WO2020103956A1 - 基于光照调控的木糖母液微生物发酵维生素 b 12 的方法 - Google Patents

基于光照调控的木糖母液微生物发酵维生素 b 12 的方法

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Publication number
WO2020103956A1
WO2020103956A1 PCT/CN2019/121085 CN2019121085W WO2020103956A1 WO 2020103956 A1 WO2020103956 A1 WO 2020103956A1 CN 2019121085 W CN2019121085 W CN 2019121085W WO 2020103956 A1 WO2020103956 A1 WO 2020103956A1
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vitamin
mother liquor
xylose mother
microbial fermentation
strain
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PCT/CN2019/121085
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English (en)
French (fr)
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石丽华
朱炫
左齐乐
张文瑶
陈杰
陈跃文
郑晓阳
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浙江华康药业股份有限公司
焦作市华康糖醇科技有限公司
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Application filed by 浙江华康药业股份有限公司, 焦作市华康糖醇科技有限公司 filed Critical 浙江华康药业股份有限公司
Publication of WO2020103956A1 publication Critical patent/WO2020103956A1/zh

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    • 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
    • C12P19/28—N-glycosides
    • C12P19/42—Cobalamins, i.e. vitamin B12, LLD factor

Definitions

  • the technical field of microbial fermentation of the present invention particularly relates to a method for microbial fermentation of vitamin B 12 by xylose mother liquor based on light regulation.
  • Vitamin B 12 in nature can only be synthesized by microorganisms, mainly bacteria and actinomycetes. Bacteria are mainly Propionibacterium and Pseudomonas denitrificans. Actinomycetes are mainly Streptomyces. Vitamin B 12 was previously extracted from antibiotic waste liquid, but the extraction efficiency is low. The chemical synthesis is extremely complicated and the cost is very high. The key is that the obtained product has low purity and a large number of by-products, which is not suitable for industrialization.
  • Chinese Patent Publication No. CN102453690A disclosed a method of mutagenesis 12 a producing strain Pseudomonas denitrificans fermentation production of vitamin B, vitamin B get 12 high-yield strain by mutagenesis screening methods, the main drawback of this method is mutagenic There are uncertainties and high technical requirements.
  • Chinese Patent No. CN108251476A discloses a method for extracting vitamin B 12 from enzyme preparation wastewater. The enzyme preparation wastewater is used as a raw material to extract vitamin B 12 after a series of treatments.
  • the main disadvantage of this method is that the enzyme preparation wastewater needs to go through The process is relatively complicated.
  • YUE YU et al. (Yue Yu, Xuan Zhu, etc, Enhancing the vitamin B 12 production and growth of Propionibacterium freudenreichii in tofu wastewater via a light-induced vitamin B 12 riboswitch, Appl Microbiol Biotechnol, DOI 10.1007 / s00253-015-6958- 6) It has been found that blue light can increase the production of vitamin B 12 in the fermentation process of tofu wastewater by exciting the vitamin B 12 ribosome switch, but in this study, cobalt sulfate was added to the tofu wastewater during the fermentation process, and previous studies have shown that cobalt compounds can Increase the synthesis of vitamin B 12 .
  • the technical problem to be solved by the present invention is to provide a method for microbial fermentation of vitamin B 12 by xylose mother liquor microorganisms based on light regulation to improve the production of vitamin B 12 in a xylose mother liquor culture medium of vitamin B 12 producing strains, with simple operation and technical difficulty Low, without adding substances that can increase the production of vitamin B 12 , use light to increase the production of vitamin B 12 .
  • the present invention is achieved in this way, and provides a method for microbial fermentation of vitamin B 12 based on xylose mother liquor based on light regulation, including the following steps:
  • Step one the seeds of vitamin B 12 production strains were cultured activated, and the activated vitamin B 12 production strain was inoculated to 5% SLB culture medium placed in the stand-by temperature expansion. 4 deg.] C;
  • Step 2 Pre-treat the xylose mother liquor by filtration and cold sterilization
  • Step 3 Centrifuge the vitamin B 12 production strain obtained by expansion in step 1, discard the supernatant to obtain the precipitated strain producing vitamin B 12 , and wash the precipitated strain;
  • Step 4 Inoculate the washed precipitated strain producing vitamin B 12 into the vitamin B 12 detection medium containing the xylose mother liquor pretreated in step two as the medium raw material, and perform fermentation treatment together with the xylose mother liquor.
