WO2020135893A1 - 一株米曲霉blcy-006及其在制备低聚半乳糖中的应用 - Google Patents

一株米曲霉blcy-006及其在制备低聚半乳糖中的应用 Download PDF

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WO2020135893A1
WO2020135893A1 PCT/CN2020/075704 CN2020075704W WO2020135893A1 WO 2020135893 A1 WO2020135893 A1 WO 2020135893A1 CN 2020075704 W CN2020075704 W CN 2020075704W WO 2020135893 A1 WO2020135893 A1 WO 2020135893A1
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aspergillus oryzae
blcy
strain
culture
medium
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李方华
干昭波
窦光朋
邵先豹
张明站
杜倩
杨腾腾
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Shandong Bailong Chuangyuan Bio Tech Co Ltd
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
    • C12N9/2471—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
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    • 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
    • C12N1/14—Fungi; Culture media therefor
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    • 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
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C—CHEMISTRY; METALLURGY
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • 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
    • C12P19/12—Disaccharides
    • 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
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
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    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01023—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/66—Aspergillus
    • C12R2001/69—Aspergillus oryzae

Definitions

  • the invention relates to an Aspergillus oryzae BLCY-006 and its application in the preparation of galactooligosaccharides, which belongs to the technical field of microorganisms.
  • Aspergillus oryzae belongs to the subfamily of Hypomycetes, Hyphomycetes, Hyphomycetes, and from the family Stenosporidae. Aspergillus is a common species of fungus, which is widely distributed, mainly distributed in food, fermented food, spoiled organic matter and soil. Aspergillus oryzae is a strain of traditional Chinese brewing food sauce and soy sauce, which can also be used to produce amylase, protease, pectinase and kojic acid.
  • Galactooligosaccharides is a kind of functional oligosaccharides, and its molecular structure is generally connected with 1-7 galactosyl groups on galactose or glucose molecules. In nature, trace amounts of GOS are present in animal milk, and human breast milk contains more GOS. The establishment of bifidobacteria in infants depends heavily on the GOS component of breast milk.
  • Galactooligosaccharides have strong acid and heat resistance. They are not digested and absorbed by human small intestine, but can be fermented by colonic flora.
  • galactose oligosaccharides can simultaneously proliferate bifidobacteria and lactic acid bacteria in the intestine, and can inhibit the growth of harmful pathogenic bacteria and spoilage bacteria.
  • galactooligosaccharides are not used by oral bacteria such as mutant streptococcus, which can reduce the incidence of dental caries.
  • Galactooligosaccharides can also promote the absorption of calcium, magnesium, and potassium, reduce sodium absorption, and reduce the levels of total cholesterol and triglycerides, which can improve lipid metabolism, and can effectively stimulate intestinal peristalsis, reduce and prevent the occurrence of constipation. Regulate intestinal microecology and promote intestinal health. The safety of galactooligosaccharides has been widely recognized. In 2010, galactooligosaccharides became the second largest functional oligosaccharide product in Japan. In September 2008, China included it in the new resource food catalog.
  • the current methods for producing galactooligosaccharides are mainly strain fermentation and enzyme conversion.
  • the strain fermentation method refers to the direct fermentation of lactose solution with ⁇ -galactosidase producing strain to produce galactooligosaccharides.
  • the disadvantage of this method is that the galactooligosaccharide produced is not of high purity, and subsequent purification is difficult.
  • the enzyme conversion method refers to cultivating the enzyme-producing strains first, and then extracting ⁇ -galactosidase for enzyme conversion to produce galactooligosaccharides.
  • the problem with this method is that the activity of the extracted enzyme is low, and the high content of the reaction in the conversion process The effect of by-product glucose results in low conversion rates and high production costs.
  • Chinese patent document CN101691538A discloses a method for preparing high-purity galactooligosaccharides, which includes product separation and purification steps such as Aspergillus oryzae fermentation, ceramic membrane ultrafiltration, and nanofiltration separation. Aspergillus oryzae isolated from soil was used as the starting strain, and aspergillus oryzae BLB-21 (Accession No. CGMCC No. 2951) was obtained by mutagenic screening. The Aspergillus oryzae was then used to directly ferment a high-concentration lactose solution.
  • the patent still has the problems of low ⁇ -galactosidase activity, low purity and yield of the prepared galactooligosaccharide.
  • the present invention provides a strain of Aspergillus oryzae BLCY-006 and its application in the preparation of galactooligosaccharides.
  • the invention also provides a method for cultivating Aspergillus oryzae BLCY-006.
  • the original strain of Aspergillus oryzae BLCY-006 of the present invention is isolated from the soil near the production site of galactooligosaccharides in Bailong Chuangyuan, Dezhou, Shandong, China, and obtained after repeated mutagenesis and screening.
