Preparation method of easy-to-purify Sanzan gum fermentation liquor

Abstract

The invention belongs to preparation of Sanzan gum, and particularly relates to a preparation method of easily purified Sanzan gum fermentation liquor. The method comprises the following steps: inoculating sphingomonas with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.19480 and the Latin name of Sphingomonas sp. Into a sterile fermentation culture medium, and carrying out aerated fermentation; the carbon source of the fermentation medium adopts one or a combination of glucose and sucrose, the inorganic salt adopts one or a combination of potassium dihydrogen phosphate and sodium dihydrogen phosphate, and ammonia water is adopted to adjust the fermentation medium and control the pH value in aerated fermentation to be 6.5-7.5. The method solves the problems of more nitrogen source residues, high ash content, low gum production rate and the like in the prior art, and has the advantages of easiness in purification, high gum production rate, low nitrogen source residues, low ash content and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the preparation of triosean gum, and particularly relates to a preparation method of triosean gum fermentation liquor easy to purify. BACKGROUND

[0002] Triosean gum, also known as sphingosine gum Ss, is a new type of microbial gum synthesized by triosean sphingomonas, has excellent thickening, gelling, emulsifying and suspension stabilizing properties, is a new variety of sphingosine gum polymer, and is also the first microbial polysaccharide independently researched and developed and industrialized produced in China.

[0003] The applicant discloses a preparation method of triosean gum fermentation liquor in a patent document with the patent number 202010346509.7, which takes sphingomonas with the Latin name Sphingomonas sp. CGMCC No. 19480 as a production strain, takes one or a combination of protein peptone, yeast extract, sodium nitrate, ammonium chloride and ammonium sulfate as a nitrogen source, and performs aeration fermentation. In practice, in order to improve the yield, an organic nitrogen source or a composite nitrogen source composed of organic nitrogen and inorganic nitrogen is usually used, and a single inorganic alkali is used to control the pH in the fermentation process. The existing problems of the above-mentioned prior art are as follows: 1. The nitrogen source residue in the final fermentation product is high (usually 2.1% to 2.4%), the ash content is high (usually 11% to 13%), and the product purity is affected; 2. The fermentation medium preparation and intermediate pH control are complex, the organic nitrogen source or the composite nitrogen source needs to be added in the fermentation medium, and inorganic salts such as dipotassium hydrogen phosphate and potassium dihydrogen phosphate are also added for buffering the pH in the fermentation process, the variety of materials is large during the preparation of the fermentation medium, the process is complicated, and the inorganic alkali needs to be added to adjust the pH in the fermentation process; 3. The gum yield is low (less than 3.4%). SUMMARY

[0004] The purpose of the present application is to provide a preparation method of triosean gum fermentation liquor easy to purify, which can effectively solve the problems of many impurities, complex fermentation medium preparation and pH control, and low gum yield in the prior art.

[0005] The overall technical concept of the present application is as follows:

[0006] The preparation method of triosean gum fermentation liquor easy to purify comprises inoculating sphingomonas with the preservation number CGMCC No. 19480 and the Latin name Sphingomonas sp. into a sterile fermentation medium for aeration fermentation; wherein:

[0007] A. The carbon source of the fermentation medium is one or a combination of glucose and sucrose, and the content is 40 g / L to 80 g / L; the inorganic salt is one or a combination of potassium dihydrogen phosphate and sodium dihydrogen phosphate, and the content is 15 g / L to 25 g / L; the pH of the fermentation medium is adjusted to 6.5 to 7.5 by using ammonia water;

[0008] B. The aeration fermentation temperature is 28℃~34℃. During fermentation, ammonia water is added to maintain the pH of the fermentation broth at 6.5~7.5. The addition of ammonia water is stopped after 50 hours of fermentation. Fermentation ends when the viscosity of the fermentation broth is ≥4000cp.

