WO2023280257A1 - 一种阿卡波糖发酵液的预处理工艺 - Google Patents

一种阿卡波糖发酵液的预处理工艺 Download PDF

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WO2023280257A1
WO2023280257A1 PCT/CN2022/104300 CN2022104300W WO2023280257A1 WO 2023280257 A1 WO2023280257 A1 WO 2023280257A1 CN 2022104300 W CN2022104300 W CN 2022104300W WO 2023280257 A1 WO2023280257 A1 WO 2023280257A1
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add
solution
acarbose
filter aid
pretreatment process
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PCT/CN2022/104300
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English (en)
French (fr)
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张亮亮
徐亚强
何志勇
肖楠
王子宝
李娜
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杭州中美华东制药江东有限公司
杭州中美华东制药有限公司
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Publication of WO2023280257A1 publication Critical patent/WO2023280257A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C07H1/08—Separation; Purification from natural products
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20—Carbocyclic rings
    • C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/045—Actinoplanes

Definitions

  • the invention relates to a treatment process for microbial fermentation products, in particular to a pretreatment process for acarbose fermentation liquid.
  • Acarbose (Acarbose) is a kind of ⁇ -glucosidase inhibitor developed by German Bayer company in the mid-1970s, because it can effectively inhibit the activity of ⁇ -glucosidase in small intestinal wall cells, and delay intestinal reaction to oligosaccharides, The degradation and absorption of disaccharides or polysaccharides has good pharmacokinetic properties and low toxicity, and is widely used in the treatment of type II diabetes clinically.
  • Acarbose Due to the complex chemical structure of acarbose, 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 the research on its anabolic pathway and fermentation process in microorganisms has always been a research hotspot of acarbose.
  • the existing pretreatment method is generally Add aluminum sulfate to coagulate, adjust the pH to 5.5-6.5, and then filter with a plate and frame filter press. The existing pretreatment method cannot normally complete the solid-liquid separation to obtain the filtrate.
  • CN109580317A discloses a pretreatment method for acarbose fermented liquid.
  • the acarbose fermented liquid is taken for the first time under the centrifugal force of 3000-5000g, and the alcoholic solution of trichloroacetic acid whose volume is 2-4 times that of the supernatant is taken and mixed with The supernatant was mixed, centrifuged twice, and the supernatant was filtered through a membrane.
  • This method uses the harmful carcinogenic reagent trichloroacetic acid, and the cost is high, so it is only suitable for laboratory research and not suitable for large-scale industrial production.
  • An object of the present invention is to overcome the problem that the existing pretreatment method cannot realize the solid-liquid separation of the bacterial concentration less than 40% and the viscosity less than 650mPa. craft.
  • the process includes the following steps:
  • a kind of pretreatment process of acarbose fermented liquid can also be:
  • the filter aids A or B are each independently selected from diatomite, perlite, coarse river sand, volcanic rock, preferably diatomite and perlite;
  • the filter aid A is preferably different from the filter aid B, and it is further preferred that the filter aid A is selected from diatomaceous earth, and the filter aid B is selected from perlite;
  • 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%.
  • the acidifying agent is oxalic acid, sulfuric acid, preferably oxalic acid.
  • the pH range of adding an acidifying agent to adjust the pH in the above process is 2.5-3.5, preferably 2.5-3.0.
  • the alkali in step (c) is an inorganic alkali solution, such as sodium hydroxide and ammonia solution; the adjusted pH range is 3.8-4.8, preferably 4.3-4.5.
  • 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 fermentation stock solution volume, preferably 8-10%.
  • 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, and an ionicity of 40-60%. Particles with a size of 80-100 mesh; more preferably 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.
  • CPAM cationic polyacrylamide
  • Another object of the present invention is to provide a kind of preparation method of CPAM solution, it comprises the following steps:
  • the organic solvent is alcohols, such as methanol and ethanol.
  • the amount of solvent used for wetting the CPAM particles is 0.1-0.3%, preferably 0.1-0.2% of the solution preparation amount; the wetting time is 10-30min, preferably 15-20min.
  • the dissolution temperature of the above CPAM is 40-55° C., preferably 45-50° C.; the stirring speed is 200-400 rpm.
  • the acarbose fermentation stock solution in the present invention is obtained by fermenting and culturing the acarbose-producing bacterium Actinomyces uthaensis.
  • the acarbose fermentation stoste is prepared as follows:
  • the culture temperature is 26-30°C.
  • the ingredients of the above shake flask seed medium per 100mL medium, carbon source 1.2-3.0g, nitrogen source 2.5-4.0g, inorganic salt 0.2-0.6g, and the rest is water;
  • the proportion of each component of the seed tank medium in the medium is: per 100mL of medium, carbon source 2.0-3.5g, nitrogen source 3.5-6.0g, inorganic salt 0.2-0.6g, and the rest is water;
  • the proportion of each component of the fermentation medium in the medium is: per 100mL of the medium, 5-10g of carbon source, 1.0-3.5g of nitrogen source, 0.2-2.0g of inorganic salt, the rest is water, and the pH value is 6.5-7.5.
  • the invention provides a pretreatment process of acarbose fermentation liquid, which can solve the problem of difficult solid-liquid separation of acarbose fermentation liquid by adding filter aid twice, adjusting pH with acid and alkali, and then using CPAM flocculation treatment Problems, especially the problem of difficult solid-liquid separation of acarbose fermentation liquid with a bacterial concentration of less than 40% and a viscosity of less than 650mPa.S, also increases the effect of partial decolorization, reduces the pressure of subsequent purification processes, and is conducive to improving product yield .