  • the strain initially appeared white and yellow, and then turned yellowish brown to pale greenish brown.
  • the conidia head is radial, with a diameter of 150-300 ⁇ m, and a few are loose columnar. The conidia are about 2mm.
  • This strain can produce ⁇ -galactosidase with high yield.
  • the enzyme activity of the enzyme can reach 300 U/ml, which is more than 50% higher than that of traditional ⁇ -galactosidase.
  • the enzyme also has the characteristics of resistance to lactose and glucose, and its application in the production of galactooligosaccharides can greatly improve the ability of lactose to be converted into galactooligosaccharides, and significantly reduce production costs.
  • the cultivation method of the above Aspergillus BLCY-006 includes the following steps:
  • step (2) Take the activated strain prepared in step (1), inoculate in seed medium, and propagate and culture for 20-30 hours under the condition of 28-35°C to prepare seed liquid;
  • step (3) Take the seed liquid prepared in step (2), inoculate the fermentation medium at a volume ratio of 2 to 10%, and expand the culture at 28 to 35°C for 25 to 35 hours to obtain the bacterial fermentation liquid.
  • the components of the seed medium in the step (2) are as follows, all of which are weight percentages:
  • the fermentation medium components in the step (3) are as follows, all in weight percent:
  • the solid medium in step (1) is a conventional PDA solid medium in the art.
  • step (b) Add the cells collected in step (a) to the pre-chilled phosphate buffer and then react with the pre-treated adsorbent.
  • the reaction time is 5 to 25 hours to fix the cells on the surface of the adsorbent to prepare ⁇ -Galactosidase;
  • step (c) Prepare a lactose solution with a mass concentration of 40 to 60%, add the ⁇ -galactosidase obtained in step (b) to the lactose solution, and prepare the crude galactose oligosaccharide solution 12 hours after the incubation reaction;
  • step (d) The crude galactooligosaccharide solution prepared in step (c) is subjected to decolorization, filtration, ion exchange, chromatographic separation, concentration, and drying to obtain galactooligosaccharide.
  • the filtering is performed by a plate and frame filter press, the working pressure is 0.3 to 0.5 MPa, and the flow rate is 5 to 10 m 3 /h.
  • the adsorbent is selected from alumina, diatomaceous earth, porous ceramics or cellulose.
  • the amount of the ⁇ -galactosidase added is 0.1 to 10 wt% based on the mass of lactose.
  • the temperature of the heat preservation reaction is 30-60°C.
  • step (d) the addition amount of activated carbon required for decolorization is 0.1 wt%, the decolorization time is 1.5 h; the operating pressure of the chromatographic separation is 0.2 MPa, the temperature is 60° C., and the water consumption ratio 1:1.2, feeding 1.8m 3 per hour.
  • a ⁇ -galactosidase comprising an adsorbent and an enzyme-containing Aspergillus oryzae immobilized on the adsorbent, the enzyme-containing Aspergillus oryzae is made from Aspergillus oryzae BLCY-006.
  • the present invention separates the Aspergillus oryzae species from the soil, undergoes mutagenesis treatment techniques such as ultraviolet mutagenesis and ion implantation mutagenesis, and finally obtains a high-yield strain producing ⁇ -galactosidase, which is named BLCY-006.
  • mutagenesis treatment techniques such as ultraviolet mutagenesis and ion implantation mutagenesis
  • BLCY-006 high-yield strain producing ⁇ -galactosidase
  • Its enzyme activity reaches 300U/ml, which is more than 50% higher than the traditional ⁇ -galactosidase activity.
  • the enzyme also has the characteristics of resistance to glucose and lactose. Its application in the production of galactooligosaccharides can greatly improve the conversion of lactose into The ability of galactooligosaccharides significantly reduces production costs.
  • the preparation of the invention obtains ⁇ -galactosidase, the use efficiency of the enzyme is improved, the utilization rate of lactose is significantly improved, and the content of galactose oligosaccharide in the crude solution of galactose oligosaccharide produced is also significantly increased. Dadi reduces the difficulty and cost of subsequent purification of galactooligosaccharides, and significantly improves the quality of galactooligosaccharides.
  • the materials and medicines involved in the present invention are ordinary commercial products.
  • the light absorption value at 420 nm was measured.
  • OD420 The light absorption value at 420 nm
  • Na 2 HPO 4 -citric acid buffer concentration: 0.1mol/L, pH5.2
  • OTP o-nitrophenol
  • One unit (1U) of ⁇ -galactosidase activity refers to the catalytic substrate o-nitrophenol- ⁇ -D-galactopyranoside (ONPG) at 60°C and pH 5.2 per minute ) The amount of enzyme required to produce 1 ⁇ mol of o-nitrophenol (ONP).