[0009] The specific technical concept of this invention also includes:

[0010] To facilitate the industrial production of Sanzan gum, the preferred technical solution is that, in step A, after the Sphingomonas sp. (CGMCC No. 19480) has been cultured on a large scale, the seed culture is inoculated into the fermentation medium at a volume percentage of 10% to 20% of the seed culture to the culture medium.

[0011] A more preferred technical solution is that the expanded culture process includes:

[0012] (1) The Sphingomonas sp. strain with preservation number CGMCC No.19480 and Latin name Sphingomonas sp. was inoculated into a primary seed culture medium and cultured under aeration to prepare a primary seed.

[0013] (2) The primary seeds are expanded and cultured to produce secondary seeds;

[0014] (3) The prepared secondary seed is inoculated into the sterile fermentation medium at an inoculation amount of 10% to 20% of the volume percentage of the secondary seed and the fermentation medium.

[0015] Furthermore, the process conditions for preparing primary seeds in step (1) of the expanded culture process are as follows: slant culture is inoculated into sterilized primary seed culture medium at a mass ratio of 1% to 2%, and primary seeds are prepared by aeration culture.

[0016] Furthermore, the process conditions for preparing secondary seeds in step (2) of the expanded culture process are as follows: the primary seeds are inoculated into the sterilized secondary seed culture medium at a ratio of 0.5% to 1% of the volume of the primary seed culture medium to the secondary seed culture medium, and then cultured with aeration to produce secondary seeds.

[0017] To facilitate seed growth, the preferred technical approach is to maintain an aerated culture temperature of 28℃~34℃, an aeration rate of 0.2vvm~0.3vvm, and transfer conditions where, under microscopic examination of the primary seed solution, the seed morphology shows uniform and irregularly distributed bacilli with a high OD500 concentration. 600 =1.2 to 1.5.

[0018] To further optimize seed cultivation, a preferred technical implementation is that each liter of primary or secondary seed culture medium comprises the following components by mass:

[0019] 10g-15g sucrose; 10g-15g soybean protein peptides; 5g-8g yeast powder; 3g-5g sodium chloride; 10ml-20ml trace element solution; pH: natural.

[0020] The trace element solution is composed of the following raw materials:

[0021] Citric acid 50g-150g; calcium hydroxide 3g-10g; magnesium hydroxide 5g-15g; manganese sulfate 0.02g-0.095g; zinc sulfate 0.02g-0.099g; copper sulfate pentahydrate 0.02g-0.095g; cobalt sulfate heptahydrate 0.01g-0.068g; magnesium sulfate 5g-9.6g; ferrous sulfate 1g-2.8g; boric acid 0.001g-0.016g; balance: water.

[0022] To avoid incomplete dissolution of solutes in the trace element solution or precipitation during preparation, a preferred technical means is to sequentially add citric acid, calcium hydroxide, magnesium hydroxide, manganese sulfate, zinc sulfate, copper sulfate pentahydrate, cobalt sulfate heptahydrate, magnesium sulfate, ferrous sulfate, and boric acid to water and mix them evenly.

[0023] To facilitate cell growth and the synthesis of secondary metabolites during fermentation, a preferred technical implementation is that the ventilation rate in step B is 1.2 vvm to 1.5 vvm.

[0024] To facilitate the measurement of the viscosity of the fermentation broth, the preferred technical means is that the viscosity of the fermentation broth in step B is measured using a Brookfield viscometer with rotor No. 4 at a speed of 60 rpm, and the viscosity is calculated as: viscosity = reading × 100.

[0025] Using the fermentation broth containing triazine gum prepared in this invention as raw material, triazine gum can be easily extracted from the fermentation broth by steps such as adjusting the isoelectric point of the fermentation broth, neutralizing, drying, and pulverizing. For example, the methods described in patent documents with application numbers 201911131488.0, 201910354160.9, and 202010346509.7 will not be elaborated upon by the applicant.

