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WO2018099479A1 - 一种高纯度d-阿洛酮糖的制备方法 - Google Patents

一种高纯度d-阿洛酮糖的制备方法 Download PDF

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WO2018099479A1
WO2018099479A1 PCT/CN2017/114289 CN2017114289W WO2018099479A1 WO 2018099479 A1 WO2018099479 A1 WO 2018099479A1 CN 2017114289 W CN2017114289 W CN 2017114289W WO 2018099479 A1 WO2018099479 A1 WO 2018099479A1
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psicose
liquid
preparation
follows
reaction
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PCT/CN2017/114289
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English (en)
French (fr)
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窦宝德
窦光朋
张明站
邵先豹
李方华
杜倩
杨腾腾
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山东百龙创园生物科技股份有限公司
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Priority to US16/466,060 priority Critical patent/US11149048B2/en
Publication of WO2018099479A1 publication Critical patent/WO2018099479A1/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
    • C07H1/08—Separation; Purification from natural products
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/02—Monosaccharides
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • 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
    • C12P19/02—Monosaccharides
    • 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
    • C12P19/24—Preparation of compounds containing saccharide radicals produced by the action of an isomerase, e.g. fructose
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y501/00—Racemaces and epimerases (5.1)
    • C12Y501/03—Racemaces and epimerases (5.1) acting on carbohydrates and derivatives (5.1.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y503/00—Intramolecular oxidoreductases (5.3)
    • C12Y503/01—Intramolecular oxidoreductases (5.3) interconverting aldoses and ketoses (5.3.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/13—Crystalline forms, e.g. polymorphs

Definitions

  • the invention relates to a preparation method of high-purity D-psicose, belonging to the field of biotechnology.
  • D-psicose is an important rare sugar found in nature in very small amounts in sugar cane molasses, dried fruit, sugar products, wheat and thorn plants. Its name stems from the isolation of a small amount of D-psicose from the antibiotic apyrone glucoside. D-psicose is absorbed into the blood circulation through the small intestine in the human body, is not metabolized into energy after absorption in the small intestine, and has low fermentation utilization for intestinal microorganisms. D-psicose has a variety of important physiological functions: neuroprotection, blood sugar, fat loss, scavenging reactive oxygen species, anti-oxidation, inhibiting cancer cell proliferation, low-calorie sweeteners, etc.
  • D-psicose can protect the nerve by increasing intracellular glutathione levels, lowering blood sugar, reducing fat, improving product quality, and higher antioxidant function; as a sweetener, D-Alo
  • the ketose sweetness is equivalent to 70% of fructose, and the energy is only 0.3% of sucrose. It produces almost no energy and no toxicity to growing mice.
  • D-psicose can inhibit liver fat synthesis in mice.
  • Enzyme activity which reduces the accumulation of abdominal fat, can be used as a sweetener in assisting weight loss;
  • D-psicose shows a strong function of scavenging reactive oxygen species, showing it in various disease prevention and treatment Out of potential medical value.
  • Chinese patent document CN105602879A (Application No. 201610051547.3) discloses a genetically engineered strain for efficiently secreting D-psicose 3-epimerase and a method for constructing the same.
  • the invention realizes RDPE in Bacillus subtilis by constructing a recombinant expression plasmid pMA5-RDPE by using D-psicose 3-epimerase gene rdpe derived from Ruminococcus sp. 5_1_39B_FAA, and then transforming Bacillus subtilis The constitutive secretory expression.
  • the optimal inducible promoter PxylA was obtained and the secretion level of RDPE was significantly increased.
  • the xylose utilization gene xylAB xylA and xylB
  • the xylose metabolism pathway of Bacillus subtilis was blocked, and the secretion of RDPE was further increased, and the optimal induction concentration of the inducer xylose was reduced from 4.0% to 0.5%.
  • the engineered strain 1A751SD-XR was evaluated in a 7.5L fermentor by means of fed-batch, and the secretion level of RDPE was up to 95U/mL and 2.6g/L.
  • the enzyme activity of the enzyme is still low and cannot meet the needs of actual production.
  • D-psicose currently has problems such as low conversion rate and easy decomposition, which greatly limits the application field of D-psicose, and it is difficult to effectively exert its own characteristics.
  • the present invention provides a method for preparing high-purity D-psicose according to the deficiencies of the prior art.
  • a method for preparing D-psicose the steps are as follows:
  • Bacillus subtilis is Bacillus subtilis BLCY-005, and was deposited on October 26, 2016 in the General Microbiology Center of China Microbial Culture Collection Management Committee, deposit number CGMCC No. 13152, Address: Chaoyang District, Beijing Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road;
  • step (3) The crude D-psicose obtained in the step (2) is subjected to decolorization, filtration, separation, chromatographic separation, concentration, and then crystallization or drying to obtain D-psicose.
  • the centrifugation conditions are: a temperature of 10 to 20 ° C, a rotation speed of 3000 r / min, and a time of 30 to 50 minutes.