  • the invention also provides a preparation method of the CPAM solution, which greatly shortens the dissolution time of the CPAM particles by pre-wetting with methanol, an organic alcohol.
  • the acarbose-producing bacterium adopted in the acarbose fermentation stoste in the examples is Actinomycetes utahensis MYIH97 (Actinoplanes utahensis MYIH97) preserved in Wuhan China Type Culture Collection Center (CCTCC for short), and the preservation number is CCTCC, NO : M2016237, the fermentation broth preparation method is as follows:
  • shake flask seed medium formula is: 20g/L of soybean powder, 10g/L of glycerin, 40g/L of cornstarch, 3g/L of calcium carbonate, and the rest is water.
  • the formula of seed tank culture medium is: soybean powder 20g/L, glycerin 10g/L, cornstarch 40g/L, calcium carbonate 3g/L and bubble enemy 0.5g/L, the rest is water, and the pH value before the culture medium is sterilized is adjusted. to 6.8-7.0.
  • the tank pressure is 0.05Mpa
  • the ventilation rate is 1:1-1:1.5
  • the stirring is 120rpm-200rpm
  • the culture is 168h at 26-30°C.
  • the formula of the fermentation tank medium is: glucose 40g/L, maltose 20g/L, soybean powder 20g/L, sodium glutamate 3g/L, calcium carbonate 2.5g/L, dipotassium hydrogen phosphate 1.5g/L, chloride Iron 0.5g/L, calcium chloride 2g/L and foam enemy 0.5g/L, the rest is water, and the pH value of the medium is adjusted to 6.8-7.0 before sterilization.
  • CPAM particles with a solid content of 95%, molecular weight of 10 million, ionicity of 50%, and a particle size of 100 mesh, wet the above CPAM particles with 3mL of methanol for 20min, and then evenly and slowly sprinkle them into 45°C water in a stirring state. and stirred at 400 rpm until completely dissolved to obtain a 0.05% CPAM solution.
  • CPAM solution used in the following examples is the prepared solution unless otherwise specified.
  • a measuring cylinder to measure 200mL of acarbose fermentation stock solution and pour it into a 500mL beaker, add solid oxalic acid to the beaker to adjust the pH to 3.0, stir for 15 minutes, then add 1mol/L sodium hydroxide solution to adjust the pH to 4.3, and then add to the fermentation
  • Add 20mL of freshly prepared 0.05% CPAM solution to the solution stir at a slow speed until flocculation is evident, then add 3g of diatomaceous earth, stir until the flocs are slightly dispersed, and pour the pretreated fermentation broth into a funnel equipped with filter paper Natural filtration, the measured filtration rate is 0.7mL/min.
  • a measuring cylinder to measure 200mL of acarbose fermentation stock solution and pour it into a 500mL beaker, add 2g of perlite, stir evenly, add solid oxalic acid to the beaker to adjust the pH to 3.0, stir for 15min, then add 1mol/L sodium hydroxide solution to Return the pH to 4.3, then add 20mL of the freshly prepared 0.05% CPAM solution to the fermentation broth, stir at a slow speed until flocculation is evident, then add 1g of perlite, stir until the flocs are slightly dispersed, pour the pretreated fermentation broth Put it into a funnel equipped with filter paper and filter naturally, and the measured filtration rate is 1.0mL/min.
  • a measuring cylinder to measure 200mL of acarbose fermentation stock solution and pour it into a 500mL beaker, add 2g of perlite, stir evenly, add solid oxalic acid to the beaker to adjust the pH to 3.0, stir for 15min, then add 1mol/L sodium hydroxide solution to Adjust the pH to 4.3, then add 20mL of the freshly prepared 0.05% CPAM solution to the fermentation broth, stir slowly until the flocculation phenomenon is obvious, then add 1g of diatomaceous earth, stir until the flocs are slightly dispersed, and the pretreated fermentation broth Pour into a funnel equipped with filter paper and filter naturally, and the measured filtration rate is 1.3mL/min.
  • Example 7 Change the kind of acidifying agent in Example 7 to be hydrochloric acid, and other condition parameters are constant, and the measured filtration rate is 0.3mL/min, and the color of the filtrate observed by naked eyes is dark brown.
  • Example 11 The amount of solid oxalic acid added in Example 11 was changed so that the adjusted pH value was 3.0, and other condition parameters were unchanged, and the measured filtration rate was 1.6mL/min.
  • Example 11 The amount of solid oxalic acid added in Example 11 was changed so that the adjusted pH value was 3.5, and other conditional parameters were unchanged, and the measured filtration rate was 1.1 mL/min.
  • Example 11 The amount of solid oxalic acid added in Example 11 was changed so that the adjusted pH value was 4.0, and other condition parameters were unchanged, and the measured filtration rate was 0.5mL/min.
  • a measuring cylinder to take 200mL of acarbose fermentation stock solution and pour it into a beaker, add 2g of diatomaceous earth, stir well, add solid oxalic acid to the beaker to adjust the pH to 3.0, stir for 15min, add 1g of perlite, stir well, and pretreat The final fermentation broth was poured into a funnel equipped with filter paper and filtered naturally, the measured filtration rate was 0.5mL/min, and the color of the filtrate was light brown by visual observation.
  • a measuring cylinder to take 200mL of acarbose fermentation stock solution into a beaker, add 2g of diatomaceous earth, stir evenly, add 20mL of 0.05% CPAM solution to the beaker, stir at a slow speed until flocculation is obvious, add 1g of perlite , stir until the flocs are slightly dispersed, pour the pretreated fermentation broth into a funnel equipped with filter paper and filter naturally, the measured filtration rate is 0.9mL/min, and the color of the filtrate is dark brown by visual observation.
