A pretreatment process for acarbose fermentation broth

Abstract

The present invention belongs to the field of microbial fermentation. Specifically, it provides a pretreatment process for acarbose fermentation broth. By adding a filter aid twice, adjusting the pH with acid and alkali, and then using CPAM flocculation treatment, the problem of the difficulty in solid-liquid separation of acarbose fermentation broth can be solved, especially the problem of the difficulty in solid-liquid separation of acarbose fermentation broth with a bacterial concentration of less than 40% and a viscosity of less than 650mPa.S. The effect of partial decolorization is increased, the pressure of subsequent purification process is reduced, and it is beneficial to improve the product yield. The present invention also provides a preparation method of CPAM solution. By wetting with organic alcohols such as methanol, the dissolution time of CPAM particles is greatly shortened.

Description

Technical Field

[0001] The invention relates to a processing technology for a microbial fermentation product, in particular to a pretreatment technology for an acarbose fermentation broth. Background Art

[0002] Acarbose is an α-glucosidase inhibitor developed by Bayer AG in Germany in the mid-1970s. It is widely used in the treatment of type 2 diabetes clinically because it can effectively inhibit the activity of α-glucosidase in small intestinal wall cells, delay the degradation and absorption of oligosaccharides, disaccharides or polysaccharides in the intestine, and has good pharmacokinetic properties and low toxicity.

[0003] Since the chemical structure of acarbose is relatively complex, its chemical synthesis steps are relatively cumbersome and costly, and the current industrial-scale production is mainly obtained through microbial fermentation. Acarbose can be extracted from the fermentation broth of Actinoplanes sp., and its synthetic metabolic pathway in microorganisms and fermentation process research have always been the research hotspots of acarbose. Experimental studies have found that the fermentation broth obtained by fermentation culture of the acarbose-producing bacteria Actinoplanes utahensis MYIH97 (Actinoplanes utahensis MYIH97) is quite different from the previous acarbose fermentation broth in terms of properties and characteristics, which are specifically manifested in bacterial concentration and viscosity. The bacterial concentration of the previous acarbose fermentation broth was greater than 50%, and the viscosity was greater than 800mPa.S, while the fermentation broth obtained by this strain had a bacterial concentration of less than 40% and a viscosity of less than 650mPa.S. The existing pretreatment method is generally to add aluminum sulfate for coagulation, adjust to pH 5.5-6.5, and then filter with a plate and frame filter press. The existing pretreatment method can no longer normally complete solid-liquid separation to obtain a filtrate.

[0004] CN109580317A discloses a pretreatment method for acarbose fermentation liquid, wherein the acarbose fermentation liquid is centrifuged at a centrifugal force of 3000-5000g for the first time, a trichloroacetic acid alcohol solution 2-4 times the volume of the supernatant is mixed with the supernatant, centrifuged for the second time, and the supernatant is filtered through a membrane. This method uses trichloroacetic acid, a harmful carcinogenic agent, and has high cost, so it is only suitable for laboratory research and is not suitable for large-scale industrial production. Summary of the invention

[0005] One object of the present invention is to overcome the problem that the existing pretreatment method cannot achieve solid-liquid separation of fermentation broth with a bacterial concentration of less than 40% and a viscosity of less than 650mPa.S, and to provide a pretreatment process for acarbose fermentation broth suitable for industrialization.

[0006] The process includes the following steps:

[0007] (a) adding filter aid A to acarbose fermentation stock solution and stirring to obtain a mixed fermentation solution;

[0008] (b) adding an acidulant to the mixed fermentation broth to adjust the pH and stirring the mixture for reaction;

[0009] (c) adjusting the pH of the acidified fermentation broth with an alkaline solution;

[0010] (d) adding a freshly prepared flocculant to the fermentation broth to adjust the pH by adding alkali, and stirring until flocculation is obviously observed;

[0011] (e) Add filter aid B to the fermentation liquid after flocculation, stir until the flocs are slightly dispersed, and separate the solid and liquid.

[0012] A pretreatment process for acarbose fermentation broth, the process steps may also be:

[0013] (a) adding filter aid A to acarbose fermentation stock solution and stirring to obtain a mixed fermentation solution;

[0014] (b1) adding the prepared flocculant to the mixed fermentation liquid and stirring until flocculation is obvious;

[0015] (c1) adding an acidulant to the flocculated fermentation liquid to adjust the pH and stirring the mixture for reaction;

[0016] (d1) Adding filter aid B to the fermentation liquid after flocculation to separate the solid and liquid.