  • Potato 200g glucose 20g, agar 15-20g, deionized water 1000ml, ammonium nitrate 0.2%; ammonium sulfate 0.1%; dipotassium hydrogen phosphate 0.1%; PH 6.5-7.0.
  • the method of making medium is as follows
  • step (1) take a large test tube containing 5ml of sterile water, take 2ml of the enriched and cultured bacterial solution in step (1) and dilute it, fully shake and disperse, use the inoculating ring to aseptically pick Take a ring of diluent and make 3 to 4 first parallel streaks on the side of the plate medium, then turn the petri dish at an angle of about 60 degrees to burn the residue on the inoculation ring. After cooling, do the same streak method For the second stroke, do the third and fourth strokes in the same way. After the streak is completed, cover the dish and put the dish upside down. After culturing at 28-35°C for 30h, pick a single colony and inoculate it on 10 inclined plane media, numbered 01-10 respectively.
  • composition of the plate medium is as follows:
  • Potato 200g glucose 20g, agar 15-20g, deionized water 1000ml, ammonium nitrate 0.2%; ammonium sulfate 0.1%; dipotassium hydrogen phosphate 0.1%; PH 6.5-7.0.
  • composition of the shake flask medium is as follows:
  • soybean cake extract juice 100ml was added soluble starch 2g, potassium dihydrogen phosphate 0.1g, magnesium sulfate 0.05g, ammonium sulfate 0.05g, agar 2g, natural pH.
  • the preparation method of the bean cake leaching juice 100 grams of bean cake, add 500ml of water, soak for 4 hours, boil for 3 to 4 hours, filter the gauze naturally, take the liquid and adjust to 5 Baume degree.
  • Ultraviolet mutagenesis was performed on No. 08 strain, which was irradiated with 20W ultraviolet lamp 15cm for irradiation time of 200s, and the resulting high-yield strains were subjected to ion implantation mutagenesis treatment to finally obtain high-yield strains with high yield of ⁇ -galactosidase.
  • It is named BLCY-006, and its enzyme activity reaches 300U/ml.
  • the strain initially appeared white and yellow, and then turned yellowish brown to pale greenish brown.
  • the conidia head is radial, with a diameter of 150-300 ⁇ m, and a few are loose columnar. The conidia are about 2mm. After identification, the strain was Aspergillus oryzae.
  • step (2) Take the activated strain prepared in step (1), inoculate in the seed culture medium, propagate and culture for 30h under the condition of 30°C to prepare the seed liquid;
  • the components of the seed medium are as follows:
  • the preparation method of the bean cake leaching juice 100g bean cake, add 500ml of water, soak for 4 hours, boil for 3 to 4 hours, filter the gauze naturally, take the liquid, and adjust to 5 Baume degree.
  • step (3) Take the seed liquid prepared in step (2), inoculate the fermentation medium at a volume ratio of 1%, and expand the culture at 30°C for 35h to obtain the bacterial fermentation liquid;
  • the components of the fermentation medium are as follows, all of which are weight percentages:
  • Aspergillus oryzae BLCY-006 was inoculated in PDA medium, and activated and cultured at 35°C for 20h to prepare an activated strain;
  • step (2) Take the activated strain prepared in step (1), inoculate in the seed medium, and propagate and culture for 20 h under the condition of 35°C to obtain a seed solution;
  • the components of the seed medium are as follows:
  • the preparation method of the bean cake leaching juice 100 grams of bean cake, add 500ml of water, soak for 4 hours, boil for 3-4 hours, filter the gauze naturally, take the liquid, and adjust to 5 Baume.
  • step (3) Take the seed liquid prepared in step (2), inoculate the fermentation medium at a volume ratio of 10%, and expand the culture at 38°C for 20h to obtain the bacterial fermentation liquid;
  • the components of the fermentation medium are as follows, all of which are weight percentages:
  • step (2) Take the activated strain prepared in step (1), inoculate in the seed culture medium, and propagate and culture for 25h under the condition of 32°C to prepare the seed liquid;
  • the components of the seed medium are as follows:
  • the preparation method of the bean cake leaching juice 100 grams of bean cake, add 500ml of water, soak for 4 hours, boil for 3-4 hours, filter the gauze naturally, take the liquid, and adjust to 5 Baume.
  • step (3) Take the seed liquid prepared in step (2), inoculate the fermentation medium at a volume ratio of 10%, and expand the culture at 38°C for 20h to obtain the bacterial fermentation liquid;
  • the components of the fermentation medium are as follows, all of which are weight percentages:
  • Chinese patent document CN101691538A (application number: 200910018452.1) discloses a method for preparing high-purity galactooligosaccharides.
  • Aspergillus oryzae BLB-21 (CGMCC No. 2951) disclosed in the patent was cultured under the same conditions as in Example 2.