  • a measuring cylinder to take 200mL of acarbose fermentation stock solution into a beaker, add 2g of diatomaceous earth, stir evenly, add 20mL of 0.05% CPAM solution to the fermentation broth, stir at a slow speed until flocculation is obvious, and pour into the beaker
  • Add solid oxalic acid to adjust the pH to 3.0, stir and react for 15 minutes, then add 1g of perlite, stir evenly, pour the pretreated fermentation broth into a funnel equipped with filter paper and filter naturally, the measured filtration rate is 1.1mL/min, observe with the naked eye The filtrate was light brown in color.
  • test results prove that the combination of acidification and flocculation process can not only accelerate the solid-liquid separation of fermentation broth, but also have a partial decolorization effect, which reduces the pressure for the subsequent treatment of fermentation broth.
  • the best effect is to acidify the fermentation broth first and then use flocculant.
  • Example 28 Change the amount of CPAM added in Example 28 to 10mL, and keep other conditional parameters unchanged, then pour the pretreated fermentation broth into a funnel equipped with filter paper and filter naturally, and the measured filtration rate is 1.1mL/min.
  • Example 28 Change the amount of CPAM added in Example 28 to 30mL, and keep other conditional parameters unchanged, then pour the pretreated fermentation broth into a funnel equipped with filter paper and filter naturally, and the measured filtration rate is 1.2mL/min.
  • Example 28 Change the amount of CPAM added in Example 28 to 40mL, and keep other conditional parameters unchanged, then pour the pretreated fermentation broth into a funnel equipped with filter paper and filter naturally, and the measured filtration rate is 0.7mL/min.
  • CPAM particles with different ion degrees from the above-mentioned examples were selected for the following pretreatment process.
  • the ionicity of the CPAM particles in Example 32 was changed to 40%, the other condition parameters were unchanged, and the measured filtration rate was 1.0 mL/min.
  • the ionicity of the CPAM particles in Example 32 was changed to 60%, the other condition parameters were unchanged, and the measured filtration rate was 1.5mL/min.
  • the test shows that in the preparation of CPAM solution, wetting with organic solvent methanol and ethanol first can accelerate 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%.