[0017] Specifically, the filter aid A or B is independently selected from diatomaceous earth, perlite, coarse river sand, volcanic stone, preferably diatomaceous earth and perlite;

[0018] Specifically, filter aid A and filter aid B are preferably different, and filter aid A is further preferably selected from diatomaceous earth, and filter aid B is selected from perlite;

[0019] The added mass of the filter aid is 0.5-1.5% (g / ml) of the volume of the fermentation stock solution, preferably 0.5-1.0%.

[0020] Specifically, the acidulant is oxalic acid or sulfuric acid, preferably oxalic acid.

[0021] Specifically, the pH range for adjusting the pH by adding an acidulant in the above process is 2.5-3.5, preferably 2.5-3.0.

[0022] Specifically, the base in step (c) is an inorganic base solution, such as sodium hydroxide or ammonia solution; the adjusted pH range is 3.8-4.8, preferably 4.3-4.5.

[0023] Specifically, the flocculant in step (d) or (b1) is added in the form of an aqueous solution, preferably cationic polyacrylamide (abbreviated as CPAM), wherein the concentration of the CPAM solution is 0.05%-0.1%, preferably 0.05%-0.06%; the amount of CPAM solution added is 5-15% (V:V) of the volume of the fermentation stock solution, preferably 8-10%.

[0024] Specifically, the flocculant in step (d) or (b1) is selected from cationic polyacrylamide (abbreviated as CPAM), preferably with a solid content greater than 93%, a molecular weight of 9-10 million, an ionicity of 40-60%, and a particle size of 80-100 mesh; more preferably, it is CPAM particles with a solid content of 95%, a molecular weight of 10 million, an ionicity of 50%, and a particle size of 100 mesh.

[0025] Another object of the present invention is to provide a method for preparing a CPAM solution, which comprises the following steps:

[0026] (1) Wetting the CPAM particles with an organic solvent in a 0.1-0.3% (V:V) solution;

[0027] (2) The wetted CPAM particles are evenly and slowly added into the stirred water, and stirred at 40-55° C. until dissolved.

[0028] Specifically, the organic solvent is alcohol, such as methanol and ethanol.

[0029] Specifically, the amount of solvent used to wet the CPAM particles is 0.1-0.3% of the prepared solution, preferably 0.1-0.2%; the wetting time is 10-30 min, preferably 15-20 min.

[0030] Specifically, the dissolution temperature of the CPAM is 40-55°C, preferably 45-50°C; the stirring speed is 200-400 rpm.

[0031] Specifically, the acarbose fermentation stock solution in the present invention is obtained by fermentation and culture of the acarbose-producing bacterium Actinomyces utahensis.

[0032] As a specific implementation manner, the acarbose fermentation stock solution is prepared as follows:

[0033] Inoculate the bacterial lawn into the shake flask seed culture medium, culture at 250 rpm for 2-3 days to obtain the shake flask seed solution;

[0034] Inoculate the shake flask seed solution into the seed tank at an inoculation rate of 0.1-0.5% of the volume of the seed tank culture medium, culture at 120-200 rpm for 2-3 days to obtain the tank seed solution;

[0035] Inoculate the tank seed liquid into the fermentation tank medium at an inoculation rate of 6-15% of the fermentation volume, at 150-200 rpm, ferment for 168 hours, and collect the fermentation liquid;

[0036] The culture temperature was 26-30°C.

[0037] The components of the shake flask seed culture medium are as follows: 1.2-3.0 g of carbon source, 2.5-4.0 g of nitrogen source, 0.2-0.6 g of inorganic salt, and the rest is water per 100 mL of culture medium;

[0038] The proportion of each component of the seed tank culture medium in the culture medium is: 2.0-3.5 g of carbon source, 3.5-6.0 g of nitrogen source, 0.2-0.6 g of inorganic salt, and the rest is water per 100 mL of culture medium;

[0039] The proportions of the components of the fermentation medium in the medium are: 5-10 g of carbon source, 1.0-3.5 g of nitrogen source, 0.2-2.0 g of inorganic salt, and the rest is water per 100 mL of the medium, with a pH value of 6.5-7.5.

[0040] Beneficial effects of the present invention:

[0041] The present invention provides a pretreatment process for acarbose fermentation broth, which can solve the problem of difficult solid-liquid separation of acarbose fermentation broth, especially the problem of difficult solid-liquid separation of acarbose fermentation broth with a bacterial concentration of less than 40% and a viscosity of less than 650mPa.S, by adding a filter aid twice in succession, adjusting the pH with acid and alkali, and then using CPAM flocculation treatment. The effect of partial decolorization is increased, the pressure of subsequent purification process is reduced, and it is beneficial to improve the product yield. The present invention also provides a preparation method of CPAM solution, which greatly shortens the dissolution time of CPAM particles by pre-wetting with organic alcohols. DETAILED DESCRIPTION

[0042] The technical solution of the present invention will be clearly and completely described below in conjunction with specific implementation methods. However, those skilled in the art will understand that the embodiments described below are only part of the embodiments of the present invention, rather than all of the embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention.