  • step (b) Add the cells collected in step (a) to the pre-chilled phosphate buffer solution and then react with the pre-treated adsorbent for a reaction time of 15h to fix the cells on the surface of diatomaceous earth to prepare ⁇ - Galactosidase;
  • step (c) Prepare a lactose solution with a mass concentration of 40% and 60%, add the ⁇ -galactosidase obtained in step (b) to the lactose solution, based on the mass of lactose, the addition amount is 5wt%, and the heat preservation reaction is 12h After the preparation of crude galactose oligosaccharide solution;
  • step (d) The crude galactooligosaccharide solution prepared in step (c) is subjected to decolorization, filtration, ion exchange, chromatographic separation, concentration, and drying to obtain galactooligosaccharide.
  • the addition amount of activated carbon required for decolorization in step (d) is 0.1wt%, the decolorization time is 1.5h; the operating pressure of chromatographic separation is 0.2MPa, the temperature is 60°C, the water consumption ratio is 1:1.2, and the feed is 1.8 per hour m 3 .

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Abstract

提供了一株米曲霉（AspergillusoryZae）BLCY-006及其在制备低聚半乳糖中的应用，所述菌株产P-半乳糖苷酶，经培养发酵后酶活可达到300U/ml，相对于传统P-半乳糖苷酶活力提高50%以上，还具有耐乳糖和葡萄糖的特性。

Description

一株米曲霉BLCY-006及其在制备低聚半乳糖中的应用 技术领域
本发明涉及一株米曲霉BLCY-006及其在制备低聚半乳糖中的应用，属于微生物技术领域。
背景技术
米曲霉属半知菌亚门，丝孢纲，丝孢目，从梗孢科。曲霉属真菌中的一个常见种，其分布甚广，主要分布在粮食、发酵食品、腐败有机物和土壤等处。米曲霉是我国传统酿造食品酱和酱油的生产菌种，其也可用于生产淀粉酶、蛋白酶、果胶酶和曲酸等。
低聚半乳糖(Galactooligosaccharides，GOS)是一种功能性低聚糖，其分子结构一般是在半乳糖或葡萄糖分子上连接1～7个半乳糖基。在自然界中，动物的乳汁中存在微量的GOS，人母乳中的GOS含量较多。婴儿体内的双歧杆菌菌群的建立很大程度上依赖母乳中的GOS成分。低聚半乳糖具有较强的耐酸性、耐热性，其不被人小肠消化吸收，但可被结肠菌群发酵。同时，低聚半乳糖对肠内的双歧杆菌和乳酸菌具有同时增殖的作用，且能抑制有害病原菌和腐败菌生长。此外，低聚半乳糖不被突变链球菌等口腔细菌利用，可降低龋齿发生率。低聚半乳糖还能促进钙、镁、钾的吸收，降低钠吸收，以及降低总胆固醇和甘油三酯水平，可改善脂质代谢，以及能有效刺激肠道蠕动，减少和防止便秘的发生，调节肠道微生态、促进肠道健康。低聚半乳糖的安全性已经受到广泛认可。2010年，低聚半乳糖在日本成为第二大功能性低聚糖产品，2008年9月，中国将它列入了新资源食品目录。
当前生产低聚半乳糖的方法主要是菌种发酵法和酶转化法。菌种发酵法是指用产β-半乳糖苷酶的菌种直接发酵乳糖溶液生产低聚半乳糖。该方法的缺点是生产的低聚半乳糖纯度不高，后续提纯困难。酶转化法是指先培养产酶菌种，然后提取β-半乳糖苷酶进行酶转化生产低聚半乳糖，该方法目前存在的问题是提取的酶活较低，转化过程中受到反应中高含量的副产物葡萄糖的影响造成转化率低，生产成本高居不下。
