WO2010020095A1

WO2010020095A1 - Method for producing high puritied fructooligosaccharide

Info

Publication number: WO2010020095A1
Application number: PCT/CN2008/072474
Authority: WO
Inventors: 魏远安; 曾昭政; 姚评佳; 曾宪纲; 谢庆武; 谢拥葵; 梁锦添; 曾宪经; 陈子健; 陈振鹏
Original assignee: 江门量子高科生物股份有限公司
Priority date: 2008-08-22
Filing date: 2008-09-23
Publication date: 2010-02-25
Also published as: CN101368195B; CN101368195A

Abstract

The invention discloses a preparation method of high purified fructooligosaccharide, in particular a method for preparing the high purified fructooligosaccharide by using immobilized enzymes. The preparation method of the invention prepares immobilized fructosyltransferase, immobilized glucose oxidase and immobilized mimic hydrogen peroxidase; then prepared enzymes are used to prepare the high purified fructooligosaccharide through an interrupted or continuous production method. In the preparation method, cheap metal porphyrin compounds are used as the mimic hydrogen peroxidase to replace expansive catalase; the fructosyltransferase, the glucose oxidase and the mimic hydrogen peroxidase are all immobilized and all can be recycled and reused; the stability and the use factor of the enzymes are improved; the production cost for preparing the high purified fructooligosaccharide is greatly reduced. The preparation method can use one step method to directly produce the high purified fructooligosaccharide from sucrose.

Description

一种高纯度低聚果糖的制备方法技术领域 Preparation method of high-purity oligofructose

本发明涉及一种高纯度低聚果糖的生产方法，尤其涉及一种利用固定化酶生产高纯度低聚果糖的方法。背景技术 The present invention relates to a method for producing high-purity oligofructose, and more particularly to a method for producing high-purity oligofructose using an immobilized enzyme. Background technique

低聚果糖（Fructooligosaccharide , F0S) , 又称蔗果低聚糖或果寡糖，是由 Fructooligosaccharide (F0S), also known as cane oligosaccharide or fructooligosaccharide, is

1 ~ 3个果糖基通过 β (2— 1 )糖苷键与蔗糖中的果糖基结合生成的蔗果三糖、蔗果四糖和蔗果五糖等的混合物。其中蔗果三糖、蔗果四糖和蔗果五糖是非消化性糖，具有双歧杆菌增殖功能，是功能性低聚糖的有效成分。迄今为止，市场上的低聚果糖产品分为两种：一种是低聚糖含量 50 %左右的低聚果糖（以下称为 FOS₅₀) , 其中低聚糖（三糖 +四糖 +五糖）含量在 50 %左右，其余为葡萄糖和蔗糖，糖尿病人和肥胖者不能接受；另一种是高纯度低聚果糖，即低聚糖含量达 75 %或更高的低聚果糖，是较理想的低聚果糖产品。 A mixture of 1 to 3 fructose groups formed by a β (2-1) glycosidic bond and a fructose group in sucrose to form a cane triose, a cane tetrasaccharide, and a cane fructose. Among them, sugar cane trisaccharide, cane fruit tetrasaccharide and cane fruit pentasaccharide are non-digestible sugars, which have the function of proliferating bifidobacteria and are effective components of functional oligosaccharides. So far, there are two kinds of oligofructose products on the market: one is oligofructose (hereinafter referred to as FOS ₅₀ ) with an oligosaccharide content of about 50%, among which oligosaccharides (trisaccharide + tetrasaccharide + pentasaccharide) ) The content is about 50%, the rest is glucose and sucrose, which is unacceptable to diabetics and obese people; the other is high-purity oligofructose, which is ideal for oligofructose with an oligosaccharide content of 75% or higher. Oligo-fructose products.

工业化生产高纯度低聚果糖的方法，国内外至今一直釆用两步法，即在微生物（酶）法获得 FOS₅₀基础上，再釆用层析分离法或者纳滤膜分离法将葡萄糖和蔗糖分离出去，得到高纯度低聚果糖产品。其中国外多用葡聚糖凝胶 SephadexG-1 0树脂来分离，其分离效果很好，但是这种树脂价格十分昂贵（约 1200美元 /公斤），使得设备投资成本太高，企业难以实现。国内生产高纯度低聚果糖的企业大多釆用截留分子量为 300Da 的膜组件进行纳滤分离，由于 300Da 的膜组件除了滤掉葡萄糖和蔗糖以外，还要把近一半数量的蔗果三糖也滤掉，其结果是低聚果糖的回收率和产品得率都很低，大约需要消耗 3 ~ 4 p屯 FOS₅₀才能获得 1 吨低聚糖含量 95 %左右的高纯度低聚果糖产品（ FOS₉₅ ), 因 FOS₅₀的市场价为每吨 8000元，光原料成本就高达 2.4 ~ 3万元 /吨，再加上设备运行时间长，能耗过高，使生产成本居高不下。 The industrial production method of high-purity oligofructose has been carried out two-step method at home and abroad, that is, based on the FOS ₅₀ obtained by the microorganism (enzyme) method, and the glucose separation method or the nanofiltration membrane separation method is used for glucose and sucrose. Separated to obtain a high-purity oligofructose product. Among them, Sephadex G-1 0 resin is widely used in foreign countries for separation. The separation effect is very good, but the price of this resin is very expensive (about 1,200 US dollars / kg), making the investment cost of equipment too high, which is difficult for enterprises to achieve. Most domestic enterprises producing high-purity oligofructose are subjected to nanofiltration separation using a membrane module with a molecular weight cut-off of 300 Da. Since the membrane module of 300 Da is filtered out of glucose and sucrose, nearly half of the amount of canetriose is also filtered. As a result, the recovery rate of oligofructose and the yield of the product are very low, and it takes about 3 ~ 4 p屯FOS ₅₀ to obtain 1 ton of high-purity oligofructose product with an oligosaccharide content of about 95% (FOS _95). ), due to the city of FOS ₅₀ The field price is 8,000 yuan per ton, and the cost of light raw materials is as high as 2.4 to 30,000 yuan/ton. Coupled with long equipment running time and high energy consumption, the production cost remains high.

中国发明专利 ZL 001 30200.0釆用截留分子量为 200Da的膜组件进行纳滤分离，虽然可以避免蔗果三糖的损失，但是该膜组件只能滤掉单糖，大部分蔗糖被截留，因此需要增加一次酶反应来转化蔗糖，而且同时需要增加一次脱盐、脱色工艺，然后进行第二次纳滤，才能获得 FOS₉₅产品，其工艺太复杂，相当于两次生产低纯度低聚果糖产品的成本加上两次纳滤的成本，其总成本与 300 D a膜组件纳滤法几乎一样高。 The Chinese invention patent ZL 001 30200.0 uses a membrane module with a molecular weight cut off of 200 Da for nanofiltration separation. Although the loss of canetriose can be avoided, the membrane module can only filter out monosaccharides, and most of the sucrose is trapped, so it needs to be increased. An enzyme reaction to convert sucrose, and at the same time need to add a desalting, decolorization process, and then a second nanofiltration, in order to obtain FOS ₉₅ products, the process is too complicated, equivalent to the cost of two low-purity oligofructose products The cost of the last two nanofiltrations is almost as high as the 300 D a membrane module nanofiltration.

除了层析法和纳滤分离法外，国内外也有过双酶法或混合酶法生产高纯度低聚果糖的探索性研究报导。 Yun J .W等于 1 993、 1 994年报导，用果糖基转移酶加葡萄糖氧化酶的双酶系，获得 90.05 %以上的 FOS产品；但这些试验由于需用过氧化氢酶配套，过氧化氢酶的成本太高，又不能回收，没有工业化应用价值。国内也有人尝试过双酶法；江南大学江波等 1 997 年报导在釆用黑曲霉单酶法将蔗糖转化为 56.64 % FOS的基础上，第二步用葡萄糖氧化酶加过氧化氢酶协同作用，将葡萄糖转化为葡萄糖酸，然后用离子交换法去掉葡萄糖酸，可获得 87 %的 FOS。这种双酶法的弱点是工业化生产难以实现，原因是葡萄糖氧化酶没有重复利用，过氧化氢酶的价格太昂贵。据 Sigma公司 2008年的报价，货号为 C351 5 的过氧化氢酶售价为 785.07元 /1 0mg。 In addition to chromatography and nanofiltration separation methods, there have been exploratory research reports on the production of high-purity fructose by double enzymatic or mixed enzymatic methods at home and abroad. Yun J.W is equal to 1 993, reported in 1994, using a double enzyme system of fructosyltransferase plus glucose oxidase to obtain more than 90.05% of FOS products; however, these experiments require the use of catalase, hydrogen peroxide. The cost of the enzyme is too high to be recycled, and there is no industrial application value. Some people have tried the double enzymatic method in China; Jiangbo University of Jiangnan et al reported in 1997 that the sucrose was converted to 56.64% FOS by the single-enzyme method of Aspergillus niger, and the second step was the synergistic effect of glucose oxidase plus catalase. , glucose is converted to gluconic acid, and then gluconic acid is removed by ion exchange to obtain 87% of FOS. The weakness of this double enzymatic method is that industrial production is difficult to achieve because the glucose oxidase is not reused and the price of catalase is too expensive. According to the quotation of Sigma in 2008, the price of catalase with the number C351 5 is 785.07 yuan / 10 mg.

