be distilled to recover ethanol. The wet finished product is dried in a blast drying oven at 105°C for 3 hours and then crushed and passed through an 80-mesh (0.175 mm) sieve to obtain the sieved material as a dry finished product. During the drying process, the material is appropriately turned over to promote drying of the material.

[0106] Example 4

[0107] This embodiment provides a method for producing xanthan gum, which is basically the same as that of embodiment 2, except that the fermentation conditions are as follows:

[0108] The temperature was 31.0±0.5°C within 20-24 hours, 31.5±0.5°C within 24-48 hours, and 32.0±0.5°C after 48 hours. Other fermentation control conditions were the same as those in Example 2. The fermentation was terminated when the residual sugar content was 0.02%. The fermentation cycle was 60.2 hours.

[0109] The extraction conditions are the same as in Example 2.

[0110] Example 5

[0111] This embodiment provides a fermentation enhancer for improving fermentation intensity and a method for producing xanthan gum using the same, comprising the following steps:

[0112] 1. The fermentation synergist used is the same as in Example 3.

[0113] 2. Weigh the above-mentioned fermentation enhancer according to the formula amount and add it to the xanthan gum fermentation medium base material. After fully dissolving, sterilize and cool it down, and use it as the fermentation medium to directly carry out the xanthan gum fed batch fermentation; in addition, weigh the above-mentioned fermentation enhancer according to the formula amount to prepare the feed medium.

[0114] The fermentation medium and feed medium were substantially the same as those in Example 3, except that the fermentation synergist content in the fermentation medium was 2 g / L, and the synergist content in the feed medium was 10 g / L.

[0115] 3. The fermentation conditions were the same as those in Example 3. Specifically, when the fermentation was continued for 12 hours, the residual sugar concentration in the fermentation liquid was detected to be 0.21%, and feeding was started according to the description in Example 3.

[0116] The fermentation was continued until the residual sugar content reached 0.01%, and the fermentation was terminated. The fermentation cycle was 62.5 h.

[0117] 4. The extraction process is the same as in Example 3.

[0118] Example 6

[0119] This embodiment provides a method for producing xanthan gum, comprising the following steps:

[0120] 1. Use the fermentation enhancer prepared in Example 1.

[0121] 2. Weigh the fermentation enhancer according to the formula amount and add it to the xanthan gum fermentation medium base material, sterilize and cool it after it is fully dissolved, and use it as the fermentation medium to directly carry out intermittent single batch aerobic fermentation of xanthan gum;

[0122] The fermentation medium of this example is the same as that of Example 1.

[0123] 3. The fermentation conditions were the same as those in Example 4. The fermentation was continued until the residual sugar content reached 0.03%, and the fermentation was terminated. The fermentation period was 60.9 h.

[0124] 4. The extraction process is the same as in Example 1.

[0125] Example 7

[0126] This embodiment provides a fermentation enhancer for improving fermentation intensity and a method for producing xanthan gum using the same, comprising the following steps:

[0127] 1. Weigh anhydrous betaine: sodium hexametaphosphate: zinc sulfate: manganese sulfate in a mass ratio of 2000:1000:2:2, and mix them thoroughly to obtain a xanthan gum fermentation enhancer.

[0128] 2. Weigh the fermentation enhancer according to the formula amount and add it to the xanthan gum fermentation medium base material, sterilize and cool it after it is fully dissolved, and use it as the fermentation medium to directly carry out intermittent single batch aerobic fermentation of xanthan gum;

[0129] The fermentation medium of this embodiment is basically the same as that of embodiment 1, except that it contains the fermentation enhancer described in step 1 of this embodiment.

[0130] 3. The fermentation conditions were the same as those in Example 1. The fermentation was continued until the residual sugar content reached 0.05%, and the fermentation was terminated. The fermentation period was 62.6 hours.

[0131] 4. The extraction process is the same as in Example 1.

[0132] Example 8

[0133] This embodiment provides a fermentation enhancer for improving fermentation intensity and a method for producing xanthan gum using the same, comprising the following steps:

[0134] 1. Weigh anhydrous betaine: sodium tripolyphosphate: zinc sulfate: manganese sulfate in a mass ratio of 1000:1000:2:2, and mix them thoroughly to obtain a xanthan gum fermentation enhancer.

[0135] 2. Weigh the above-mentioned fermentation enhancer according to the formula amount and add it to the xanthan gum fermentation medium base material. After fully dissolving, sterilize and cool it down, and use it as the fermentation medium to directly carry out the xanthan gum fed batch fermentation; in addition, weigh the above-mentioned fermentation enhancer according to the formula amount to prepare the feed medium.