[0043] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0044] The acarbose-producing bacteria used in the acarbose fermentation stock solution in the embodiment is Actinoplanes utahensis MYIH97, which is deposited in Wuhan China Center for Type Culture Collection (CCTCC for short), with a deposit number of CCTCC, NO: M2016237. The fermentation solution preparation method is as follows:

[0045] (1) Inoculate the frozen bacterial solution in the glycerol tube directly into the seed culture medium of the shake flask, and culture at 26-30°C and 250 rpm for 44-48 hours;

[0046] The formula of the shake flask seed culture medium is: 20g / L soybean powder, 10g / L glycerol, 40g / L corn starch, 3g / L calcium carbonate, and the rest is water.

[0047] (2) Inoculate the cultured seed solution from the shake flask into the seed tank. The ventilation rate is 1:1-1:1.5, the tank pressure is 0.05Mpa, the stirring is 120rpm-200rpm, and the culture is carried out at 26-30℃ for 44-48h.

[0048] The formula of the seed tank culture medium is: 20g / L soybean powder, 10g / L glycerol, 40g / L corn starch, 3g / L calcium carbonate and 0.5g / L foetida, and the rest is water. The pH value of the culture medium is adjusted to 6.8-7.0 before sterilization.

[0049] (3) The cultured tank seed liquid is connected to the fermentation tank, the tank pressure is 0.05Mpa, the ventilation volume is 1:1 to 1:1.5, the stirring is 120rpm to 200rpm, and the culture is carried out at 26 to 30°C for 120-168h.

[0050] The formula of the fermentation tank culture medium is: 40g / L glucose, 20g / L maltose, 20g / L soybean powder, 3g / L sodium glutamate, 2.5g / L calcium carbonate, 1.5g / L dipotassium hydrogen phosphate, 0.5g / L ferric chloride, 2g / L calcium chloride and 0.5g / L foetida, and the rest is water. The pH value of the culture medium is adjusted to 6.8-7.0 before sterilization.

[0051] The preparation method of the CPAM solution in the specific embodiment is:

[0052] (1) Wetting the CPAM particles with a 0.1-0.3% (V:V) solution of an organic solvent for 10-30 min;

[0053] (2) The wetted CPAM particles are evenly and slowly added into the stirred water, and stirred at 40-55° C. and 200-400 rpm for 20-50 min until they are completely dissolved.

[0054] Take the preparation of 1L 0.05% CPAM solution as an example:

[0055] Weigh 0.5 g of CPAM particles with a solid content of 95%, a molecular weight of 10 million, an ionicity of 50%, and a particle size of 100 mesh, moisten the above CPAM particles with 3 mL of methanol for 20 minutes, and then slowly and evenly sprinkle them into 45°C water in a stirring state, keep the temperature constant and stir at 400 rpm until completely dissolved to obtain a 0.05% CPAM solution.

[0056] The CPAM solutions used in the following examples are prepared solutions unless otherwise specified.

[0057] Example 1

[0058] 200 mL of acarbose fermentation stock solution was measured with a measuring cylinder and poured into a 500 mL beaker, 3 g of diatomaceous earth (1.5% g / mL of the fermentation stock solution volume ratio) was added, and the mixture was stirred evenly. Solid oxalic acid was added into the beaker to adjust the pH to 3.0, and the mixture was stirred for 15 min. Then, 1 mol / L sodium hydroxide solution was added to adjust the pH to 4.3, and 20 mL of freshly prepared 0.05% CPAM solution was added into the fermentation liquid, and the mixture was stirred slowly until flocculation was obviously observed and the flocculants were slightly dispersed. The pretreated fermentation liquid was poured into a funnel equipped with filter paper for natural filtration, and the filtration rate was measured to be 0.7 mL / min.

[0059] Example 2

[0060] Use a measuring cylinder to measure 200 mL of acarbose fermentation stock solution and pour it into a 500 mL beaker. Add solid oxalic acid to the beaker to adjust the pH to 3.0, stir and react for 15 minutes, then add 1 mol / L sodium hydroxide solution to adjust the pH back to 4.3, then add 20 mL of freshly prepared 0.05% CPAM solution to the fermentation liquid, stir slowly until flocculation is obvious, then add 3 g of diatomaceous earth, stir until the floccules are slightly dispersed, pour the pretreated fermentation liquid into a funnel equipped with filter paper for natural filtration, and measure the filtration rate to be 0.7 mL / min.