  • the homogenization conditions are: a temperature of 10 to 20 ° C, a pressure of 30 mPa to 50 mPa, and a time of 10 to 20 min.
  • the preparation method of the fermentation broth of Bacillus subtilis is as follows:
  • Bacillus subtilis BLCY-005 is inoculated into a seed culture medium, and cultured at 30 to 38 ° C for 6 to 12 hours to prepare a seed liquid;
  • the seed medium components are as follows, all in weight percentage:
  • Peptone 1% peptone 1%, yeast dipping powder 0.5%, sodium chloride 1%, anhydrous magnesium sulfate 0.01%, potassium dihydrogen phosphate 0.02%, balance water, pH 6.0-7.0;
  • the seed liquid prepared in the step I is inoculated into the fermentation medium at a ratio of 1 to 10% by volume, and fermented at 30 to 38 ° C for 30 to 48 hours to prepare a fermentation liquid of Bacillus subtilis;
  • the fermentation medium components are as follows, all in weight percentage:
  • Yeast extract powder 3% corn syrup powder 2%, glucose 1%, anhydrous magnesium sulfate 0.01%, diammonium phosphate 0.02%, ammonium sulfate 0.02%, balance water, pH 6.0-7.0.
  • the mixed solution containing D-psicose 3-epimerase is added in an amount of 5% by volume based on the fructose solution.
  • the decolorization step is as follows:
  • the crude D-psicose prepared in the step (2) is added to the activated carbon in a ratio of 0.5 to 1% by mass, and stirred at 80 to 85 ° C for 30 to 40 minutes.
  • the filtration is carried out by plate and frame filtration, and the filtration pressure is 0.2 to 0.4 MPa, and the water flow rate is 5.0 to 6.0 t/h.
  • the leaving step is as follows:
  • the decolorized and filtered crude sugar solution was passed through a continuous ion exchange system at a flow rate of 3 times the resin volume per hour at 35 to 55 ° C. Desalination is carried out, and the transmittance of the liquid after detachment is ⁇ 98%.
  • the treated liquid is clear and transparent, and has no odor.
  • the chromatographic separation step is as follows:
  • the chromatographic operating pressure is 0.20 to 0.30 MPa
  • the temperature is 60 to 70 ° C
  • the water consumption ratio is 1: (1.3 to 1.6)
  • the feed is 1.5 to 2.0 m 3 per hour
  • D-psicose is collected.
  • the four-effect falling film evaporator is used for concentration
  • the degree of vacuum is 0.06-0.09 MPa
  • the temperature of the feed liquid is 50-85 ° C
  • the concentration is 60-75% of the original volume.
  • the drying is spray drying, and the steps are as follows:
  • the concentrated sugar liquid enters the drying tower, the inlet air temperature is 130-150 ° C, the atomizer is activated, and the liquid is spray-dried into a powdery solid.
  • the crystallization reaction conditions are:
  • the concentration of the sugar liquid is 70-85%, the temperature is 50-70 °C, the seed crystal with the solute mass is 10-30%, and the mixture is evenly stirred. It is allowed to stand at 50-70 ° C for 8-16 hours, and then decreased by 1 ° C according to 3-6 h. The rate is slowly lowered while stirring slowly until a large amount of uniform, regular crystal grains are formed in the solution and separated to obtain D-psicose.
  • the present invention separates Bacillus subtilis from soil, and is subjected to mutagenesis treatment techniques such as ultraviolet mutagenesis and nitrosoguanidine mutagenesis treatment, and finally obtains a high-yield strain of high-yield D-psicose epimerase.
  • mutagenesis treatment techniques such as ultraviolet mutagenesis and nitrosoguanidine mutagenesis treatment
  • a high-yield strain of high-yield D-psicose epimerase For BLCY-005, its enzyme activity reaches 143U/ml, which is more than 50% higher than the traditional D-psicose epimerase activity, which greatly improves the ability of sucrose to be converted into D-psicose.
  • the method of adding fructose makes the D-psicose content of the final product reach 99%, which is significantly better than the products obtained by the prior preparation, and the production cost is significantly reduced.
  • the optimum pH value is 5.5-6.5, and the
  • the invention adopts the direct production of the fermentation liquid of Bacillus subtilis, and the process of extracting the enzyme is omitted, the production cost is greatly reduced, and the cost is reduced by about 25% compared with the traditional production method, so that the competitiveness of the product is greatly enhanced;
  • the present invention obtains high-purity D-psicose, D-alloxone, by adjusting the process steps and parameters by using D-psicose epimerase which is significantly higher than the existing enzyme activity.
  • a low-calorie sweetener sugar can be widely used in food and beverage, cosmetics, medicine and other fields to achieve large-scale industrial production of D-psicose, which significantly reduces production costs and expands the application of D-psicose. range.