  • static media 12 detects the vitamin B in a darkened environment 65h ⁇ 80h, and at 10 ⁇ 50cm is provided at the light source of vitamin B 12 assay medium from said source of vitamin B 12 assay medium is irradiated intermittently, the light source Each light time lasts 10min ⁇ 80min, and the ratio of light time to dark time (non-light time) is 1: 2 ⁇ 5: 1;
  • Step 5 The fermentation solution after the fermentation treatment in Step 4 is subjected to cell disruption treatment, and then the fermentation broth after the cell disruption treatment is subjected to low-temperature centrifugation. After centrifugation, a supernatant containing vitamin B 12 is obtained, and the supernatant is added to the supernatant. Dilute sodium cyanide solution, and then extract the treated supernatant through SPE column to extract vitamin B 12 , collect the eluent containing vitamin B 12 after extraction, and blow dry with nitrogen. The product after the eluent is dried is Vitamin B 12 .
  • vitamin B 12 production seed strain was cultured activation means: vitamin B 12 production strains
  • the seeds were inoculated from 1% to 500 mL of MRS liquid culture medium in a preservation tube and placed in an incubator for anaerobic culture for 10 h to 14 h.
  • the composition of the MRS liquid culture medium includes (g / L): peptone 10.0, beef Dipping powder 5.0, yeast extract powder 4.0, glucose 20.0, dipotassium hydrogen phosphate 2.0, sodium acetate 5.0, triammonium citrate 2.0, magnesium sulfate 0.2, manganese sulfate 0.05, Tween-80 1.0, pH 6.5 ⁇ 0.2.
  • the expansion method is to place the vitamin B 12 production strain in the SLB medium, and culture in a incubator for 2h ⁇ 9h, so that the colony forming unit in the medium reaches 10 8 CFU / mL
  • the components of SLB medium include (weight ratio): 1% tryptone, 1% yeast extract, 1.5% glucose, 0.25 ⁇ K 2 HPO 4 , 0.05 ⁇ MnSO 4 , 0.1% Tween-80, adjust pH 6.5 ⁇ 0.2.
  • the tools used in the cold filtration sterilization method include: a syringe, a filter, and a 0.22 ⁇ m filter membrane.
  • the operation steps of the cold filtration sterilization method include: attach the filter and the filter membrane tightly , Tighten the filter of the assembled filter and the syringe, load the xylose mother liquor to be sterilized into the syringe, and then slowly push the syringe to make the xylose mother liquor in the syringe flow through the filter and then sterilize. Operate on a sterile operating table.
  • step three the condition of the centrifugal treatment is centrifugation at 3000 r / min for 10 min at 4 ° C.
  • the method for washing the precipitated strain is to wash the precipitated strain repeatedly three times with 0.9% sodium chloride solution.
  • the components of the vitamin B 12 detection medium include (g / L): acid hydrolyzed casein 12.0, riboflavin 0.002, Tween-80 2.0, dipotassium hydrogen phosphate 1.0, diphosphate Potassium hydrogen 1.0, magnesium sulfate 0.4, ferrous sulfate 0.02, manganese sulfate 0.02, 1% by weight xylose mother liquor, pH 6.3 ⁇ 0.2.
  • the light source is an incandescent lamp with a power of 15-100W.
  • the cell disruption process refers to the ultrasonic disruption of the fermentation solution using a cell disruption instrument.
  • the operating power of the cell disruption instrument is 400 W, the working time is 3 s, and the intermittent time is 3 s, repeated 99 times.
  • the low-temperature centrifugal treatment refers to centrifuging the fermentation broth at 4 ° C for 8 minutes at a rotational speed of 6000 r / min.
  • the step of the SPE column extraction method includes: activating the SPE column with 5 mL of 90% methanol, washing the methanol three times with 15 mL of ultra-pure water, equilibrating the column, and passing the treated supernatant through After the SPE column, the impurities adsorbed on the SPE column are washed with 5 mL of ultrapure water, and then the liquid is pumped to dry, and then the SPE column is washed with methanol, and the eluent is collected for subsequent processing.
  • the method of microbial fermentation of vitamin B 12 based on xylose mother liquor microorganisms of the present invention expands the vitamin B 12 production strain obtained by cultivating and activating the seeds of the vitamin B 12 production strain in the SLB medium
  • the vitamin B 12 producing strain was centrifuged to produce the vitamin B 12 producing strain, which was washed and inoculated in the vitamin B 12 detection medium, and the filtered cold sterilization method was used in the vitamin B 12 detection medium.
  • the pretreated xylose mother liquor is used as a raw material.
  • intermittently applying light treatment to the fermentation broth in the vitamin B 12 detection medium can significantly improve the vitamin B 12 production strain vitamin B 12 Yield.
  • the invention also has the characteristics of simple operation and low technical difficulty.