[0026] To verify the utilization rate of total nitrogen and the performance of the prepared triazine gel by the method of the present invention, the applicant conducted the following experiments:

[0027] I. Glue yield test, the method is as follows:

[0028] (1) Weigh 100 grams of fermentation liquid in a beaker;

[0029] (2) Add about 200 ml of hydrochloric acid solution (hydrochloric acid concentration 1:10 to 1:20), stir with a glass rod until sedimentation is complete, there is no slurry, and the filter cloth is squeezed dry;

[0030] (3) Squeeze the material dry and put it into a petri dish, tear it into pieces and dry it under a heat lamp (about 4 hours);

[0031] (4) Then transfer it to a 105℃ oven and dry it until constant weight (about 4 hours), then take it out and weigh it on a balance;

[0032] (5) The calculation formula is as follows: Glue yield = (sample weight × 1.08) ÷ volume × 100%.

[0033] II. Total Nitrogen Content

[0034] The Kjeldahl method was used for nitrogen determination.

[0035] III. Determination of Ash Content

[0036] 1. The sample was first dried at 105℃±1℃ for 4 hours;

[0037] 2. Determine the ash content according to the method described in GB5009.4.

[0038] The essential features and significant technical advancements achieved by this invention are as follows:

[0039] 1. Since ammonia water is easily volatilized and leaves no residue during post-fermentation treatment, the fermentation broth of Sanzanjiao prepared by the method described in this invention is easy to purify. The applicant's tests have confirmed that it fundamentally reduces the ash and impurities in Sanzanjiao products. The applicant's tests show that the ash content is reduced by no less than about 37%, and the purity is significantly higher than that of Sanzanjiao products prepared by existing processes.

[0040] 2. Compared with existing fermentation processes that use organic or compound nitrogen sources, the total nitrogen residue after fermentation in this application is reduced by at least 24%, the nitrogen source utilization rate is high, the fermentation broth is easy to purify, and the raw material consumption is effectively reduced, thus improving the input-output ratio.

[0041] 3. Compared with existing fermentation processes that use organic or compound nitrogen sources, using ammonia as a fed-batch nitrogen source has two advantages: First, it enables effective pH control while adding the nitrogen source, simplifying the process steps of nitrogen source selection and pH control in existing processes. Second, it enables effective control of the amount of ammonia added through real-time pH monitoring, facilitating automated control of ammonia addition.

[0042] 4. The present invention optimizes the micronutrient ratio in seed preparation to facilitate strain growth; rationally selects the nitrogen source in the fermentation process to meet the needs of strain growth and secondary metabolite synthesis; and assists in the optimized fermentation process. The above technical measures not only benefit strain growth but also facilitate secondary metabolite synthesis, thereby improving the gum yield of the present invention. The applicant's tests have confirmed that the gum yield is increased by no less than 2.9% compared with the existing process. Detailed Implementation

[0043] The present invention will be further described below with reference to embodiments, but this is not intended to limit the scope of the invention. The scope of protection of the present invention is determined by the contents of the claims, and any equivalent technical means substitutions made in accordance with the specification do not depart from the scope of protection of the present invention. Methods in the embodiments of the present invention that do not specify specific conditions are performed according to commonly used technical means in the art or conditions recommended by the manufacturer.

[0044] Example 1

[0045] A method for preparing easily purified tristigin fermentation broth includes inoculating *Sphingomonas* sp. (CGMCC No. 19480) into a sterile fermentation medium and aerating the medium for fermentation; wherein:

[0046] A. The carbon source of the fermentation medium is glucose or sucrose or a combination thereof, at a concentration of 40 g / L; the inorganic salt is potassium dihydrogen phosphate or sodium dihydrogen phosphate or a combination thereof, at a concentration of 15 g / L; ammonia water is used to adjust the pH of the fermentation medium to 6.5–7.5.

[0047] B. The aeration fermentation temperature is 28℃. During fermentation, ammonia water is added to maintain the pH of the fermentation broth at 6.5-7.5. The addition of ammonia water is stopped after 50 hours of fermentation. Fermentation ends when the viscosity of the fermentation broth is ≥4000cp.