  • CPAM particles with a solid content of 95%, molecular weight of 9 million, ionicity of 50%, and a particle size of 100 mesh.
  • the acarbose fermentation stock solution can be a general fermentation liquid, that is, the bacterial concentration is greater than 50%, and the viscosity is greater than 800mPa.S; it can also be a fermentation liquid with a bacterial concentration and viscosity that is less than 40 %, a fermentation broth with a viscosity less than 650mPa.S.
  • the simple replacement and replacement of the acarbose pretreatment process steps in the present invention all belong to the category of the present invention.

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Abstract

本发明属于微生物发酵领域，具体地，提供了一种阿卡波糖发酵液的预处理工艺，通过先后分两次添加助滤剂、酸碱调节pH后使用CPAM絮凝处理，可以解决阿卡波糖发酵液固液分离困难的问题，尤其是菌浓小于40％、粘度小于650mPa.S的阿卡波糖发酵液固液分离困难的问题，还增加了部分脱色的效果，减轻了后续纯化工艺压力，有利于提高产品得率。本发明还提供了一种CPAM溶液的配制方法，通过使用有机醇类如甲醇润湿，大大缩短了CPAM颗粒溶解的时间。

Description

一种阿卡波糖发酵液的预处理工艺 技术领域
本发明涉及微生物发酵产品的处理工艺，特别地涉及一种阿卡波糖发酵液的预处理工艺。
背景技术
阿卡波糖(Acarbose)是德国Bayer公司于上世纪70年代中期研制开发的一种α-葡萄糖苷酶抑制剂，因其能够有效抑制小肠壁细胞α糖苷酶活性，延缓肠道对寡糖、双糖或多糖的降解和吸收，且具有良好的药动学性质和低毒性质，临床上被广泛用于II型糖尿病的治疗。
由于阿卡波糖的化学结构比较复杂，其化学合成步骤相对繁琐且成本高昂，目前工业规模的生产主要是通过微生物发酵获得的。阿卡波糖可从由犹他游动放线菌(Actinoplanes sp.)的发酵液中提取得到，其在微生物体内的合成代谢路径以及发酵工艺研究一直是阿卡波糖的研究热点。实验研究发现采用阿卡波糖产生菌犹他游动放线菌MYIH97(Actinoplanes utahensis MYIH97)发酵培养得到的发酵液与之前阿卡波糖发酵液性质特征差异较大，具体表现在菌浓和粘度上，之前阿卡波糖发酵液的菌浓大于50％，粘度大于800mPa.S，而采用此菌种得到的发酵液菌浓小于40％，粘度小于650mPa.S，现有的预处理方法一般是加硫酸铝凝聚，调至pH 5.5～6.5，再用板框压滤机过滤，采用现有的预处理方法已经无法正常完成固液分离得到滤液。
CN109580317A公开了一种阿卡波糖发酵液的前处理方法，取阿卡波糖发酵液于3000～5000g的离心力下初次离心，取上清液体积2～4倍的三氯乙酸的醇溶液与上清液混合，二次离心，取上清液过滤膜。该方法使用了有害致癌试剂三氯乙酸，且成本高，只适于实验室研究，不适用大规模工业化生产。
发明内容
本发明的一个目的在于克服现有预处理方法无法实现菌浓小于40％、粘度小于650mPa.S发酵液固液分离的问题，提供了一种适合产业化的阿卡波糖发酵液的预处理工艺。
该工艺包括以下步骤：
(a)在阿卡波糖发酵原液中添加助滤剂A，搅拌均匀得到混合发酵液；
(b)在上述混合发酵液中添加酸化剂调pH，搅拌反应；
(c)将上述酸化后的发酵液用碱溶液回调pH；
(d)在上述加碱回调pH的发酵液中加入现配的絮凝剂，搅拌至明显出现絮凝现象；
(e)在上述絮凝后的发酵液中添加助滤剂B，搅拌至絮凝物稍分散，固液分离。
一种阿卡波糖发酵液的预处理工艺，该工艺步骤还可以是：
(a)在阿卡波糖发酵原液中添加助滤剂A，搅拌均匀得到混合发酵液；
(b1)在上述混合发酵液中加入现配的絮凝剂，搅拌至明显出现絮凝现象；
(c1)在上述絮凝后的发酵液中添加酸化剂调pH，搅拌反应；
(d1)在上述絮凝后的发酵液中添加助滤剂B，固液分离。
具体地，所述助滤剂A或B各自独立选自硅藻土、珍珠岩、粗河砂粒、火山石，优选硅藻土、珍珠岩；
具体地，助滤剂A与助滤剂B优选为不同，进一步优选为助滤剂A选自硅藻土，助滤剂B选自珍珠岩；
所述助滤剂的添加质量为发酵原液体积的0.5-1.5％(g/ml)，优选0.5-1.0％。
具体地，所述酸化剂为草酸、硫酸，优选草酸。
具体地，上述工艺中所述添加酸化剂调pH的pH范围为2.5-3.5，优选2.5-3.0。
具体地，步骤(c)所述碱为无机碱溶液，比如氢氧化钠、氨水溶液；回调pH范围为3.8-4.8，优选为4.3-4.5。
具体地，步骤(d)或(b1)所述絮凝剂以水溶液的形式添加，优选阳离子型聚丙烯酰胺(简写为CPAM)，其中CPAM溶液的浓度为0.05％-0.1％，优选0.05％-0.06％；CPAM溶液的添加量为发酵原液体积的5-15％(V:V)，优选8-10％。
具体地，步骤(d)或(b1)所述絮凝剂选自阳离子型聚丙烯酰胺(简写为CPAM)，优选为固含量大于93％，分子量900-1000万，离子度40-60％，颗粒大小80-100目的颗粒；更优选地为固含量为95％、分子量1000万、离子度50％、粒径100目的CPAM颗粒。