  • a strain of Bacillus subtilis BLCY-005 was deposited on October 26, 2016 in the General Microbiology Center of the China Microbial Culture Collection Management Committee. Address: Microbiology Research, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing , Deposit No. CGMCC No. 13152;
  • the original strain of Bacillus subtilis BLCY-005 of the present invention is isolated from the soil near the production workshop of Bailong Chuangyuan in Dezhou, Shandong province, and obtained after mutagenesis.
  • the specific separation process is as follows:
  • a scribing separation method take a large test tube containing 5 ml of sterile water, take 2 ml of the bacterial solution enriched and cultured in step (1), dilute it, shake well, and use an inoculating ring to aseptically pick Take the first step of the dilution solution and make the first parallel scribe line 3-4 on the side of the plate medium. Then rotate the culture dish at an angle of about 60 degrees to burn off the residue on the inoculation ring. After cooling, the same line method is used. The second line is crossed, and the same method is used to make the third and fourth lines in sequence. After the scribing is completed, the dish cover is covered, the culture dish is inverted, and after 24 hours at 28-38 ° C, a single colony is picked and inoculated on 10 slant medium, numbered 01-10.
  • the 01-10 slant seed was inoculated in a shake flask medium and cultured at 28-38 ° C for 24 h.
  • the conversion of D-psicose to D-fructose was measured on the 0-10 shake flask fermentation broth, and the flask was transformed into No. 06 shake flask. The highest rate reached 26.12%.
  • Plate medium composition peptone 1%, yeast dipping powder 0.5%, sodium chloride 1%, pH natural;
  • Shake flask medium composition yeast dip powder: 3%, corn syrup powder 2%, glucose 1%, anhydrous magnesium sulfate: 0.01%, diammonium hydrogen phosphate: 0.02%, ammonium sulfate 0.02%, pH 6.0-7.0;
  • UV-induced mutagenesis of No. 06 strain UV-induced mutagenesis was carried out by using a 15W ultraviolet lamp at 20cm, and the irradiation time was 120s.
  • the obtained high-yield strain was subjected to nitrosoguanidine mutagenesis treatment to finally obtain high conversion D-alloxone.
  • the saccharide isomerase producing strain was named BLCY-005, and its D-psicose 3-epimerase activity reached 143 U/ml, which was significantly higher than that of the wild strain of 75 U/ml.
  • Enzyme activity determination method 1 ml of the reaction system, add 800 ⁇ l of phosphate buffer 50 ml, pH 7.0, D-fructose dissolved concentration of 100 g / L, 200 ⁇ l of fermentation broth, 55 ° C for 10 min, then boil for 10 min to terminate Enzyme reaction.
  • Enzyme unit (U) The amount of enzyme required to catalyze the production of 1 ⁇ mol D-psicose per minute.
  • the colonies are stained white, translucent, the edges of the colonies are irregular, wavy, and the center is highly convex. Observed by microscopy, the strain is about 1.0-1.5 microns long and about 0.6-0.9 microns wide. It has no capsule and does not produce flagella.
  • the spores are located in the center of the cells or slightly polarized. After the spores are formed, they are not inflated.
  • Example 1 The method for culturing Bacillus subtilis BLCY-005 described in Example 1 is as follows:
  • Bacillus subtilis BLCY-005 was inoculated into LB medium, and activated at 35 ° C for 12 h to prepare an activated strain;
  • step (b) taking the activated strain obtained in the step (a), inoculation in a seed culture medium, and proliferating for 12 hours at 35 ° C to obtain a seed liquid;
  • the seed medium components are as follows, all in weight percentage:
  • step (c) taking the seed liquid prepared in the step (b), inoculated in a fermentation medium at a ratio of 5% by volume, and expanding the culture at 35 ° C for 48 hours to obtain a fermentation broth of Bacillus subtilis;
  • the fermentation medium components are as follows, all in weight percentage:
  • Yeast extract powder 3% corn syrup powder 2%, glucose 1%, anhydrous magnesium sulfate 0.01%, diammonium hydrogen phosphate 0.02%, ammonium sulfate 0.02%, balance water, pH 6.8.
  • a method for preparing D-psicose the steps are as follows:
  • Example 2 The fermentation broth of Bacillus subtilis prepared in Example 2 was centrifuged at 20 ° C and a rotation speed of 3000 r/min for 30 minutes, and then the cells were homogenized at a temperature of 20 ° C and a pressure of 30 mPa for 10 min. a mixture containing D-psicose 3-epimerase;
  • the decolorization step is as follows:
  • the crude D-psicose prepared in the step (2) is added to the activated carbon at a ratio of 0.5% by mass, and stirred at 85 ° C for 30 min;
  • the plate frame is filtered, the filtration pressure is 0.4 Mpa, and the water flow rate is 5.0 t/h;
  • the decolorized and filtered crude sugar liquid was subjected to separation and desalting by a continuous ion exchange system at a flow rate of 3 times the resin volume/hour at 55 ° C, and the transmittance of the liquid after the separation was ⁇ 98%;
  • the chromatographic operating pressure is 0.20 MPa, the temperature is 70 ° C, the water consumption ratio is 1:1.3, and the feed is 2.0 m 3 per hour, and D-psicose is collected;
  • the concentration adopts a four-effect falling film evaporator, the degree of vacuum is 0.06 MPa, the temperature of the feed liquid is 85 ° C, and is concentrated to 60% of the original volume;
  • the drying is spray drying, and the steps are as follows:
  • the concentrated sugar liquid enters the drying tower, the inlet air temperature is 150 ° C, the atomizer is started, and the liquid is spray-dried to obtain D-psicose.
