黑曲霉乳糖酶固定化

实验一黑曲霉孢子固定化及其对淀粉糖化的效果分析一、目的要求1. 学习、掌握用包埋法固定微生物细胞的技术及固定化细胞增殖技术。

2. 了解固定化细胞的应用技术。

3. 掌握评价固定化细胞应用效果的方法。

二、试剂①马铃薯斜面培养基：马铃薯200g，葡萄糖20g，琼脂15~20g，定容至1000ml，pH值自然。

②生长培养基：同上（不加琼脂），200ml，灭菌。

③发酵培养基：配制1%、2%、3%、4%的可溶性淀粉溶液各200ml，pH=5.0，灭菌。

④3g海藻酸钠溶于80ml蒸馏水中，灭菌。

⑤0.1M CaCl2溶液250ml，灭菌。

⑥无菌水，500ml，灭菌。

三、实验步骤1. 制备黑曲霉孢子悬液将斜面黑曲霉孢子刮下，用无菌水制成5×108个/ ml孢子的悬液10 ml。

2. 制备海藻酸钠菌悬液将10 ml孢子悬液加入到80ml海藻酸钠溶液中，加入10ml无菌水定容至100ml，配成海藻酸钠浓度为3%、黑曲霉孢子浓度为5×107个/ ml的海藻酸钠菌悬液。

3. 制粒和固定用制粒装置将海藻酸钠菌悬液滴入0.1M CaCl2溶液中使之成粒。

固定化球直径为3mm，室温下固定5小时。

4. 固定化细胞预培养固定化球用无菌水冲洗三次，称重，每瓶15-20g装入生长培养基，25~28℃，220r/min震荡预培养36小时。

5. 摇床发酵培养：固定化球过滤，无菌水冲洗后转入发酵培养基。

对照：0.5%、1%、2%可溶性淀粉，28℃，220r/min震荡培养。

处理：含有15-20g固定化凝胶球的相同浓度的可溶性淀粉，50~60℃，220r/min，24小时6. 还原糖测定用3,5-二硝基水杨酸比色法测还原糖生成量。

取3支25ml刻度试管，编号（对照1管，处理每个浓度2管），按下表精确加入待测液和试剂。

单位：ml对照 处理① 处理② 待测液（？%淀粉）1 1 1 蒸馏水1 1 1 3,5-二硝基水杨酸 1.5 1.5 1.5摇匀，沸水浴中，5分钟，取出后立即放入盛有冷水的烧杯中冷却至室温，蒸馏水定容至25ml 。

乳糖酶的生产技术及其在食品工业应用研究进展摘要：乳糖酶亦称β-半乳糖苷酶，在工业生产中有广泛的应用，本文通过来源及其性质、基础研究与应用等方面对乳糖酶进行综述。

关键词：乳糖酶；固定化；应用乳是各种哺乳动物哺育其幼仔最理想的天然食物。

它富含优质蛋白质、乳脂、乳糖等营养成分和钙、磷、钾等矿物质以及多种维生素，还含有多种免疫物质、酶、激素等具有生理活性调节功能的生物活性物质。

乳糖是哺乳动物乳汁中特有的糖类，它是由一分子葡萄糖和一分子半乳糖组成的双糖，其合成步骤为：以葡萄糖为前体物质，一部分葡萄糖先转化为半乳糖，然后经乳糖合成酶催化。

半乳糖与葡萄糖结合，形成乳糖。

人体摄入乳糖后，在消化过程中，经乳糖酶催化，分解为葡萄糖和半乳糖。

乳糖是矿物质的载体，能促进钙、磷吸收及整理肠道，其分解产物半乳糖是婴儿脑发育的必需物质，参与脑组织及其神经系统的构成。

但是，机体却不能直接利用乳糖，乳糖必须经乳糖酶分解为单糖后才能被吸收和利用（杨卉新等，2014）。

若乳糖酶缺乏者一次摄入较多乳糖，乳糖未能及时被消化吸收，进入结肠后被肠道细菌分解，产生大量乳酸、甲酸等短链脂肪酸和氢气，造成渗透压升高，使肠腔中的水分增多，引起腹涨、肠鸣、肠绞痛直至发生水泻等症状，总称为乳糖不耐受症。

乳糖不耐受症状，在中国人群中发生率很高，因此限制了很大一部分国人对牛奶的摄入，而牛奶又是人类良好的优质蛋白、矿物质及维生素的天然来源，故乳糖酶缺乏问题显得尤为突出（张玉英，2014）。