  • a preferred embodiment of the method for microbial fermentation of vitamin B 12 based on xylose mother liquor microbial regulation by the present invention includes the following steps:
  • Step 1 The seeds of the vitamin B 12 production strain are cultured and activated, and then the activated vitamin B 12 production strain is inoculated with 5% into the SLB medium for expansion and placed at 4 ° C for use.
  • Step two Pre-treat the xylose mother liquor by filtration and cold sterilization.
  • Step 3 Centrifuge the vitamin B 12 producing strain obtained in step 1 and discard the supernatant medium to obtain the precipitated strain producing vitamin B 12 ; wash the precipitated strain and thoroughly wash away the SLB attached to its surface Culture medium.
  • Step 4 Inoculate the washed precipitated strain producing vitamin B 12 into the vitamin B 12 detection medium containing the xylose mother liquor pretreated in step two as the medium raw material, and perform fermentation treatment together with the xylose mother liquor.
  • static media 12 detects the vitamin B in a darkened environment 65h ⁇ 80h, and at 10 ⁇ 50cm is provided at the light source of vitamin B 12 assay medium from said source of vitamin B 12 assay medium is irradiated intermittently, the light source Each light time lasts 10min ⁇ 80min, and the ratio of light time to dark time (non-light time) is 1: 2 ⁇ 5: 1.
  • Step 5 The fermentation solution after the fermentation treatment in Step 4 is subjected to cell disruption treatment, and then the fermentation broth after the cell disruption treatment is subjected to low-temperature centrifugation. After centrifugation, a supernatant containing vitamin B 12 is obtained, and the supernatant is added to the supernatant. Dilute sodium cyanide solution, and then extract the treated supernatant through SPE column to extract vitamin B 12 , collect the eluent containing vitamin B 12 after extraction, and blow dry with nitrogen. The product after the eluent is dried is Vitamin B 12 .
  • the dilute sodium cyanide solution is added to the supernatant in order to convert other forms of vitamin B 12 into cyanocobalamin, to avoid the degradation of the resulting gland cobalamin, methylcobalamin, or hydroxycobalamin due to instability.
  • the dilute sodium cyanide solution is a 0.1M dipotassium hydrogen phosphate solution, pH 6.0, containing 0.1 mg / mL sodium cyanide.
  • the vitamin B 12 production seed strain is Propionibacterium freudenreichii or Propionibacterium acnes Xie
  • the vitamin B 12 production seed strain was cultured activation means: the seeds of vitamin B 12 from the production strains Inoculate 1% to 500 mL of MRS liquid culture medium in a preservation tube and place it in a 37 ° C / 30 ° C incubator for anaerobic culture for 10h to 14h.
  • the composition of the MRS liquid culture medium includes (g / L): peptone 10.0 , Beef powder 5.0, yeast powder 4.0, glucose 20.0, dipotassium phosphate 2.0, sodium acetate 5.0, triammonium citrate 2.0, magnesium sulfate 0.2, manganese sulfate 0.05, Tween-80 1.0, pH 6.5 ⁇ 0.2.
  • the expansion method is to place the vitamin B 12 producing strain in the SLB medium, and culture in a 37 ° C / 30 ° C incubator for 2h ⁇ 9h, so that the colony forming unit in the medium reaches 10 8 CFU / mL, where the components of SLB medium include (weight ratio): 1% tryptone, 1% yeast extract, 1.5% glucose, 0.25 ⁇ K 2 HPO 4 , 0.05 ⁇ MnSO 4 , 0.1% Tween-80, adjust pH6 .5 ⁇ 0.2.
  • the tools used in the cold filtration sterilization method include: a syringe, a filter, and a 0.22 ⁇ m filter membrane.
  • the operation steps of the cold filtration sterilization method include: attaching the filter to the filter membrane tightly, and then assembling Tighten the filter of the filter membrane and the syringe, load the xylose mother liquor to be sterilized into the syringe, and then slowly push the syringe to make the xylose mother liquor in the syringe flow through the filter and sterilize.
  • the whole process is aseptic operation Operation on stage.
  • the xylose mother liquor contains carbohydrates and proteins, and the Maillard reaction will occur under high temperature conditions, which affects the sugar concentration in the xylose mother liquor. Therefore, the xylose mother liquor must be cold sterilized.
  • step three the condition of centrifugal treatment is centrifugation at 3000r / min for 10 minutes at 4 ° C.
  • the method for washing the precipitated strain is to wash the precipitated strain repeatedly three times with 0.9% sodium chloride solution to thoroughly remove the surface of the attached bacteria SLB medium.
  • the components of the vitamin B 12 detection medium include (g / L): acid hydrolyzed casein 12.0, riboflavin 0.002, Tween-80 2.0, dipotassium hydrogen phosphate 1.0, potassium dihydrogen phosphate 1.0 , Magnesium sulfate 0.4, Ferrous sulfate 0.02, Manganese sulfate 0.02, 1% by weight of xylose mother liquor, pH is 6.3 ⁇ 0.2.