  • a method for preparing D-psicose the steps are as follows:
  • Example 2 The fermentation broth of Bacillus subtilis prepared in Example 2 was centrifuged at 10 ° C and a rotation speed of 3000 r/min for 50 minutes, and then the cells were homogenized at a temperature of 10 ° C and a pressure of 50 mPa for 20 min. a mixture containing D-psicose 3-epimerase;
  • the decolorization step is as follows:
  • the crude D-psicose prepared in the step (2) is added to the activated carbon in a proportion of 1% by mass, and stirred at 80 ° C for 40 minutes;
  • the plate frame is filtered, the filtration pressure is 0.2 Mpa, and the water flow rate is 6.0 t/h;
  • the decolorized and filtered crude sugar liquid was subjected to separation and desalting by a continuous ion exchange system at a flow rate of 3 times the resin volume/hour at 35 ° C, and the transmittance of the liquid after the separation was ⁇ 98%;
  • the chromatographic operating pressure is 0.30 MPa, the temperature is 60 ° C, the water consumption ratio is 1:1.6, and the feed is 1.5 m 3 per hour, and D-psicose is collected;
  • the concentration adopts a four-effect falling film evaporator, the degree of vacuum is 0.09Mpa, the temperature of the feed liquid is 50 ° C, and is concentrated to 75% of the original volume;
  • the concentration of the sugar liquid is 70%, the temperature is 70 ° C, the seed crystal with 10% solute mass is added, stirred evenly, and allowed to stand at 70 ° C for 8 hours, then slowly cooled at a rate of 1 ° C at 6 h, while slowly stirring until the solution is formed. A large number of uniform, regular grains are separated and D-psicose is obtained.

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Abstract

一种高纯度D-阿洛酮糖的制备方法，步骤如下：（1）将枯草芽孢杆菌的发酵液经离心后，取菌体经均质处理，得到含有D-阿洛酮糖3-差向异构酶的混合液；（2）配制果糖溶液，加入含有D-阿洛酮糖3-差向异构酶的混合液，调pH值，添加氯化钴，保温反应，然后向反应液中流加果糖溶液，继续反应，停止反应，制得D-阿洛酮糖粗液；（3）将D-阿洛酮糖粗液经脱色、过滤、离交、色谱分离、浓缩后，再经过结晶或干燥，制得D-阿洛酮糖。

Description

一种高纯度D-阿洛酮糖的制备方法 技术领域
本发明涉及一种高纯度D-阿洛酮糖的制备方法，属于生物技术领域。