[0136] The fermentation medium and feed medium of this embodiment are basically the same as those of embodiment 3, except that they contain the fermentation enhancer described in step 1 of this embodiment.

[0137] 3. The fermentation conditions were the same as those in Example 3. Specifically, when the fermentation was continued for 12 hours, the residual sugar concentration in the fermentation liquid was detected to be 0.16%, and feeding was started according to the description in Example 3.

[0138] The fermentation was run until the residual sugar reached 0.08%, and the fermentation was terminated. The fermentation cycle was 63.1 h.

[0139] 4. The extraction process is the same as in Example 3.

[0140] Example 9

[0141] This embodiment provides a method for producing xanthan gum, which is basically the same as that of Embodiment 5, except that the fermentation strain used is CGMCC1.3408.

[0142] Example 10

[0143] This embodiment provides a fermentation enhancer for improving fermentation intensity and a method for producing xanthan gum using the same, comprising the following steps:

[0144] 1. Prepare the fermentation enhancer according to the method of Example 3.

[0145] 2. Prepare fermentation medium and feed medium according to the method of Example 3.

[0146] 3. The fermentation conditions were basically the same as those in Example 3, except that the temperature was controlled at 31.0±0.5° C. within 20-24 hours, 31.5±0.5° C. within 24-48 hours, and 32.0±0.5° C. after 48 hours. Specifically, when the residual sugar concentration of the fermentation liquid was detected to be 0.11% after 12 hours of fermentation, feeding was started according to the description of Example 3.

[0147] The fermentation was continued until the residual sugar content reached 0.02%, and the fermentation was terminated. The fermentation cycle was 60.1 h.

[0148] 4. The extraction process is the same as in Example 3.

[0149] Comparative Example 1

[0150] This comparative example provides a method for producing xanthan gum by fermentation, comprising the following steps:

[0151] 1. The fermentation medium of this comparative example is substantially the same as that of Example 1, except that the fermentation synergist is not added.

[0152] 2. The fermentation conditions were the same as in Example 1.

[0153] 3. The extraction process is the same as in Example 1.

[0154] Comparative Example 2

[0155] This comparative example provides a method for producing xanthan gum by fermentation, comprising the following steps:

[0156] 1. The fermentation medium of this comparative example is substantially the same as that of Example 4, except that the fermentation synergist is not added.

[0157] 2. Fermentation conditions were the same as in Example 4.

[0158] 3. The extraction process is the same as in Example 4.

[0159] Comparative Example 3

[0160] This comparative example provides a method for producing xanthan gum by fermentation, comprising the following steps:

[0161] 1. The fermentation medium and feed medium of this comparative example are basically the same as those of Example 3, except that the fermentation synergist is not added to the fermentation medium and feed medium.

[0162] 2. Fermentation conditions were the same as in Example 3.

[0163] 3. The extraction process is the same as in Example 3.

[0164] Comparative Example 4

[0165] This comparative example provides a method for producing xanthan gum by fermentation, comprising the following steps:

[0166] 1. The fermentation medium of this comparative example adopts a formula substantially the same as that of Example 2, the only difference being that the content of the fermentation enhancer in the fermentation medium is 7 g / L.

[0167] 2. Fermentation conditions were the same as in Example 2.

[0168] 3. The extraction process is the same as in Example 2.

[0169] Comparative Example 5

[0170] This comparative example provides a method for producing xanthan gum by fermentation, comprising the following steps:

[0171] 1. The fermentation medium of this comparative example is basically the same as that of Example 1, except that the glucose content in the fermentation medium formula is 50 g / L, and the fermentation synergist is not added.

[0172] 2. The fermentation conditions were the same as in Example 1.

[0173] 3. The extraction process is the same as in Example 1.

[0174] Comparative Example 6

[0175] This comparative example provides a method for producing xanthan gum by fermentation, comprising the following steps:

[0176] 1. The fermentation medium of this comparative example is basically the same as that of Example 3, except that no fermentation synergist is added to the fermentation medium;

[0177] The feed medium formulation was (g / L): glucose, 400, pH 7.10;

[0178] 2. The fermentation conditions were the same as those in Example 3. Specifically, when the fermentation was continued for 12 hours, the residual sugar concentration in the fermentation liquid was detected to be 0.12%, and feeding was started according to the description in Example 3.

[0179] The fermentation was continued until the residual sugar content reached 0.29%, and the fermentation was terminated. The fermentation cycle was 60.2 h.