  • the light source is an incandescent lamp with a power of 15-100W.
  • the power of the incandescent lamp is preferably 60 W
  • the distance is preferably 30 cm
  • the ratio of light time / dark time is preferably 1: 1
  • the time of each light is preferably 30 min.
  • the cell disruption process refers to the ultrasonic disruption of the fermentation solution using a cell disruption instrument.
  • the working power of the cell disruption instrument is 400 W, the working time is 3 s, and the intermittent time is 3 s, repeated 99 times.
  • the low-temperature centrifugal treatment refers to centrifuging the fermentation broth at 4 ° C for 8 minutes at a rotational speed of 6000 r / min.
  • the steps of the SPE column extraction method include: activating the SPE column with 5 mL of 90% methanol, washing the methanol three times with 15 mL of ultrapure water, equilibrating the column, and processing the supernatant after passing through the SPE column , Wash the impurities adsorbed on the SPE column with 5mL of ultrapure water first, and then use a vacuum pump to drain the liquid, and then wash the SPE column with methanol. The cyanocobalamin adsorbed on the SPE column is eluted. Subsequent processing.
  • cyanocobalamin 12 is chemically most stable form of vitamin B.
  • the treated supernatant flows through the SPE column, the material in the supernatant is absorbed in the column. There are differences in the adsorption of different substances on the column packing. When washing with ultrapure water, impurities will be eluted preferentially, while methanol has an elution effect on cyanocobalamin, thereby extracting cyanocobalamin.
  • vitamin B 12 In addition to SPE column extraction of vitamin B 12 , it can be replaced by ion exchange resin method and organic solvent extraction method.
  • the present invention sets a light group and a control group for comparative experiments.
  • the production of vitamin B 12 using the method of the present invention is called the light group, while the control group refers to the production process conditions except that the vitamin B 12 detection medium is placed in a dark environment for 72h in step four, and no light is applied throughout the process. , The other conditions are exactly the same as the lighting group.