中国专利文献CN101691538A公开了一种高纯度低聚半乳糖的制备方法，包括米曲霉发酵，陶瓷膜超滤、纳滤分离等产品分离纯化步骤。其采用土壤中分离的Aspergillus oryzae米曲霉为出发菌株，经诱变筛选得到米曲霉BLB-21(保藏号CGMCCNo.2951)。然后该米曲霉被用于直接发酵高浓度乳糖溶液。不过，该专利仍然存在β-半乳糖苷酶活性较低，制备的低聚半乳糖纯度和收率较低等问题。
发明内容
本发明针对现有技术的不足，提供一株米曲霉BLCY-006及其在制备低聚半乳糖中的应用。
本发明还提供一种米曲霉BLCY-006的培养方法。
本发明的技术方案如下：
一株米曲霉(Aspergillus oryzae)BLCY-006，2018年12月5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.16965，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。
本发明所述米曲霉(Aspergillus oryzae)BLCY-006的原始菌株分离于中国山东德州百龙创园低聚半乳糖生产车间附近的土壤，并经过反复多次诱变、筛选，获得。
该菌株初呈白色、黄色，后转黄褐色至淡绿褐色。分生孢子头呈放射状，直径150～300μm，也有少数为疏松柱状。分生孢子梗2mm左右。该菌株可高产β-半乳糖苷酶，经培养发酵后该酶的酶活可达到300U/ml，相对于传统β-半乳糖苷酶活力提高50％以上。同时，该酶还具有耐乳糖和葡萄糖的特性，其应用于低聚半乳糖生产中可大大提高乳糖转化成低聚半乳糖的能力，显著降低生产成本。
上述米曲霉(Aspergillus oryzae)BLCY-006的培养方法包括以下步骤：
(1)取米曲霉(Aspergillus oryzae)BLCY-006接种于固体培养基中，在28～35℃的条件下，活化培养20～30h，制得活化菌株；
(2)取步骤(1)制得的活化菌株，接种于种子培养基中，在28～35℃的条件下，增殖培养20～30h，制得种子液；
(3)取步骤(2)制得的种子液，按体积比2～10％的比例接种于发酵培养基中，在28～35℃，扩大培养25～35h，即得菌体发酵液。
根据本发明优选的，所述步骤(2)中的种子培养基组分如下，均为重量百分比：
硝酸铵0.2％，硫酸铵0.1％，磷酸二氢钾0.1％，尿素0.05％，蛋白胨1％，蔗糖2％，葡萄糖5％，余量水，pH为4.5～6.5。
根据本发明优选的，所述步骤(3)中的发酵培养基组分如下，均为重量百分比：
蔗糖5％，葡萄糖5％，蛋白胨1％，硫酸铵0.1％，磷酸二氢钾0.1％，余量水。
根据本发明优选的，所述步骤(1)中的固体培养基为本领域常规PDA固体培养基。
上述米曲霉(Aspergillus oryzae)BLCY-006在制备低聚半乳糖中的应用，其特征在于，步骤包括：
(a)按照上述米曲霉(Aspergillus oryzae)BLCY-006的培养方法制备得到菌体发酵液，经过滤收集菌体；
(b)将步骤(a)收集到的菌体加入预冷的磷酸缓冲液然后与经过预处理的吸附剂进行反应，反应时间为5～25h，使菌体固定在吸附剂表面，制得β-半乳糖苷酶；
(c)配制质量浓度为40～60％的乳糖溶液，将步骤(b)得到的β-半乳糖苷酶加入到乳糖溶液中，保温反应后12h后，制得低聚半乳糖粗溶液；
(d)将步骤(c)制得的低聚半乳糖粗溶液经脱色、过滤、离子交换、色谱分离、浓缩、干燥得到低聚半乳糖。
根据本发明优选的，所述步骤(a)中，过滤采用板框式压滤机过滤，工作压力为0.3～0.5MPa，流速为5～10m 3/h。
根据本发明优选的，所述步骤(b)中，吸附剂选自氧化铝、硅藻土、多孔陶瓷或纤维素。
根据本发明优选的，步骤(c)中，所述β-半乳糖苷酶的加入量，以乳糖质量计，为0.1～10wt％。
根据本发明优选的，步骤(c)中，所述保温反应的温度为30～60℃。
根据本发明优选的，步骤(d)中，所述脱色所需活性炭添加量为0.1wt％，脱色时间为1.5h；所述色谱分离的运行压力为0.2MPa，温度为60℃，水耗比1:1.2，每小时进料1.8m 3。
本发明未详细说明的实验步骤可按照文献记载或现有技术进行。
在一些方面，提供一种β-半乳糖苷酶，包括吸附剂和固定在吸附剂上的含酶米曲霉，所述含酶米曲霉由米曲霉BLCY-006制得。
有益效果
1.本发明从土壤中分离出米曲霉菌种，在经过紫外诱变、离子注入诱变处理等诱变处理技术，最后获得高产β-半乳糖苷酶的高产菌株，命名为BLCY-006。其酶活达到300U/ml，相对于传统β-半乳糖苷酶活力提高50％以上，同时该酶还具有耐葡萄糖、乳糖的特性，其应用于低聚半乳糖生产中可大大提高乳糖转化成低聚半乳糖的能力，显著降低生产成本。
