WO2022017390A1 - 一种维生素b12粗品的制备方法 - Google Patents

一种维生素b12粗品的制备方法 Download PDF

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WO2022017390A1
WO2022017390A1 PCT/CN2021/107398 CN2021107398W WO2022017390A1 WO 2022017390 A1 WO2022017390 A1 WO 2022017390A1 CN 2021107398 W CN2021107398 W CN 2021107398W WO 2022017390 A1 WO2022017390 A1 WO 2022017390A1
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acetone
vitamin
filtrate
concentrate
leaching
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PCT/CN2021/107398
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English (en)
French (fr)
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户鹏东
裴立忠
高宏伟
沈毅
马琼
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宁夏金维制药股份有限公司
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Publication of WO2022017390A1 publication Critical patent/WO2022017390A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H23/00—Compounds containing boron, silicon, or a metal, e.g. chelates, vitamin B12
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • 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

Definitions

  • the invention belongs to the technical field of biological fermentation and extraction, in particular to a method for preparing a crude vitamin B12 product.
  • Vitamin B12 (VB12), English name Vitamin B12, also known as cobalamin or cyanocobalamin, is a B vitamin composed of cobalt-containing porphyrin compounds, including adenosylcobalamin (deoxyadenosylcobalamin), cyanocobalamin (cyanocobalamin), methylcobalamin (methylcobalamin), hydroxycobalamin (hydroxycobalamin), the trivalent cobalt contained is located in the center of the corrin ring plane similar to porphyrin.
  • vitamin B12 generally refers specifically to cyanocobalamin, and its molecular characteristics contain a complex formed by cobalt atoms and cyano groups.
  • Vitamin B12 is the largest and most complex vitamin molecule that has been found so far, and it is also the only vitamin that contains metal ions; plants do not contain VB12, nor can they produce VB12. Liver is the best source of VB12, followed by milk, meat, eggs, fish, etc. VB12 is an essential coenzyme in the synthesis of ribonucleic acid and deoxyribonucleic acid. VB12 deficiency in vivo can cause pathological changes in the nervous system such as peripheral nerves and central encephalopathy.
  • the molecular formula of VB12 is C 63 H 88 CoN 14 O 14 P, the molecular weight is 1355.38, and its structural formula is:
  • VB12 is a dark red crystal or crystalline powder, insoluble at 300°C; hygroscopic, can absorb 12% water in the air, and its hydrated crystal is stable in the air. The color becomes darker at 210 ⁇ 220°C; it is soluble in water and alcohol, but insoluble in acetone, chloroform and ether; its solution is neutral, the most stable at pH 4.5 ⁇ 5, and can be used in alkaline or strong acid solution Slowly hydrolyzed.
  • Pseudomonas denitrificans is generally used for aerobic fermentation to produce adenosylcobalamin fermentation broth, which is then extracted, transformed and purified to obtain VB12 product.
  • the process of removing the deoxyadenosylcobalamin group from the cobalt atom in the adenosylcobalamin molecule, and then combining with the -CN group to become cyanocobalamin is called transformation.
  • the conversion is achieved by adding sodium cyanide or potassium cyanide.
  • Adenosylcobalamin is an intracellular fermentation product. Adenosylcobalamin mainly exists in mycelial cells. In order to improve the release rate, it is necessary to soak the filter residue after the plate and frame filtration with water for many times, and then rotate at high speed by a centrifuge to break the wall. . Because the centrifuge is easily polluted, it needs to be disassembled and washed many times, which affects the continuity of operation, and generates a lot of waste water, which increases the pressure of environmental protection treatment.
  • Sodium cyanide and potassium cyanide are highly toxic drugs with high safety risks and high management costs. Special supervision needs to be carried out in procurement, transportation, use, storage, packaging barrel recycling, etc., and the waste gas, waste water, waste solids, etc. generated during the conversion process need to be treated harmlessly to ensure the safety of the ecological environment.
  • the technical problem to be solved by the present invention is to overcome the above-mentioned defects of the prior art, and to provide a kind of continuous production that can realize continuous production, has no toxic effect, is safe and reliable, reduces the generation of sewage, reduces environmental protection pressure, and can effectively improve the conversion yield.
  • a preparation method of a crude vitamin B12 product comprising the following process:
  • the acetone composite solvent is used for leaching 2 to 3 times, and each time is 3 to 5 hours.
  • the secondary flocculation precipitation and filtration refers to adding 0.1-0.3% w/v polysilicic acid to the conversion reaction solution Aluminum and iron, stir for 15-20min, and then filter through plate and frame filter and liquid filter in turn.