1889年荷兰生物学家，Beijerineek首次报道了乳糖酶可水解乳糖以来，人们对于乳糖酶的研究日趋完整（蒋世琼，2000）。

目前，解决乳糖不耐受的最佳方法是用乳糖酶水解乳糖来生产低乳糖或无乳糖乳制品。

而现在商业乳糖酶中乳糖酶的最适温度在37℃左右或者更高（P Nicholas，2002）。

国外学者经多年研究，已成功地找到产乳糖酶的微生物，并研制了一系列乳糖酶商品，现已投入市场。

乳糖酶的工业生产原理
乳糖酶是一种酶，它能够将乳糖分解成葡萄糖和半乳糖。

乳糖酶的工业生产原理主要包括以下几个步骤：
1. 菌种培养：首先，培养乳糖酶产生菌株。

常用的菌株有放线菌、大肠杆菌等。

这些菌株经过筛选和改良，具有较高的乳糖酶产量。

2. 酶的提取与纯化：将培养得到的菌株进行破碎、离心等操作，获取到含有乳糖酶的菌液。

接下来，通过过滤、酶解和纯化等步骤将乳糖酶从菌液中分离出来，获得较纯的乳糖酶。

3. 乳糖酶的固定化：为了提高乳糖酶的稳定性和重复使用性，常常将乳糖酶固定在固体载体上。

常用的固体载体有树脂、硅胶、海藻酸钙等。

将乳糖酶与固体载体相混合，通过吸附、交联等方法将酶固定在载体上，形成固定化乳糖酶。

4. 生产过程的优化和控制：在乳糖酶的工业生产过程中，需要优化和控制多种因素，包括合适的菌种培养条件、培养基的配方和浓度、酶的提取条件、固定化乳糖酶的反应条件等。

5. 产品的提取和纯化：最后，将固定化乳糖酶的反应体系进行分离和纯化，以获得高纯度的乳糖酶产品。

乳糖酶的工业生产原理可以根据具体的生产需求和工艺技术进行调整和改进。

一、实验目的1. 了解乳糖酶的性质和作用。

2. 掌握乳糖酶固定化的基本原理和方法。

3. 研究不同固定化方法对乳糖酶活性的影响。

4. 探讨固定化乳糖酶在乳糖水解中的应用。

二、实验原理乳糖酶（β-半乳糖苷酶）是一种能够催化乳糖水解为葡萄糖和半乳糖的酶。

乳糖不耐受症患者由于体内缺乏乳糖酶，无法消化乳糖，导致食用乳制品后出现腹胀、腹泻等症状。

固定化乳糖酶可以克服传统酶制剂的缺点，如酶活性不稳定、易失活等，从而提高乳糖水解效率。

本实验采用化学结合法将乳糖酶固定化在载体上，通过比较不同固定化方法对酶活性的影响，筛选出最佳的固定化方法。

三、实验材料与仪器材料：1. 乳糖酶2. 载体：壳聚糖、明胶、海藻酸钠3. 乳糖4. 磷酸盐缓冲液5. pH计6. 离心机7. 酶标仪仪器：1. 烧杯2. 移液器3. 恒温水浴锅4. 电子天平5. 显微镜四、实验步骤1. 乳糖酶溶液的制备：将乳糖酶用磷酸盐缓冲液溶解，配制成一定浓度的酶溶液。