背景技术
D-阿洛酮糖是一种重要的稀有糖，在自然界中极少量地存在于甘蔗糖蜜、水果干、糖制品、小麦和鼠刺属植物中。其名称起源于从抗菌素阿洛酮糖腺苷中也可以分离到少量的D-阿洛酮糖。D-阿洛酮糖在人体内是通过小肠吸收进入血液循环中，在小肠吸收后不会被代谢成能量，且对于肠道微生物具有较低的发酵利用度。D-阿洛酮糖具有多种重要的生理功能：神经保护作用，将血糖、减脂，清除活性氧簇，抗氧化，抑制癌细胞增殖，低卡路里甜味剂等。
D-阿洛酮糖可通过提高细胞内谷胱甘肽水平，起到保护神经的作用，降血糖、减脂、改善产品品质、较高的抗氧化功能；作为甜味剂，D-阿洛酮糖甜度相当于果糖的70％，能量只有蔗糖的0.3％，对生长小鼠几乎不产生能量，没有毒性；与果糖和果糖相比，D-阿洛酮糖能抑制小鼠肝脏脂肪合成酶活性，减少腹脂积累，可以作为一种甜味剂在辅助减肥中使用；D-阿洛酮糖显示出较强的清除活性氧簇的功能，使其在多种疾病预防和治疗中显示出潜在的医疗价值。
中国专利文献CN105602879A(申请号201610051547.3)公开了一株高效分泌D-阿洛酮糖3-差向异构酶的基因工程菌株及其构建方法。该发明通过利用由瘤胃菌Ruminococcus sp.5_1_39B_FAA来源的D-阿洛酮糖3-差向异构酶基因rdpe构建重组表达质粒pMA5-RDPE，然后转化枯草芽孢杆菌，实现了RDPE在枯草芽孢杆菌中组成型分泌表达。通过比较三个糖诱导型启动子，得到最优诱导型启动子PxylA，并明显提高了RDPE的分泌水平。通过敲除木糖利用基因xylAB(xylA和xylB)，阻断了枯草芽孢杆菌的木糖代谢途径，进一步提高了RDPE的分泌量，并使诱导剂木糖的最优诱导浓度由4.0％降低为0.5％。最终，采用分批补料方式，在7.5L发酵罐中对工程菌株1A751SD-XR进行评价，RDPE的分泌水平最高可以达到95U/mL和2.6g/L。但该酶的酶活仍然较低，无法满足实际生产的需要。
但目前D-阿洛酮糖存在转化率低、易分解等问题，这样大大限制了D-阿洛酮糖的应用领域，产品本身特性难以有效发挥。
发明内容
本发明针对现有技术的不足，提供一种高纯度D-阿洛酮糖的制备方法。
本发明技术方案如下：
一种D-阿洛酮糖的制备方法，步骤如下：
(1)将枯草芽孢杆菌的发酵液经离心后，取菌体经均质处理，得到含有D-阿洛酮糖3- 差向异构酶的混合液；
所述枯草芽孢杆菌为枯草芽孢杆菌(Bacillus subtilis)BLCY-005，于2016年10月26日保存于中国微生物菌种保藏管理委员会普通微生物中心，保藏号CGMCC No.13152，地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所；
(2)配制质量浓度为20％～60％的果糖溶液，加入含有D-阿洛酮糖3-差向异构酶的混合液，调pH值5.5～6.5，按质量百分比0.001％～0.005％的比例添加氯化钴，在40～60℃条件下，保温反应10～30小时，然后向反应液中流加果糖溶液，维持反应体系中果糖质量浓度为20％～60％，继续反应10～30h，停止反应，制得D-阿洛酮糖粗液；
(3)将步骤(2)制得的D-阿洛酮糖粗液经脱色、过滤、离交、色谱分离、浓缩后，再经过结晶或干燥，制得D-阿洛酮糖。
根据本发明优选的，所述步骤(1)中，离心条件为：温度10～20℃，转速3000r/min，时间30～50分钟。
根据本发明优选的，所述步骤(1)中，均质的条件为：温度10～20℃，压力30mpa～50mpa，时间10～20min。
根据本发明优选的，所述步骤(1)中，枯草芽孢杆菌的发酵液的制备方法如下：
I、将枯草芽孢杆菌(Bacillus subtilis)BLCY-005接种于种子培养基中，在30～38℃的条件下，增殖培养6～12h，制得种子液；
所述种子培养基组分如下，均为重量百分比：
蛋白胨1％，酵母浸粉0.5％，氯化钠1％，无水硫酸镁0.01％，磷酸二氢钾0.02％，余量水，pH6.0～7.0；
II、将步骤I制得的种子液按体积比1～10％的比例接种于发酵培养基中，在30～38℃发酵培养30～48h，制得枯草芽孢杆菌发酵液；
所述发酵培养基组分如下，均为重量百分比：
酵母浸粉3％，玉米浆粉2％，葡萄糖1％，无水硫酸镁0.01％，磷酸氢二铵0.02％，硫酸铵0.02％，余量水，pH6.0～7.0。
根据本发明优选的，所述步骤(2)中，含有D-阿洛酮糖3-差向异构酶的混合液的加入量为果糖溶液体积百分比的5％。
根据本发明优选的，所述步骤(3)中，脱色步骤如下：
将步骤(2)制得的D-阿洛酮糖粗液，按质量百分比0.5～1％的比例加入活性炭，80～85℃搅拌30～40min。