本发明的另一个目的在于提供了一种CPAM溶液的配制方法，其包括以下步骤：
(1)用0.1-0.3％(V:V)溶液配制量的有机溶剂润湿CPAM颗粒；
(2)将所述润湿后的CPAM颗粒均匀缓慢加入搅拌的水中，在40-55℃搅拌至溶解。
具体地，有机溶剂为醇类，例如甲醇、乙醇。
具体地，上述润湿CPAM颗粒的溶剂用量为溶液配制量的0.1-0.3％，优选为0.1-0.2％；润湿时间为10-30min，优选为15-20min。
具体地，上述CPAM的溶解温度为40-55℃，优选为45-50℃；搅拌转速为200-400rpm。
具体地，本发明中阿卡波糖发酵原液为阿卡波糖产生菌犹他游动放线菌经过发酵培养所 得。
作为一种具体实施方式，所述的阿卡波糖发酵原液按如下方法制备：
将菌苔接入摇瓶种子培养基，250rpm，培养2-3天，得摇瓶种子液；
将摇瓶种子液按种子罐培养基体积0.1-0.5％的接种量接种于种子罐，120-200rpm，培养2-3天，得罐种子液；
将罐种子液按发酵体积的6-15％的接种量接种于发酵罐培养基，150-200rpm，发酵培养168h，收集发酵液；
其中培养温度均为26～30℃。
上述摇瓶种子培养基成分：每100mL培养基中，碳源1.2～3.0g，氮源2.5～4.0g，无机盐0.2～0.6g，其余为水；
种子罐培养基各组分在培养基中的比例为：每100mL培养基中，碳源2.0～3.5g，氮源3.5～6.0g，无机盐0.2～0.6g，其余为水；
发酵培养基各组分在培养基中的比例为：每100mL培养基中，碳源5～10g，氮源1.0～3.5g，无机盐0.2～2.0g，其余为水，pH值6.5～7.5。
本发明的有益效果：
本发明提供了一种阿卡波糖发酵液的预处理工艺，通过先后分两次添加助滤剂、酸碱调节pH后使用CPAM絮凝处理，可以解决阿卡波糖发酵液固液分离困难的问题，尤其是菌浓小于40％、粘度小于650mPa.S的阿卡波糖发酵液固液分离困难的问题，还增加了部分脱色的效果，减轻了后续纯化工艺压力，有利于提高产品得率。本发明还提供了一种CPAM溶液的配制方法，通过预先使用有机醇类甲醇润湿，大大缩短了CPAM颗粒溶解的时间。
具体实施方式
下面将结合具体实施方式对本发明的技术方案进行清楚、完整地描述，但是本领域技术人员将会理解，下列所描述的实施例是本发明一部分实施例，而不是全部的实施例，仅用于说明本发明，而不应视为限制本发明的范围。
基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
实施例中阿卡波糖发酵原液采用的阿卡波糖产生菌为犹他游动放线菌MYIH97 (Actinoplanes utahensis MYIH97)保藏于武汉中国典型培养物保藏中心(简称CCTCC)，保藏编号为CCTCC，NO:M2016237，发酵液制备方法如下：
(1)将甘油管冷冻保存的菌液直接接种到摇瓶种子培养基中，温度26～30℃、转速250rpm振荡培养48h；
其中摇瓶种子培养基配方为：黄豆粉20g/L、甘油10g/L、玉米淀粉40g/L、碳酸钙3g/L，其余为水。
(2)将培养好的摇瓶种子液接种至种子罐。通气量1:1～1:1.5，罐压0.05Mpa，搅拌120rpm～200rpm，26～30℃培养48h。
其中种子罐培养基配方为：黄豆粉20g/L、甘油10g/L、玉米淀粉40g/L、碳酸钙3g/L和泡敌0.5g/L，其余为水，培养基灭菌前pH值调至6.8-7.0。
(3)将培养好的罐种子液接入发酵罐，罐压0.05Mpa，通气量1:1～1:1.5，搅拌120rpm～200rpm，26～30℃培养168h。
其中发酵罐培养基配方为：葡萄糖40g/L，麦芽糖20g/L，黄豆粉20g/L，谷氨酸钠3g/L，碳酸钙2.5g/L，磷酸氢二钾1.5g/L，氯化铁0.5g/L，氯化钙2g/L和泡敌0.5g/L，其余为水，培养基灭菌前pH值调至6.8-7.0。
具体实施例中的CPAM溶液的配制方法为：
(1)用0.1-0.3％(V:V)溶液配制量的有机溶剂润湿CPAM颗粒10-30min；
(2)将所述润湿后的CPAM颗粒均匀缓慢加入搅拌的水中，在40-55℃，200-400rpm条件下搅拌至完全溶解。
以配制1L 0.05％的CPAM溶液为例：
称取0.5g固含量为95％、分子量1000万、离子度50％、粒径100目的CPAM颗粒，用3mL甲醇润湿上述CPAM颗粒20min，再均匀缓慢撒入处于搅拌状态的45℃水中，恒温并在400rpm转速下搅拌至完全溶解，得到0.05％的CPAM溶液。
下述实施例中使用的CPAM溶液如无特别说明均为此配制溶液。
实施例1
用量筒量取200mL阿卡波糖发酵原液倒入500mL烧杯中，加入3g硅藻土(发酵原液体积比的1.5％g/mL)，搅拌均匀，向烧杯中添加固体草酸调pH至3.0，搅拌反应15min，再加入1mol/L氢氧化钠溶液将pH回调至4.3，再向发酵液中加入20mL现配的0.05％CPAM溶液，慢速搅拌至明显出现絮凝现象且絮凝物稍分散，将预处理后的发酵液倒入装有滤纸的漏斗中自然过滤，测量滤速为0.7mL/min。
实施例2
用量筒量取200mL阿卡波糖发酵原液倒入500mL烧杯中，向烧杯中添加固体草酸调pH至3.0，搅拌反应15min，再加入1mol/L氢氧化钠溶液将pH回调至4.3，再向发酵液中加入20mL现配的0.05％CPAM溶液，慢速搅拌至明显出现絮凝现象，再添加3g硅藻土，搅拌至絮凝物稍分散，将预处理后的发酵液倒入装有滤纸的漏斗中自然过滤，测量滤速为0.7mL/min。
实施例3