  • the added amount of the acetone crystals in terms of volume, is 8 to 10 times the total volume of the vitamin B12 filtrate.
  • the leaching concentrate is mixed with the filtrate, adsorbed by the macroporous resin, and then subjected to a concentration of 30%, 40% and 50% by volume percentage.
  • the acetone aqueous solution was decomposed and concentrated under reduced pressure to obtain adenosylcobalamin desorption concentrate.
  • the invention adopts the acetone composite solvent composed of acetone, isopropanol and water for leaching and extraction of adenosylcobalamin, which changes the polarity brought by single use of water, improves the ester solubility, increases the affinity of the mycelium cell wall, and is conducive to entering the Adenosylcobalamin is extracted from the hyphal cells.
  • the invention has high yield, for example, an increase of 3-5%, and has strong production and operation continuity and produces less waste water.
  • the transformation process replaces sodium cyanide (NaCN), potassium cyanide (KCN), cyanamide (CNNH 2 ) etc. with cyanamide in the transformation process, because the cyanamide is weakly acidic in the aqueous solution, the transformation process is not easy to cause.
  • VB12 is hydrolyzed, while sodium cyanide and potassium cyanide are hydrolyzed to show strong alkalinity, and local over-alkali is easy to occur in the conversion process, resulting in hydrolysis of VB12. Therefore, using cyanamide as a conversion agent, the conversion yield is higher; at the same time, sodium cyanide, Potassium cyanide is highly toxic, while cyanamide is less toxic. Therefore, replacing sodium cyanide and potassium cyanide with cyanamide in the conversion process can reduce the pressure on safety and environmental protection, and is conducive to the safe and orderly daily production and operation of enterprises.
  • Pseudomonas denitrificans Pseudomonas denitrificans
  • acidification is used for
  • the acid used is hydrochloric acid or sulfuric acid
  • the volume of the input water during hydrolysis is 1-1.5 times the volume of the fermentation broth
  • the heating temperature of the water is 75-95°C.
  • W or w represents the mass of the substance
  • V or v represents the volume of the substance.
  • the leaching solution and the filtrate were combined to obtain a mixed solution.
  • the above mixed solution was put into a macroporous resin adsorption column, and 30% (v/v), 40% (v/v), and 50% (v/v) acetone aqueous solution was used for analysis in sequence, and the analysis solution was collected and concentrated under reduced pressure to obtain glands. Glycobalamin analytical concentrate.
  • the above VB12 filtrate was put into a crystallizing tank, and at room temperature, 8 times the volume of VB12 filtrate was added with acetone to crystallize VB12, suction filtration, and drying to obtain VB12 crude product with a yield of 95%.
  • the overall yield was 76.1%.
  • the total amount of wastewater is 2173L.
  • the leaching solution and the filtrate were combined to obtain a mixed solution.
  • the above mixed solution was put into a macroporous resin adsorption column, and 30% (v/v), 40% (v/v), and 50% (v/v) acetone aqueous solution was used for analysis in sequence, and the analysis solution was collected and concentrated under reduced pressure to obtain glands. Glycobalamin analytical concentrate.
  • the above VB12 filtrate was put into a crystallizing tank, and at room temperature, acetone of 9 times the volume of the VB12 filtrate was added to crystallize the VB12, suction filtration, and drying to obtain a crude VB12 product.
  • the overall yield was 75.2%.
  • the total amount of wastewater is 2275L.
  • the leaching solution and the filtrate were combined to obtain a mixed solution.
  • the above mixed solution was put into a macroporous resin adsorption column, and 30% (v/v), 40% (v/v), and 50% (v/v) acetone aqueous solution was used for analysis in sequence, and the analysis solution was collected and concentrated under reduced pressure to obtain glands. Glycobalamin analytical concentrate.
  • the transformation reaction is over, in the transformation reaction solution, add 0.3% (w/v) polymeric aluminum ferric silicate, stir for 15 to 20 minutes, let stand for 15 minutes, then filter through plate and frame, liquid filter (1 ⁇ m), Wash to obtain VB12 filtrate.
  • the above VB12 filtrate was put into a crystallizing tank, and at room temperature, 10 times the volume of VB12 filtrate was added with acetone to precipitate VB12, suction filtration, and drying to obtain a crude VB12 product.
  • the overall yield was 76.7%.
  • the total amount of wastewater is 2415L.