[0180] 3. The extraction process is the same as in Example 3.

[0181] Comparative Example 7

[0182] This comparative example provides a method for producing xanthan gum by fermentation, comprising the following steps:

[0183] 1. The fermentation medium of this comparative example adopts the formula of comparative example 5;

[0184] 2. Fermentation conditions were the same as in Example 4.

[0185] 3. The extraction process is the same as in Example 4.

[0186] Comparative Example 8

[0187] This comparative example provides a method for producing xanthan gum by fermentation, comprising the following steps:

[0188] 1. The fermentation medium and feed medium of this comparative example both adopt the same formula as that of Example 9, except that no fermentation enhancer is added;

[0189] 2. The strain and fermentation conditions were the same as those in Example 9.

[0190] 3. The extraction process is the same as in Example 9.

[0191] Comparative Example 9

[0192] This comparative example provides a method for producing xanthan gum by fermentation, comprising the following steps:

[0193] 1. The fermentation medium of this comparative example adopts the same formula as that of Example 7, except that no betaine is added to the fermentation synergist;

[0194] 2. The fermentation and extraction conditions were the same as in Example 7.

[0195] Comparative Example 10

[0196] This comparative example provides a method for producing xanthan gum by fermentation, comprising the following steps:

[0197] 1. The fermentation medium of this comparative example adopts the same formula as that of Example 7, except that no phosphate substances are added to the fermentation synergist;

[0198] 2. The fermentation and extraction conditions were the same as in Example 7.

[0199] Comparative Example 11

[0200] This comparative example provides a method for producing xanthan gum by fermentation, comprising the following steps:

[0201] 1. The fermentation medium of this comparative example adopts a formula substantially the same as that of Example 1, except that manganese sulfate is replaced with an equal amount of ferrous sulfate in the fermentation synergist;

[0202] 2. The fermentation and extraction conditions were the same as in Example 1.

[0203] Comparative Example 12

[0204] This comparative example provides a method for producing xanthan gum by fermentation, comprising the following steps:

[0205] 1. The fermentation medium of this comparative example adopts a formula substantially the same as that of Example 1, except that zinc sulfate is replaced by an equal amount of ferrous sulfate in the fermentation synergist;

[0206] 2. The fermentation and extraction conditions were the same as in Example 1.

[0207] Experimental example

[0208] In order to further verify the difference in product performance, the present invention adopts the following method to evaluate product performance:

[0209] 1) Conventional physical and chemical index tests of xanthan gum samples

[0210] Conventional physical and chemical indicators such as viscosity of xanthan gum samples were measured with reference to GB1886.41-2015;

[0211] 2) Determination of pyruvic acid content in xanthan gum samples

[0212] The pyruvic acid content of xanthan gum was determined using the following method: HPLC system, UV-detector, Bio-Rad 300×7.8 mm HPX-87H column to detect the pyruvyl content, 5 mM H2SO4 as mobile phase, flow rate 0.6 mL / min, detection wavelength 210 nm, room temperature, injection volume 20 μL;

[0213] Plotting of pyruvic acid standard curve: Prepare 15, 30, 45, 75, 150, 200 mg / L pyruvic acid solutions respectively, measure the elution time and peak area of ​​pyruvic acid, and obtain the standard curve based on the peak area;

[0214] Xanthan gum sample preparation and detection: Prepare 0.5% (m / m) xanthan gum aqueous solution, 90℃ water bath for 1h, mix several times during the process; ultrasonic crush, once every 5s, 10s interval each time, each sample ultrasonic 24 times; take 1mL of the ultrasonic solution into a 5mL EP tube, add 1mL of 0.1M phosphoric acid, seal after mixing, heat at 90℃ for 90min, and add ultrapure water to 3mL. Take the above xanthan gum mixed solution and filter it with a 0.22μm filter before sampling. The results are queried from the standard curve and calculated according to the corresponding dilution multiple.

[0215] 3) Calculation of fermentation conversion rate:

[0216] After the xanthan gum fermentation is completed, the fermentation conversion rate of a single batch is calculated according to the single tank production and carbon source input, using the following formula:

[0217]

[0218] 4) Detection of process OD

[0219] During the fermentation process, a small amount of fermentation liquid was sampled at 0, 4, 8, 12, 16, 20, and 24 h of fermentation and diluted 10 times with 0.1 M dilute hydrochloric acid. The absorbance of the diluted samples was then measured at a wavelength of 600 nm. The growth curve before 24 h of fermentation was plotted with the fermentation time as the horizontal axis and the net increase in absorbance after 0 h converted and subtracted as the vertical axis.