用量筒量取200mL阿卡波糖发酵原液倒入500mL烧杯中，加入2g硅藻土(发酵原液体积比的1％g/mL)，搅拌均匀，向烧杯中添加固体草酸调pH至3.0，搅拌反应15min，再加入1mol/L氢氧化钠溶液将pH回调至4.3，再向发酵液中加入20mL现配的0.05％CPAM溶液，慢速搅拌至明显出现絮凝现象，再添加硅藻土1g(发酵原液体积比的0.5％g/mL)，搅拌至絮凝物稍分散，将预处理后的发酵液倒入装有滤纸的漏斗中自然过滤，测量滤速为0.9mL/min。
实施例4
用量筒量取200mL阿卡波糖发酵原液倒入500mL烧杯中，加入2g硅藻土(发酵原液体积比的1％g/mL)，搅拌均匀，向烧杯中添加固体草酸调pH至3.0，搅拌反应15min，再加入1mol/L氢氧化钠溶液将pH回调至4.3，再向发酵液中加入20mL现配的0.05％CPAM溶液，慢速搅拌至明显出现絮凝现象，再添加珍珠岩1g(发酵原液体积比的0.5％g/mL)，搅拌至絮凝物稍分散，将预处理后的发酵液倒入装有滤纸的漏斗中自然过滤，测量滤速为2.0mL/min，滤液颜色为浅褐色。
实施例5
用量筒量取200mL阿卡波糖发酵原液倒入500mL烧杯中，加入2g珍珠岩，搅拌均匀，向烧杯中添加固体草酸调pH至3.0，搅拌反应15min，再加入1mol/L氢氧化钠溶液将pH回调至4.3，再向发酵液中加入20mL现配的0.05％CPAM溶液，慢速搅拌至明显出现絮凝现象，再添加1g珍珠岩，搅拌至絮凝物稍分散，将预处理后的发酵液倒入装有滤纸的漏斗中自然过滤，测量滤速为1.0mL/min。
实施例6
用量筒量取200mL阿卡波糖发酵原液倒入500mL烧杯中，加入2g珍珠岩，搅拌均匀，向烧杯中添加固体草酸调pH至3.0，搅拌反应15min，再加入1mol/L氢氧化钠溶液将pH回调至4.3，再向发酵液中加入20mL现配的0.05％CPAM溶液，慢速搅拌至明显出现絮凝现象， 再添加1g硅藻土，搅拌至絮凝物稍分散，将预处理后的发酵液倒入装有滤纸的漏斗中自然过滤，测量滤速为1.3mL/min。
实施例7
用量筒量取200mL阿卡波糖发酵原液，加入2g硅藻土(发酵原液体积比的1％g/mL)，搅拌均匀，向烧杯中添加固体草酸调pH至3.0，搅拌反应15min，再加入1mol/L氢氧化钠溶液将pH回调至4.3，再向发酵液中加入16mL现配的0.05％CPAM溶液，慢速搅拌至明显出现絮凝现象，再添加珍珠岩1g(发酵原液体积比的0.5％g/mL)，搅拌至絮凝物稍分散，将预处理后的发酵液倒入装有滤纸的漏斗中自然过滤，测量滤速为2.1mL/min，肉眼观察到滤液颜色变为浅褐色。
实施例8
改变实施例7中酸化剂种类为盐酸，其他条件参数不变，测量滤速为0.3mL/min，肉眼观察到滤液颜色为黑褐色。
实施例9
改变实施例7中酸化剂种类为硫酸，其他条件参数不变，测量滤速为1.2mL/min，肉眼观察到滤液颜色为黑褐色。
实施例10
改变实施例7中酸化剂种类为磷酸，其他条件参数不变，测量滤速为0.8mL/min，肉眼观察到滤液颜色为褐色。
实施例11
用量筒量取200mL阿卡波糖发酵原液，加入2g硅藻土，搅拌均匀，向烧杯中添加固体草酸调pH至2.5，搅拌反应15min，再加入1mol/L氢氧化钠溶液将pH回调至4.5，再向发酵液中加入10mL现配的0.05％CPAM溶液，慢速搅拌至明显出现絮凝现象，再添加珍珠岩1g，搅拌至絮凝物稍分散，将预处理后的发酵液倒入装有滤纸的漏斗中自然过滤，测量滤速为1.4mL/min。
实施例12
改变实施例11中添加固体草酸的量，使得调节pH的值为3.0，其他条件参数不变，测量滤速为1.6mL/min。
实施例13
改变实施例11中添加固体草酸的量，使得调节pH的值为3.5，其他条件参数不变，测量滤速为1.1mL/min。
实施例14
改变实施例11中添加固体草酸的量，使得调节pH的值为4.0，其他条件参数不变，测量滤速为0.5mL/min。
实施例15
用量筒量取200mL阿卡波糖发酵液，加入2g硅藻土，搅拌均匀，向烧杯中添加固体草酸调pH至3.0，搅拌反应15min后加入1mol/L氢氧化钠溶液将pH回调至3.8；向发酵液中加入现配的20mL 0.05％CPAM溶液，慢速搅拌至明显出现絮凝现象，然后添加1g珍珠岩，搅拌至絮凝物稍分散，将预处理后的发酵液倒入装有滤纸的漏斗中自然过滤，测量滤速为1.2mL/min。
实施例16
用量筒量取200mL阿卡波糖发酵液，加入2g硅藻土，搅拌均匀，向烧杯中添加固体草酸调pH至3.0，搅拌反应15min后加入1mol/L氢氧化钠溶液将pH回调至4.8；向发酵液中加入现配的20mL 0.05％CPAM溶液，慢速搅拌至明显出现絮凝现象，然后添加1g珍珠岩，搅拌至絮凝物稍分散，将预处理后的发酵液倒入装有滤纸的漏斗中自然过滤，测量滤速为1.1mL/min。
实施例17
用量筒量取200mL阿卡波糖发酵液，加入2g硅藻土，搅拌均匀，向烧杯中添加固体草酸调pH至3.0，搅拌反应15min后加入1mol/L氢氧化钠溶液将pH回调至5.3；向发酵液中加入现配的20mL 0.05％CPAM溶液，慢速搅拌至明显出现絮凝现象，然后添加1g珍珠岩，搅拌至絮凝物稍分散，将预处理后的发酵液倒入装有滤纸的漏斗中自然过滤，测量滤速为0.6mL/min。
实施例18
用量筒取200mL阿卡波糖发酵液，加入2g硅藻土，搅拌均匀，向烧杯中分别添加固体草酸调pH至3.0，搅拌反应15min，再加入1mol/L氢氧化钠溶液将pH回调至4.3；向发酵液中加入现配的4mL 0.2％硫酸铝溶液，添加1g珍珠岩，搅拌至絮凝物稍分散，将预处理后的发酵液倒入装有滤纸的漏斗中自然过滤，测量滤速为0.2mL/min。
实施例19
用量筒取200mL阿卡波糖发酵液，加入2g硅藻土，搅拌均匀，向烧杯中分别添加固体草酸调pH至3.0，搅拌反应15min，再加入1mol/L氢氧化钠溶液将pH回调至4.3；向发酵液中加入现配的4mL 0.2％氯化铁溶液，添加1g珍珠岩，搅拌至絮凝物稍分散，将预处理后 的发酵液倒入装有滤纸的漏斗中自然过滤，测量滤速为0.1mL/min。