根据本发明优选的，所述步骤(3)中，过滤采用板框过滤，过滤压力0.2～0.4Mpa，水流量5.0～6.0t/h。
根据本发明优选的，所述步骤(3)中，离交步骤如下：
将脱色过滤后的粗糖液以3倍树脂体积/小时的流速，在35～55℃通过连续离子交换系 统，进行离交脱盐，离交后料液透光率≥98％。处理后的料液清澈透明，无异味。
根据本发明优选的，所述步骤(3)中，色谱分离步骤如下：
色谱运行压力0.20～0.30MPa，温度60～70℃，水耗比1:(1.3～1.6)，每小时进料1.5～2.0m3，收集D-阿洛酮糖。
根据本发明优选的，所述步骤(3)中，浓缩采用四效降膜蒸发器，真空度为0.06-0.09Mpa，料液温度50～85℃，浓缩至原体积的60～75％。
根据本发明优选的，所述步骤(3)中，干燥为喷雾干燥，步骤如下：
浓缩后的糖液进入到干燥塔中，进风温度130～150℃，启动雾化器，液体经喷雾干燥为粉末状固体。
根据本发明优选的，所述步骤(3)中，结晶反应条件为：
糖液质量浓度70～85％，温度50～70℃，添加溶质质量10～30％的晶种，搅拌均匀，于50～70℃静置8～16小时，随后按照3～6h降1℃的速率缓慢降温，同时缓慢搅拌，直至溶液中形成大量均匀、规则的晶粒，分离，制得D-阿洛酮糖。
有益效果
1、本发明从土壤中分离出枯草芽孢杆菌，在经过紫外诱变、亚硝基胍诱变处理等诱变处理技术，最后获得高产D-阿洛酮糖差向异构酶的高产菌株命名为BLCY-005，其酶活达到143U/ml，较传统D-阿洛酮糖差向异构酶活提高50％以上，大大提高了蔗糖转化成D-阿洛酮糖的能力，同时采用流加果糖的方法，使终产品中D-阿洛酮糖含量达到99％，显著优于现有制备获得的产品，显著降低生产成本，最适pH值为5.5～6.5，与传统菌株产D-阿洛酮糖差向异构酶最适pH偏中性相比，有利于生产中对污染的控制。
2、本发明采用枯草芽孢杆菌发酵液直接生产，省去了提取酶的过程，使生产成本大大降低，较传统生产方法降低成本达25％左右，使产品的竞争力大大增强；
3、本发明由于采用显著高于现有酶活的D-阿洛酮糖差向异构酶，通过调整工艺步骤和参数，制得了高纯度的D-阿洛酮糖，D-阿洛酮糖作为低热量甜味剂，可广泛应用于食品饮料、化妆品、医药等领域，实现了D-阿洛酮糖大规模工业生产，显著降低了生产成本，扩大了D-阿洛酮糖的应用范围。
具体实施方式
下面结合实施例对本发明的技术方案做进一步阐述，但本发明所保护范围不限于此。
实施例1
一株枯草芽孢杆菌(Bacillus subtilis)BLCY-005，2016年10月26日保存于中国微生物菌种保藏管理委员会普通微生物中心，地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏号保藏号CGMCC No.13152；
本发明所述枯草芽孢杆菌(Bacillus subtilis)BLCY-005的原始菌株分离于山东德州百龙创园生产车间附近的土壤，并经过诱变后获得，具体分离过程如下：
富集培养
选取山东德州百龙创园研发中试车间附近的土壤，用小铲子除去表土，取离地面5-15cm处的土壤约10g，用无菌水稀释10倍，加入LB培养基进行富集培养，30～38℃培养24h。
纯种分离
采用划线分离法，取一支盛有5ml无菌水的大试管，取步骤(1)中富集培养后的菌液2ml放入其中稀释，充分振荡分散，用接种环以无菌操作挑取稀释液一环先在平板培养基一边做第一次平行划线3-4条，再转动培养皿约60度角，将接种环上剩余物烧掉，待冷却后同一次划线方法做第二次划线，同法依次做第三次和第四次划线。划线完毕，盖上皿盖，将培养皿倒置，28～38℃培养24h后，挑取单个菌落接种于10个斜面培养基上，分别编号01-10。
将01-10斜面种子接种于摇瓶培养基中培养28～38℃培养24h，对01-10摇瓶发酵液进行D-阿洛酮糖对D-果糖的转化率测定，06号摇瓶转化率最高，达到26.12％。
平板培养基成分：蛋白胨1％，酵母浸粉0.5％，氯化钠1％，pH自然；
摇瓶培养基成分：酵母浸粉：3％,玉米浆粉2％，葡萄糖1％，无水硫酸镁：0.01％，磷酸氢二铵：0.02％，硫酸铵0.02％，pH为6.0-7.0；
诱变筛选
对06号菌种进行紫外线诱变，紫外线诱变采用15W紫外线灯20cm照射，照射时间为120s，得到的高产菌种再进行亚硝基胍诱变处理，最终得到高转化率D-阿洛酮糖差向异构酶的产生菌株命名为BLCY-005，其产D-阿洛酮糖3-差向异构酶酶活达到143U/ml，显著高于野生菌株的75U/ml。
酶活测定方法：1ml的反应体系中，加入800μl的磷酸盐缓冲液50ml，pH7.0，D-果糖的溶解浓度为100g/L，200μl的发酵液，55℃保温10min，然后煮沸10min以终止酶反应。