[0220] The test results of some processes of the above embodiments and comparative examples and the test results of the obtained xanthan gum samples are shown in Table 1. The comparison results of the bacterial growth in the first 24 hours of fermentation of some embodiments and comparative examples are shown in Table 1. Figure 1-Figure 2 shown.

[0221] Table 1

[0222]

[0223]

[0224] From the fermentation process and results of each embodiment and comparative example, the fermentation synergist of the present invention is added to the culture medium, under the same conditions, the fermentation cycle is significantly shortened and the sugar consumption rate and bacterial growth rate before 24 hours of fermentation are accelerated. From the bacterial growth before 24 hours of fermentation, the temperature has a positive effect on the growth of the bacteria, as shown in the comparison between Example 1 and Comparative Example 1, the comparison between Example 3 and Comparative Example 3, the comparison between Example 4 and Comparative Example 2, and the comparison between Comparative Example 5 and Comparative Example 7; at the same time, the addition of the fermentation synergist enables the strain to tolerate a higher temperature than conventional fermentation, as shown in the comparison between Example 2 and Example 4 and Comparative Example 2.

[0225] According to xanthan gum fermentation experience, raising the temperature will cause the microbial cell growth rate to increase, but its colloid production ability and conversion rate will be significantly reduced. In the present invention, from the perspective of the cell growth of 24h before fermentation, the embodiment of the fermentation synergist is added, and the increase in temperature does increase the cell growth rate, but does not have a negative impact on the fermentation conversion rate, as shown in Example 2 compared with Example 4. Similarly, it can be found by comparing Example 3 and Example 10 that the increase in temperature does not have a significant negative impact on the fermentation conversion rate. Although colloid production has begun after 24h, the OD determination of the cell itself has a certain impact, so it is impossible to accurately reflect the cell growth situation by net increase OD, but it can be seen from Comparative Example 2 and Comparative Example 2 that the comparative example 2 without adding the fermentation synergist, after fermenting 20h, the temperature is increased, which directly promotes the rapid growth of the cell, thereby affecting the final conversion rate of the fermentation. This shows that the addition of the fermentation synergist can improve the cell's tolerance to temperature.

[0226] It was also found in the study of the present invention that the addition of fermentation synergists helps microorganisms to tolerate higher concentrations of substrate sugar concentrations, thereby ensuring the fermentation conversion rate. High substrate sugar concentrations show a significant inhibitory effect on the growth of the early stage of the bacteria (see Comparative Example 1 and Example 1 for comparison), which may be the reason for the long final fermentation cycle; although the growth rate of the bacteria in the later stage is significantly faster (shown as a significant increase in the net increase in OD in 24h), for microorganisms, too fast bacterial growth rate may cause a significant reduction in conversion rate and rapid aging. Although it is not easy to accurately judge the net increase in OD after 24h of fermentation because the fermentation product becomes sticky and affects the OD detection, it can be predicted according to the fermentation experience of each product that the sugar consumption and gum production capacity of the product in the later stage may be significantly reduced, which may be the reason for the low conversion rate and high residual sugar at the end of the fermentation in the corresponding batch results. For high-concentration carbon source fermentation without adding fermentation synergist (Comparative Examples 1-3), the culture results with the addition of fermentation synergist showed lower residual sugar concentration in the final fermentation broth and better conversion rate (Examples 1-3). Each example showed a more suitable bacterial growth rate and gel production rate, thereby ensuring the stability of the fermentation conversion rate. The same conclusion can be obtained for intermittent single batch aerobic fermentation or fed batch fermentation.

[0227] It was also found that after adding the above-mentioned enhancers, the pyruvic acid content of the fermentation products was generally higher, and the viscosity of the final product was also improved. This shows that the synergistic effect of the above-mentioned enhancers not only improved the microorganisms' tolerance to temperature, but also improved their tolerance to high-concentration substrates, thereby significantly improving their fermentation intensity and product performance.

[0228] It is worth noting that in Comparative Example 3 where no fermentation synergist was added, due to the addition of feed medium, the net increase in OD of the bacteria was slightly reduced, and then secondary growth occurred, which may be caused by the dilution effect of the feed medium on the system. It is also obvious that the addition of the fermentation synergist of the present invention can effectively reduce the influence of the feed medium on the system and maintain the stability of bacterial growth (see Example 3 and Comparative Example 3).