鉴于现有技术的上述缺陷，现有必要提供一种能明显降低成本的工业化生产高纯度低聚果糖的方法。发明内容 In view of the above-mentioned drawbacks of the prior art, it is necessary to provide a method for industrially producing high-purity oligofructose which can significantly reduce the cost. Summary of the invention

本发明提供一种能明显降低成本的工业化生产高纯度低聚果糖的方法。本发明是利用可重复使用的固定化酶来生产高纯度低聚果糖。本发明的高纯度低聚果糖的制备方法包括以下两个步骤： The present invention provides a method for industrially producing high-purity oligofructose which can significantly reduce cost. The present invention utilizes a reusable immobilized enzyme to produce high purity fructooligosaccharides. The preparation method of the high-purity oligofructose of the invention comprises the following two steps:

( 1 ) 制备固定化果糖基转移酶、固定化葡萄糖氧化酶和固定化过氧化氢模拟酶； (1) preparing an immobilized fructosyltransferase, an immobilized glucose oxidase, and an immobilized hydrogen peroxide modeling enzyme;

(2) 利用步骤（ 1 ) 制备的酶用间歇式或连续式的生产方法生产高纯度低聚果糖。 (2) The enzyme prepared by the step (1) is produced by a batch or continuous production method to produce high-purity oligofructose.

其中，固定化果糖基转移酶的制备： Among them, the preparation of immobilized fructosyltransferase:

选择具有能分泌果糖基转移酶的优良菌株，在合适的培养基中培养，然后将培养得到的大量菌丝体进行破壁，用离心法将酶分离，用固定化试剂将酶固定下来。 An excellent strain capable of secreting fructosyltransferase is selected, cultured in a suitable medium, and then a large number of cultured myceliums are disrupted, and the enzyme is separated by centrifugation, and the enzyme is immobilized by an immobilization reagent.

上面所述的具有能分泌果糖基转移酶的优良菌株经本发明人进行实验和筛选，有以下几种可达到要求，它们是：米曲霉（Aspergillus oryzae)、黑曲霉 (Aspergillus niger)和 )¾土曲霉 (Aspergillus terricola) 。 The above-mentioned excellent strains capable of secreting fructosyltransferase are subjected to experiments and screening by the present inventors, and there are several kinds of achievable requirements: Aspergillus oryzae, Aspergillus niger, and 3⁄4 Aspergillus terricola.

上面所述的合适的培养基包括以下几种物盾： 1-10%蔗糖， 1-5%豆粕粉， 0.1-1% 玉米粉，其培养方法是在 25-35°C, 通风比 1:0.1-1:1, 搅拌速度 100-300rpm条件下，经过 15-45小时，获得大量菌丝体。 Suitable media as described above include the following shields: 1-10% sucrose, 1-5% soybean meal, 0.1-1% corn flour, cultured at 25-35 ° C, ventilation ratio 1: 0.1-1:1, agitating speed of 100-300 rpm, after 15 to 45 hours, a large amount of mycelium was obtained.

上面得到的菌丝体必须先进行破壁，收集这些菌体细胞的胞内酶，才获得较好的果糖基转移酶，所述的菌体破壁可用已知的方法如机械破壁法、冻融法、超声波破碎法、溶菌酶法等获得果糖基转移酶的混合液，再用离心法将含酶的溶液分离。 The mycelium obtained above must first be broken, and the intracellular enzymes of these bacterial cells are collected to obtain a better fructosyltransferase. The cell wall can be broken by a known method such as mechanical wall breaking method. A mixture of fructosyltransferase is obtained by freeze-thaw method, ultrasonication method, lysozyme method, etc., and the enzyme-containing solution is separated by centrifugation.

上面所述的用试剂将酶固定化的方法，采用化学偶联法，固定化试剂包括载体和交联剂，用来固定酶的载体是大孔树脂，所用的交联剂为戊二醛。利用偶联反应可使吸附在载体上的果糖基转移酶之间以化学键连接起来，达到固定化的目的。所用戊二醛浓度在 0.1 -0.9% , 过低或过高都影响固定化的效果。 The above method for immobilizing an enzyme with a reagent employs a chemical coupling method, and the immobilizing reagent includes a carrier and a crosslinking agent, and the carrier for immobilizing the enzyme is a macroporous resin, and the crosslinking agent used is glutaraldehyde. By using the coupling reaction, the fructosyltransferase adsorbed on the carrier can be chemically bonded to each other to achieve the purpose of immobilization. The concentration of glutaraldehyde used is between 0.1 and 0.9%. Too low or too high affects the immobilization effect.

固定化过程中，酶和载体的比例范围是每克载体需酶 15-95U, 当酶的用量 < 15u/g载体时，固定化酶的酶活太低，酶促作用很难发挥出来，但当酶活〉 95u/g载体时，酶的固定化效率太低，部分呈游离酶状态，使用一次后就流失掉。固定化的条件是： pH4-8, 温度 0-1CTC, 搅拌速度 15-60rpm。固定化操作：果糖基转移酶经分离后，用高效液相色谱法（HPLC)测定其酶活力，按一定的酶 /载体比例加入载体，混合吸附 5~24h后，加入一定浓度的戊二醛溶液，在上述条件下，交联反应 10-24h, 充分洗涤、离心甩千，可得到固定化果糖基转移酶，于 0-1CTC保存。 During the immobilization process, the ratio of enzyme to carrier is 15-95 U per gram of carrier. When the amount of enzyme is < 15 u/g, the enzyme activity of immobilized enzyme is too low, and enzymatic action is difficult to exert, but When the enzyme activity is >95 u/g, the immobilization efficiency of the enzyme is too low, and part of it is in the state of free enzyme, which is lost after one use. The conditions for immobilization are: pH 4-8, temperature 0-1 CTC, stirring speed 15-60 rpm. Immobilization: After separation of fructosyltransferase, the enzyme activity was determined by high performance liquid chromatography (HPLC), added to the carrier at a certain enzyme/carrier ratio, and mixed for 5~24 hours, then a certain concentration of glutaraldehyde was added. The solution, under the above conditions, is cross-linked for 10-24 hours, and is sufficiently washed and centrifuged to obtain an immobilized fructosyltransferase, which is stored at 0-1 CTC.

在本发明的另外的实施例中，固定化果糖基转移酶的制备方法，可按照 ZL01128345.9中公开的方法制备，此处不再赘述。 In a further embodiment of the present invention, the preparation method of the immobilized fructosyltransferase can be prepared according to the method disclosed in ZL01128345.9, and will not be described herein.

本发明中固定化葡萄糖氧化酶的制备： Preparation of immobilized glucose oxidase in the present invention:

将游离葡萄糖氧化酶用化学偶联法与载体交联，所用的载体是大孔树脂、琼脂糖凝胶、烯丙基葡聚糖凝胶或壳聚糖凝胶等，所用交联剂为戊二醛。 The free glucose oxidase is cross-linked with a carrier by a chemical coupling method, and the carrier used is a macroporous resin, an agarose gel, an allyl dextran gel or a chitosan gel, and the crosslinking agent used is pentane. Dialdehyde.

固定化的条件是：酶和载体的配比是每克载体需酶 100-300 U,在 p H 5.0~ 7.0、温度 10~50°C、搅拌速度 10~ 100r/min、戊二醛的终浓度为 0.05-0.3% 的条件下反应 3~ 10h。 The conditions for immobilization are: the ratio of enzyme to carrier is 100-300 U per gram of carrier, at p H 5.0~7.0, temperature 10~50 °C, stirring speed 10~100r/min, end of glutaraldehyde The reaction was carried out for 3 to 10 hours at a concentration of 0.05-0.3%.

所用的游离葡萄糖氧化酶可以是商业酶，也可以用自行筛选和诱变的黑曲霉菌株（Aspergillus carbonarius OLG2 ), 经过种子培养和发酵培养获得菌体后，再经过破壁、离心分离而提取的葡萄糖氧化酶。该菌株的发酵培养基组成是（以 g/L计）：蔗糖 30 - 50, 琼月旨 10-20, NaN0₃ 、 MgS0₄ - 7H₂0 、 KCI 、 FeS0₄ - 4H₂0 和 K₂HP0₄各 0.01 - 3.0, 调节 pH为 5.5 ~ 7.5。 The free glucose oxidase used may be a commercial enzyme, or may be obtained by self-screening and mutagenesis of Aspergillus carbonarius OLG2, obtained by seed culture and fermentation culture, and then subjected to cell wall separation and centrifugation. Glucose oxidase. The fermentation medium composition of this strain is (in g/L): sucrose 30 - 50, Qiongyue 10-20, NaN0 ₃ , MgS0 ₄ - 7H ₂ 0 , KCI, FeS0 ₄ - 4H ₂ 0 and K ₂ HP0 ₄ each 0.01 - 3.0, adjust the pH to 5.5 ~ 7.5.