  • the leaching solution and the filtrate were combined to obtain a mixed solution.
  • the above mixed solution was put into a macroporous resin adsorption column, and 30% (v/v), 40% (v/v), and 50% (v/v) acetone aqueous solution was used for analysis in sequence, and the analysis solution was collected and concentrated under reduced pressure to obtain glands. Glycobalamin analytical concentrate.
  • the above VB12 filtrate was put into a crystallizing tank, and at room temperature, acetone of 9 times the volume of the VB12 filtrate was added to crystallize the VB12, suction filtration, and drying to obtain a crude VB12 product.
  • the overall yield was 77.8%.
  • the total amount of wastewater is 3025L.
  • the leaching solution and the filtrate were combined to obtain a mixed solution.
  • the above mixed solution was put into a macroporous resin adsorption column, and 30% (v/v), 40% (v/v), and 50% (v/v) acetone aqueous solution was used for analysis in sequence, and the analysis solution was collected and concentrated under reduced pressure to obtain glands. Glycobalamin analytical concentrate.
  • the above VB12 filtrate was put into a crystallizing tank, and at room temperature, acetone of 9 times the volume of the VB12 filtrate was added to crystallize the VB12, suction filtration, and drying to obtain a crude VB12 product.
  • the overall yield was 76.9%.
  • the total amount of wastewater is 2642L.
  • the leaching solution and the filtrate were combined to obtain a mixed solution.
  • the above mixed solution was put into a macroporous resin adsorption column, and 30% (v/v), 40% (v/v), and 50% (v/v) acetone aqueous solution was used for analysis in sequence, and the analysis solution was collected and concentrated under reduced pressure to obtain glands. Glycobalamin analytical concentrate.
  • VB12 filtrate add the acetone of 9 times of VB12 filtrate volume, make VB12 crystallize out, suction filter, dry, obtain VB12 crude product.