[0061] Example 3

[0062] 200 mL of acarbose fermentation stock solution was measured with a measuring cylinder and poured into a 500 mL beaker, 2 g of diatomaceous earth (1% g / mL of the fermentation stock solution by volume) was added, and the mixture was stirred evenly. Solid oxalic acid was added to the beaker to adjust the pH to 3.0, and the mixture was stirred for 15 min. A 1 mol / L sodium hydroxide solution was then added to adjust the pH back to 4.3, and 20 mL of freshly prepared 0.05% CPAM solution was added to the fermentation liquid, and the mixture was stirred slowly until obvious flocculation occurred. 1 g of diatomaceous earth (0.5% g / mL of the fermentation stock solution by volume) was added, and the mixture was stirred until the flocs were slightly dispersed. The pretreated fermentation liquid was poured into a funnel equipped with filter paper for natural filtration, and the filtration rate was measured to be 0.9 mL / min.

[0063] Example 4

[0064] 200 mL of acarbose fermentation stock solution was measured with a measuring cylinder and poured into a 500 mL beaker, 2 g of diatomaceous earth (1% g / mL of the fermentation stock solution by volume) was added, and the mixture was stirred evenly. Solid oxalic acid was added into the beaker to adjust the pH to 3.0, and the mixture was stirred for 15 min. Then, 1 mol / L sodium hydroxide solution was added to adjust the pH to 4.3. Then, 20 mL of freshly prepared 0.05% CPAM solution was added into the fermentation liquid, and the mixture was stirred slowly until obvious flocculation occurred. Then, 1 g of perlite (0.5% g / mL of the fermentation stock solution by volume) was added, and the mixture was stirred until the flocs were slightly dispersed. The pretreated fermentation liquid was poured into a funnel equipped with filter paper for natural filtration, and the filtration rate was measured to be 2.0 mL / min, and the color of the filtrate was light brown.

[0065] Example 5

[0066] Use a measuring cylinder to measure 200 mL of acarbose fermentation stock solution and pour it into a 500 mL beaker, add 2 g of perlite, stir evenly, add solid oxalic acid to the beaker to adjust the pH to 3.0, stir and react for 15 minutes, then add 1 mol / L sodium hydroxide solution to adjust the pH back to 4.3, then add 20 mL of freshly prepared 0.05% CPAM solution to the fermentation liquid, stir slowly until flocculation is obvious, then add 1 g of perlite, stir until the flocculants are slightly dispersed, pour the pretreated fermentation liquid into a funnel equipped with filter paper for natural filtration, and measure the filtration rate to be 1.0 mL / min.

[0067] Example 6

[0068] Use a measuring cylinder to measure 200 mL of acarbose fermentation stock solution and pour it into a 500 mL beaker, add 2 g of perlite, stir evenly, add solid oxalic acid to the beaker to adjust the pH to 3.0, stir and react for 15 minutes, then add 1 mol / L sodium hydroxide solution to adjust the pH back to 4.3, then add 20 mL of freshly prepared 0.05% CPAM solution to the fermentation liquid, stir slowly until flocculation is obvious, then add 1 g of diatomaceous earth, stir until the floccules are slightly dispersed, pour the pretreated fermentation liquid into a funnel equipped with filter paper for natural filtration, and measure the filtration rate to be 1.3 mL / min.

[0069] Example 7

[0070] 200 mL of acarbose fermentation stock solution was measured with a measuring cylinder, 2 g of diatomaceous earth (1% g / mL of the fermentation stock solution by volume) was added, and the mixture was stirred evenly. Solid oxalic acid was added to a beaker to adjust the pH to 3.0, and the mixture was stirred for 15 min. A 1 mol / L sodium hydroxide solution was then added to adjust the pH to 4.3. 16 mL of a freshly prepared 0.05% CPAM solution was then added to the fermentation liquid, and the mixture was stirred slowly until obvious flocculation occurred. 1 g of perlite (0.5% g / mL of the fermentation stock solution by volume) was added, and the mixture was stirred until the flocs were slightly dispersed. The pretreated fermentation liquid was poured into a funnel equipped with filter paper for natural filtration. The filtration rate was measured to be 2.1 mL / min, and the color of the filtrate turned light brown when observed with the naked eye.

[0071] Example 8

[0072] The type of acidifier in Example 7 was changed to hydrochloric acid, and other parameters remained unchanged. The filtration rate was measured to be 0.3 mL / min, and the color of the filtrate was observed to be dark brown by naked eyes.