固定化葡萄糖氧化酶的制备也可釆用壳聚糖和海藻酸盐的界面凝固 -交联耦合固定化法。先将含有发泡剂的海藻酸钠溶液滴入氯化钙溶液，形成一种具有很多微孔的海藻酸钙凝胶珠；多孔海藻酸钙凝胶珠再和壳聚糖反应形成多孔微球。由于壳聚糖分子链上有大量的伯氨基，而海藻酸盐的分子链上有大量的羧基，所以壳聚糖和海藻酸钠可以通过正、负电荷的吸引形成聚电解质膜，这样制备的微胶嚢其球性度和光洁度很好，而且具有很多微孔，其比表面较大，很适合于作为葡萄糖氧化酶的固定化载体，这样首先解决了固定化载体选择和成型问题。其次，由于这种多孔微球中每个微孔的表面是一层壳聚糖凝胶，再利用带有两个醛基的双功能交联剂戊二醛与壳聚糖和葡萄糖氧化酶通过 Sch iff反应而将酶固定在微胶嚢的各个微孔上。这样酶与载体的结合十分牢固，不易流失，而且只分布在多孔微球中微孔的表面，空间屏障和酶促反应的传盾阻力都很小，提高了固定化酶的活性。 The preparation of immobilized glucose oxidase can also be carried out by interfacial coagulation-crosslinking coupling immobilization of chitosan and alginate. First, a sodium alginate solution containing a foaming agent is dropped into a calcium chloride solution to form a calcium alginate gel bead having a plurality of micropores; the porous calcium alginate gel beads are further reacted with chitosan to form a porous microsphere. . Since there are a large number of primary amino groups on the molecular chain of chitosan and a large number of carboxyl groups in the molecular chain of alginate, chitosan and sodium alginate can form a polyelectrolyte membrane by positive and negative charge attraction. Microcapsules have good sphericity and smoothness, and have many micropores, which are larger than the surface and are very suitable for The immobilization carrier of glucose oxidase first solves the problem of immobilization carrier selection and molding. Secondly, since the surface of each microporous in the porous microsphere is a layer of chitosan gel, the bifunctional cross-linking agent with two aldehyde groups, glutaraldehyde and chitosan and glucose oxidase are used. The Schiff reaction is used to immobilize the enzyme on each microwell of the microcapsule. The binding of the enzyme to the carrier is very strong, not easy to lose, and is only distributed on the surface of the micropores in the porous microspheres, and the resistance of the space barrier and the enzymatic reaction are small, and the activity of the immobilized enzyme is increased.

固定化的条件是： The conditions for immobilization are:

将含有 0.01~0.20%NaHCO3 (盾量分数）的 1·0~2·0% (盾量分数）海藻酸钠溶液在蠕动泵加压下通过 Φ 1 - 2mm针头滴入 1.0-2.0% (质量分数）的 CaCI₂溶液，室温放置 2 ~4h, 得到白色的海藻酸钙多孔微球。用 5%醋酸溶液配制 0.1~0.5% (盾量分数）的壳聚糖溶液，按 2份溶液： 1 份微球（盾量比）投入海藻酸钙多孔 4 球，在 30~40°C、 200rpm的恒温摇床中摇动 20~40min, 用 0.5~ 1.0% (盾量分数）的 CaCI₂溶液洗涤，获得海藻酸钙 -壳聚糖多孔微球。取海藻酸钙 -壳聚糖多孔微球 100g, 加 5L 5%戊二醛溶液，在 25~35°C、 200rpm的恒温摇床中摇动 4~6h, 蒸馏水反复洗涤以除去残存的戊二醛。按每克海藻酸钙 - 壳聚糖多孔微球载体需葡萄糖氧化酶 200 ~ 500 U, 在 25~35°C、 200rpm的恒温摇床中摇动 2~4h, 放入水箱中于 4°C下静置过夜，离心、沉淀，用蒸馏水反复冲洗，过滤即得固定化葡萄糖氧化酶。 The sodium alginate solution containing 0.01 to 0.20% NaHCO3 (shield fraction) of 1.0% to 2.0% (shield fraction) was instilled through a Φ 1 - 2 mm needle under a peristaltic pump pressure of 1.0-2.0% (mass The fractional) CaCI ₂ solution was allowed to stand at room temperature for 2 to 4 hours to obtain white calcium alginate porous microspheres. Prepare a chitosan solution of 0.1~0.5% (shield fraction) with 5% acetic acid solution, and put 2 pieces of microalgae (shield ratio) into the porous 4 balls of calcium alginate at 30~40 °C. The calcium alginate-chitosan porous microspheres were obtained by shaking in a 200 rpm constant temperature shaker for 20 to 40 minutes and washing with 0.5 to 1.0% (shield fraction) of CaCI ₂ solution. Take 100 g of calcium alginate-chitosan porous microspheres, add 5 L of 5% glutaraldehyde solution, shake for 4-6 h at 25-35 ° C, 200 rpm shaker, and wash repeatedly with distilled water to remove residual glutaraldehyde. . Calcium oxidase 200-500 U per gram of calcium alginate-chitosan porous microsphere carrier, shake for 2~4h in a constant temperature shaker at 25~35 °C, 200 rpm, placed in a water tank at 4 ° C Allow to stand overnight, centrifuge, precipitate, rinse repeatedly with distilled water, and filter to obtain immobilized glucose oxidase.

本发明中固定化过氧化氢模拟酶的制备： Preparation of immobilized hydrogen peroxide mimic enzyme in the present invention:

将金属卟啉化合物氯化血红素或血红蛋白用化学偶联法与载体交联，所用的载体为琼脂糖凝胶、烯丙基葡聚糖凝胶或壳聚糖凝胶等；所用交联剂为戊二醛。固定化的条件是： p H 8.0-12.0, 温度 10~50°C, 搅拌速度 10 ~ 100r/min, 金属卟啉化合物和载体的质量比是每克载体需金属卟啉化合物 0.1 ~0.3克，交联剂戊二醛的终浓度为 0.05-0.3%, 反应时间 3~ 10h。反应结束后，用水洗涤，并测定其过氧化氢酶活性。 The metalloporphyrin compound hemin or hemoglobin is cross-linked with a carrier by a chemical coupling method, and the carrier used is an agarose gel, an allyl dextran gel or a chitosan gel; It is glutaraldehyde. The conditions for immobilization are: p H 8.0-12.0, temperature 10~50 ° C, stirring speed 10 ~ 100r / min, mass ratio of metal porphyrin compound to carrier is 0.1 ~ 0.3 g per gram of carrier metal porphyrin compound, The final concentration of the cross-linking agent glutaraldehyde is 0.05-0.3%, and the reaction time is 3-10 h. After the reaction is finished, wash with water, and The catalase activity was measured.

过氧化氢酶的活性中心是铁（III) 卟啉。用具有铁卟啉环的天然提取物氯化血红素（卟啉铁）或血红蛋白作为过氧化氢酶模拟酶，其优点是： 1、与过氧化氢酶结构相似，模拟酶的性能好； 2、属于天然产物，安全无毒，特别适于食品工业的使用； 3、材料来源于动物血，资源丰富，提取工艺筒便，价格不高。 The active center of catalase is iron (III) porphyrin. The use of a natural extract of iron porphyrin ring, hemin (porphyrin iron) or hemoglobin as a catalase mimic enzyme, has the following advantages: 1. Similar to the structure of catalase, the performance of the mimetic enzyme is good; It is a natural product, safe and non-toxic, and is especially suitable for use in the food industry. 3. The material is derived from animal blood, rich in resources, and the extraction process is convenient, and the price is not high.

用间歇式或连续式的生产方法生产高纯度低聚果糖，其中间歇式的分批法生产高纯度低聚果糖的方法是： High-purity oligofructose is produced by a batch or continuous production method, wherein the batch method for producing high-purity oligofructose is:

在反应罐中，加入 20 ~ 55% (盾量百分数）的蔗糖溶液，按每 Kg蔗糖（千）同时加入 1000 ~ 4000 U固定化果糖基转移酶、 1000 ~ 4000 U固定化葡萄糖氧化酶和 1000-4000U固定化过氧化氢模拟酶的酶量，以 1.0-3.0 L/min的流速通入空气，用 CaC0₃粉末控制反应液的 pH值，使其维持在 4.0-7.0之间，温度在 10~50°C和搅拌速度为 50 ~ 200r/min的条件下反应 10 ~ 50h, 以 H PLC检测低聚果糖含量达标后，用筛网过滤反应液，分离回收固定化酶（固定化果糖基转移酶、固定化葡萄糖氧化酶和固定化过氧化氢模拟酶），并用水充分洗涤后作下一批原料的生产之用。滤液经过精滤、离子交换和喷雾干燥成为高纯度低聚果糖产品。 In the reaction tank, add 20 ~ 55% (% of the shield) sucrose solution, add 1000 ~ 4000 U immobilized fructosyltransferase, 1000 ~ 4000 U immobilized glucose oxidase and 1000 per Kg sucrose (thousands). -4000U immobilized hydrogen peroxide mimic enzyme enzyme amount, air is introduced at a flow rate of 1.0-3.0 L/min, and the pH of the reaction solution is controlled by CaC0 ₃ powder to maintain the temperature between 4.0 and 7.0, and the temperature is 10 The reaction was carried out at ~50 ° C and stirring speed of 50 ~ 200r / min for 10 ~ 50h. After detecting the content of oligofructose by H PLC, the reaction solution was filtered through a sieve to separate and recover the immobilized enzyme (immobilized fructosyl transfer). The enzyme, immobilized glucose oxidase and immobilized hydrogen peroxide mimic enzyme) are washed thoroughly with water and used for the production of the next batch of raw materials. The filtrate is subjected to fine filtration, ion exchange and spray drying to form a high purity oligofructose product.