  • the dried product prepared in Step 5 was dissolved in 1 mL of ultrapure water, and detected by HPLC method.
  • the concentration of cyanocobalamin in the control group was 2.57mg / L
  • the concentration in the light group was 5.08mg / L
  • the concentration in the light group was about twice that of the control group, indicating that the fermentation broth containing the xylose mother liquor could be significantly increased after intermittent light
  • the production of vitamin B 12 producing vitamin B 12 achieves the object of the present invention.
  • the HPLC chromatographic conditions of the HPLC detection method are: chromatographic column C18 4.6 * 250 mm, mobile phase 0.1% formic acid aqueous solution-acetonitrile, flow rate 1.0 mL / min, injection volume 20 ⁇ L, detection wavelength 361 nm.

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Abstract

一种基于光照调控的木糖母液微生物发酵维生素B 12的方法，将生产菌株的种子进行培养活化，在SLB培养基中扩培，离心处理得到沉淀菌株，洗涤后接种在维生素B 12检测培养基中，并在维生素B 12检测培养基中使用经过过滤冷灭菌方法预处理的木糖母液作为原料，在生产维生素B 12的沉淀菌株发酵过程中，间歇性地对维生素B 12检测培养基中发酵液实施光照处理。

Description

基于光照调控的木糖母液微生物发酵维生素B 12 的方法 技术领域
本发明微生物发酵技术领域，特别涉及一种基于光照调控的木糖母液微生物发酵维生素B 12的方法。
背景技术
自然界中维生素B 12只能由微生物合成，主要是细菌和放线菌，细菌以丙酸杆菌、脱氮假单胞菌为主，放线菌主要是链霉菌属。维生素B 12早先是从抗生素废液中提取的，但是提取的效率低。化学合成又极其复杂，成本很高，关键是得到的产物纯度低，具有大量副产物，不适合产业化。
目前工业化生产维生素B 12主要使用微生物发酵法，因此一种提高产量对于发酵生产维生素B 12具有重大意义。公布号CN102453690A的中国专利中公开了一种脱氮假单胞菌发酵生产维生素B 12高产菌株的诱变方法，通过诱变筛选的方法获取维生素B 12高产菌株，该方法的主要缺点是诱变存在不确定性，且技术要求高。公告号CN108251476A的中国专利中公开了从酶制剂废水中提取维生素B 12的方法，以酶制剂废水为原料，经过一系列处理后提取维生素B 12，该方法主要的缺点是酶制剂废水需经过多道工序处理，相对比较复杂。YUE YU等人（Yue Yu、Xuan Zhu、etc，Enhancing the vitamin B 12 production and growth of Propionibacterium freudenreichii in tofu wastewater via a light-induced vitamin B 12 riboswitch，Appl Microbiol Biotechnol，DOI 10.1007/s00253-015-6958-6）已经研究发现，蓝光通过激发维生素B 12核糖体开关可以提高豆腐废水发酵过程中维生素B 12的产量，但是该研究发酵过程中在豆腐废水中添加了硫酸钴，而已有研究表明钴化合物能够增加维生素B 12的合成。
技术问题
本发明所要解决的技术问题在于，提供一种基于光照调控的木糖母液微生物发酵维生素B 12的方法，提高维生素B 12生产菌株在木糖母液培养基中维生素B 12产量，操作简单、技术难度低，在不添加能够增加维生素B 12产量物质的情况下，利用光照提高维生素B 12的产量。
技术解决方案
本发明是这样实现的，提供一种基于光照调控的木糖母液微生物发酵维生素B 12的方法，包括如下步骤：
步骤一、将维生素B 12生产菌株的种子进行培养活化，然后将活化后的维生素B 12生产菌株接种5%至SLB培养基中扩培后置于4℃温度下待用；