[0048] Step A involves inoculating the sphingomonas sp. (CGMCC No. 19480, Latin name: Sphingomonas sp.) into the fermentation medium after expanding its culture, with the seed culture being inoculated at a volume percentage of 10% of the culture medium to the seed culture.

[0049] The expanded culture process includes:

[0050] (1) The Sphingomonas sp. strain with preservation number CGMCC No.19480 and Latin name Sphingomonas sp. was inoculated into a primary seed culture medium and cultured under aeration to prepare a primary seed.

[0051] (2) The primary seeds are expanded and cultured to produce secondary seeds;

[0052] (3) The prepared secondary seed is inoculated into the sterile fermentation medium at an inoculation amount of 10% of the volume percentage of the secondary seed and the fermentation medium.

[0053] The process conditions for preparing primary seeds in step (1) of the expanded culture process are as follows: slant culture is inoculated into sterilized primary seed culture medium at a mass ratio of 1%, and primary seeds are prepared by aeration culture.

[0054] The process conditions for preparing secondary seeds in step (2) of the expanded culture process are as follows: the primary seeds are inoculated into the sterilized secondary seed culture medium at a ratio of 0.5% to the volume ratio of primary seeds to secondary seed culture medium, and then cultured with aeration to produce secondary seeds.

[0055] During the expanded culture process, the aeration temperature was 28℃, the ventilation rate was 0.2 vvm, and the transfer conditions were as follows: microscopic examination of the primary seed culture revealed that the seed bacteria were uniform in color, irregularly distributed, and had a high OD500 concentration. 600 =1.2 to 1.5.

[0056] Each liter of primary or secondary seed culture medium comprises the following components by mass:

[0057] 10g sucrose; 10g soybean protein peptides; 5g yeast powder; 3g sodium chloride; 10ml trace element solution; pH: natural.

[0058] The trace element solution is composed of the following raw materials:

[0059] Citric acid 50g; calcium hydroxide 3g; magnesium hydroxide 5g; manganese sulfate 0.02g; zinc sulfate 0.02g; copper sulfate pentahydrate 0.02g; cobalt sulfate heptahydrate 0.01g; magnesium sulfate 5g; ferrous sulfate 1g; boric acid 0.001g; balance is water.

[0060] The trace element solution is prepared by sequentially adding citric acid, calcium hydroxide, magnesium hydroxide, manganese sulfate, zinc sulfate, copper sulfate pentahydrate, cobalt sulfate heptahydrate, magnesium sulfate, ferrous sulfate, and boric acid to water and mixing them thoroughly.

[0061] The ventilation volume in step B is 1.2vvm.

[0062] To facilitate the measurement of the viscosity of the fermentation broth, the preferred technical means is that the viscosity of the fermentation broth in step B is measured using a Brookfield viscometer with rotor No. 4 at a speed of 60 rpm, and the viscosity is calculated as: viscosity = reading × 100.

[0063] Example 2

[0064] The difference between this embodiment and Embodiment 1 is that:

[0065] A method for preparing easily purified tristigin fermentation broth includes inoculating *Sphingomonas* sp. (CGMCC No. 19480) into a sterile fermentation medium and aerating the medium for fermentation; wherein:

[0066] A. The carbon source of the fermentation medium is glucose or sucrose or a combination thereof, with a content of 80 g / L; the inorganic salt is potassium dihydrogen phosphate or sodium dihydrogen phosphate or a combination thereof, with a content of 25 g / L; ammonia water is used to adjust the pH of the fermentation medium to 6.5-7.5.

[0067] B. The aeration fermentation temperature is 34℃. During fermentation, ammonia water is added to maintain the pH of the fermentation broth at 6.5-7.5. The addition of ammonia water is stopped after 50 hours of fermentation. Fermentation ends when the viscosity of the fermentation broth is ≥4000cp.