连续式的柱式反应法（即一步法）生产高纯度低聚果糖的方法是： The continuous column reaction method (ie, one-step method) produces high-purity oligofructose by:

将固定化果糖基转移酶、固定化葡萄糖氧化酶和固定化过氧化氢模拟酶按酶活比 1.0-4.0: 1.0 - 3.0: 1.0-4.0混合，再和 2~8% (质量百分数）的碳酸钙颗粒混匀，装入柱式反应器中，以 10~60% (盾量百分数）的蔗糖溶液在 pH4.0~7.0, 温度 10~ 5CTC下反复通过此柱，以 HPLC检测低聚果糖含量达标后过滤，经离子交换和喷雾千燥成为高纯度低聚果糖产品。 The immobilized fructosyltransferase, immobilized glucose oxidase and immobilized hydrogen peroxide mimic enzyme are mixed at an enzyme activity ratio of 1.0-4.0: 1.0 - 3.0: 1.0-4.0, and 2 to 8% (mass percent) of carbonic acid. The calcium particles are mixed and placed in a column reactor. The sucrose solution of 10~60% (% by weight) is repeatedly passed through the column at pH 4.0~7.0, temperature 10~5CTC, and the oligofructose content is detected by HPLC. After reaching the standard, it is filtered, and ion-exchanged and spray-dried to become a high-purity oligofructose product.

与现有的高纯度低聚果糖的制备方法相比较，本发明的果糖基转移酶、葡萄糖氧化酶和过氧化氢模拟酶都经过固定化处理，提高了酶的稳定性，并可以重复使用，提高了酶的利用效率。另外，用廉价的金属卟啉化合物作为过氧化氢模拟酶代替學贵的过氧化氢酶，其成本约只有过氧化氢酶的十分之一，这样能大幅度降低制备高纯度低聚果糖的生产成本。再者，与国外现行的两步法的高纯度低聚果糖制备方法比较，本发明的制备方法可以用一步法从蔗糖直接生产高纯度的低聚果糖。本发明的工艺流程筒短、技术成熟、可操作性强，而且生产成本大大降低，适于工业化生产。 Compared with the preparation method of the existing high-purity oligofructose, the fructosyltransferase, the glucose oxidase and the hydrogen peroxide mimic enzyme of the present invention are all immobilized, thereby improving the stability of the enzyme and being reusable. Increased enzyme utilization efficiency. In addition, an inexpensive metal porphyrin compound is used as a hydrogen peroxide simulation. The enzyme replaces the expensive catalase, and its cost is only about one-tenth of that of catalase, which can greatly reduce the production cost of preparing high-purity oligofructose. Furthermore, the preparation method of the present invention can directly produce high-purity oligofructose from sucrose in a one-step process as compared with the conventional two-step high-purity oligofructose preparation method. The process flow of the invention is short, the technology is mature, the operability is strong, and the production cost is greatly reduced, which is suitable for industrial production.

说明：（ 1 ) 本发明所称的糖含量均为盾量百分数，低聚糖含量指三糖 +四糖 +五糖的总和，各组分糖的含量由高效液相色谱（ HPLC)的归一化法计算而得。 Description: (1) The sugar content referred to in the present invention is the percentage of shield. The oligosaccharide content refers to the sum of trisaccharide + tetrasaccharide + pentasaccharide, and the content of sugar in each component is determined by high performance liquid chromatography (HPLC). The calculation method is obtained.

(2) 本发明所称的 "低纯度低聚果糖" 是指低聚糖含量在 50%左右，所称的 "高纯度低聚果糖" 是指低聚糖含量在 75%以上（含）。 (2) The "low-purity oligofructose" referred to in the present invention means that the oligosaccharide content is about 50%, and the so-called "high-purity oligofructose" means that the oligosaccharide content is 75% or more (inclusive).

( 3) 本发明所接触三种酶的酶活性定义分别如下： (3) The enzyme activities of the three enzymes contacted by the present invention are defined as follows:

果糖基转移酶（fructosytransferase, 缩写 FTS, 酶分类号 EG2.4 1.9)：测定方法按国家轻工行业标准 QB 2583-2003的附录 A, 酶活力定义是根据酶供应者标识的最佳酶化反应的条件下，将蔗糖转化为低聚果糖每分钟产生 1 μ ΓΠθΙ蔗果三糖所需酶量为一个酶活力单位（ U )。 Fructose transferase (fructosy transferase, abbreviation FTS, enzyme classification number EG2.4 1.9): Determination method according to national light industry standard QB 2583-2003 Appendix A, enzyme activity is defined according to the enzyme enzyme identification of the best enzymatic reaction Under the conditions, the amount of enzyme required to produce sucrose fructose per minute to produce sucrose fructose is one enzyme activity unit (U).

葡萄糖氧化酶（ glucose oxidase, 缩写 GOD, 酶分类号 EC1.1 3.4): 酶活力测定方法按滴定法（见邬显章著《酶的工业生产技术》，吉林科学技术出版社， 1988 年 49 ~ 53页），酶活力定义是在 35°C的反应温度和所规定的条件下每分钟生产 1 μ mol葡萄糖酸所需酶量为一个酶活力单位（ U )。 Glucose oxidase (Gold oxidase, abbreviated as GOD, enzyme classification number EC1.1 3.4): Determination of enzyme activity by titration (see "Industrial Production Technology of Enzymes" by Yan Xianzhang, Jilin Science and Technology Press, 1988, pp. 49 ~ 53) The enzyme activity is defined as the amount of enzyme required to produce 1 μmol of gluconic acid per minute at a reaction temperature of 35 ° C and the specified conditions is an enzyme activity unit ( U ).

过氧化氢模拟酶：酶活力测定方法按滴定法（见 B.施特尔马赫著，钱嘉渊译《酶的测定方法》，中国轻工业出版社， 1992年第一版），酶活力定义是在 35°C的反应温度和所规定的条件下每分钟分解 1 μ mol过氧化氢所需酶量为一个酶活力单位（ U )。 Hydrogen peroxide mimic enzyme: Determination of enzyme activity by titration (see B. Stermacher, Qian Jiayuan, "Method for the determination of enzymes", China Light Industry Press, 1992, first edition), definition of enzyme activity is The amount of enzyme required to decompose 1 μmol of hydrogen peroxide per minute at a reaction temperature of 35 ° C and the specified conditions is an enzyme activity unit ( U ).

下面将结合实施例详细介绍本发明的高纯度低聚果糖的制备方法。以下的实施例仅用来解释本发明，而不应以此来限定本发明的保护范围，根据本发明的本盾所做的等同变化仍属于本发明的保护范围。具体实施方式 The preparation method of the high-purity oligofructose of the present invention will be described in detail below with reference to the examples. The following examples are only intended to explain the present invention, and should not be construed as limiting the scope of the present invention. The equivalent changes made by the present invention in accordance with the present invention are still within the scope of the present invention. detailed description

实施例一 Embodiment 1

(一）固定化果糖基转移酶的制备： (1) Preparation of immobilized fructosyltransferase:

选取柄土曲霉（Aspergillus terricola)菌种接入含 5%蔗糖、 3%豆粕粉、 0.3%玉米粉培养基的 200升发酵罐中，于 33°C发酵 24小时，得种子培养液。在 5000升发酵罐中，加入培养基溶液，其中含蔗糖 5%、豆粕粉 3%、玉米粉 0.3%。于 121°C灭菌 40min, 加入种子培养液 150升，在 33°C、 220rpm的条件下，培养 24h, 得到含有菌丝体的发酵液，经离心机离心，分离收集菌丝体，洗涤、破壁、离心取酶液，经 HPLC测定酶活力后，按每 40u酶液加入 1g载体的比例，在 2000升反应罐中加入酶液和大孔树脂，搅拌均勾，冷却至 8°C , 吸附 12h。加入戊二醛，使其最终浓度为 0.2%, 在 8°C下反应 16小时，过滤取固态物，充分洗涤，离心甩千水分，即得固定化果糖基转移酶制剂。 The Aspergillus terricola strain was introduced into a 200 liter fermentor containing 5% sucrose, 3% soybean meal, and 0.3% corn flour medium, and fermented at 33 ° C for 24 hours to obtain a seed culture solution. In a 5000 liter fermentor, a medium solution containing 5% sucrose, 3% soybean meal, and 0.3% corn flour was added. After sterilization at 121 ° C for 40 min, 150 liters of seed culture solution was added, and cultured at 33 ° C and 220 rpm for 24 hours to obtain a fermentation broth containing mycelium, which was centrifuged to separate mycelium, washed, and washed. Broken wall, centrifuge to take the enzyme solution, after HPLC determination of enzyme activity, according to the ratio of 1g carrier per 40u enzyme solution, add enzyme solution and macroporous resin in 2000 liter reaction tank, stir and hook, cool to 8 ° C, Adsorption for 12h. Glutaraldehyde was added to have a final concentration of 0.2%, and reacted at 8 ° C for 16 hours, and the solid matter was collected by filtration, washed thoroughly, and centrifuged to obtain a fixed fructosyltransferase preparation.