步骤二、采用过滤冷灭菌方法对木糖母液先进行预处理；
步骤三、将步骤一中扩培得到的维生素B 12生产菌株进行离心处理，弃上清液得到生产维生素B 12的沉淀菌株，将该沉淀菌株进行洗涤；
步骤四、将洗涤后的生产维生素B 12的沉淀菌株接种至含有步骤二预处理后的木糖母液为培养基原料的维生素B 12检测培养基中，与木糖母液一起进行发酵处理，同时将该维生素B 12检测培养基静置于黑暗环境中65h~80h，而且，在距离维生素B 12检测培养基10~50cm处设置光源，所述光源对维生素B 12检测培养基进行间歇性照射，光源每次光照时间持续10min~80min，其光照时间与黑暗时间（非光照时间）的比例为1:2~5:1；
步骤五、将步骤四发酵处理后的发酵溶液进行细胞破碎处理，再将细胞破碎处理后的发酵液进行低温离心处理，离心后得到含维生素B 12的上清液，向该上清液中加入稀氰化钠溶液，再将处理后的上清液通过SPE柱萃取方式萃取维生素B 12，收集萃取后含有维生素B 12的洗脱液，用氮气吹干，洗脱液干燥后的产物即为维生素B 12。
进一步地，在步骤一中，所述维生素B 12生产菌株的种子为费氏丙酸杆菌或谢氏丙酸杆菌，将维生素B 12生产菌株的种子进行培养活化是指：将维生素B 12生产菌株的种子从保藏管中接种1%至500mL MRS液体培养基中，置于培养箱中厌氧培养10h~14h，其中，所述MRS液体培养基的成分包括（g/L）：蛋白胨10.0，牛肉浸粉5.0，酵母浸粉4.0，葡萄糖20.0，磷酸氢二钾2.0，乙酸钠5.0，柠檬酸三铵2.0，硫酸镁0.2，硫酸锰0.05，吐温-80 1.0，pH为6.5±0.2。
进一步地，在步骤一中，扩培方式是将维生素B 12生产菌株置于SLB培养基中，在培养箱中静置培养2h~9h，使培养基中菌落形成单位达10 8CFU/mL，其中，SLB培养基成分包括（重量比）：1%胰蛋白胨，1%酵母提取物，1.5%葡萄糖，0.25‰K 2HPO 4，0.05‰MnSO 4，0.1%吐温-80，调节pH 6.5±0.2。
进一步地，在步骤二中，所述过滤冷灭菌方法使用的工具包括：注射器、过滤器和0.22 μm滤膜，过滤冷灭菌方法的操作步骤包括：将过滤器加上滤膜后盖紧，将组装好滤膜的过滤器与注射器旋紧，将待灭菌的木糖母液装入注射器中，然后缓慢推动注射器，使注射器里的木糖母液从过滤器中流过后灭菌，整个过程在无菌操作台上操作。
进一步地，在步骤三中，离心处理的条件是在4℃、3000r/min下离心10min，洗涤该沉淀菌株的方式是用0.9%氯化钠溶液将沉淀菌株反复洗涤三遍。
进一步地，在步骤四中，所述维生素B 12检测培养基的成分包括（g/L）：酸水解酪蛋白12.0，核黄素0.002，吐温-80 2.0，磷酸氢二钾1.0，磷酸二氢钾1.0，硫酸镁0.4，硫酸亚铁0.02，硫酸锰0.02，重量比1%的木糖母液，pH为6.3±0.2。
进一步地，在步骤四中，所述光源为白炽灯，其功率为15~100W。
进一步地，在步骤五中，所述细胞破碎处理是指使用细胞破碎仪对发酵溶液进行超声破碎，细胞破碎仪的工作功率为400W，工作时间3s，间歇时间3s，重复99次。
进一步地，在步骤五中，所述低温离心处理是指将发酵液于4℃条件下离心8min，转速6000 r/min。
进一步地，在步骤五中，所述SPE柱萃取方式的步骤包括：用5mL 90%的甲醇活化SPE柱子，再用15mL超纯水分三次洗涤甲醇，平衡柱子，处理后的上清液流过SPE柱后，先用5mL超纯水洗涤SPE柱吸附的杂质，再用真空泵抽干液体，然后用甲醇洗涤SPE柱，收集洗脱液留待后续处理。
 有益效果
与现有技术相比，本发明的基于光照调控的木糖母液微生物发酵维生素B 12的方法，将利用维生素B 12生产菌株的种子进行培养活化得到的维生素B 12生产菌株扩培在SLB培养基中，将维生素B 12生产菌株进行离心处理得到的生产维生素B 12的沉淀菌株经过洗涤后接种在维生素B 12检测培养基中，并在维生素B 12检测培养基中使用了经过过滤冷灭菌方法预处理的木糖母液作为原料，在生产维生素B 12的沉淀菌株发酵过程中，间歇性地对维生素B 12检测培养基中发酵液实施光照处理，能够明显提高维生素B 12生产菌株维生素B 12的产量。本发明还具有操作简单、技术难度低等特点。
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在此处键入附图说明描述段落。
 本发明的最佳实施方式
为了使本发明所要解决的技术问题、技术方案及有益效果更加清楚明白，以下结合实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
本发明基于光照调控的木糖母液微生物发酵维生素B 12的方法的较佳实施例，包括如下步骤：
步骤一、将维生素B 12生产菌株的种子进行培养活化，然后将活化后的维生素B 12生产菌株接种5%至SLB培养基中扩培后置于4℃温度下待用。
步骤二、采用过滤冷灭菌方法对木糖母液先进行预处理。
步骤三、将步骤一中扩培得到的维生素B 12生产菌株进行离心处理，弃上清液培养基得到生产维生素B 12的沉淀菌株，将该沉淀菌株进行洗涤，彻底洗去附着其表面的SLB培养基。