2.本发明制备获得了β-半乳糖苷酶，酶的使用效率有所提高，对乳糖的利用率显著提高，所产低聚半乳糖粗溶液中低聚半乳糖的含量也显著提升，极大地降低后续低聚半乳糖提纯的难度和成本，并显著提高低聚半乳糖成品的质量。
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。
本发明中所涉及材料及药品均为普通市售产品。
β-半乳糖苷酶酶活力测定方法：
准确称取邻硝基苯酚-β-D-吡喃半乳糖苷(ONPG)底物0.1g，溶于40mL Na 2HPO 4-柠檬酸缓冲液(pH5.2，0.1mol/L)，即为浓度为0.25％(W/V)的ONPG溶液。使用pH5.2，0.1mol/L的Na 2HPO 4-柠檬酸将待测菌体发酵液稀释至合适倍数，吸取800μl的底物溶液加入试管中，于60℃水浴锅中预热2min，加入200μl稀释后酶液混匀，反应15min后依次加入2ml 1mol/L Na 2CO 3溶液终止反应。测定420nm处的光吸收值(OD420)。以加入Na 2HPO 4-柠檬酸缓冲液(浓度为0.1mol/L，pH5.2)作为空白对照，利用标准曲线，计算反应生成的邻硝基酚(ONP)的量，进而计算出β-半乳糖苷酶的酶活力。
酶活力单位定义：一个单位(1U)的β-半乳糖苷酶活性指在60℃，pH5.2条件下，每分钟催化底物邻硝基苯酚-β-D-吡喃半乳糖苷(ONPG)生成1μmol邻硝基酚(ONP)所需的酶量。
生物材料：
一株米曲霉(Aspergillus oryzae)BLCY-006，2018年12月5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CGMCCNo.16965，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。
实施例1
米曲霉(Aspergillus oryzae)BLCY-006的筛选过程如下：
(1)富集培养
选取山东德州百龙创园低聚半乳糖生产车间附近的土壤，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PDA培养基进行富集培养，培养基成分如下：
马铃薯200克、葡萄糖20克、琼脂15～20克、去离子水1000毫升、硝酸铵0.2％；硫酸铵0.1％；磷酸氢二钾0.1％；PH6.5～7.0。
培养基制作方法如下
200g马铃薯切成小块，加水煮烂(煮沸20～30分钟，能被玻璃棒戳破即可)，用八层纱布过滤，加热，再据实际实验需要加1～10g琼脂，硝酸铵0.2％；硫酸铵0.1％；磷酸氢二钾0.1％继续加热搅拌混匀，待琼脂溶解完后，加入葡萄糖，搅拌均匀，稍冷却后再补足去离子水分至1000毫升，分装试管或者锥形瓶，加塞、包扎，(121℃)灭菌20分钟左右后取出试管摆斜面或者摇匀，冷却后贮存备用。
(2)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，28～35℃培养30h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28～35℃培养30h，对01～10摇瓶发酵液的β-半乳糖苷酶酶活进行测定，08号摇瓶酶活最高，达到105U/ml。
平板培养基成分如下：
马铃薯200克、葡萄糖20克、琼脂15～20克、去离子水1000毫升、硝酸铵0.2％；硫酸铵0.1％；磷酸氢二钾0.1％；PH6.5～7.0。
摇瓶培养基成分如下：
100ml豆饼浸出汁中加入可溶性淀粉2克，磷酸二氢钾0.1克，硫酸镁0.05克，硫酸铵0.05克，琼脂2克，自然pH。
所述豆饼浸出汁制作方法：100克豆饼，加水500ml，浸泡4小时，煮沸3～4小时，纱布自然过滤，取液，调整至5波美度。
(3)诱变筛选
对08号菌种进行紫外线诱变，紫外线诱变采用20W紫外线灯15cm照射，照射时间为200s，得到的高产菌种再进行离子注入诱变处理，最终得到高产β-半乳糖苷酶的高产菌株命名为BLCY-006，其酶活达到300U/ml。该菌株初呈白色、黄色，后转黄褐色至淡绿褐色。分生孢子头呈放射状，直径150～300μm，也有少数为疏松柱状。分生孢子梗2mm左右。经鉴定，该菌株为米曲霉(Aspergillus oryzae)。
上述米曲霉(Aspergillus oryzae)，命名为BLCY-006，2018年12月5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CGMCCNo.16965，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。
实施例2
实施例1所述的米曲霉(Aspergillus oryzae)BLCY-006的培养方法，步骤包括：