(二）固定化葡萄糖氧化酶的制备： (ii) Preparation of immobilized glucose oxidase:

诱变黑曲霉 ( Aspergillus carbonarius 0LG2 ) 菌种接入含 2 ~ 3%蔗糖， 1 - 2% 琼月旨， 1 ~ 2%酵母提取物和 NaN0₃ 、 MgS0₄ - 7H₂0 、 KCI 、 FeS0₄ · 4H₂0和 K₂HP0₄各 0.001 ~ 0.3%培养基的 100升种子发酵罐中，调节 pH为 5.5 ~ 7.0, 于 33°C 培养 16 h,得种子培养液。在 3000升发酵罐中，加入培养基溶液，其中含 3 ~ 5%蔗糖， 1 ~2%琼月旨， NaN0₃ 、 MgS0₄ - 7H₂0 、 KCI 、 FeS0₄ · 4H₂0和 K₂HP0₄各 0.001 - 0.3%, 于 120 °C 灭菌 30 min, 加入种子培养液 80升，在 33 °C , 200r/min的条件下培养 30 h, 得含菌丝体的发酵液，离心分离收集菌丝体，洗涤、破壁、离心取酶液，超滤浓缩得到萄萄糖氧化酶的粗酶液，用滴定法测定其酶活力。 Mutagenesis of Aspergillus carbonarius 0LG2 strains containing 2 ~ 3% sucrose, 1 - 2% Qiongyue, 1 ~ 2% yeast extract and NaN0 ₃ , MgS0 ₄ - 7H ₂ 0 , KCI, FeS0 ₄ · In a 100 liter seed fermenter of 4H ₂ 0 and K ₂ HP0 ₄ each of 0.001 ~ 0.3% medium, the pH was adjusted to 5.5 ~ 7.0, and cultured at 33 ° C for 16 h to obtain a seed culture solution. In a 3000 liter fermentor, a medium solution containing 3 to 5% sucrose, 1 to 2% Qiongyue, NaN0 ₃ , MgS0 ₄ - 7H ₂ 0 , KCI, FeS0 ₄ · 4H ₂ 0 and K ₂ HP0 was added. ₄ 0.001 - 0.3% each, sterilized at 120 °C for 30 min, added 80 liters of seed culture solution, and cultured at 33 °C, 200r/min for 30 h to obtain mycelium-containing fermentation broth, collected by centrifugation. The mycelium was washed, broken, centrifuged to obtain an enzyme solution, and concentrated by ultrafiltration to obtain a crude enzyme solution of glucose oxidase, and the enzyme activity was measured by a titration method.

在 100升反应罐中， 30-4CTC下，按每克载体（湿）加入 100-300 U 酶液的比例，加入 10 Kg 的壳聚糖凝胶（湿）和 100 ~ 300万 U 的酶液，搅拌混匀后加入戊二醛，使其终浓度为 0.05~ 0.2%, 在 pH 5.0-7.0, 30 ~ 4CTC下反应 4 ~ 6 h , 过滤取固态物，用去离子水充分洗涤，离心甩千、成型，即得固定化萄糖氧酶制剂。 In a 100 liter reaction tank, add 10 Kg of chitosan gel (wet) and 100 to 3 million U of enzyme solution at a ratio of 100-300 U per gram of carrier (wet) at 30-4 CTC. After stirring and mixing, add glutaraldehyde to a final concentration of 0.05-0.2%, and react at pH 5.0-7.0, 30 ~ 4CTC for 4-6 h. The solid matter is filtered, washed thoroughly with deionized water, centrifuged, and shaped to obtain a fixed glucose oxidase preparation.

(三）固定化过氧化氢模拟酶的制备 (III) Preparation of immobilized hydrogen peroxide mimic enzyme

在 100升反应罐中，加入 0.1mol/L 氢氧化钠溶液 50L, 30 ~ 4CTC下加入卟啉铁 2.5Kg搅拌至完全溶解。按卟啉铁：壳聚糖凝胶 = 1.0 ~ 3.0: 10.0的盾量比，加入 10 ~25Kg的壳聚糖凝胶（湿），搅拌混匀后加入戊二醛，使其终浓度为 0.05 ~ 0.2%%, 在 pH 9.0- 12.0, 30~40°C下反应 4~8 h , 过滤取固态物，用去离子水充分洗涤，离心甩千、成型，即得固定化过氧化氢模拟酶。用滴定法测定其酶活力。 In a 100 liter reaction tank, 50 L of a 0.1 mol/L sodium hydroxide solution was added, and 2.5 Kg of porphyrin iron was added at 30 to 4 CTC until it was completely dissolved. According to the porphyrin iron: chitosan gel = 1.0 ~ 3.0: 10.0 shield ratio, add 10 ~ 25Kg of chitosan gel (wet), stir and mix, add glutaraldehyde, the final concentration is 0.05 ~ 0.2%%, reacted at pH 9.0- 12.0, 30~40 °C for 4~8 h, filtered to take solids, washed thoroughly with deionized water, centrifuged, and shaped to obtain immobilized hydrogen peroxide mimic enzyme . The enzyme activity was determined by titration.

(四）固定化酶法生产高纯度低聚果糖 (iv) Production of high-purity oligofructose by immobilized enzymatic method

在 2.2M³ 的反应罐中，力。入 50 ~ 55% (盾量百分数）蔗糖溶液 2000 Kg, 加入固定化果糖基转移酶制剂 125 ~ 380万 U、固定化萄糖氧酶制剂 100 ~ 300万 U和固定化过氧化氢模拟酶 150-400万 U, 以 1.0- 3.0 L/min的流速通入空气，用 CaC0₃粉末控制反应液的 pH值，使其维持在 5.0 ~ 7.0之间，在 30~45°C和搅拌速度为 50~ 200r/min的条件下反应 12~30h, 以 HPLC检测低聚果糖含量达标后，用筛网过滤反应液，分离回收固定化果糖基转移酶、固定化葡萄糖氧化酶和固定化过氧化氢模拟酶，并用水充分洗涤后作下一批原料的生产之用。滤液经过精滤、离子交换和喷雾千燥成为高纯度低聚果糖产品。 In a 2.2M ³ reaction tank, force. 50 ~ 55% (% of shield) sucrose solution 2000 Kg, added immobilized fructosyltransferase preparation 125 ~ 3.8 million U, immobilized glucooxygenase preparation 100 ~ 3 million U and immobilized hydrogen peroxide mimic enzyme 150 -4 million U, air is introduced at a flow rate of 1.0-3.0 L/min, and the pH of the reaction solution is controlled with CaC0 ₃ powder to maintain a pH between 5.0 and 7.0, at 30 to 45 ° C and a stirring speed of 50 The reaction was carried out at ~200r/min for 12~30h. After HPLC detection of oligofructose content reached the standard, the reaction solution was filtered through a sieve, and the immobilized fructosyltransferase, immobilized glucose oxidase and immobilized hydrogen peroxide were separated and recovered. The enzyme is washed thoroughly with water and used for the production of the next batch of raw materials. The filtrate is subjected to fine filtration, ion exchange and spray drying to form a high-purity oligofructose product.

实施例二 Embodiment 2

(一）固定化果糖基转移酶的制备 (1) Preparation of immobilized fructosyltransferase

选取柄土曲霉（Aspergillus terricola)菌种接入含 5%蔗糖、 3%豆粕粉、 0.3%玉米粉培养基的 200升发酵罐中，于 33°C发酵 24小时，得种子培养液。在 5000升发酵罐中，加入培养基溶液，其中含蔗糖 5%、豆粕粉 3%、玉米粉 0.3%。于 121°C灭菌 40min, 加入种子培养液 150升，在 33°C、 220rpm的条件下，培养 24h, 得到含有菌丝体的发酵液，经离心机离心，分离收集菌丝体，洗涤，破壁，离心取酶液，经 HPLC测定酶活力后，按每 40u酶液加入 1g载体的比例，在 2000升反应罐中加入酶液和大孔树脂，搅拌均勾，冷却至 8°C , 吸附 12h。加入戊二醛，使其最终浓度为The Aspergillus terricola strain was selected into a 200 liter fermentor containing 5% sucrose, 3% soybean meal, and 0.3% corn flour medium, and fermented at 33 ° C for 24 hours to obtain a seed culture solution. In a 5000 liter fermentor, a medium solution containing 5% sucrose, 3% soybean meal, and 0.3% corn flour was added. After sterilization at 121 ° C for 40 min, 150 liters of seed culture solution was added, and cultured at 33 ° C and 220 rpm for 24 hours to obtain a fermentation broth containing mycelium, which was centrifuged, and the mycelium was collected and washed. Broken wall, centrifuge to take the enzyme solution, after HPLC determination of enzyme activity, add 1g carrier per 40u enzyme solution, add enzyme in 2000 liter reaction tank The liquid and macroporous resin were stirred and stirred, cooled to 8 ° C, and adsorbed for 12 h. Add glutaraldehyde to a final concentration of

0.2%, 在 8°C下反应 16小时，过滤取固态物，充分洗涤，离心甩千水分，即得固定化果糖基转移酶制剂。 0.2%, reacted at 8 ° C for 16 hours, filtered to take the solid matter, washed thoroughly, and centrifuged to obtain thousands of water to obtain an immobilized fructosyltransferase preparation.

诱变黑曲霉 ( Aspergillus carbonarius OLG2 ) 菌种接入含 2~3%蔗糖， 1 - 2%琼脂， 1 ~ 2%酵母提取物和 NaN0₃ 、 MgS0₄-7H₂0 、 KCI 、 FeS0₄-4H₂0 和 K₂HP0₄各 0.001 ~ 0.3%培养基的 100升种子发酵罐中，调节 pH为 5.5 ~ 7. Q 于 33°C培养 16 h,得种子培养液。在 3000升发酵罐中，加入培养基溶液，其中含 3~5%蔗糖， 1 ~2%琼月旨， NaN0₃ 、 MgS0₄-7H₂0 、 KCI 、 FeS0₄4H₂0和 K₂HP0₄各 0.001 ~ 0.3%, 于 120 °C灭菌 30 min, 加入种子培养液 80升，在 33Mutagenesis of Aspergillus carbonarius OLG2 strains containing 2~3% sucrose, 1-2% agar, 1-2% yeast extract and NaN0 ₃ , MgS0 ₄ -7H ₂ 0 , KCI , FeS0 ₄ -4H _In a 100 liter seed fermenter of 0.001 to 0.3% medium of ₂₀ and K ₂ HP0 ₄ , the pH was adjusted to 5.5 ~ 7. Q and cultured at 33 ° C for 16 h to obtain a seed culture solution. In a 3000 liter fermentor, a medium solution containing 3 to 5% sucrose, 1 to 2% Qiongyue, NaN0 ₃ , MgS0 ₄ -7H ₂ 0 , KCI, FeS0 ₄ 4H ₂ 0 and K ₂ HP0 _{4 was added.} 0.001 ~ 0.3% each, sterilized at 120 °C for 30 min, added seed broth 80 liters, at 33

°C, 200r/min的条件下培养 30 h, 得含菌丝体的发酵液，离心分离收集菌丝体，洗涤、破壁、离心取酶液，超滤浓缩得到萄糖氧化酶的粗酶液，用滴定法测定其酶活力。 After incubating at 200 °C for 30 h, the mycelium-containing fermentation broth was obtained, and the mycelium was collected by centrifugation, washed, broken, centrifuged to obtain the enzyme solution, and concentrated by ultrafiltration to obtain the crude enzyme of glucose oxidase. Liquid, the enzyme activity was determined by titration.