[0073] Example 9

[0074] The type of acidifier in Example 7 was changed to sulfuric acid, and other parameters remained unchanged. The filtration rate was measured to be 1.2 mL / min, and the color of the filtrate was observed to be dark brown by naked eyes.

[0075] Example 10

[0076] The type of acidifier in Example 7 was changed to phosphoric acid, and other parameters remained unchanged. The filtration rate was measured to be 0.8 mL / min, and the color of the filtrate was observed to be brown by naked eyes.

[0077] Embodiment 11

[0078] Use a measuring cylinder to measure 200 mL of acarbose fermentation stock solution, add 2 g of diatomaceous earth, stir evenly, add solid oxalic acid to the beaker to adjust the pH to 2.5, stir and react for 15 minutes, then add 1 mol / L sodium hydroxide solution to adjust the pH back to 4.5, then add 10 mL of freshly prepared 0.05% CPAM solution to the fermentation liquid, stir slowly until flocculation is obvious, then add 1 g of perlite, stir until the flocculants are slightly dispersed, pour the pretreated fermentation liquid into a funnel equipped with filter paper for natural filtration, and measure the filtration rate to be 1.4 mL / min.

[0079] Example 12

[0080] The amount of solid oxalic acid added in Example 11 was changed so that the pH value was adjusted to 3.0. Other parameters remained unchanged and the filtration rate was measured to be 1.6 mL / min.

[0081] Embodiment 13

[0082] The amount of solid oxalic acid added in Example 11 was changed so that the pH value was adjusted to 3.5. Other parameters remained unchanged and the filtration rate was measured to be 1.1 mL / min.

[0083] Embodiment 14

[0084] The amount of solid oxalic acid added in Example 11 was changed so that the pH value was adjusted to 4.0. Other parameters remained unchanged and the filtration rate was measured to be 0.5 mL / min.

[0085] Embodiment 15

[0086] 200 mL of acarbose fermentation broth was measured with a measuring cylinder, 2 g of diatomaceous earth was added, and the mixture was stirred evenly. Solid oxalic acid was added to a beaker to adjust the pH to 3.0. After stirring for 15 min, 1 mol / L sodium hydroxide solution was added to adjust the pH back to 3.8. 20 mL of the freshly prepared 0.05% CPAM solution was added to the fermentation broth, and the mixture was stirred slowly until flocculation was obviously observed. Then 1 g of perlite was added and the mixture was stirred until the flocculation was slightly dispersed. The pretreated fermentation broth was poured into a funnel equipped with filter paper for natural filtration, and the filtration rate was measured to be 1.2 mL / min.

[0087] Example 16

[0088] 200 mL of acarbose fermentation broth was measured with a measuring cylinder, 2 g of diatomaceous earth was added, and the mixture was stirred evenly. Solid oxalic acid was added to a beaker to adjust the pH to 3.0. After stirring for 15 min, 1 mol / L sodium hydroxide solution was added to adjust the pH back to 4.8. 20 mL of freshly prepared 0.05% CPAM solution was added to the fermentation broth, and the mixture was stirred slowly until obvious flocculation occurred. Then 1 g of perlite was added and the mixture was stirred until the floccules were slightly dispersed. The pretreated fermentation broth was poured into a funnel equipped with filter paper for natural filtration, and the filtration rate was measured to be 1.1 mL / min.

[0089] Embodiment 17

[0090] 200 mL of acarbose fermentation broth was measured with a measuring cylinder, 2 g of diatomaceous earth was added, and the mixture was stirred evenly. Solid oxalic acid was added to a beaker to adjust the pH to 3.0. After stirring for 15 min, 1 mol / L sodium hydroxide solution was added to adjust the pH back to 5.3. 20 mL of the freshly prepared 0.05% CPAM solution was added to the fermentation broth, and the mixture was stirred slowly until flocculation was obviously observed. Then 1 g of perlite was added and the mixture was stirred until the flocculation was slightly dispersed. The pretreated fermentation broth was poured into a funnel equipped with filter paper for natural filtration, and the filtration rate was measured to be 0.6 mL / min.

[0091] Embodiment 18

[0092] Use a measuring cylinder to take 200 mL of acarbose fermentation broth, add 2 g of diatomaceous earth, stir evenly, add solid oxalic acid to the beaker to adjust the pH to 3.0, stir and react for 15 minutes, and then add 1 mol / L sodium hydroxide solution to adjust the pH back to 4.3; add 4 mL of freshly prepared 0.2% aluminum sulfate solution to the fermentation broth, add 1 g of perlite, stir until the flocs are slightly dispersed, pour the pretreated fermentation broth into a funnel equipped with filter paper for natural filtration, and measure the filtration rate to be 0.2 mL / min.