(1)取米曲霉(Aspergillus oryzae)BLCY-006接种于PDA培养基中，在30℃的条件下，活化培养30h，制得活化菌株；
(2)取步骤(1)制得的活化菌株，接种于种子培养基中，在30℃的条件下，增殖培养30h，制得种子液；
所述种子培养基组分如下：
100ml豆饼浸出汁中加入可溶性淀粉2克，磷酸二氢钾0.1克，硫酸镁0.05克，硫酸铵0.05克，琼脂2克，自然pH；
所述豆饼浸出汁制作方法：100g豆饼，加水500ml，浸泡4小时，煮沸3～4小时，纱布自然过滤，取液，调整至5波美度。
(3)取步骤(2)制得的种子液，按体积比1％的比例接种于发酵培养基中，在30℃，扩大培养35h，即得菌体发酵液；
所述发酵培养基组分如下，均为重量百分比：
蔗糖10％，麸皮2％，酵母膏1％，硝酸钠0.3％，MgSO 4·7H 2O0.05％，余量水。
实施例3
实施例1所述的米曲霉(Aspergillus oryzae)BLCY-006的培养方法，步骤包括：
(1)取米曲霉(Aspergillus oryzae)BLCY-006接种于PDA培养基中，在35℃的条件下，活化培养20h，制得活化菌株；
(2)取步骤(1)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20h，制得种子液；
所述种子培养基组分如下：
100ml豆饼浸出汁中加入可溶性淀粉2克，磷酸二氢钾0.1克，硫酸镁0.05克，硫酸铵0.05克，琼脂2克，自然pH；
所述豆饼浸出汁制作方法：100克豆饼，加水500ml，浸泡4小时，煮沸3-4小时，纱布自然过滤，取液，调整至5波美度。
(3)取步骤(2)制得的种子液，按体积比10％的比例接种于发酵培养基中，在38℃，扩大培养20h，即得菌体发酵液；
所述发酵培养基组分如下，均为重量百分比：
蔗糖10％，麸皮2％，酵母膏1％，硝酸钠0.3％，MgSO 4·7H 2O0.05％，余量水。
实施例4
实施例1所述的米曲霉(Aspergillus oryzae)BLCY-006的培养方法，步骤包括：
(1)取米曲霉(Aspergillus oryzae)BLCY-006接种于PDA培养基中，在32℃的条件下，活化培养25h，制得活化菌株；
(2)取步骤(1)制得的活化菌株，接种于种子培养基中，在32℃的条件下，增殖培养25h，制得种子液；
所述种子培养基组分如下：
100ml豆饼浸出汁中加入可溶性淀粉2克，磷酸二氢钾0.1克，硫酸镁0.05克，硫酸铵0.05克，琼脂2克，自然pH；
所述豆饼浸出汁制作方法：100克豆饼，加水500ml，浸泡4小时，煮沸3-4小时，纱布自然过滤，取液，调整至5波美度。
(3)取步骤(2)制得的种子液，按体积比10％的比例接种于发酵培养基中，在38℃，扩大培养20h，即得菌体发酵液；
所述发酵培养基组分如下，均为重量百分比：
蔗糖10％，麸皮2％，酵母膏1％，硝酸钠0.3％，MgSO 4·7H 2O0.05％，余量水。
对比例1
取从土壤中得到、但还未做诱变、筛选前的米曲霉原始菌株进行培养，培养条件与实施例2相同。
对比例2
中国专利文献CN101691538A(申请号：200910018452.1)公开了一种高纯度低聚半乳糖的制备方法。取该专利公开的米曲霉BLB-21(保藏号CGMCCNo.2951)进行培养，培养条件与实施例2相同。
实验例1
取实施例2～4以及对比例1～2培养得到的菌体发酵液，检测发酵液中β-半乳糖苷酶的酶活结果如下表1：
表1菌体发酵液酶活
组别 β-半乳糖苷酶酶活
实施例2 305U/mL
实施例3 311U/mL
实施例4 315U/mL
对比例1 162U/mL
对比例2 189U/mL
由表1数据可以看出，实施例2-4与对比例1-2相比，通过本发明提供的米曲霉BLCY-006制备的菌体发酵液中β-半乳糖苷酶酶活有显著的提高。
实验例2
米曲霉(Aspergillus oryzae)BLCY-006在制备β-半乳糖苷酶中的应用，步骤包括：
(a)取实施例2～4与对比例1～2制备的菌体发酵液经板框式压滤机过滤，工作压力为0.3～0.5MPa，流速为5～10m 3/h，收集菌体；
(b)将步骤(a)收集到的菌体加入预冷的磷酸缓冲液然后与经过预处理的吸附剂进行反应，反应时间为15h，使菌体固定在硅藻土表面，制得β-半乳糖苷酶；
(c)配制质量浓度为40％和60％的乳糖溶液，将步骤(b)得到的β-半乳糖苷酶加入到乳糖溶液中，以乳糖质量计，加入量为5wt％，保温反应后12h后，制得低聚半乳糖粗溶液；
(d)将步骤(c)制得的低聚半乳糖粗溶液经脱色、过滤、离子交换、色谱分离、浓缩、干燥得到低聚半乳糖。
其中，步骤(d)中脱色所需活性炭添加量为0.1wt％，脱色时间为1.5h；色谱分离的运行压力为0.2MPa，温度为60℃，水耗比1:1.2，每小时进料1.8m 3。