[0229] Of course, the present invention also sees the phenomenon of "too much is as bad as too little". Comparative Example 4 is an experiment of adding dosage during the experiment. However, compared with Example 2, although the amount of fermentation synergist added has a small change range, the expected continuous improvement result is not obtained. From the fermentation conversion rate, it can be seen that its impact is a relatively obvious decline, which is not in line with expectations. In addition, Comparative Examples 5-7 adopt conventional fermentation methods in the industry, which reduces the input amount of total sugar in fermentation, thereby shortening the fermentation cycle, and the performance of its final product is not significantly affected, and can meet the final product standard. However, it is found that compared with the experimental results of adding fermentation synergist, its conversion rate is not ideal, and is only equivalent to conventional fermentation. Therefore, it is not ideal to improve the fermentation intensity only by limiting the input of carbon source, and it is necessary to make appropriate changes to the culture medium components or culture conditions. The synergist of the present invention can effectively make up for the shortcomings of conventional fermentation.

[0230] In order to verify the universality of the fermentation enhancer to the strains, the present invention also selected other strains for comparative experiments, see Example 9 and Comparative Example 8. The results show that although the fermentation intensity and product performance of different strains are different, the enhancer still has a relatively obvious effect of improving the conversion rate and shortening the cycle.

[0231] In addition, the present invention also found that the components in the above-mentioned fermentation enhancer have a synergistic effect. Comparative Example 9 did not add any betaine substances but only added phosphates. From the final fermentation results, it can be seen that although it can also tolerate high sugar concentrations, its conversion rate is significantly reduced, the cycle is significantly shortened, and the level of gum production is reduced. Comparative Example 10 does not add phosphates. Although it can tolerate high concentrations of substrate sugar, its conversion rate is also not significantly improved, and its sugar consumption capacity seems to have decreased; at the same time, from the perspective of Comparative Examples 11-12, the replacement of some components has not achieved the effect of further improving the fermentation intensity. The viscosity of the products of the above 4 comparative examples is also mediocre, and no significant improvement is seen, and the fermentation results are not ideal. At the same time, Example 7 shows better results, not only in terms of fermentation indicators, but also from the bacterial growth curve before fermentation 24h, the OD net increase rate of Example 7 does not show obvious excessive or low phenomena. It shows that the fermentation enhancer has an ideal effect on controlling bacterial growth and improving conversion rate.

[0232] From the final test results of the products, the viscosity of each product obtained in the embodiments of the present invention and the comparative examples meets the standard of "GB1886.41-2015 Food Additive Xanthan Gum", and from the test results of the pyruvic acid content in the products, the pyruvic acid content in the products generally increases when the fermentation synergist of the present invention is added, and the product viscosity is also generally improved. The pyruvic acid content may cause the improvement of the viscosity of the xanthan gum product, but the addition of the fermentation synergist of the present invention can cause the increase of the pyruvic acid content, which is the first discovery of the present invention.

[0233] Whether from the perspective of shortening the fermentation cycle, improving the fermentation conversion rate, improving the tolerance of the strain to high-concentration carbon sources, and increasing the pyruvic acid content and improving the viscosity of the product after fermentation, the fermentation enhancer of the present invention can be applied to the fermentation of xanthan gum, thereby improving the fermentation intensity of xanthan gum and has great production application space.

[0234] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for producing xanthan gum by fermentation, characterized in that: The fermentation medium includes a synergist, which includes: betaine substances, phosphates or polyphosphates, zinc sulfate and manganese sulfate; the betaine substances include one or more of betaine, betaine hydrates or betaine inorganic salts; When intermittent single batch aerobic fermentation is used during fermentation, the concentration of the synergist in the fermentation medium is 2 to 6 g / L; When the fermentation is carried out in fed-batch fermentation, the concentration of the synergist in the fermentation medium is 1-2 g / L.

2. The method according to claim 1, characterized in that: When the synergist includes betaine, phosphate, zinc sulfate and manganese sulfate, the mass ratio of the betaine, phosphate, zinc sulfate and manganese sulfate is (1000-2000): (1000-2000): (1-2): (0.5-1); When the synergist includes betaine, polyphosphate, zinc sulfate and manganese sulfate, the mass ratio of the betaine, polyphosphate, zinc sulfate and manganese sulfate is (1000-2000): (900-1100): (1.8-2.2): (1.8-2.2); And / or, the betaine substance includes one or more of anhydrous betaine, monohydrate betaine, betaine hydrochloride, betaine sulfate, betaine citrate, and betaine phosphate; the phosphate includes one or more of dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, potassium phosphate, sodium phosphate, and hydrates thereof; The polyphosphate includes one or more of tripolyphosphate, hexametaphosphate and hydrates thereof.