  • the overall yield was 75.6%.
  • the total amount of wastewater is 5151L.
  • the leaching solution and the filtrate were combined to obtain a mixed solution.
  • the above mixed solution was put into a macroporous resin adsorption column, and 30% (v/v), 40% (v/v), and 50% (v/v) acetone aqueous solution was used for analysis in sequence, and the analysis solution was collected and concentrated under reduced pressure to obtain glands.
  • the volume of the concentrated solution of glycocobalamin analysis was 1100 L (the titer was 272 ⁇ g/ml), and the yield was 96.6%.
  • VB12 filtrate acetone of 9 times the volume of the VB12 filtrate was added to make VB12 crystallize out, suction filtration, and drying to obtain a crude VB12 product.
  • the overall yield was 75.7%.
  • the total amount of wastewater is 5091L.

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Abstract

一种维生素B12粗品的制备方法，其是将脱氮假单孢杆菌经酸化、水解、絮凝沉淀后过滤，分别收集滤渣和滤液；其中滤渣用丙酮复合溶媒浸提后离心处理，减压浓缩得到浸提浓缩液；将上述浸提浓缩液同滤液混合，经大孔树脂吸附、丙酮水溶液解析、减压浓缩得到腺苷钴胺解析浓缩液；加入氰胺水溶液转化，之后二次絮凝沉淀、过滤得到维生素B12过滤液；在维生素B12过滤液加入丙酮结晶，抽滤，干燥即可得到维生素B12粗品。采用丙酮复合溶媒进行浸提提取腺苷钴胺，改变了单一使用水带来的极性，提高了酯溶性，增加菌丝细胞壁亲和性，比单纯用水浸泡、离心机高速破壁提取方式，收率高，生产操作连贯性强，产生废水量少。

Description

一种维生素B12粗品的制备方法 技术领域
本发明属于生物发酵与提取技术领域，特别是涉及一种维生素B12粗品的制备方法。
背景技术
维生素B12(VB12)，英文名称Vitamin B12，又称钴胺素或氰钴素，是一种由含钴的卟啉类化合物组成的B族维生素，包括腺苷钴胺(deoxyadenosylcobalamin)、氰钴胺(cyanocobalamin)、甲钴胺(methylcobalamin)、羟钴胺(hydroxycobalamin)，所含的三价钴位于类似卟啉的咕啉环平面的中心。现有技术中，维生素B12一般特指氰钴胺，其分子的特点含有钴原子及氰基团而形成的一种复合体。
维生素B12(VB12)是目前已发现的最大、最复杂的维生素分子，也是唯一含有金属离子的维生素；植物不含VB12，也不能制造VB12。肝脏是VB12的最好来源，其次为奶、肉、蛋、鱼等。VB12是核糖核酸和脱氧核糖核酸合成过程中必需的一种辅酶，体内VB12缺乏可以引起周围神经和中枢性脑病等神经系统的病理性改变。
VB12的分子式为C 63H 88CoN 14O 14P，分子量1355.38，其结构式为：
Figure PCTCN2021107398-appb-000001
VB12系深红色结晶或结晶性粉，300℃不溶化；有吸湿性，在空气中能吸收12％水分，其含水结晶在空气中稳定。在210～220℃时颜色变深；能溶于水和醇，不溶于丙酮、氯仿和醚；其溶液呈中性，在pH4.5～5时最稳定， 在碱性或强酸性溶液中可缓慢水解。