[0068] Step A involves inoculating the sphingomonas sp. strain (CGMCC No. 19480, Latin name: Sphingomonas sp.) into the fermentation medium after expansion culture, with an inoculation rate of 20% of the seed culture to the culture medium volume.

[0069] The process conditions for preparing primary seeds in step (1) of the expanded culture process are as follows: slant culture is inoculated into sterilized primary seed culture medium at a mass ratio of 2%, and primary seeds are prepared by aeration culture.

[0070] The process conditions for preparing secondary seeds in step (2) of the expanded culture process are as follows: the primary seeds are inoculated into the sterilized secondary seed culture medium at a ratio of 1% to the volume ratio of primary seeds to secondary seed culture medium, and then cultured with aeration to produce secondary seeds.

[0071] The aerated culture temperature was 34℃, the ventilation rate was 0.3 vvm, and the transfer conditions were as follows: microscopic examination of the primary seed culture revealed that the seed bacteria were uniform in color, irregularly distributed, and had a high OD500 concentration. 600 =1.2 to 1.5.

[0072] Each liter of primary or secondary seed culture medium comprises the following components by mass:

[0073] 15g sucrose; 15g soybean protein peptides; 8g yeast powder; 5g sodium chloride; 20ml trace element solution; pH: natural.

[0074] The trace element solution is composed of the following raw materials:

[0075] Citric acid 150g; calcium hydroxide 10g; magnesium hydroxide 15g; manganese sulfate 0.095g; zinc sulfate 0.099g; copper sulfate pentahydrate 0.095g; cobalt sulfate heptahydrate 0.068g; magnesium sulfate 9.6g; ferrous sulfate 2.8g; boric acid 0.016g; balance is water.

[0076] In step B, the ventilation volume is 1.5vvm.

[0077] The rest of the content is as described above.

[0078] Example 3

[0079] The difference between this embodiment and Embodiment 1 is that:

[0080] A method for preparing easily purified tristigin fermentation broth includes inoculating *Sphingomonas* sp. (CGMCC No. 19480) into a sterile fermentation medium and aerating the medium for fermentation; wherein:

[0081] A. The carbon source of the fermentation medium is glucose or sucrose or a combination thereof, with a content of 60 g / L; the inorganic salt is potassium dihydrogen phosphate or sodium dihydrogen phosphate or a combination thereof, with a content of 20 g / L; ammonia water is used to adjust the pH of the fermentation medium to 6.5-7.5.

[0082] B. The aeration fermentation temperature is 31℃. During fermentation, ammonia water is added to maintain the pH of the fermentation broth at 6.5-7.5. The addition of ammonia water is stopped after 50 hours of fermentation. Fermentation ends when the viscosity of the fermentation broth is ≥4000cp.

[0083] Step A involves inoculating the sphingomonas sp. strain (CGMCC No. 19480, Latin name: Sphingomonas sp.) into the fermentation medium after expansion culture, with an inoculation rate of 15% (seed culture to medium volume percentage).

[0084] The process conditions for preparing primary seeds in step (1) of the expanded culture process are as follows: the slant culture is inoculated into the sterilized primary seed culture medium at a mass ratio of 1.5%, and the primary seeds are prepared by aeration culture.

[0085] The process conditions for preparing secondary seeds in step (2) of the expanded culture process are as follows: the primary seeds are inoculated into the sterilized secondary seed culture medium at a ratio of 0.8% of the volume of the primary seed culture medium to the secondary seed culture medium, and then cultured by aeration to produce secondary seeds.

[0086] The aerated culture temperature was 31℃, the ventilation rate was 0.25 vvm, and the transfer conditions were as follows: microscopic examination of the primary seed culture revealed that the seed bacteria were uniform in color, irregularly distributed, and the bacterial concentration OD was [not specified]. 600=1.2 to 1.5.