[0093] Embodiment 19

[0094] Use a measuring cylinder to take 200 mL of acarbose fermentation broth, add 2 g of diatomaceous earth, stir evenly, add solid oxalic acid to the beaker to adjust the pH to 3.0, stir and react for 15 minutes, and then add 1 mol / L sodium hydroxide solution to adjust the pH back to 4.3; add 4 mL of freshly prepared 0.2% ferric chloride solution to the fermentation broth, add 1 g of perlite, stir until the flocs are slightly dispersed, pour the pretreated fermentation broth into a funnel equipped with filter paper for natural filtration, and measure the filtration rate to be 0.1 mL / min.

[0095] Embodiment 20

[0096] Use a measuring cylinder to take 200 mL of acarbose fermentation broth, add 2 g of diatomaceous earth, stir evenly, add solid oxalic acid to the beaker to adjust the pH to 3.0, stir and react for 15 minutes, and then add 1 mol / L sodium hydroxide solution to adjust the pH back to 4.3; add 4 mL of freshly prepared 0.2% polyaluminium chloride solution to the fermentation broth, add 1 g of perlite, stir until the flocs are slightly dispersed, pour the pretreated fermentation broth into a funnel equipped with filter paper for natural filtration, and measure the filtration rate to be 0.2 mL / min.

[0097] Embodiment 21

[0098] Use a measuring cylinder to take 200 mL of acarbose fermentation broth, add 2 g of diatomaceous earth, stir evenly, add solid oxalic acid to the beaker to adjust the pH to 3.0, stir and react for 15 minutes, and then add 1 mol / L sodium hydroxide solution to adjust the pH back to 4.3; add 4 mL of freshly prepared 0.2% polyaluminum ferric chloride solution to the fermentation broth, add 1 g of perlite, stir until the flocs are slightly dispersed, pour the pretreated fermentation broth into a funnel equipped with filter paper for natural filtration, and measure the filtration rate to be 0.4 mL / min.

[0099] Embodiment 22

[0100] Use a measuring cylinder to take 200 mL of acarbose fermentation broth, add 2 g of diatomaceous earth, stir evenly, add solid oxalic acid to the beaker to adjust the pH to 3.0, stir and react for 15 minutes, and then add 1 mol / L sodium hydroxide solution to adjust the pH back to 4.3; add 16 mL of freshly prepared 0.05% polyammonium chloride solution to the fermentation broth, add 1 g of perlite, stir until the flocs are slightly dispersed, pour the pretreated fermentation broth into a funnel equipped with filter paper for natural filtration, and the filtration rate is measured to be 0.9 mL / min.

[0101] Embodiment 23

[0102] Use a measuring cylinder to take 200 mL of acarbose fermentation broth, add 2 g of diatomaceous earth, stir evenly, add solid oxalic acid to the beaker to adjust the pH to 3.0, stir and react for 15 minutes, and then add 1 mol / L sodium hydroxide solution to adjust the pH back to 4.3; add 16 mL of freshly prepared 0.05% anionic polyacrylamide solution to the fermentation broth, add 1 g of perlite, stir until the flocculants are slightly dispersed, pour the pretreated fermentation broth into a funnel equipped with filter paper for natural filtration, and measure the filtration rate to be 0.1 mL / min.

[0103] Embodiment 24

[0104] Use a measuring cylinder to take 200 mL of acarbose fermentation broth, add 2 g of diatomaceous earth, stir evenly, add solid oxalic acid to the beaker to adjust the pH to 3.0, stir and react for 15 minutes, and then add 1 mol / L sodium hydroxide solution to adjust the pH back to 4.3; add 16 mL of freshly prepared 0.05% non-ionic polyacrylamide solution to the fermentation broth, add 1 g of perlite, stir until the flocculants are slightly dispersed, pour the pretreated fermentation broth into a funnel equipped with filter paper for natural filtration, and measure the filtration rate to be 0.1 mL / min.

[0105] Embodiment 25

[0106] Use a measuring cylinder to take 200 mL of acarbose fermentation stock solution and pour it into a beaker, add 2 g of diatomaceous earth and stir evenly, add solid oxalic acid to the beaker to adjust the pH to 3.0, stir and react for 15 minutes, add 1 g of perlite and stir evenly, pour the pretreated fermentation liquid into a funnel equipped with filter paper for natural filtration, the filtration rate is measured to be 0.5 mL / min, and the color of the filtrate is light brown when observed with the naked eye.