将含有 0.01~0.20%NaHCO₃(盾量分数）的 1.0-2.0% (盾量分数）海藻酸钠溶液，在蠕动泵加压下通过 φ 1 ~2mm针头滴入 1·0~2·0% (盾量分数）的 CaCI₂溶液，室温放置 2 ~ 4h, 得到白色的海藻酸钙多孔微球。用 5%醋酸溶液配制 0.1~0.5% (盾量分数）的壳聚糖溶液，按 2份溶液： 1 份微球（盾量比）投入海藻酸钙多孔 4 球，在 30~40°C、 200rpm的恒温摇床中摇动 20~40min, 用 0.5~ 1.0% (盾量分数）的 CaCI₂溶液洗涤，获得海藻酸钙 -壳聚糖多孔微球。取该微球 100g, 力。 5L 5%戊二醛溶液，在 25~35°C、 200rpm的恒温摇床中摇动 4 ~ 6h , 蒸馏水反复洗涤，以除去残存的戊二醛。 1.0-2.0% (shield fraction) sodium alginate solution containing 0.01~0.20% NaHCO ₃ (shield fraction), and drip 1·0~2·0% through φ 1 ~2mm needle under peristaltic pump pressure The CaCI ₂ solution (shield fraction) was placed at room temperature for 2 to 4 hours to obtain white calcium alginate porous microspheres. Prepare a chitosan solution of 0.1~0.5% (shield fraction) with 5% acetic acid solution, and put 2 pieces of microalgae (shield ratio) into the porous 4 balls of calcium alginate at 30~40 °C. The calcium alginate-chitosan porous microspheres were obtained by shaking in a 200 rpm constant temperature shaker for 20 to 40 minutes and washing with 0.5 to 1.0% (shield fraction) of CaCI ₂ solution. Take the microsphere 100g, force. 5L 5% glutaraldehyde solution was shaken for 4-6 hours in a constant temperature shaker at 25-35 ° C and 200 rpm, and distilled water was repeatedly washed to remove residual glutaraldehyde.

在 100升反应罐中， 30~40°C下，按每克多孔微球载体（湿）加入 200 500 U 酶液的比例，加入 10 Kg 的多孔敫球载体（湿）和 200 ~ 500万 U 的酶液，在 25 ~ 35°C、 200rpm的恒温摇床中摇动 2~4h, 放入水拒中于 4°C下静置过夜，离心，沉淀用去离子水反复冲洗，离心甩千，即得固定化葡萄糖氧化酶制剂。 In a 100 liter reaction tank, add 10 Kg of porous spheroidal carrier (wet) and 2 to 5 million U per gram of porous microsphere carrier (wet) at a ratio of 200 500 U of enzyme solution at 30 to 40 ° C. The enzyme solution was shaken for 2 to 4 hours in a constant temperature shaker at 25 ~ 35 ° C and 200 rpm, placed in a water rejection, allowed to stand at 4 ° C overnight, centrifuged, and sunk. The mixture is repeatedly rinsed with deionized water, and centrifuged for thousands of times to obtain an immobilized glucose oxidase preparation.

在 100升反应罐中，加入去离子水 50L, 30~40°C下加入血红蛋白 3Kg搅拌至完全溶解。按血红蛋白：壳聚糖凝胶 = 2.0 ~ 3.0: 10.0的盾量比，力。入 10~ 15Kg的壳聚糖凝胶（湿），搅拌混匀后加入戊二醛，使其终浓度为 0.05- 0.2%%, 在 pH 5.0 - 7.0, 30~40°C下反应 4~8 h , 过滤取固态物，用去离子水充分洗涤，离心甩千、成型，即得固定化过氧化氢模拟酶。用滴定法测定其酶活力。 In a 100 liter reaction tank, 50 L of deionized water was added, and 3 kg of hemoglobin was added at 30 to 40 ° C until it was completely dissolved. According to hemoglobin: chitosan gel = 2.0 ~ 3.0: 10.0 shield ratio, force. Add 10~15Kg of chitosan gel (wet), stir and mix, add glutaraldehyde to the final concentration of 0.05-0.2%%, react at pH 5.0 - 7.0, 30~40 °C 4~8 h, filter the solid matter, wash it thoroughly with deionized water, centrifuge it, and shape it to obtain immobilized hydrogen peroxide mimic enzyme. The enzyme activity was determined by titration.

在 3.5M³ 的反应罐中，力。入 50 ~ 55% (盾量百分数）蔗糖溶液 3000 Kg, 加入固定化果糖基转移酶制剂 200 ~ 550万 U、固定化萄糖氧酶制剂 150 ~ 450万 U和固定化过氧化氢模拟酶 220 ~ 560万 U, 以 1.0-2.0 L/min的流速通入空气，用 CaC0₃粉末控制反应液的 pH值维持在 4.5 ~ 7.0之间，在 30 ~ 45 °C和搅拌速度为 100 ~ 200r/min的条件下反应 12~28h, 以 HPLC检测低聚果糖含量达标后，用筛网过滤反应液，分离回收固定化酶一固定化果糖基转移酶、固定化葡萄糖氧化酶和固定化过氧化氢模拟酶，并用水洗涤后作下一批原料的生产之用。滤液经过精滤、离子交换和喷雾千燥成为高纯度低聚果糖产品。实施例三 In a 3.5M ³ reaction tank, force. Into 50 ~ 55% (% of shield) sucrose solution 3000 Kg, added immobilized fructosyltransferase preparation 200 ~ 5.5 million U, immobilized glucooxygenase preparation 150 ~ 4.5 million U and immobilized hydrogen peroxide mimic enzyme 220 ~ 5.6 million U, air is introduced at a flow rate of 1.0-2.0 L/min, and the pH of the reaction solution is maintained between 4.5 and 7.0 with CaC0 ₃ powder, at 30 to 45 ° C and a stirring speed of 100 to 200 r / The reaction was carried out for 12~28h under the condition of min. After HPLC detection of the oligofructose content reached the standard, the reaction solution was filtered through a sieve, and the immobilized enzyme-immobilized fructosyltransferase, immobilized glucose oxidase and immobilized hydrogen peroxide were separated and recovered. The enzyme was simulated and washed with water for the production of the next batch of raw materials. The filtrate is subjected to fine filtration, ion exchange and spray drying to form a high-purity oligofructose product. Embodiment 3

(一）固定化果糖基转移酶的制备，按 ZL 01128345.9的实施例一操作。 (i) Preparation of immobilized fructosyltransferase, operating as in Example 1 of ZL 01128345.9.

固定化载体按实施例二方法制备，取市售商业萄糖氧化酶的粗酶液，经滴定法测定酶活力后，在 100升反应罐中， 30-4CTC下，按每 Kg多孔微球载体（湿）加入 20万〜 50万 U 酶液的比例，力。入 20 Kg 的多孔微球载体（湿）和 40万〜 100万 U 的酶液，在 25~35°C、 200rpm的恒温摇床中摇动 2~4h, 放入水拒中于 4°C下静置过夜，离心、沉淀，用去离子水反复冲洗，离心甩千，即得固定化葡萄糖氧化酶制剂。 (三）固定化过氧化氢模拟酶的制备 The immobilized carrier is prepared according to the method of the second embodiment, and the crude enzyme solution of commercial sugar oxidase is commercially available. After the enzyme activity is determined by titration, in a 100 liter reaction tank, 30-4 CTC, per Kg porous microsphere carrier (wet) Add the ratio of 200,000 ~ 500,000 U enzyme solution, force. Into the 20 Kg porous microsphere carrier (wet) and 400,000 ~ 1 million U of enzyme solution, shake in a constant temperature shaker at 25 ~ 35 ° C, 200 rpm for 2 ~ 4h, put in water to reject at 4 ° C After standing overnight, centrifugation, sedimentation, repeated washing with deionized water, and centrifugation, the immobilized glucose oxidase preparation was obtained. (III) Preparation of immobilized hydrogen peroxide mimic enzyme

在 200升反应罐中，加入 0.1mol/L 氢氧化钠溶液 100L, 30 ~ 4CTC下加入卟啉铁 5Kg搅拌至完全溶解。按卟啉铁：壳聚糖凝胶 = 1.0 ~ 3.0: 10.0的盾量比，加入 20 ~50Kg的壳聚糖凝胶（湿），搅拌混匀后加入戊二醛，使其终浓度为 0.05 ~ 0.2%%, 在 pH 9.0- 12.0, 30~40°C下反应 4~8 h , 过滤取固态物，用去离子水充分洗涤，离心甩千、成型，即得固定化过氧化氢模拟酶。用滴定法测定其酶活力。 In a 200 liter reaction tank, 100 L of a 0.1 mol/L sodium hydroxide solution was added, and 5 Kg of porphyrin iron was added at 30 to 4 CTC until it was completely dissolved. According to the porphyrin iron: chitosan gel = 1.0 ~ 3.0: 10.0 shield ratio, add 20 ~ 50Kg chitosan gel (wet), stir and mix, add glutaraldehyde, the final concentration is 0.05 ~ 0.2%%, reacted at pH 9.0- 12.0, 30~40 °C for 4~8 h, filtered to take solids, washed thoroughly with deionized water, centrifuged, and shaped to obtain immobilized hydrogen peroxide mimic enzyme . The enzyme activity was determined by titration.