工业生产中，一般采用脱氮假单孢菌(Pseμdomonas denitrificans)进行有氧发酵生产腺苷钴胺发酵液，再经提取、转化、纯化得到VB12产品。其中，将腺苷钴胺分子中的钴原子上脱去脱氧腺苷钴胺基团，再结合上-CN基团成为氰钴胺的过程，称为转化。一般生产工艺中，通过加入氰化钠或氰化钾，实现转化。这种工艺存在的问题是：
1、腺苷钴胺为胞内发酵产物，腺苷钴胺主要存在于菌丝细胞中，为提高释放率，需要将板框过滤后的滤渣多次加水浸泡，再经离心机高速旋转破壁。由于离心机易污染，需要多次拆洗，影响连贯性作业，且产生废水多，增加了环保处理压力。
2、氰化钠、氰化钾为剧毒药品，安全风险大，管理费用高。需在采购、运输、使用、贮存、包装桶回收等环节，进行特别监管，而且转化过程产生的废气、废水、废固等需进行无害化处理，以确保生态环境的安全。
引证文件列表
专利文献
专利文献1CN 111073922 A。
发明内容
技术问题
有鉴于此，本发明要解决的技术问题是克服上述现有技术的缺陷，提供一种可实现连续化生产，无毒害作用，安全可靠，减少污水产生，降低环保压力，且能有效提高转化收率的维生素B12粗品的制备方法。
解决方案
为了解决上述技术问题，根据本发明的一实施例，提供了一种维生素B12粗品的制备方法，包含如下过程：
1)将脱氮假单孢杆菌发酵液经酸化、水解和絮凝沉淀后过滤，分别收集滤渣和滤液；
2)将滤渣用丙酮复合溶媒浸提后离心处理，减压浓缩得到浸提浓缩液，所述丙酮复合溶媒为丙酮、异丙醇和水的混合物，其体积比为：V丙酮：V异丙醇：V水＝5.5～6.5:1.5～2.5:5.5～6.5，投料比为W 滤渣：W 丙酮复合溶媒＝1:3.5～ 5.5；
3)将上述浸提浓缩液同滤液混合，经大孔树脂吸附、丙酮水溶液解析和减压浓缩得到腺苷钴胺解析浓缩液；
4)在腺苷钴胺解析浓缩液加入氰胺水溶液进行转化，之后二次絮凝沉淀和过滤得到维生素B12过滤液；
5)在维生素B12过滤液加入丙酮结晶，抽滤，干燥即可得到维生素B12粗品。
对于上述制备方法，在一种可能的实现方式中，过程2)中，采用丙酮复合溶媒浸提2～3次，每次3～5小时。
对于上述制备方法，在一种可能的实现方式中，过程4)中，所述转化是指在30～35℃温度条件下，在腺苷钴胺解析浓缩液中加入质量浓度40～45％的氰胺水溶液，搅拌反应90～120min，氰胺加入量为W 腺苷钴胺解析浓缩液：W 氰 胺＝1:0.028～0.029。
对于上述制备方法，在一种可能的实现方式中，过程4)中，所述二次絮凝沉淀和过滤是指在转化反应液中，加入以w/v计的0.1～0.3％的聚合硅酸铝铁，搅拌15～20min，然后依次经板框过滤和液体过滤器过滤。
对于上述制备方法，在一种可能的实现方式中，过程5)中，所述丙酮结晶的加入量，以体积计，为维生素B12过滤液的总体积的8～10倍。
对于上述制备方法，在一种可能的实现方式中，过程3)中，浸提浓缩液同滤液混合，经大孔树脂吸附，再经以体积百分比计，浓度为30％、40％和50％的丙酮水溶液解析，减压浓缩，得到腺苷钴胺解析浓缩液。
有益效果
本发明采用丙酮、异丙醇和水组成的丙酮复合溶媒进行浸提提取腺苷钴胺，改变了单一使用水带来的极性，提高了酯溶性，增加菌丝细胞壁亲和性，有利于进入菌丝细胞内将腺苷钴胺提取出来。本发明比单纯用水浸泡、离心机高速破壁提取方式，收率高，例如提高3～5％，而且生产操作连贯性强，产生废水量少。
更进一步优选地，本发明中转化工艺用氰胺替代氰化钠(NaCN)、氰化钾(KCN)，氰胺(CNNH 2)等，由于氰胺在水溶液中显弱酸性，转化过程不易造成VB12水解，而氰化钠、氰化钾水解呈较强碱性，转化过程易发生局 部过碱，造成VB12水解，因而，用氰胺作转化剂，转化收率较高；同时氰化钠、氰化钾是剧毒，而氰胺毒性较低，因此，转化工艺用氰胺替代氰化钠、氰化钾，能减少安全、环保压力，有利于企业安全有序开展日常生产经营。
根据下面对示例性实施例的详细说明，本发明的其它特征及方面将变得清楚。
具体实施方式
在这里专用的词“示例性”意为“用作例子、实施例或说明性”。这里作为“示例性”所说明的任何实施例不必解释为优于或好于其它实施例。
另外，为了更好的说明本发明，在下文的具体实施方式中给出了众多的具体细节。应该理解的是，具体实施方式是用于说明本发明而不是对本发明的限制。本领域技术人员应当理解，没有某些具体细节，本发明同样可以实施。在另外一些实例中，对于本领域技术人员熟知的方法、手段、元件和电路未作详细描述，以便于凸显本发明的主旨。