[0107] Embodiment 26

[0108] Use a measuring cylinder to take 200 mL of acarbose fermentation stock solution and pour it into a beaker, add 2 g of diatomaceous earth, stir evenly, add 20 mL of freshly prepared 0.05% CPAM solution into the beaker, stir slowly until flocculation is obvious, add 1 g of perlite, stir until the flocculants are slightly dispersed, pour the pretreated fermentation liquid into a funnel equipped with filter paper for natural filtration, the filtration rate is measured to be 0.9 mL / min, and the color of the filtrate is dark brown when observed with the naked eye.

[0109] Embodiment 27

[0110] Use a measuring cylinder to take 200 mL of acarbose fermentation stock solution and pour it into a beaker, add 2 g of diatomaceous earth, stir evenly, add 20 mL of freshly prepared 0.05% CPAM solution to the fermentation liquid, stir slowly until flocculation is obvious, add solid oxalic acid to the beaker to adjust the pH to 3.0, stir and react for 15 minutes, then add 1 g of perlite, stir evenly, pour the pretreated fermentation liquid into a funnel equipped with filter paper for natural filtration, the filtration rate is measured to be 1.1 mL / min, and the color of the filtrate is light brown when observed with the naked eye.

[0111] The test results show that the combination of acidification and flocculation process can not only accelerate the solid-liquid separation of the fermentation broth, but also has a partial decolorization effect, which reduces the pressure on the subsequent treatment of the fermentation broth. Among them, the best effect is to acidify the fermentation broth first and then use a flocculant.

[0112] Embodiment 28

[0113] Use a measuring cylinder to take 200 mL of acarbose fermentation broth, add 2 g of diatomaceous earth, stir evenly, add solid oxalic acid to the beaker to adjust the pH to 3.0, stir and react for 15 minutes, and then add 1 mol / L sodium hydroxide solution to adjust the pH back to 4.3; add 4 mL of freshly prepared 0.05% CPAM solution to the fermentation broth, add 1 g of perlite, stir until the flocs are slightly dispersed, pour the pretreated fermentation broth into a funnel equipped with filter paper for natural filtration, and the filtration rate is measured to be 0.5 mL / min.

[0114] Embodiment 29

[0115] The amount of CPAM added in Example 28 was changed to 10 mL, and other condition parameters remained unchanged. Then, the pretreated fermentation broth was poured into a funnel filled with filter paper for natural filtration, and the filtration rate was measured to be 1.1 mL / min.

[0116] Embodiment 30

[0117] The amount of CPAM added in Example 28 was changed to 30 mL, and other condition parameters remained unchanged. Then, the pretreated fermentation liquid was poured into a funnel filled with filter paper for natural filtration, and the filtration rate was measured to be 1.2 mL / min.

[0118] Embodiment 31

[0119] The amount of CPAM added in Example 28 was changed to 40 mL, and other parameters remained unchanged. The pretreated fermentation broth was then poured into a funnel filled with filter paper for natural filtration, and the filtration rate was measured to be 0.7 mL / min.

[0120] Embodiment 32

[0121] CPAM particles with different ionicity from the above embodiment are selected to carry out the following pretreatment process.

[0122] Use a measuring cylinder to measure 200 mL of acarbose fermentation stock solution and pour it into a beaker, add 2 g of diatomaceous earth, stir evenly, add solid oxalic acid to the beaker to adjust the pH to 3.0, stir and react for 15 minutes, then add 1 mol / L sodium hydroxide solution to adjust the pH back to 4.3, then add 20 mL of freshly prepared 0.05% CPAM solution (CPAM particles with a solid content of 95%, a molecular weight of 10 million, a particle size of 100 mesh, but an ionicity of 30%) to the fermentation liquid, stir slowly until flocculation is obvious, then add 1 g of perlite, stir until the flocculants are slightly dispersed, pour the pretreated fermentation liquid into a funnel equipped with filter paper for natural filtration, and measure the filtration rate to be 0.7 mL / min.

[0123] Embodiment 33

[0124] The ionicity of the CPAM particles in Example 32 was changed to 40%, and other parameters remained unchanged. The filtration rate was measured to be 1.0 mL / min.

[0125] Embodiment 34

[0126] The ionicity of the CPAM particles in Example 32 was changed to 60%, and other parameters remained unchanged. The filtration rate was measured to be 1.5 mL / min.