将固定化果糖基转移酶、固定化葡萄糖氧化酶和固定化过氧化氢模拟酶的酶活比按： 1.2-3.8: 1.0 - 3.0: 1.5-4.0混合，再和 2~8% (质量百分数）的碳酸钙颗粒混匀，装入柱式反应器中，以 10~50% (盾量百分数）的蔗糖溶液在 pH4.8~ 7.0, 温度 30~ 5CTC下反复通过此柱，以 HPLC检测低聚果糖含量达标后，过滤回收固定化果糖基转移酶、固定化葡萄糖氧化酶和固定化过氧化氢模拟酶，滤液经离子交换和喷雾千燥成为高纯度低聚果糖产品。 The enzyme activity ratio of immobilized fructosyltransferase, immobilized glucose oxidase and immobilized hydrogen peroxide mimic enzyme is: 1.2-3.8: 1.0 - 3.0: 1.5-4.0, and then 2 to 8% (mass percentage) The calcium carbonate particles are mixed and placed in a column reactor. The sucrose solution of 10~50% (% by weight) is repeatedly passed through the column at a temperature of 30~5CTC to detect oligomerization by HPLC. After the fructose content reached the standard, the immobilized fructosyltransferase, the immobilized glucose oxidase and the immobilized hydrogen peroxide mimic enzyme were recovered by filtration, and the filtrate was dried by ion exchange and spray to become a high-purity oligofructose product.

Claims

权利要求 Rights request

1、一种高纯度低聚果糖的生产方法，其包括以下步骤： A method for producing high-purity oligofructose, comprising the steps of:

2、如权利要求 1 所述的高纯度低聚果糖的生产方法，其特征在于，所述制备固定化果糖基转移酶的方法为：选择具有能分泌杲糖基转移酶的优良菌株，在合适的培养基和培养条件中培养，然后将培养得到的菌丝体进行破壁，用离心法将酶分离，用固定化试剂将酶固定下来，其中，所述的优良菌株为米曲霉、黑曲霉或柄土曲霉。 The method for producing high-purity oligofructose according to claim 1, wherein the method for preparing the immobilized fructosyltransferase is: selecting an excellent strain capable of secreting a sucrose-based transferase, at a suitable The culture medium and the culture conditions are cultured, and then the cultured mycelium is broken, the enzyme is separated by centrifugation, and the enzyme is immobilized by an immobilization reagent, wherein the excellent strain is Aspergillus oryzae, Aspergillus niger Or Aspergillus terreus.

3、如权利要求 2所述的高纯度低聚果糖的生产方法，其特征在于，所述的合适的培养基包括以下几种物盾 1 - 10% (盾量分数）蔗糖， 1 ~5% (盾量分数）豆粕粉， 0.1 ~ 1% (盾量分数）玉米粉，所述的培养条件包括培养温度 25 ~35°C, 通风比为 1:0.1— 1:1, 搅拌速度 100~ 300rpm。 3. The method for producing high-purity oligofructose according to claim 2, wherein the suitable medium comprises the following materials: 1 - 10% (shield fraction) sucrose, 1 to 5% (Shield fraction) Soybean meal, 0.1 ~ 1% (shield fraction) corn flour, the culture conditions include culture temperature 25 ~ 35 ° C, ventilation ratio is 1: 0.1 - 1:1, stirring speed 100 ~ 300rpm .

4、如权利要求 2 所述的高纯度低聚果糖的生产方法，其特征在于，所述的用固定化试剂将酶固定下来，所用的固定化方法为化学偶联法，所述的固定化试剂包括载体和交联剂，其中所述载体为大孔树脂，所述交联剂为戊二醛。 The method for producing high-purity oligofructose according to claim 2, wherein the immobilization reagent immobilizes the enzyme, and the immobilization method used is a chemical coupling method, and the immobilization is performed. The reagent includes a carrier and a crosslinking agent, wherein the carrier is a macroporous resin, and the crosslinking agent is glutaraldehyde.

5、如权利要求 4所述的高纯度低聚果糖的生产方法，其特征在于，固定化过程中酶和载体的比例范围是每克载体对应 15~95u的酶，固定化条件是 pH4~8, 温度 0~ 10°C, 搅拌速度 15~60rpm, 其中固定化包括以下操作步骤：果糖基转移酶经分离后，测定其酶活力，按上述的酶 /载体比例加入载体，混合吸附 5~24h后，加入 0.1 ~ 0.9%浓度的戊二醛溶液，在上述条件下，交联反应 10~24h, 洗涤、离心甩千，可得到固定化果糖基转移酶，于 0~ 10°C保存。 The method for producing high-purity oligofructose according to claim 4, wherein the ratio of the enzyme to the carrier in the immobilization process ranges from 15 to 95 u per gram of the carrier, and the immobilization condition is pH 4 to 8. The temperature is 0~10°C, and the stirring speed is 15~60rpm. The immobilization includes the following steps: After the fructosyltransferase is separated, the enzyme activity is determined, and the carrier is added according to the above enzyme/carrier ratio, and the adsorption is mixed for 5~24h. After adding 0.1~0.9% glutaraldehyde solution, under the above conditions, cross-linking reaction for 10~24h, washing and centrifuging Thousands, an immobilized fructosyltransferase can be obtained and stored at 0 to 10 °C.

6、如权利要求 1所述的高纯度低聚果糖的生产方法，其特征在于，固定化葡萄糖氧化酶是将游离的葡萄糖氧化酶用化学偶联法与载体交联，所用的载体是天然的或合成的高聚物，所用交联剂为戊二醛。 The method for producing high-purity oligofructose according to claim 1, wherein the immobilized glucose oxidase crosslinks the free glucose oxidase with a carrier by a chemical coupling method, and the carrier used is natural. Or a synthetic high polymer, the crosslinking agent used is glutaraldehyde.

7、如权利要求 6 所述的高纯度低聚果糖的生产方法，其特征在于，所述载体为交联琼脂糖凝胶、交联烯丙基葡聚糖凝胶或者交联壳聚糖凝胶。 7. The method for producing high-purity oligofructose according to claim 6, wherein the carrier is a crosslinked agarose gel, a crosslinked allyl dextran gel or a crosslinked chitosan condensate. gum.

8、如权利要求 6或 7所述的高纯度低聚果糖的生产方法，其特征在于，固定化过程中酶和载体的配比是每克载体对应 100~ 300 U的葡萄糖氧化酶，固定化是在 pH 5.0 ~ 7.0、温度 10~50°C、搅拌速度 10 ~ 100r/min、戊二醛的终浓度为 0.05 ~ 0.3%和反应 3~ 10h的条件下进行的。 The method for producing high-purity oligofructose according to claim 6 or 7, wherein the ratio of the enzyme to the carrier in the immobilization process is 100 to 300 U of glucose oxidase per gram of the carrier, and is immobilized. It is carried out under the conditions of pH 5.0 ~ 7.0, temperature 10~50 °C, stirring speed 10 ~ 100r/min, final concentration of glutaraldehyde 0.05~0.3% and reaction 3~10h.

9、如权利要求 1所述的高纯度低聚果糖的生产方法，其特征在于，固定化葡萄糖氧化酶的制备方法为：壳聚糖和海藻酸盐的界面凝固一交联耦合固定化法。 The method for producing high-purity oligofructose according to claim 1, wherein the method for preparing the immobilized glucose oxidase is: an interfacial solidification-crosslinking coupling immobilization method of chitosan and alginate.

10、如权利要求 9所述的高纯度低聚果糖的生产方法，其特征在于，所述的壳聚糖和海藻酸盐的界面凝固一交联耦合固定化法是：先将含有发泡剂的海藻酸钠溶液滴入氯化钙溶液，形成一种具有很多微孔的海藻酸钙凝胶珠；多孔海藻酸钙凝胶珠再和壳聚糖反应形成具有很多微孔的微球；再用交联剂戊二醛与壳聚糖和葡萄糖氧化酶通过 Schiff反应而将酶固定在微球的微孔上。 The method for producing high-purity oligofructose according to claim 9, wherein the interfacial coagulation-crosslinking coupling immobilization method of the chitosan and the alginate is: first containing a foaming agent The sodium alginate solution is dropped into the calcium chloride solution to form a calcium alginate gel bead having a plurality of micropores; the porous calcium alginate gel beads are further reacted with chitosan to form microspheres having many micropores; The enzyme was immobilized on the micropores of the microspheres by a Schiff reaction using a cross-linking agent glutaraldehyde with chitosan and glucose oxidase.