实施例20
用量筒取200mL阿卡波糖发酵液，加入2g硅藻土，搅拌均匀，向烧杯中分别添加固体草酸调pH至3.0，搅拌反应15min，再加入1mol/L氢氧化钠溶液将pH回调至4.3；向发酵液中加入现配的4mL 0.2％聚合氯化铝溶液，添加1g珍珠岩，搅拌至絮凝物稍分散，将预处理后的发酵液倒入装有滤纸的漏斗中自然过滤，测量滤速为0.2mL/min。
实施例21
用量筒取200mL阿卡波糖发酵液，加入2g硅藻土，搅拌均匀，向烧杯中分别添加固体草酸调pH至3.0，搅拌反应15min，再加入1mol/L氢氧化钠溶液将pH回调至4.3；向发酵液中加入现配的4mL 0.2％聚合氯化铝铁溶液，添加1g珍珠岩，搅拌至絮凝物稍分散，将预处理后的发酵液倒入装有滤纸的漏斗中自然过滤，测量滤速为0.4mL/min。
实施例22
用量筒取200mL阿卡波糖发酵液，加入2g硅藻土，搅拌均匀，向烧杯中分别添加固体草酸调pH至3.0，搅拌反应15min，再加入1mol/L氢氧化钠溶液将pH回调至4.3；向发酵液中加入现配的16mL 0.05％聚合氯化铵溶液，添加1g珍珠岩，搅拌至絮凝物稍分散，将预处理后的发酵液倒入装有滤纸的漏斗中自然过滤，测量滤速为0.9mL/min。
实施例23
用量筒取200mL阿卡波糖发酵液，加入2g硅藻土，搅拌均匀，向烧杯中分别添加固体草酸调pH至3.0，搅拌反应15min，再加入1mol/L氢氧化钠溶液将pH回调至4.3；向发酵液中加入现配的16mL 0.05％阴离子型聚丙烯酰胺溶液，添加1g珍珠岩，搅拌至絮凝物稍分散，将预处理后的发酵液倒入装有滤纸的漏斗中自然过滤，测量滤速为0.1mL/min。
实施例24
用量筒取200mL阿卡波糖发酵液，加入2g硅藻土，搅拌均匀，向烧杯中分别添加固体草酸调pH至3.0，搅拌反应15min，再加入1mol/L氢氧化钠溶液将pH回调至4.3；向发酵液中加入现配的16mL 0.05％非离子型聚丙烯酰胺溶液，添加1g珍珠岩，搅拌至絮凝物稍分散，将预处理后的发酵液倒入装有滤纸的漏斗中自然过滤，测量滤速为0.1mL/min。
实施例25
用量筒取200mL阿卡波糖发酵原液倒入烧杯中，加入2g硅藻土，搅拌均匀，向烧杯中添加固体草酸调pH至3.0，搅拌反应15min，添加1g珍珠岩，搅拌均匀，将预处理后的发酵液倒入装有滤纸的漏斗中自然过滤，测量滤速为0.5mL/min，肉眼观察滤液颜色为浅褐色。
实施例26
用量筒取200mL阿卡波糖发酵原液倒入烧杯中，加入2g硅藻土，搅拌均匀，向烧杯中添加20mL现配的0.05％CPAM溶液，慢速搅拌至明显出现絮凝现象，添加1g珍珠岩，搅拌至絮凝物稍分散，将预处理后的发酵液倒入装有滤纸的漏斗中自然过滤，测量滤速为0.9mL/min，肉眼观察滤液颜色为黑褐色。
实施例27
用量筒取200mL阿卡波糖发酵原液倒入烧杯中，加入2g硅藻土，搅拌均匀，向发酵液中加入20mL现配的0.05％CPAM溶液，慢速搅拌至明显出现絮凝现象，向烧杯中添加固体草酸调pH至3.0，搅拌反应15min，再添加1g珍珠岩，搅拌均匀后将预处理后的发酵液倒入装有滤纸的漏斗中自然过滤，测量滤速为1.1mL/min，肉眼观察滤液颜色为浅褐色。
试验结果证明酸化和絮凝工艺合用不仅可以加速发酵液的固液分离，而且有部分脱色效果，为后续发酵液的处理减轻了压力，其中先将发酵液酸化再使用絮凝剂效果最好。
实施例28
用量筒取200mL阿卡波糖发酵液，加入2g硅藻土，搅拌均匀，向烧杯中分别添加固体草酸调pH至3.0，搅拌反应15min，再加入1mol/L氢氧化钠溶液将pH回调至4.3；向发酵液中加入现配的4mL 0.05％CPAM溶液，添加1g珍珠岩，搅拌至絮凝物稍分散，将预处理后的发酵液倒入装有滤纸的漏斗中自然过滤，测量滤速为0.5mL/min。
实施例29
改变实施例28中CPAM的添加量为10mL，其他条件参数不变，然后将预处理后的发酵液倒入装有滤纸的漏斗中自然过滤，测量滤速为1.1mL/min。
实施例30
改变实施例28中CPAM的添加量为30mL，其他条件参数不变，然后将预处理后的发酵液倒入装有滤纸的漏斗中自然过滤，测量滤速为1.2mL/min。
实施例31
改变实施例28中CPAM的添加量为40mL，其他条件参数不变，然后将预处理后的发酵液倒入装有滤纸的漏斗中自然过滤，测量滤速为0.7mL/min。
实施例32
选用与上述实施例不同离子度的CPAM颗粒进行如下预处理工艺。
用量筒量取200mL阿卡波糖发酵原液倒入烧杯中，加入2g硅藻土，搅拌均匀，向烧杯中添加固体草酸调pH至3.0，搅拌反应15min，再加入1mol/L氢氧化钠溶液将pH回调至 4.3，再向发酵液中加入20mL现配的0.05％CPAM溶液(CPAM颗粒选用固含量为95％、分子量1000万、粒径100目，但离子度为30％)，慢速搅拌至明显出现絮凝现象，再添加珍珠岩1g，搅拌至絮凝物稍分散，将预处理后的发酵液倒入装有滤纸的漏斗中自然过滤，测量滤速为0.7mL/min。
实施例33
改变实施例32中CPAM颗粒的离子度为40％，其他条件参数不变，测量滤速为1.0mL/min。
实施例34
改变实施例32中CPAM颗粒的离子度为60％，其他条件参数不变，测量滤速为1.5mL/min。
实施例35
0.05％CPAM溶液的配制方法：
选用5g固含量为95％、分子量1000万、离子度50％、粒径100目的CPAM颗粒分5组试验，其中4组分别用1mL的甲醇(试剂级)、3mL的甲醇(试剂级)、1mL的乙醇(试剂级)、3mL的乙醇(试剂级)，润湿CPAM颗粒15min，再分别均匀缓慢撒入处于搅拌状态的45℃的995g水中，恒温并在300rpm转速下搅拌至完全溶解，记录溶解所需时间；第五组CPAM颗粒不润湿，直接均匀缓慢撒入处于搅拌状态的45℃水中，恒温并在300rpm转速下搅拌至完全溶解，记录溶解所需时间，结果如下：
表1.