[0127] Embodiment 35

[0128] Preparation method of 0.05% CPAM solution:

[0129] 5g of CPAM particles with a solid content of 95%, a molecular weight of 10 million, an ionicity of 50%, and a particle size of 100 mesh were selected and divided into 5 groups for testing. Four of the groups were respectively wetted with 1mL of methanol (reagent grade), 3mL of methanol (reagent grade), 1mL of ethanol (reagent grade), and 3mL of ethanol (reagent grade) to wet the CPAM particles for 15 minutes, and then were evenly and slowly sprinkled into 995g of water at 45°C in a stirring state, and the temperature was kept constant and stirred at a speed of 300rpm until completely dissolved, and the time required for dissolution was recorded; the fifth group of CPAM particles were not wetted, but were directly and evenly and slowly sprinkled into 45°C water in a stirring state, and the temperature was kept constant and stirred at a speed of 300rpm until completely dissolved, and the time required for dissolution was recorded. The results are as follows:

[0130] Table 1

[0131]

[0132] The experiment shows that wetting the CPAM solution with organic solvents such as methanol and ethanol first can speed up the dissolution of CPAM particles. Compared with the time-consuming direct dissolution of CPAM particles without wetting, the dissolution time of CPAM particles can be shortened by 44%-63%.

[0133] Embodiment 36

[0134] Measure 20L of acarbose fermentation liquid and pour it into a beaker, add 200g of diatomaceous earth, stir evenly, add 200g of solid oxalic acid to the beaker to adjust the pH to 2.8, stir and react for 20min, then add 1mol / L sodium hydroxide solution to adjust the pH back to 4.0, then add 1L of freshly prepared 0.1% CPAM solution to the fermentation liquid, stir slowly until flocculation is obvious, add 100g of perlite, stir until the floccules are slightly dispersed, and filter the pretreated fermentation liquid with a plate and frame filter press. The measured filtration rate is 21L / m 2 / h, the filter cake was observed to be formed and could be squeezed dry, and the color of the filtrate changed from the initial dark brown to light brown.

[0135] The method for preparing 1L of 0.1% CPAM solution is as follows:

[0136] Weigh 1 g of CPAM particles with a solid content of 95%, a molecular weight of 9 million, an ionicity of 50%, and a particle size of 100 mesh, moisten the CPAM particles with 3 mL of methanol for 25 minutes, and then slowly and evenly sprinkle them into 45°C water in a stirring state. Keep the temperature constant and stir at 400 rpm for 40 minutes until they are completely dissolved.

[0137] In the present invention, the acarbose fermentation stock solution can be a general fermentation solution, i.e., a fermentation solution with a bacterial concentration greater than 50% and a viscosity greater than 800 mPa.S; or a fermentation solution with a lower bacterial concentration and viscosity, i.e., a fermentation solution with a bacterial concentration less than 40% and a viscosity less than 650 mPa.S.

[0138] Simple replacement or substitution of the acarbose pretreatment process steps in the present invention all fall within the scope of the present invention.

Claims

1. A pretreatment process for acarbose fermentation broth, comprising the following steps: a. Add filter aid A to the acarbose fermentation solution and stir to obtain a mixed fermentation solution; b. Adding an acidifier to the mixed fermentation broth to adjust the pH, stirring the reaction to obtain an acidified fermentation broth; c. The acidified fermentation broth is adjusted to pH with an alkaline substance; d. Adding a flocculant to the fermentation broth after adjusting the pH above; e. Add filter aid B to the fermentation broth after flocculation, solid-liquid separation; Filter aid A is selected from diatomaceous earth, and filter aid B is selected from perlite; The added mass of filter aid A or B is 0.5-1.0% g / mL based on the volume of the fermentation stock solution; Step b, the pH range is 2.5-3.0; Step c, adjusting the pH range to 4.3-4.5; Step d, the flocculant is cationic polyacrylamide; The acidulant is oxalic acid; The flocculant is added in the form of an aqueous solution; The flocculant aqueous solution preparation process is: 1) Wetting the cationic polyacrylamide particles with an organic solvent; 2) Add the wetted particles into water and stir at 40-55°C until completely dissolved; The organic solvent is selected from methanol and ethanol; calculated by volume ratio, the amount of the organic solvent is 0.1-0.2% of the solution preparation amount; The wetting time is 15-20 minutes, the dissolving temperature is 45-50°C, and the stirring speed is 200-400 rpm.

2. The pretreatment process according to claim 1, characterized in that: In step c, the alkaline substance is added in the form of an aqueous solution, and the alkaline substance is sodium hydroxide or ammonia water.

3. The pretreatment process according to claim 2, characterized in that: The cationic polyacrylamide is selected from particles with a solid content of 95%, a molecular weight of 10 million, an ionicity of 50%, and a particle size of 100 meshes.

4. The pretreatment process according to claim 3, characterized in that: The concentration of the flocculant aqueous solution is 0.05%-0.06%; the added volume is 8-10% of the volume of the fermentation stock solution.

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