对脱氮假单孢杆菌发酵液进行酸化和水解处理得到脱氮假单孢杆菌酸化水解液，其中脱氮假单孢杆菌(Pseudomonas denitrificans)为应用于工业生产维生素B12的生产菌，其中酸化所用的酸为盐酸或者硫酸，水解时入水的体积为发酵液体积的1～1.5倍，水的加热温度为75～95℃。
如无特殊说明，在所示出的比例中，W或w表示物质的质量，V或v表示物质的体积。
取脱氮假单孢杆菌70000L，分成7份，分别进行以下实验：
实施例1
取脱氮假单孢杆菌发酵液10000L，按其体积加入1倍水，搅拌下，加盐酸调PH4.8，将酸化液经蒸汽喷射塔快速加热，进行酸化水解，控制水解温度75℃，得到酸化水解液，分别按水解液体积，加入0.4％(w/v)聚合氯化铝和0.2％(w/v)珍珠岩，搅拌30分钟，静置，进板框过滤，得到滤液和滤渣。
将以上滤渣，投入溶媒浸提罐，按投料比(滤渣(W)：复合溶媒(V)＝1：3.5)，加入丙酮复合溶媒(丙酮(V)：异丙醇(V)：水(V)＝5.5:1.5:5.5)，控制搅拌转速60rpm，室温下进行浸提，浸提3hr后，进板框过滤，收集滤渣， 照前进行第二次浸提，合并两次浸提液，减压浓缩，得到浸提浓缩液。
将浸提液与滤液合并得到混合液。
将以上混合液进大孔树脂吸附柱，依次用30％(v/v)、40％(v/v)、50％(v/v)丙酮水溶液解析，收集解析液，减压浓缩，得到腺苷钴胺解析浓缩液。
将以上腺苷钴胺解析浓缩液投入转化罐中，控制温度30～32℃，搅拌转速60rpm，按W 腺苷钴胺解析浓缩液：W 氰胺＝1:0.028，加入质量浓度40％(w/v)氰胺水溶液，转化反应90min。
转化反应结束，在转化反应液中，加入0.1％(w/v)聚合硅酸铝铁，搅拌15～20min，静置15分钟，依次经板框过滤、液体过滤器过滤(1μm)，水顶洗，得到VB12过滤液。
将以上VB12过滤液投入结晶罐中，室温下，加入8倍VB12过滤液体积的丙酮，使VB12结晶析出，抽滤、干燥，得到VB12粗品，收率95％。总收率76.1％。废水总量2173L。
实施例2
取脱氮假单孢杆菌发酵液10000L，按其体积加入1.5倍水，搅拌下，加盐酸调PH5.0，将酸化液经蒸汽喷射塔快速加热，进行酸化水解，控制水解温度80℃，得到酸化水解液，分别按水解液体积，加入0.4％(w/v)聚合氯化铝和0.2％(w/v)珍珠岩，搅拌30分钟，静置，进板框过滤，得到滤液和滤渣。
将以上滤渣，投入溶媒浸提罐，按投料比(滤渣(W)：复合溶媒(V)＝1：4.0)，加入丙酮复合溶媒(丙酮(V)：异丙醇(V)：水(V)＝6.5:2.5:6.5)，控制搅拌转速60rpm，室温下进行浸提，浸提6hr后，进板框过滤，收集滤渣，照前进行第二次浸提，合并两次浸提液，减压浓缩，得到浸提浓缩液。
将浸提液与滤液合并得到混合液。
将以上混合液进大孔树脂吸附柱，依次用30％(v/v)、40％(v/v)、50％(v/v)丙酮水溶液解析，收集解析液，减压浓缩，得到腺苷钴胺解析浓缩液。
将以上腺苷钴胺解析浓缩液投入转化罐中，控制温度32～34℃，搅拌转速70rpm，按W 腺苷钴胺解析浓缩液：W 氰胺＝1:0.028，加入42％(w/w)氰胺水溶液，转化反应100min。
转化反应结束，在转化反应液中，加入0.2％(w/v)聚合硅酸铝铁，搅拌 15～20min，静置15分钟，依次经板框过滤、液体过滤器过滤(1μm)，水顶洗，得到VB12过滤液。
将以上VB12过滤液投入结晶罐中，室温下，加入9倍VB12过滤液体积的丙酮，使VB12结晶析出，抽滤、干燥，得到VB12粗品。总收率75.2％。废水总量2275L。
实施例3
取脱氮假单孢杆菌发酵液10000L，按其体积加入1.5倍水，搅拌下，加硫酸调PH5.2，将酸化液经蒸汽喷射塔快速加热，进行酸化水解，控制水解温度85℃，得到酸化水解液，分别按水解液体积，加入0.4％(w/v)聚合氯化铝和0.2％(w/v)珍珠岩，搅拌30分钟，静置，进板框过滤，得到滤液和滤渣。
将以上滤渣，投入溶媒浸提罐，按投料比(滤渣(W)：复合溶媒(V)＝1：4.5)，加入丙酮复合溶媒(丙酮(v)：异丙醇(v)：水(V)＝6.0:2.0:6.0)，控制搅拌转速60rpm，室温下进行浸提，浸提5hr后，进板框过滤，收集滤渣，照前进行第二次浸提，合并两次浸提液，减压浓缩，得到浸提浓缩液。
将浸提液与滤液合并得到混合液。
将以上混合液进大孔树脂吸附柱，依次用30％(v/v)、40％(v/v)、50％(v/v)丙酮水溶液解析，收集解析液，减压浓缩，得到腺苷钴胺解析浓缩液。