取步骤(c)制备得到的低聚半乳糖粗溶液，检测其中的葡萄糖含量、乳糖含量、半乳糖含量以及低聚半乳糖含量；取步骤(d)制得的低聚半乳糖检测低聚半乳糖纯度及收率，结果如表2～3所示。
表2 40％乳糖溶液制备低聚半乳糖的各项指标
Figure PCTCN2020075704-appb-000001
表3 60％乳糖溶液制备低聚半乳糖的各项指标
Figure PCTCN2020075704-appb-000002
Figure PCTCN2020075704-appb-000003
通过以上数据可以看出，应用本发明提供的米曲霉(Aspergillus oryzae)BLCY-006制备的菌体发酵液，实施例2～4制备的低聚半乳糖粗溶液中对乳糖的利用率不低于88％，低聚半乳糖含量达到了61％以上；而对比例1～2对乳糖的利用率仅有81％左右，低聚半乳糖粗溶液中低聚半乳糖含量仅为40％左右。通过对比可以发现应用本发明提供的米曲霉用于制备低聚半乳糖，对乳糖的利用率显著提高，所产低聚半乳糖粗溶液中低聚半乳糖的含量也显著提升。
从最后得到的低聚半乳糖产物中分析，实施例2～4制备的低聚半乳糖的纯度均在85％以上，对比例1～2制备的低聚半乳糖的纯度均不超过75％。利用实施例2～4的菌体发酵液制备的低聚半乳糖收率为92％左右，而利用对比例1～2的菌体发酵液制备的低聚半乳糖收率仅为75％左右。实施例2～4相比于对比例1～2中低聚半乳糖的纯度和收率都有显著提高。

Claims (11)

  1. 一株米曲霉(Aspergillus oryzae)BLCY-006，2018年12月5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.16965，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. 权利要求1所述的米曲霉(Aspergillus oryzae)BLCY-006的培养方法，其特征在于，步骤包括：(1)取米曲霉(Aspergillus oryzae)BLCY-006接种于固体培养基中，在28～35℃的条件下，活化培养20～30h，制得活化菌株；(2)取步骤(1)制得的活化菌株，接种于种子培养基中，在28～35℃的条件下，增殖培养20～30h，制得种子液；(3)取步骤(2)制得的种子液，按体积比2～10％的比例接种于发酵培养基中，在28～35℃，扩大培养25～35h，即得菌体发酵液。
  3. 如权利要求2所述的培养方法，其特征在于，步骤(2)中所述种子培养基组分如下，均为重量百分比：硝酸铵0.2％；硫酸铵0.1％；磷酸二氢钾0.1％；尿素0.05％；蛋白胨1％；蔗糖2％；葡萄糖5％，余量水，pH为4.5～6.5。
  4. 如权利要求2所述的培养方法，其特征在于，步骤(3)中所述发酵培养基组分如下，均为重量百分比：蔗糖5％，葡萄糖5％，蛋白胨1％，硫酸铵0.1％；磷酸二氢钾0.1％，余量水。
  5. 如权利要求2所述的培养方法，其特征在于，步骤(1)中所述固体培养基为常规PDA固体培养基。
  6. 权利要求1所述的米曲霉(Aspergillus oryzae)BLCY-006在制备低聚半乳糖中的应用，其特征在于，包括如下步骤：(a)按照权利要求2所述的米曲霉(Aspergillus oryzae)BLCY-006的培养方法制备得到菌体发酵液，经过滤收集菌体；(b)将步骤(a)收集到的菌体加入预冷的磷酸缓冲液然后与经过预处理的吸附剂进行反应，反应时间为5～25h，使菌体固定在吸附剂表面，制得β-半乳糖苷酶；(c)配制质量浓度为40～60％的乳糖溶液，将步骤(b)得到的β-半乳糖苷酶加入到乳糖溶液中，保温反应后12h后，制得低聚半乳糖粗溶液；(d)将步骤(c)制得的低聚半乳糖粗溶液经脱色、过滤、离子交换、色谱分离、浓缩、干燥得到低聚半乳糖。
  7. 如权利要求6所述的应用，其特征在于，步骤(a)中所述过滤采用板框式压滤机过滤，工作压力为0.3～0.5MPa，流速为5～10m 3/h。
  8. 如权利要求6所述的应用，其特征在于，步骤(b)中所述吸附剂选自氧化铝、硅藻土、多孔陶瓷或纤维素。
  9. 如权利要求6所述的应用，其特征在于，步骤(c)中所述β-半乳糖苷酶的加入量，以乳糖质量计，为0.1～10wt％；所述保温反应的温度为30～60℃。
  10. 如权利要求6所述的应用，其特征在于，步骤(d)中所述脱色所需活性炭添加量为0.1wt％，脱色时间为1.5h；所述色谱分离的运行压力为0.2MPa，温度为60℃，水耗比1:1.2，每小时进料1.8m 3。
  11. 一种β-半乳糖苷酶，包括吸附剂和固定在吸附剂上的含酶米曲霉，所述含酶米曲霉由权利要求1所述的米曲霉BLCY-006制得。
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