[0087] Each liter of primary or secondary seed culture medium comprises the following components by mass:

[0088] 13g sucrose; 13g soybean protein peptides; 6.5g yeast powder; 4g sodium chloride; 15ml trace element solution; pH: natural.

[0089] The trace element solution is composed of the following raw materials:

[0090] Citric acid 100g; calcium hydroxide 6.5g; magnesium hydroxide 10g; manganese sulfate 0.05g; zinc sulfate 0.05g; copper sulfate pentahydrate 0.05g; cobalt sulfate heptahydrate 0.04g; magnesium sulfate 7.2g; ferrous sulfate 1.7g; boric acid 0.007g; balance: water.

[0091] The ventilation volume in step B is 1.35vvm.

[0092] The rest of the content is as described above.

[0093] Example 4

[0094] The difference between this embodiment and Embodiment 1 is that:

[0095] A method for preparing easily purified tristigin fermentation broth includes inoculating *Sphingomonas* sp. (CGMCC No. 19480) into a sterile fermentation medium and aerating the medium for fermentation; wherein:

[0096] A. The carbon source of the fermentation medium is glucose or sucrose or a combination thereof, at a concentration of 50 g / L; the inorganic salt is potassium dihydrogen phosphate or sodium dihydrogen phosphate or a combination thereof, at a concentration of 18 g / L; ammonia water is used to adjust the pH of the fermentation medium to 6.5–7.5.

[0097] B. The aeration fermentation temperature is 29℃. During fermentation, ammonia water is added to maintain the pH of the fermentation broth at 6.5-7.5. The addition of ammonia water is stopped after 50 hours of fermentation. Fermentation ends when the viscosity of the fermentation broth is ≥4000cp.

[0098] Step A involves inoculating the sphingomonas sp. (CGMCC No. 19480, Latin name: Sphingomonas sp.) into the fermentation medium after expanding its culture, with the seed culture being inoculated at a volume percentage of 13% to the culture medium.

[0099] The process conditions for preparing primary seeds in step (1) of the expanded culture process are as follows: the slant culture is inoculated into the sterilized primary seed culture medium at a mass ratio of 1.2%, and the primary seeds are prepared by aeration culture.

[0100] The process conditions for preparing secondary seeds in step (2) of the expanded culture process are as follows: the primary seeds are inoculated into the sterilized secondary seed culture medium at a ratio of 0.6% of the volume of the primary seed culture medium to the secondary seed culture medium, and then cultured by aeration to produce secondary seeds.

[0101] The aerated culture temperature was 29℃, the ventilation rate was 0.23 vvm, and the transfer conditions were as follows: microscopic examination of the primary seed culture revealed that the seed bacteria were uniform in color, irregularly distributed, and had a high OD500 concentration. 600 =1.2 to 1.5.

[0102] Each liter of primary or secondary seed culture medium comprises the following components by mass:

[0103] 11g sucrose; 12g soybean protein peptides; 6g yeast powder; 3.5g sodium chloride; 12ml trace element solution; pH: natural.

[0104] The trace element solution is composed of the following raw materials:

[0105] Citric acid 80g; calcium hydroxide 4g; magnesium hydroxide 8g; manganese sulfate 0.04g; zinc sulfate 0.03g; copper sulfate pentahydrate 0.04g; cobalt sulfate heptahydrate 0.03g; magnesium sulfate 6.5g; ferrous sulfate 1.5g; boric acid 0.004g; balance is water.

[0106] The ventilation volume in step B is 1.3vvm.

[0107] The rest of the content is as described above.

[0108] Example 5

[0109] The difference between this embodiment and Embodiment 1 is that:

[0110] A method for preparing easily purified tristigin fermentation broth includes inoculating *Sphingomonas* sp. (CGMCC No. 19480) into a sterile fermentation medium and aerating the medium for fermentation; wherein:

[0111] A. The carbon source of the fermentation medium is glucose or sucrose or a combination thereof, with a content of 70 g / L; the inorganic salt is potassium dihydrogen phosphate or sodium dihydrogen phosphate or a combination thereof, with a content of 23 g / L; ammonia water is used to adjust the pH of the fermentation medium to 6.5-7.5.