11、如权利要求 10所述的高纯度低聚果糖的生产方法，其特征在于，所述的壳聚糖和海藻酸盐的界面凝固一交联耦合固定化法详细过程为：将含有 0.01~0.20%NaHCO₃ (盾量分数）的 1 ·0 ~ 2.0% (盾量分数）海藻酸钠溶液在蠕动泵加压下滴入 1·0~2·0% (盾量分数）的 CaCI₂溶液，室温放置 2~4h, 得到白色的海藻酸钙多孔微球；用 5%醋酸溶液配制 0.1~0.5% (盾量分数）的壳聚糖溶液，按 2份溶液：The method for producing high-purity oligofructose according to claim 10, wherein the interfacial solidification-crosslinking coupling immobilization method of the chitosan and the alginate is as follows: 0.20% NaHCO ₃ (shield fraction) 1 ·0 ~ 2.0% (shield fraction) sodium alginate solution was dropped into the 1.00~2·0% (shield fraction) CaCI ₂ solution under peristaltic pump pressure , placed at room temperature for 2~4h, to obtain white calcium alginate porous microspheres; prepare 0.1~0.5% (shield fraction) of chitosan solution with 5% acetic acid solution, according to 2 parts solution:

1份微球（盾量比）投入海藻酸钙多孔微球，在 30 ~ 40°C、 200rpm的恒温摇床中摇动 20~40min, 用 0.5 ~ 1.0% (盾量分数）的 CaCI₂溶液洗涤，获得海藻酸钙 -壳聚糖多孔微球；取海藻酸钙 -壳聚糖多孔微球 100g, 加 5L5% (盾量分数）戊二醛溶液，在 25~35°C、 200rpm的恒温摇床中摇动 4~6h, 蒸馏水洗涤除去残存的戊二醛；按每克海藻酸钙 -壳聚糖多孔微球载体需葡萄糖氧化酶 200 ~ 500 U的量加入葡萄糖氧化酶，在 25~35°C、 200rpm的恒温摇床中摇动 2~4h, 放入水箱中于 4°C下静置过夜，离心，沉淀用蒸馏水冲洗，过滤即得固定化葡萄糖氧化酶。 1 microsphere (shield ratio) is put into the calcium alginate porous microsphere, shaken in a constant temperature shaker at 30 ~ 40 °C, 200 rpm for 20~40min, and washed with 0.5 ~ 1.0% (shield fraction) CaCI ₂ solution , obtaining calcium alginate-chitosan Porous microspheres; take calcium alginate-chitosan porous microspheres 100g, add 5L5% (shield fraction) glutaraldehyde solution, shake in 4~6h at 25~35°C, 200rpm constant temperature shaker, wash with distilled water Remove residual glutaraldehyde; add glucose oxidase per gram of calcium alginate-chitosan porous microsphere carrier in an amount of 200 ~ 500 U of glucose oxidase, shake in a constant temperature shaker at 25-35 ° C, 200 rpm 2~4h, placed in a water tank and allowed to stand at 4 ° C overnight, centrifuged, and the precipitate was washed with distilled water, and filtered to obtain immobilized glucose oxidase.

12、如权利要求 1所述的高纯度低聚果糖的生产方法，其特征在于，固定化过氧化氢模拟酶的制备方法为：金属卟啉化合物用化学偶联法与载体交联，其中所用的载体是天然或合成高聚物，所用交联剂为戊二醛。 The method for producing high-purity oligofructose according to claim 1, wherein the method for preparing the immobilized hydrogen peroxide mimic enzyme is: the metal porphyrin compound is cross-linked with a carrier by a chemical coupling method, wherein The carrier is a natural or synthetic polymer and the crosslinking agent used is glutaraldehyde.

13、如权利要求 12所述的高纯度低聚果糖的生产方法，其特征在于，所述载体为交联琼脂糖凝胶、交联烯丙基葡聚糖凝胶或者交联壳聚糖凝胶，所述金属卟啉化合物为氯化血红素或者血红蛋白。 The method for producing high-purity oligofructose according to claim 12, wherein the carrier is a crosslinked agarose gel, a crosslinked allyl dextran gel or a crosslinked chitosan condensate a gum, the metalloporphyrin compound being hemin or hemoglobin.

14、如权利要求 12所述的高纯度低聚果糖的生产方法，其特征在于，固定化过氧化氢模拟酶的制备是在 pH 8.0 ~ 12.0, 温度 10-5CTC, 搅拌速度 10 ~ 10Or/min条件下进行，其中，金属卟啉化合物和载体的盾量比是每克载体需金属卟啉化合物 0.1 ~0.3克，交联剂戊二醛的终浓度为 0.05- 0.3%, 反应时间 3~ 10h, 反应结束后，洗涤，测酶活性。 The method for producing high-purity oligofructose according to claim 12, wherein the immobilized hydrogen peroxide mimic enzyme is prepared at a pH of 8.0 to 12.0, a temperature of 10-5 CTC, and a stirring speed of 10 to 10 Or/min. Under the conditions, wherein the shield ratio of the metalloporphyrin compound and the carrier is 0.1 to 0.3 g per gram of the metal porphyrin compound, the final concentration of the crosslinking agent glutaraldehyde is 0.05 to 0.3%, and the reaction time is 3 to 10 hours. After the reaction is completed, wash and measure the enzyme activity.

15、如权利要求 1 所述的高纯度低聚果糖的制备方法，其特征在于，所述的间歇式生产方法是：在反应罐中，加入蔗糖溶液，同时加入固定化果糖基转移酶、固定化葡萄糖氧化酶和固定化过氧化氢模拟酶，通入空气，控制反应液的 pH值，在一定温度和搅拌速度下反应 10~ 50h, 监测反应液中低聚果糖的含量，果糖含量达标后，过滤反应液，分离回收固定化酶，然后滤液进一步精滤、离子交换和喷雾千燥成为高纯度低聚果糖产品。 The method for preparing high-purity oligofructose according to claim 1, wherein the batch production method comprises: adding a sucrose solution to the reaction tank, adding the immobilized fructosyltransferase, and fixing Glucose oxidase and immobilized hydrogen peroxide mimic enzyme, pass air, control the pH value of the reaction solution, react at a certain temperature and stirring speed for 10~50h, monitor the content of oligofructose in the reaction solution, and the fructose content reaches the standard The reaction solution is filtered, and the immobilized enzyme is separated and recovered, and then the filtrate is further finely filtered, ion-exchanged, and spray-dried to become a high-purity oligofructose product.

16、如权利要求 15 所述的高纯度低聚果糖的制备方法，其特征在于，往反应罐中加入的蔗糖和固定化酶的含量为：每 Kg蔗糖加入 1000-4000U 固定化果糖基转移酶、 1000-4000U固定化葡萄糖氧化酶和 1000-4000U 固定化过氧化氢模拟酶。 The method for preparing high-purity oligofructose according to claim 15, wherein the content of the sucrose and the immobilized enzyme added to the reaction tank is: 1000-4000 U per Kg of sucrose. Glycosyltransferase, 1000-4000 U immobilized glucose oxidase and 1000-4000 U immobilized hydrogen peroxide mimic enzyme.

17、如权利要求 16 所述的高纯度低聚果糖的制备方法，其特征在于，所述通入空气的量为 1.0-3.0 L/min₀ 17. The method for producing fructooligosaccharides of high purity according to claim 16, characterized in that said air flux was 1.0-3.0 L / min ₀

18、如权利要求 15 所述的高纯度低聚果糖的制备方法，其特征在于，是用 CaC0₃来控制反应液的 pH值在 4.0-7.0之间。 The method for producing high-purity oligofructose according to claim 15, wherein the pH of the reaction solution is controlled by CaCO ₃ to be between 4.0 and 7.0.

19、如权利要求 1 所述的高纯度低聚果糖的制备方法，其特征在于，所述的连续式反应法是：将固定化果糖基转移酶、固定化葡萄糖氧化酶和固定化过氧化氢模拟酶按比例混合，再和碳酸钙颗粒混勾 ,装入柱式反应器中，以 10 ~ 30% (质量百分数）的蔗糖溶液在 pH4.0~7.0、温度 10-4CTC下反复通过此柱，监测低聚果糖的含量，低聚果糖含量达标后，过滤反应液，经离子交换和喷雾千燥成为高纯度低聚果糖产品。 The method for producing high-purity oligofructose according to claim 1, wherein the continuous reaction method comprises: immobilizing fructosyltransferase, immobilized glucose oxidase, and immobilized hydrogen peroxide The simulated enzyme is mixed in proportion, and then mixed with calcium carbonate particles, and charged into a column reactor, and repeatedly passed through the column at a pH of 4.0 to 7.0 and a temperature of 10 to 4 CTC in a sucrose solution of 10 to 30% (mass percent). The oligofructose content is monitored, and after the oligofructose content reaches the standard, the reaction solution is filtered, and ion-exchanged and spray-dried to become a high-purity oligofructose product.

20、如权利要求 19 所述的高纯度低聚果糖的制备方法，其特征在于，柱式反应器中，固定化果糖基转移酶、固定化葡萄糖氧化酶和固定化过氧化氢模拟酶的比例是： 1.0-4.0: 1.0-3.0: 1.0-4.0; 所述碳酸钙的盾量百分数是 2 ~ 8 %。 The method for producing high-purity oligofructose according to claim 19, wherein the proportion of immobilized fructosyltransferase, immobilized glucose oxidase, and immobilized hydrogen peroxide mimic enzyme in the column reactor Yes: 1.0-4.0: 1.0-3.0: 1.0-4.0; The percentage of shield of calcium carbonate is 2-8%.