将以上腺苷钴胺解析浓缩液投入转化罐中，控制温度34～35℃，搅拌转速60rpm，按W 腺苷钴胺解析浓缩液：W 氰胺＝1:0.0285，加入42.5％(w/w)氰胺水溶液，转化反应110min；
转化反应结束，在转化反应液中，加入0.3％(w/v)聚合硅酸铝铁，搅拌15～20min，静置15分钟，依次经板框过滤、液体过滤器过滤(1μm)，水顶洗，得到VB12过滤液。
将以上VB12过滤液投入结晶罐中，室温下，加入10倍VB12过滤液体积的丙酮，使VB12结晶析出，抽滤、干燥，得到VB12粗品。总收率76.7％。废水总量2415L。
实施例4
取脱氮假单孢杆菌发酵液10000L，按其体积加入1倍水，搅拌下，加硫酸调PH5.0，将酸化液经蒸汽喷射塔快速加热，进行酸化水解，控制水解温 度90℃，得到酸化水解液，分别按水解液体积，加入0.4％(w/v)聚合氯化铝和0.2％(w/v)珍珠岩，搅拌30分钟，静置，进板框过滤，得到滤液和滤渣。
将以上滤渣，投入溶媒浸提罐，按投料比(滤渣(W)：复合溶媒(V)＝1：5.0)，加入丙酮复合溶媒(丙酮(v)：异丙醇(v)：水(V)＝6.0:1.5:6.5)，控制搅拌转速60rpm，室温下进行浸提，浸提3hr后，进板框过滤，收集滤渣，照前进行第二次、第三次浸提，合并三次浸提液，减压浓缩，得到浸提浓缩液。
将浸提液与滤液合并得到混合液。
将以上混合液进大孔树脂吸附柱，依次用30％(v/v)、40％(v/v)、50％(v/v)丙酮水溶液解析，收集解析液，减压浓缩，得到腺苷钴胺解析浓缩液。
将以上腺苷钴胺解析浓缩液投入转化罐中，控制温度31～33℃，搅拌转速60rpm，按W 腺苷钴胺解析浓缩液：W 氰胺＝1:0.029，加入45％(w/w)氰胺水溶液，转化反应120min。
转化反应结束，在转化反应液中，加入0.2％(w/v)聚合硅酸铝铁，搅拌15～20min，静置15分钟，依次经板框过滤、液体过滤器过滤(1μm)，水顶洗，得到VB12过滤液。
将以上VB12过滤液投入结晶罐中，室温下，加入9倍VB12过滤液体积的丙酮，使VB12结晶析出，抽滤、干燥，得到VB12粗品。总收率77.8％。废水总量3025L。
实施例5
取脱氮假单孢杆菌发酵液10000L，按其体积加入1倍水，搅拌下，加盐酸调PH5.2，将酸化液经蒸汽喷射塔快速加热，进行酸化水解，控制水解温度95℃，得到酸化水解液，分别按水解液体积，加入0.4％(w/v)聚合氯化铝和0.2％(w/v)珍珠岩，搅拌30分钟，静置，进板框过滤，得到滤液和滤渣。
将以上滤渣，投入溶媒浸提罐，按投料比(滤渣(W)：复合溶媒(V)＝1：5.5)，加入丙酮复合溶媒(丙酮(v)：异丙醇(v)：水(V)＝6.0:2.5:6.0)，控制搅拌转速60rpm，室温下进行浸提，浸提3hr后，进板框过滤，收集滤渣，照前进行第二次浸提，合并两次浸提液，减压浓缩，得到浸提浓缩液。
将浸提液与滤液合并得到混合液。
将以上混合液进大孔树脂吸附柱，依次用30％(v/v)、40％(v/v)、50％(v/v)丙酮水溶液解析，收集解析液，减压浓缩，得到腺苷钴胺解析浓缩液。
将以上腺苷钴胺解析浓缩液投入转化罐中，控制温度31～33℃，搅拌转速60rpm，按W 腺苷钴胺解析浓缩液：W 氰胺＝1:0.029，加入42.5％(w/w)氰胺水溶液，转化反应105min；
转化反应结束，在转化反应液中，加入0.2％(w/v)聚合硅酸铝铁，搅拌15～20min，静置15分钟，依次经板框过滤、液体过滤器过滤(1μm)，水顶洗，得到VB12过滤液。
将以上VB12过滤液投入结晶罐中，室温下，加入9倍VB12过滤液体积的丙酮，使VB12结晶析出，抽滤、干燥，得到VB12粗品。总收率76.9％。废水总量2642L。
对比例1
取脱氮假单孢杆菌发酵液10000L，按其体积加入1倍水，搅拌下，加盐酸调PH5.0，将酸化液经蒸汽喷射塔快速加热，进行酸化水解，控制水解温度75℃，得到酸化水解液，分别按水解液体积，加入0.4％(w/v)聚合氯化铝和0.2％(w/v)珍珠岩，搅拌30分钟，静置，进板框过滤，得到滤液和滤渣。
将以上滤渣，投入水浸提罐中，按投料比(滤渣(w)：水(v)＝1：5.0)，加入水，控制搅拌转速80rpm，室温下进行浸泡，浸泡3hr后，进板框过滤，收集滤渣，照前再进行二次浸提，合并三次浸提液。
将浸提液与滤液合并得到混合液。
将以上混合液进大孔树脂吸附柱，依次用30％(v/v)、40％(v/v)、50％(v/v)丙酮水溶液解析，收集解析液，减压浓缩，得到腺苷钴胺解析浓缩液。