[0112] B. The aeration fermentation temperature is 32℃. During fermentation, ammonia water is added to maintain the pH of the fermentation broth at 6.5-7.5. The addition of ammonia water is stopped after 50 hours of fermentation. Fermentation ends when the viscosity of the fermentation broth is ≥4000cp.

[0113] Step A involves inoculating the sphingomonas sp. (CGMCC No. 19480, Latin name: Sphingomonas sp.) into the fermentation medium after expanding its culture, with the seed culture being inoculated at a volume percentage of 18% to the culture medium.

[0114] The process conditions for preparing primary seeds in step (1) of the expanded culture process are as follows: the slant culture is inoculated into the sterilized primary seed culture medium at a mass ratio of 1.8%, and the primary seeds are prepared by aeration culture.

[0115] The process conditions for preparing secondary seeds in step (2) of the expanded culture process are as follows: the primary seeds are inoculated into the sterilized secondary seed culture medium at a ratio of 0.9% to the volume ratio of primary seeds to secondary seed culture medium, and then cultured with aeration to produce secondary seeds.

[0116] The aerated culture temperature was 33℃, the ventilation rate was 0.28 vvm, and the transfer conditions were as follows: microscopic examination of the primary seed culture revealed that the seed bacteria were uniform in color, irregularly distributed, and had a high OD500 concentration. 600 =1.2 to 1.5.

[0117] Each liter of primary or secondary seed culture medium comprises the following components by mass:

[0118] 14g sucrose; 14g soybean protein peptides; 7.5g yeast powder; 4.5g sodium chloride; 18ml trace element solution; pH: natural.

[0119] The trace element solution is composed of the following raw materials:

[0120] Citric acid 120g; calcium hydroxide 9g; magnesium hydroxide 12g; manganese sulfate 0.08g; zinc sulfate 0.08g; copper sulfate pentahydrate 0.08g; cobalt sulfate heptahydrate 0.055g; magnesium sulfate 8g; ferrous sulfate 2.5g; boric acid 0.013g; balance: water.

[0121] The ventilation volume in step B is 1.4vvm.

[0122] The rest of the content is as described above.

[0123] The applicant tested the gum yield, total nitrogen of the prepared fermentation broth, and ash content of the purified triazine gum product in Examples 1-5, and the results are as follows:

[0124] Example 1 Example 2 Example 3 Example 4 Example 5 Total nitrogen 1.32％ 1.60％ 1.46％ 1.42％ 1.58％ Ash content 6.01％ 6.92％ 6.94％ 6.20％ 6.45％ Tackifying rate (%) 3.52％ 3.58％ 3.61％ 3.5％ 3.52％

[0125] It is easy to see from the above comparison that the gum production rate in Examples 1 to 5 of the present invention is better than that of the prior art. The total nitrogen of the fermentation broth and the ash content of the purified Sanzan gum product are significantly better than those of the prior art. Moreover, the use of ammonia water flow addition to achieve nitrogen source addition and pH control greatly simplifies the fermentation control process and subsequent purification process.

Claims

1. A method for preparing easily purified tristigin fermentation broth, comprising inoculating *Sphingomonas* sp. (CGMCC No. 19480, Latin name: *Sphingomonas* sp.) into a sterile fermentation medium and aerating the medium for fermentation; characterized in that: A. The carbon source of the fermentation medium is glucose or sucrose or a combination thereof, with a content of 40 g / L to 80 g / L; the inorganic salt is potassium dihydrogen phosphate or sodium dihydrogen phosphate or a combination thereof, with a content of 15 g / L to 25 g / L; ammonia water is used to adjust the pH of the fermentation medium to 6.5 to 7.