用HPLC检测D-阿洛酮糖的生产量，计算酶活。酶活单位(U)：每分钟催化产生1μmol D-psicose所需要的酶的量。
生态学形态
菌落呈污白色，半透明，菌落边缘不规整，呈波浪状，中心高凸。经显微镜观察，该菌株长约1.0～1.5微米，宽约0.6～0.9微米，无荚膜，不产鞭毛，芽孢位于菌体中央或稍偏，芽孢形成后具体不膨大。
实施例2
实施例1所述的枯草芽孢杆菌(Bacillus subtilis)BLCY-005的培养方法，步骤如下：
(a)取枯草芽孢杆菌(Bacillus subtilis)BLCY-005接种于LB培养基中，在35℃的条件下，活化培养12h，制得活化菌株；
(b)取步骤(a)制得的活化菌株，接种于种子培养基中，在35℃的条件下，增殖培养12h，制得种子液；
所述种子培养基组分如下，均为重量百分比：
蛋白胨1％，酵母浸粉0.5％，氯化钠1％，无水硫酸镁0.01％，磷酸二氢钾0.02％，余量水，pH6.8；
(c)取步骤(b)制得的种子液，按体积比5％的比例接种于发酵培养基中，在35℃，扩大培养48h，即得枯草芽孢杆菌的发酵液；
所述发酵培养基组分如下，均为重量百分比：
酵母浸粉3％，玉米浆粉2％，葡萄糖1％，无水硫酸镁0.01％，磷酸氢二铵0.02％，硫酸铵0.02％，余量水，pH6.8。
实施例3
一种D-阿洛酮糖的制备方法，步骤如下：
(1)将实施例2制得的枯草芽孢杆菌的发酵液经20℃、转速3000r/min的条件下离心30分钟后，取菌体在温度20℃、压力30mpa的条件下均质10min，得到含有D-阿洛酮糖3-差向异构酶的混合液；
(2)配制质量浓度为60％的果糖溶液，加入含有D-阿洛酮糖3-差向异构酶的混合液，含有D-阿洛酮糖3-差向异构酶的混合液的加入量为果糖溶液体积百分比的5％，调pH值5.5，按质量百分比0.005％的比例添加氯化钴，在40℃条件下，保温反应30小时，然后向反应液中流加果糖溶液，维持反应液中果糖质量浓度为20％，继续反应30h，停止反应，制得D-阿洛酮糖粗液；
(3)将步骤(2)制得的D-阿洛酮糖粗液经脱色、板框过滤、离交、色谱分离、浓缩后，再经过结晶或干燥，制得D-阿洛酮糖；
所述脱色步骤如下：
将步骤(2)制得的D-阿洛酮糖粗液，按质量百分比0.5％的比例加入活性炭，85℃搅拌30min；
所述的板框过滤，过滤压力0.4Mpa，水流量5.0t/h；
所述离交，步骤如下：
将脱色过滤后的粗糖液以3倍树脂体积/小时的流速，在55℃通过连续离子交换系统，进行离交脱盐，离交后料液透光率≥98％；
所述色谱分离步骤如下：
色谱运行压力0.20MPa，温度70℃，水耗比1:1.3，每小时进料2.0m3，收集D-阿洛酮糖；
所述浓缩采用四效降膜蒸发器，真空度为0.06Mpa，料液温度85℃，浓缩至原体积的60％；
所述干燥为喷雾干燥，步骤如下：
浓缩后的糖液进入到干燥塔中，进风温度150℃，启动雾化器，液体经喷雾干燥，制得D-阿洛酮糖。
经检测，测定其转化率为42.58％，该转化率远高于原始菌株的26.12％。
实施例4
一种D-阿洛酮糖的制备方法，步骤如下：
(1)将实施例2制得的枯草芽孢杆菌的发酵液经10℃、转速3000r/min的条件下离心50分钟后，取菌体在温度10℃、压力50mpa的条件下均质20min，得到含有D-阿洛酮糖3-差向异构酶的混合液；
(2)配制质量浓度为20％的果糖溶液，加入含有D-阿洛酮糖3-差向异构酶的混合液，含有D-阿洛酮糖3-差向异构酶的混合液的加入量为果糖溶液体积百分比的5％，调pH值6.5，按质量百分比0.001％的比例添加氯化钴，在60℃条件下，保温反应10小时，然后向反应液中流加果糖溶液，维持反应液中果糖质量浓度为60％，继续反应10h，停止反应，制得D-阿洛酮糖粗液；
(3)将步骤(2)制得的D-阿洛酮糖粗液经脱色、板框过滤、离交、色谱分离、浓缩后，再经过结晶或干燥，制得D-阿洛酮糖；
所述脱色步骤如下：
将步骤(2)制得的D-阿洛酮糖粗液，按质量百分比1％的比例加入活性炭，80℃搅拌40min；
所述的板框过滤，过滤压力0.2Mpa，水流量6.0t/h；
所述离交，步骤如下：
将脱色过滤后的粗糖液以3倍树脂体积/小时的流速，在35℃通过连续离子交换系统，进行离交脱盐，离交后料液透光率≥98％；
所述色谱分离步骤如下：
色谱运行压力0.30MPa，温度60℃，水耗比1:1.6，每小时进料1.5m3，收集D-阿洛酮糖；
所述浓缩采用四效降膜蒸发器，真空度为0.09Mpa，料液温度50℃，浓缩至原体积的75％；
所述结晶，反应条件为：