将以上腺苷钴胺解析浓缩液投入转化罐中，控制温度70℃，搅拌下，将腺苷钴胺浓缩液中，按W 腺苷钴胺解析浓缩液：W NaCN＝1:0.032，加入30％(w/w)氰化钠水溶液，氰化转化反应90min；
氰化转化反应液中，加入0.3％(w/v)聚合硫酸铝，搅拌20min，依次经板框过滤、液体过滤器过滤(1μm)，水顶洗，得到VB12过滤液。
上述VB12过滤液中，加入9倍VB12过滤液体积的丙酮，使VB12结晶析 出，抽滤、干燥，得到VB12粗品。总收率75.6％。废水总量5151L。
对比例2
取脱氮假单孢杆菌发酵液10000L，按其体积加入1倍水，搅拌下，加硫酸调PH5.0，将酸化液经蒸汽喷射塔快速加热，进行酸化水解，控制水解温度95℃，得到酸化水解液，分别按水解液体积，加入0.4％(w/v)聚合氯化铝和0.2％(w/v)珍珠岩，搅拌30分钟，静置，进板框过滤，得到滤液和滤渣。
将以上滤渣，投入水浸提罐中，按投料比(滤渣(w)：水(v)＝1：5.0)，加入水，控制搅拌转速80rpm，室温下进行浸泡，浸泡3hr后，进板框过滤，收集滤渣，照前再进行二次浸提，合并三次浸提液。
将浸提液与滤液合并得到混合液。
将以上混合液进大孔树脂吸附柱，依次用30％(v/v)、40％(v/v)、50％(v/v)丙酮水溶液解析，收集解析液，减压浓缩，得到腺苷钴胺解析浓缩液体积1100L(效价272μg/ml)，收率96.6％。
将以上腺苷钴胺解析浓缩液投入转化罐中，控制温度70℃，搅拌下，将腺苷钴胺浓缩液中，按W 腺苷钴胺解析浓缩液：W KCN＝1:0.043，加入30％(w/w)氰化钾水溶液，氰化转化反应90min；
氰化转化反应液中，加入0.3％(w/v)聚合硫酸铝，搅拌20min，依次经板框过滤、液体过滤器过滤(1μm)，水顶洗，得到VB12过滤液。
上述VB12过滤液中，加入9倍VB12过滤液体积的丙酮，使VB12结晶析出，抽滤、干燥，得到VB12粗品。总收率75.7％。废水总量5091L。
Figure PCTCN2021107398-appb-000002
以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。

Claims (6)

  1. 一种维生素B12粗品的制备方法，其特征在于，包含如下过程：
    1)将脱氮假单孢杆菌发酵液经酸化、水解和絮凝沉淀后过滤，分别收集滤渣和滤液；
    2)将滤渣用丙酮复合溶媒浸提后离心处理，减压浓缩得到浸提浓缩液，所述丙酮复合溶媒为丙酮、异丙醇和水的混合物，其体积比为：V丙酮：V异丙醇：V水＝5.5～6.5:1.5～2.5:5.5～6.5，投料比为W 滤渣：W 丙酮复合溶媒＝1:3.5～5.5；
    3)将上述浸提浓缩液同滤液混合，经大孔树脂吸附、丙酮水溶液解析和减压浓缩得到腺苷钴胺解析浓缩液；
    4)在腺苷钴胺解析浓缩液加入氰胺水溶液进行转化，之后二次絮凝沉淀和过滤得到维生素B12过滤液；
    5)在维生素B12过滤液加入丙酮结晶，抽滤，干燥即可得到维生素B12粗品。
  2. 根据权利要求1所述的维生素B12粗品的制备方法，其特征在于，过程2)中，采用丙酮复合溶媒浸提2～3次，每次3～5小时。
  3. 根据权利要求1或2所述的维生素B12粗品的制备方法，其特征在于，过程4)中，所述转化是指在30～35℃温度条件下，在腺苷钴胺解析浓缩液中加入质量浓度40～45％的氰胺水溶液，搅拌反应90～120min，氰胺加入量为W 腺苷钴胺解析浓缩液：W 氰胺＝1:0.028～0.029。
  4. 根据权利要求1-3任一项所述的维生素B12粗品的制备方法，其特征在于，过程4)中，所述二次絮凝沉淀和过滤是指在转化反应液中，加入以w/v计的0.1～0.3％的聚合硅酸铝铁，搅拌15～20min，然后依次经板框过滤和液体过滤器过滤。
  5. 根据权利要求1-4任一项所述的维生素B12粗品的制备方法，其特征在于，过程5)中，所述丙酮结晶的加入量，以体积计，为维生素B12过滤液的总体积的8～10倍。
  6. 按照权利要求1-5任一项所述的维生素B12粗品的制备方法，其特征在于，过程3)中，浸提浓缩液同滤液混合，经大孔树脂吸附，再经以体积百分比计，浓度为30％、40％和50％的丙酮水溶液解析，减压浓缩，得到腺苷钴胺解析浓缩液。
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