5. B. The aeration fermentation temperature is 28℃~34℃. During fermentation, ammonia water is added to maintain the pH of the fermentation broth at 6.5~7.

5. The addition of ammonia water is stopped after 50 hours of fermentation. Fermentation ends when the viscosity of the fermentation broth is ≥4000cp.

2. The method for preparing easily purified triazine fermentation broth according to claim 1, characterized in that... Step A involves inoculating the sphingomonas sp. (CGMCC No. 19480, Latin name: Sphingomonas sp.) into the fermentation medium after expanding its culture, with the seed culture being inoculated at a volume percentage of 10% to 20% of the culture medium.

3. The method for preparing easily purified triazine fermentation broth according to claim 2, characterized in that... The aforementioned scale-up culture process includes: (1) The Sphingomonas sp. strain with preservation number CGMCC No.19480 and Latin name Sphingomonas sp. was inoculated into a primary seed culture medium and cultured under aeration to prepare a primary seed. (2) The primary seeds are expanded and cultured to produce secondary seeds; (3) The prepared secondary seed is inoculated into the sterile fermentation medium at an inoculation amount of 10% to 20% of the volume percentage of the secondary seed and the fermentation medium.

4. The method for preparing easily purified triazine fermentation broth according to claim 3, characterized in that... The process conditions for preparing primary seeds in step (1) of the expanded culture process are as follows: slant culture is inoculated into sterilized primary seed culture medium at a mass ratio of 1% to 2%, and primary seeds are prepared by aeration culture.

5. The method for preparing easily purified triazine fermentation broth according to claim 3, characterized in that... The process conditions for preparing secondary seeds in step (2) of the expanded culture process are as follows: the primary seeds are inoculated into the sterilized secondary seed culture medium at a ratio of 0.5% to 1% of the volume of the primary seed culture medium to the secondary seed culture medium, and then cultured with aeration to produce secondary seeds.

6. The method for preparing easily purified triazine fermentation broth according to claim 3 or 4, characterized in that... The aerated culture temperature was 28℃~34℃, and the ventilation rate was 0.2vvm~0.3vvm. The transfer conditions were as follows: microscopic examination of the primary seed culture revealed uniform color and irregular distribution of the bacilli in the seeds, with a high OD500 concentration. 600 =1.2 to 1.

5.

7. The method for preparing easily purified triazine fermentation broth according to claim 3 or 4, characterized in that... Each liter of primary or secondary seed culture medium comprises the following components by mass: 10g-15g sucrose; 10g-15g soybean protein peptides; 5g-8g yeast powder; 3g-5g sodium chloride; 10ml-20ml trace element solution; pH: natural. The trace element solution is composed of the following raw materials: Citric acid 50g-150g; calcium hydroxide 3g-10g; magnesium hydroxide 5g-15g; manganese sulfate 0.02g-0.095g; zinc sulfate 0.02g-0.099g; copper sulfate pentahydrate 0.02g-0.095g; cobalt sulfate heptahydrate 0.01g-0.068g; magnesium sulfate 5g-9.6g; ferrous sulfate 1g-2.8g; boric acid 0.001g-0.016g; balance: water.

8. The method for preparing easily purified triazine fermentation broth according to claim 7, characterized in that... The trace element solution is prepared by sequentially adding citric acid, calcium hydroxide, magnesium hydroxide, manganese sulfate, zinc sulfate, copper sulfate pentahydrate, cobalt sulfate heptahydrate, magnesium sulfate, ferrous sulfate, and boric acid to water and mixing them thoroughly.

9. The method for preparing easily purified triazine fermentation broth according to claim 1, characterized in that... In step B, the ventilation volume is 1.2vvm to 1.5vvm.

10. The method for preparing easily purified triazine fermentation broth according to claim 1, characterized in that... In step B, the viscosity of the fermentation broth is measured using a Brookfield viscometer with rotor No. 4 at a speed of 60 rpm. Viscosity = reading × 100.