糖液质量浓度70％，温度70℃，添加溶质质量10％的晶种，搅拌均匀，于70℃静置8小时，随后按照6h降1℃的速率缓慢降温，同时缓慢搅拌，直至溶液中形成大量均匀、规则的晶粒，分离，制得D-阿洛酮糖。
经检测，测定其转化率为43.12％，该转化率远高于原始菌株的26.12％。

Claims (10)

  1. 一种D-阿洛酮糖的制备方法，其特征在于，步骤如下：
    (1)将枯草芽孢杆菌的发酵液经离心后，取菌体经均质处理，得到含有D-阿洛酮糖3-差向异构酶的混合液；
    所述枯草芽孢杆菌为枯草芽孢杆菌(Bacillus subtilis)BLCY-005，于2016年10月26日保存于中国微生物菌种保藏管理委员会普通微生物中心，保藏号CGMCC No.13152，地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所；
    (2)配制质量浓度为20％～60％的果糖溶液，加入含有D-阿洛酮糖3-差向异构酶的混合液，调pH值5.5～6.5，按质量百分比0.001％～0.005％的比例添加氯化钴，在40～60℃条件下，保温反应10～30小时，然后向反应液中流加果糖溶液，维持反应体系中果糖质量浓度为20％～60％，继续反应10～30h，停止反应，制得D-阿洛酮糖粗液；
    (3)将步骤(2)制得的D-阿洛酮糖粗液经脱色、过滤、离交、色谱分离、浓缩后，再经过结晶或干燥，制得D-阿洛酮糖。
  2. 如权利要求1所述的制备方法，其特征在于，所述步骤(1)中，离心条件为：温度10～20℃，转速3000r/min，时间30～50分钟。
  3. 如权利要求1所述的制备方法，其特征在于，所述步骤(1)中，均质的条件为：温度10～20℃，压力30mpa～50mpa，时间10～20min。
  4. 如权利要求1所述的制备方法，其特征在于，所述步骤(1)中，枯草芽孢杆菌的发酵液的制备方法如下：
    I、将枯草芽孢杆菌(Bacillus subtilis)BLCY-005接种于种子培养基中，在30～38℃的条件下，增殖培养6～12h，制得种子液；
    所述种子培养基组分如下，均为重量百分比：
    蛋白胨1％，酵母浸粉0.5％，氯化钠1％，无水硫酸镁0.01％，磷酸二氢钾0.02％，余量水，pH6.0～7.0；
    II、将步骤I制得的种子液按体积比1～10％的比例接种于发酵培养基中，在30～38℃发酵培养30～48h，制得枯草芽孢杆菌发酵液；
    所述发酵培养基组分如下，均为重量百分比：
    酵母浸粉3％，玉米浆粉2％，葡萄糖1％，无水硫酸镁0.01％，磷酸氢二铵0.02％，硫酸铵0.02％，余量水，pH6.0～7.0。
  5. 如权利要求1所述的制备方法，其特征在于，所述步骤(2)中，含有D-阿洛酮糖3-差向异构酶的混合液的加入量为果糖溶液体积百分比的5％。
  6. 如权利要求1所述的制备方法，其特征在于，所述步骤(3)中，脱色步骤如下：
    将步骤(2)制得的D-阿洛酮糖粗液，按质量百分比0.5～1％的比例加入活性炭，80～85℃搅拌30～40min；
    优选的，所述步骤(3)中，过滤采用板框过滤，过滤压力0.2～0.4Mpa，水流量5.0～6.0t/h。
  7. 如权利要求1所述的制备方法，其特征在于，所述步骤(3)中，离交步骤如下：
    将脱色过滤后的粗糖液以3倍树脂体积/小时的流速，在35～55℃通过连续离子交换系统，进行离交脱盐，离交后料液透光率≥98％；
    优选的，所述步骤(3)中，色谱分离步骤如下：
    色谱运行压力0.20～0.30MPa，温度60～70℃，水耗比1:(1.3～1.6)，每小时进料1.5～2.0m3，收集D-阿洛酮糖。
  8. 如权利要求1所述的制备方法，其特征在于，所述步骤(3)中，浓缩采用四效降膜蒸发器，真空度为0.06-0.09Mpa，料液温度50～85℃，浓缩至原体积的60～75％。
  9. 如权利要求1所述的制备方法，其特征在于，所述步骤(3)中，干燥为喷雾干燥，步骤如下：
    浓缩后的糖液进入到干燥塔中，进风温度130～150℃，启动雾化器，液体经喷雾干燥为粉末状固体。
  10. 如权利要求1所述的制备方法，其特征在于，所述步骤(3)中，结晶反应条件为：
    糖液质量浓度70～85％，温度50～70℃，添加溶质质量10～30％的晶种，搅拌均匀，于50～70℃静置8～16小时，随后按照3～6h降1℃的速率缓慢降温，同时缓慢搅拌，直至溶液中形成大量均匀、规则的晶粒，分离，制得D-阿洛酮糖。
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