步骤四、将洗涤后的生产维生素B 12的沉淀菌株接种至含有步骤二预处理后的木糖母液为培养基原料的维生素B 12检测培养基中，与木糖母液一起进行发酵处理，同时将该维生素B 12检测培养基静置于黑暗环境中65h~80h，而且，在距离维生素B 12检测培养基10~50cm处设置光源，所述光源对维生素B 12检测培养基进行间歇性照射，光源每次光照时间持续10min~80min，其光照时间与黑暗时间（非光照时间）的比例为1:2~5:1。
步骤五、将步骤四发酵处理后的发酵溶液进行细胞破碎处理，再将细胞破碎处理后的发酵液进行低温离心处理，离心后得到含维生素B 12的上清液，向该上清液中加入稀氰化钠溶液，再将处理后的上清液通过SPE柱萃取方式萃取维生素B 12，收集萃取后含有维生素B 12的洗脱液，用氮气吹干，洗脱液干燥后的产物即为维生素B 12。在上清液中加入稀氰化钠溶液，旨在把其他形式的维生素B 12转化成氰钴胺，避免产生的腺钴胺、甲钴胺或羟钴胺因不稳定而降解。所述稀氰化钠溶液为0.1M磷酸氢二钾溶液，pH6.0，含0.1mg/mL氰化钠。
本发明的实施方式
在步骤一中，所述维生素B 12生产菌株的种子为费氏丙酸杆菌或谢氏丙酸杆菌，将维生素B 12生产菌株的种子进行培养活化是指：将维生素B 12生产菌株的种子从保藏管中接种1%至500mL MRS液体培养基中，置于37℃/30℃培养箱中厌氧培养10h~14h，其中，所述MRS液体培养基的成分包括（g/L）：蛋白胨10.0，牛肉浸粉5.0，酵母浸粉4.0，葡萄糖20.0，磷酸氢二钾2.0，乙酸钠5.0，柠檬酸三铵2.0，硫酸镁0.2，硫酸锰0.05，吐温-80 1.0，pH为6.5±0.2。
在步骤一中，扩培方式是将维生素B 12生产菌株置于SLB培养基中，在37℃/30℃培养箱中静置培养2h~9h，使培养基中菌落形成单位达10 8CFU/mL，其中，SLB培养基成分包括（重量比）：1%胰蛋白胨，1%酵母提取物，1.5%葡萄糖，0.25‰K 2HPO 4，0.05‰MnSO 4，0.1%吐温-80，调节pH6.5±0.2。
在步骤二中，所述过滤冷灭菌方法使用的工具包括：注射器、过滤器和0.22μm滤膜，过滤冷灭菌方法的操作步骤包括：将过滤器加上滤膜后盖紧，将组装好滤膜的过滤器与注射器旋紧，将待灭菌的木糖母液装入注射器中，然后缓慢推动注射器，使注射器里的木糖母液从过滤器中流过后灭菌，整个过程在无菌操作台上操作。
木糖母液中含有糖类物质和蛋白质，在高温条件下会发生美拉德反应，影响木糖母液中的糖浓度，因此对于木糖母液要进行冷灭菌处理。
在步骤三中，离心处理的条件是在4℃、3000r/min下离心10min，洗涤该沉淀菌株的方式是用0.9%氯化钠溶液将沉淀菌株反复洗涤三遍，彻底洗去附着菌体表面的SLB培养基。
在步骤四中，所述维生素B 12检测培养基的成分包括（g/L）：酸水解酪蛋白12.0，核黄素0.002，吐温-80 2.0，磷酸氢二钾1.0，磷酸二氢钾1.0，硫酸镁0.4，硫酸亚铁0.02，硫酸锰0.02，重量比1%的木糖母液，pH为6.3±0.2。
在步骤四中，所述光源为白炽灯，其功率为15~100W。白炽灯的功率优选60W，距离优选30cm，光照时间/黑暗时间比例优选1:1，每次光照时间优选30min。
在步骤五中，所述细胞破碎处理是指使用细胞破碎仪对发酵溶液进行超声破碎，细胞破碎仪的工作功率为400W，工作时间3s，间歇时间3s，重复99次。
在步骤五中，所述低温离心处理是指将发酵液于4℃条件下离心8min，转速6000r/min。
在步骤五中，所述SPE柱萃取方式的步骤包括：用5mL 90%的甲醇活化SPE柱子，再用15mL超纯水分三次洗涤甲醇，平衡柱子，处理后的上清液流过SPE柱后，先用5mL超纯水洗涤SPE柱吸附的杂质，再用真空泵抽干液体，然后用甲醇洗涤SPE柱，SPE柱吸附的氰钴胺被洗脱出来，收集含氰钴胺的洗脱液留待后续处理。因为，上清液加入稀氰化钠溶液处理后，所有形式的维生素B 12都转化为氰钴胺，氰钴胺是维生素B 12最稳定的化学形式。处理后的上清液流过SPE柱时，上清液中的物质吸附在柱子中。柱子填料对不同物质的吸附力存在差异，当超纯水洗涤时，杂质会优先被洗脱，而甲醇则对氰钴胺有洗脱作用，从而将氰钴胺萃取出来。
除了采用SPE柱萃取维生素B 12外，可以用离子交换树脂法、有机溶剂提取法进行代替。
为了说明本发明的方法的效果，本发明设置了光照组和对照组进行对比实验。采用本发明方法进行维生素B 12生产的称为光照组，而对照组是指其生产过程条件除了在步骤四中将维生素B 12检测培养基静置于黑暗环境中72h，全程不进行光照之外，其它条件与光照组的完全一致。最后将步骤五制得的干燥后的产物用1mL超纯水溶解，采用HPLC方法进行检测。对照组的氰钴胺浓度为2.57mg/L，光照组的浓度为5.08mg/L，光照组的浓度大约是对照组的两倍，表明含有木糖母液的发酵液经过间隙光照后能够明显提高维生素B 12生产菌株维生素B 12的产量，实现本发明的目的。
其中，HPLC的检测方法的HPLC色谱条件是：色谱柱C18 4.6*250 mm，流动相0.1%甲酸水溶液-乙腈，流速1.0mL/min，进样体积20μL，检测波长361nm。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。