3. The method according to claim 1 or 2, characterized in that: The fermentation is carried out by intermittent single batch aerobic fermentation, wherein the fermentation medium comprises: glucose, 90-100 g / L; fish peptone, 8-10 g / L; corn steep liquor, 8-10 g / L; MgSO4·7H2O, 3-4 g / L; calcium carbonate, 3-4 g / L; the synergist, 2-6 g / L; and the pH is 7.2±0.1; Alternatively, the fermentation is carried out by fed-batch fermentation, wherein the fermentation medium comprises: glucose, 10-20 g / L; fish peptone, 8-10 g / L; corn steep liquor, 8-10 g / L; MgSO4·7H2O, 3-4 g / L; calcium carbonate, 3-4 g / L; fermentation enhancer, 1-2 g / L; pH is 7.2±0.1; When the residual sugar in the fermentation broth is less than 0.3% in the fed-batch fermentation, the feed medium is fed at a rate of 600-800 mL / h, and the total volume of the feed is 9-11% of the volume of the initial fermentation broth; The feed medium comprises: The synergist is 10-30 g / L; glucose is 500-800 g / L; and the pH is 7.1±0.

1.

4. The method according to any one of claims 1 to 3, characterized in that: The fermentation conditions are: Within 0-20h, air volume 0.4-0.6vvm, temperature 29.5±0.5℃, stirring speed 200-400rpm; Within 20-24h, air volume 0.8~1.0vvm, temperature 30.0±0.5℃~31.0±0.5℃, stirring speed 400-500rpm; Within 24-48h, air volume 1.0~1.1vvm, temperature 30±0.5℃~31.5±0.5℃, stirring speed 500-650rpm; After 48h, air volume 1.1~0.8vvm, temperature 30±0.5℃~32.0±0.5℃, stirring speed 650-700rpm; During fermentation, the pH of the fermentation liquid is controlled at 6.5-7.2, and the fermentation is terminated when the residual sugar content in the fermentation liquid no longer changes or is less than 0.1%.

5. The method according to any one of claims 1 to 4, characterized in that: The step of obtaining xanthan gum from the fermentation liquid after the fermentation is completed is also included; The step of obtaining xanthan gum comprises: (1) mixing the fermentation liquid after fermentation with a soluble inorganic salt and then allowing the mixture to stand to obtain an extract; (2) extracting the extract with ethanol, wherein the final ethanol concentration of the system during the extraction is 50-55%, to obtain a first material containing xanthan gum fibers; (3) separating the first material to obtain a crude wet xanthan gum product; (4) extracting the wet xanthan gum crude product with ethanol, wherein the final ethanol concentration of the system during the extraction is 70-80%, to obtain a second material; (5) separating the second material to obtain a wet xanthan gum product; (6) drying and crushing the wet xanthan gum product.

6. The method according to claim 5, characterized in that The mass volume ratio of the soluble inorganic salt to the fermentation liquid is (0.2-1): 100 g / ml; And / or, the soluble inorganic salt is one or more of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, and ferrous chloride; And / or, in steps (2) and (4), the concentration of ethanol is 92-95%; And / or, in step (6), the drying temperature is 60-105° C. and the drying time is 3-10 h.

7. The method according to any one of claims 1 to 6, characterized in that: The seed culture medium of the fermentation bacteria includes: sucrose, 15-20 g / L; peptone, 3-4 g / L; yeast powder, 1-2 g / L; beef extract, 4-5 g / L; pH is 7.0±0.1; And / or, the fermentation bacteria is Xanthomonas campestris.

8. A synergist, characterized in that The enhancer is as described in claim 1 or 2.

9. Use of the method according to any one of claims 1 to 7 or the synergist according to claim 8 in any of the following aspects: (1) Improve the conversion rate of xanthan gum production by fermentation; (2) Shorten the fermentation cycle of xanthan gum; (3) Improve the tolerance of fermentation bacteria to high concentrations of carbon sources; (4) increasing the pyruvic acid content in xanthan gum produced by fermentation; (5) Improve the viscosity of xanthan gum produced by fermentation; (6) Improve the tolerance of fermentation bacteria to fermentation temperature.

10. A xanthan gum, characterized in that Prepared by the method described in any one of claims 1-7; preferably, the content of pyruvic acid in the xanthan gum is ≥4.7%.

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