Figure PCTCN2022104300-appb-000001
试验说明CPAM溶液配制中先用有机溶剂甲醇、乙醇润湿，可以加快CPAM颗粒溶解，对比CPAM颗粒不润湿直接溶解耗费时间，缩短CPAM颗粒溶解时间达44％-63％。
实施例36
量取20L阿卡波糖发酵液倒入烧杯中，加入200g硅藻土，搅拌均匀，向烧杯中添加200g固体草酸调pH至2.8，搅拌反应20min，再加入1mol/L氢氧化钠溶液将pH回调至4.0，再向发酵液中加入1L现配的0.1％CPAM溶液，慢速搅拌至明显出现絮凝现象，添加100g珍珠 岩，搅拌至絮凝物稍分散，将预处理后的发酵液用板框压滤机过滤。测量滤速为21L/m 2/h，肉眼观察到滤饼成型且能压干，滤液颜色由初始的黑褐色变为浅褐色。
其中现配1L 0.1％CPAM溶液方法如下：
称取1g固含量为95％、分子量900万、离子度50％、粒径100目的CPAM颗粒，用3mL甲醇润湿上述CPAM颗粒25min，再均匀缓慢撒入处于搅拌状态的45℃水中，恒温并在400rpm转速下搅拌40min至完全溶解。
在本发明中，所述阿卡波糖发酵原液可以是一般发酵液，即菌浓大于50％，粘度大于800mPa.S；也可以是菌浓和粘度较小的发酵液，即菌浓小于40％，粘度小于650mPa.S的发酵液。本发明中关于阿卡波糖预处理工艺步骤的简单置换、替换，均属于本发明范畴。

Claims (10)

  1. 一种阿卡波糖发酵液的预处理工艺，包括以下步骤：
    a.在阿卡波糖发酵原液中添加助滤剂A，搅拌均匀得到混合发酵液；
    b.在上述混合发酵液中添加酸化剂调pH，搅拌反应得到酸化后的发酵液；
    c.将上述酸化后的发酵液用碱性物质回调pH；
    d.在上述回调pH后的发酵液中加入絮凝剂；
    e.在上述絮凝后的发酵液中添加助滤剂B，固液分离。
  2. 一种阿卡波糖发酵液的预处理工艺，包括以下步骤：
    a.在阿卡波糖发酵原液中添加助滤剂A，搅拌均匀得到混合发酵液；
    b1.在上述混合发酵液中加入絮凝剂；
    c1.在上述絮凝后的发酵液中添加酸化剂调pH；
    d1.在上述絮凝后的发酵液中添加助滤剂B，固液分离。
  3. 根据权利要求1或2所述的预处理工艺，其特征在于，助滤剂A与助滤剂B各自独立选自硅藻土、珍珠岩、粗河砂粒、火山石，优选硅藻土、珍珠岩；
    优选为助滤剂A与助滤剂B不同，进一步优选为助滤剂A选自硅藻土，助滤剂B选自珍珠岩；
    和/或者
    助滤剂A或B的添加质量以所述发酵原液体积计为0.5-1.5％g/mL，优选0.5-1.0％g/mL。
  4. 根据权利要求1或2所述的预处理工艺，其特征在于，所述酸化剂为草酸、硫酸，优选草酸。
  5. 根据权利要求1或2所述的预处理工艺，其特征在于，步骤b或c1中，所述pH范围为2.5-3.5，优选2.5-3.0。
  6. 根据权利要求1所述的预处理工艺，其特征在于，步骤c所述碱性物质以水溶液的形式加入，所述的碱性物质为无机碱，优选为氢氧化钠、氨水；回调pH范围为3.8-4.8，优选为4.3-4.5。
  7. 根据权利要求1或2所述的预处理工艺，其特征在于，步骤d或c1所述的絮凝剂为阳离子型聚丙烯酰胺，所述阳离子型聚丙烯酰胺选自固含量大于93％，分子量900-1000万，离子度40％-60％，颗粒大小为80-100目的颗粒；优选固含量95％，分子量1000万，离子度50％，颗粒大小为100目的颗粒。
  8. 根据权利要求7所述的预处理工艺，其特征在于，所述的阳离子型聚丙烯酰胺以水溶液的形式添加，水溶液的浓度为0.05％-0.1％，优选0.05％-0.06％；添加体积为发酵原液体积的5-15％，优选8-10％。
  9. 根据权利要求7或8所述的预处理工艺，其特征在于，所述的絮凝剂水溶液配制过程为：
    1)用0.1-0.3％(V:V)溶液配制量的有机溶剂润湿阳离子型聚丙烯酰胺颗粒；
    2)将上述润湿后的颗粒加入水中，在40-55℃，搅拌至完全溶解。
  10. 根据权利要求9所述的预处理工艺，其特征在于，所述的有机溶剂选自醇类，例如甲醇、乙醇，其用量为溶液配制量的0.1-0.2％(V:V)；润湿时间为15-20min，溶解温度为45-50℃；搅拌转速为200-400rpm。
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