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Claims (10)

  1. 一种基于光照调控的木糖母液微生物发酵维生素B 12的方法，其特征在于，包括如下步骤：
    步骤一、将维生素B 12生产菌株的种子进行培养活化，然后将活化后的维生素B 12生产菌株接种5%至SLB培养基中扩培后置于4℃温度下待用；
    步骤二、采用过滤冷灭菌方法对木糖母液先进行预处理；
    步骤三、将步骤一中扩培得到的维生素B 12生产菌株进行离心处理，弃上清液得到生产维生素B 12的沉淀菌株，将该沉淀菌株进行洗涤；
    步骤四、将洗涤后的生产维生素B 12的沉淀菌株接种至含有步骤二预处理后的木糖母液为培养基原料的维生素B 12检测培养基中，与木糖母液一起进行发酵处理，同时将该维生素B 12检测培养基静置于黑暗环境中65h~80h，而且，在距离维生素B 12检测培养基10~50cm处设置光源，所述光源对维生素B 12检测培养基进行间歇性照射，光源每次光照时间持续10min~80min，其光照时间与黑暗时间的比例为1:2~5:1；
    步骤五、将步骤四发酵处理后的发酵溶液进行细胞破碎处理，再将细胞破碎处理后的发酵液进行低温离心处理，离心后得到含维生素B 12的上清液，向该上清液中加入稀氰化钠溶液，再将处理后的上清液通过SPE柱萃取方式萃取维生素B 12，收集萃取后含有维生素B 12的洗脱液，用氮气吹干，洗脱液干燥后的产物即为维生素B 12。
  2. 如权利要求1所述的基于光照调控的木糖母液微生物发酵维生素B 12的方法，其特征在于，在步骤一中，所述维生素B 12生产菌株的种子为费氏丙酸杆菌或谢氏丙酸杆菌，将维生素B 12生产菌株的种子进行培养活化是指：将维生素B 12生产菌株的种子从保藏管中接种1%至500mL MRS液体培养基中，置于培养箱中厌氧培养10h~14h，其中，所述MRS液体培养基的成分包括（g/L）：蛋白胨10.0，牛肉浸粉5.0，酵母浸粉4.0，葡萄糖20.0，磷酸氢二钾2.0，乙酸钠5.0，柠檬酸三铵2.0，硫酸镁0.2，硫酸锰0.05，吐温-80 1.0，pH 为6.5±0.2。
  3. 如权利要求1所述的基于光照调控的木糖母液微生物发酵维生素B 12的方法，其特征在于，在步骤一中，扩培方式是将维生素B 12生产菌株置于SLB培养基中，在培养箱中静置培养2h~9h，使培养基中菌落形成单位达10 8CFU/mL，其中，SLB培养基成分包括（重量比）：1%胰蛋白胨，1%酵母提取物，1.5%葡萄糖，0.25‰K 2HPO 4，0.05‰MnSO 4，0.1%吐温-80，调节pH 6.5±0.2。
  4. 如权利要求1所述的基于光照调控的木糖母液微生物发酵维生素B 12的方法，其特征在于，在步骤二中，所述过滤冷灭菌方法使用的工具包括：注射器、过滤器和0.22μm滤膜，过滤冷灭菌方法的操作步骤包括：将过滤器加上滤膜后盖紧，将组装好滤膜的过滤器与注射器旋紧，将待灭菌的木糖母液装入注射器中，然后缓慢推动注射器，使注射器里的木糖母液从过滤器中流过后灭菌。
  5. 如权利要求1所述的基于光照调控的木糖母液微生物发酵维生素B 12的方法，其特征在于，在步骤三中，离心处理的条件是在4℃、3000r/min下离心10min，洗涤该沉淀菌株的方式是用0.9%氯化钠溶液将沉淀菌株反复洗涤三遍。
  6. 如权利要求1所述的基于光照调控的木糖母液微生物发酵维生素B 12的方法，其特征在于，在步骤四中，所述维生素B 12检测培养基的成分包括（g/L）：酸水解酪蛋白12.0，核黄素0.002，吐温-80 2.0，磷酸氢二钾1.0，磷酸二氢钾1.0，硫酸镁0.4，硫酸亚铁0.02，硫酸锰0.02，重量比1%的木糖母液，pH为6.3±0.2。
  7. 如权利要求1所述的基于光照调控的木糖母液微生物发酵维生素B 12的方法，其特征在于，在步骤四中，所述光源为白炽灯，其功率为15~100W。
  8. 如权利要求1所述的基于光照调控的木糖母液微生物发酵维生素B 12的方法，其特征在于，在步骤五中，所述细胞破碎处理是指使用细胞破碎仪对发酵溶液进行超声破碎，细胞破碎仪的工作功率为400W，工作时间3s，间歇时间3s，重复99次。
  9. 如权利要求1所述的基于光照调控的木糖母液微生物发酵维生素B 12的方法，其特征在于，在步骤五中，所述低温离心处理是指将发酵液于4℃条件下离心8min，转速6000r/min。
  10. 如权利要求1所述的基于光照调控的木糖母液微生物发酵维生素B 12的方法，其特征在于，在步骤五中，所述SPE柱萃取方式的步骤包括：用5mL 90%的甲醇活化SPE柱子，再用15mL超纯水分三次洗涤甲醇，平衡柱子，处理后的上清液流过SPE柱后，先用5mL超纯水洗涤SPE柱吸附的杂质，再用真空泵抽干液体，然后用甲醇洗涤SPE柱，收集洗脱液留待后续处理。
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