2. 载体的制备：a. 壳聚糖：将壳聚糖用磷酸盐缓冲液溶解，配制成一定浓度的溶液。

b. 明胶：将明胶用磷酸盐缓冲液溶解，配制成一定浓度的溶液。

c. 海藻酸钠：将海藻酸钠用磷酸盐缓冲液溶解，配制成一定浓度的溶液。

3. 乳糖酶固定化：a. 壳聚糖固定化：将壳聚糖溶液与乳糖酶溶液混合，搅拌均匀，加入一定量的交联剂戊二醛，反应一定时间后，用离心机分离固定化酶。

b. 明胶固定化：将明胶溶液与乳糖酶溶液混合，搅拌均匀，加入一定量的交联剂戊二醛，反应一定时间后，用离心机分离固定化酶。

c. 海藻酸钠固定化：将海藻酸钠溶液与乳糖酶溶液混合，搅拌均匀，加入一定量的交联剂戊二醛，反应一定时间后，用离心机分离固定化酶。

4. 固定化酶的活性测定：将固定化酶分别与乳糖溶液混合，在一定条件下进行水解反应，通过测定水解产物的浓度来计算酶的活性。

5. 结果分析：比较不同固定化方法对乳糖酶活性的影响，筛选出最佳的固定化方法。

五、实验结果与分析1. 固定化酶的活性：通过实验发现，壳聚糖固定化酶的活性最高，明胶固定化酶的活性次之，海藻酸钠固定化酶的活性最低。

Removal of heavy metals from industrial wastewater by free and immobilized cells of Aspergillus nigerK.Tsekova a ,*,D.Todorova a ,S.Ganeva ba Stephan Angeloff Institute of Microbiology e Bulgarian Academy of Sciences,Acad.G.Bonchev,Str.,bl.26,1113So fia,Bulgaria bFaculty of Chemistry e So fia University,1164So fia,Bulgariaa r t i c l e i n f oArticle history:Received 24March 2010Received in revised form 5May 2010Accepted 9May 2010Available online 7June 2010Keywords:Biosorption Heavy metals Wastewater Immobilization Aspergillus nigera b s t r a c tAspergillus niger ,strain B 77,was immobilized by inclusion in two different polymers:polyvinyl e alcohol hydrogel (PVA)and Ca e alginate.The biomass/polymer matrices were formed into equal size unites of the cubes and spheres,and the resulting biomass/polymer matrices were used to remove heavy metals (Cu 2þ,Mn 2þ,Zn 2þ,Ni 2þ,Fe 3þ,Pb 2þ,Cd 2þ)from wastewater in shake flask experiments.Total biosorption capacities of the biosorbents were in the following order:free cells (33.3mg/g)<PVA e biomass (39.8mg/g)<Ca alginate e biomass (44.6mg/g).The metal removal ef ficiencies of the beads Ca alginate e biomass were 96.2%for Cd 2þ;90.0%for Pb 2þ;80.0%for Fe 3þ;72.8%for Cu 2þ;55.4%for Zn 2þ;54.4%for Ni 2þand 52.3%for Mn 2þ,while the removal ef ficiencies of cubes PVA e biomass for the same heavy metals ions were:95.0%;88.0%;80.0%;67.1%;58.5%;48.9%and 44.6%,respectively.The results obtained from these experiments,were compared with those using dispersed biomass as a sorbent.Promising results were obtained in the laboratory,as effective metal removals were observed.Ó2010Elsevier Ltd.All rights reserved.1.IntroductionThe increase use of heavy metals in difference industrial activ-ities causes their existence in wastewater.The discharge of heavy metal ions in industrial ef fluent is of great concern because of their toxic effect on living species,even at very low concentrations.The detoxi fication of metal ions from industrial ef fluent using biosorption processes is an area of extensive research during the last years (Volesky and Holan,1995;Tripathi et al.,2007).Biosorption is an innovative and low cost effective method for the removal of toxic substances from wastewaters (Zouboulis et al.,2003;Silva et al.,2009;Chatterjee et al.,2010).Fungi are recognized for their superior ability to produce a large variety of extra cellular proteins,organic acids,enzymes and other metabolites,and their waste biomass may be used as effective biosorbent for removal,reduction and detoxi fication of industrial ef fluents ingredients (Gupta and Mukerji,2001;Christian et al.,2005).Various fungal species under the genus Aspergillus ,Penicil-lium and Rhizopus have been shown to be effective in biosorption of heavy metals from polluted ef fluents both as immobilized cells and in the mobilized state (Kapoor and Viraraghvan,1995;Leitão,2009;Tsekova et al.,2010a,b ).During the last decade many research works have been focused on the development of immobilized systems of microorganisms into polymeric matrices suitable for metal ions uptake applications (Zouboulis et al.,2003;Tsekova et al.,2008;Mata et al.,2009).Biosorption has been considered as a promising technology for the removal of low levels of toxic metals from industrials ef fluents and natural waters.In view of potential applications in remediation of heavy metals from aqueous solutions the immobilization of the biomass is generally necessary.Immobilized cells are usually easier to handle,require less complex separation systems,allow a high biomass density to be maintained and provide a greater opportu-nity for reuse and recovery.Despite the current interest in microbial detoxi fication of ef fluents,relatively little work has been concerned with charac-terization of metal uptake by filamentous fungi,particularly when the heavy metals present at different and low concentrations.The purpose of this study was to determine the ability of Aspergillus niger (free and immobilized biomass)to remove toxic metals from an industrial wastewater by batch system.2.Materials and methods 2.1.Sample collection siteWastewater samples were from copper production factory in Pirdop,Bulgaria.The samples were collected for a relative long*Corresponding author.Tel.:þ35929793167;fax:þ35928700109.E-mail addresses:ktsekova@microbio.bas.bg (K.Tsekova),sganeva@chem.uni-so fia.bg (S.Ganeva).Contents lists available at ScienceDirectInternational Biodeterioration &Biodegradationjou rn al homepage:/locate/ibiod0964-8305/$e see front matter Ó2010Elsevier Ltd.All rights reserved.doi:10.1016/j.ibiod.2010.05.003International Biodeterioration &Biodegradation 64(2010)447e 451period of time(April e November2009)in high e grade plastic bottles of1.5l capacity and stored in a refrigerator at4 C.From all taken wastes a representative sample was prepared mixing150ml from each separate bottle.2.2.Determination of heavy metal concentrationsThe initial concentration of the metals in the wastewater and samples after biosorption treatment were determined using an Atomic Absorption Spectrophotometer Perkin Elmer Analyst400, air-acetyleneflame.Cadmium and lead after biosorption were determined using electrothermal atomic absorption spectrom-eter Zeeman Perkin Elmer3030,pyrolytically coated graphite tubes and optimized temperature program for modifier freeETAAS.2.3.Preparation of biosorbents2.3.1.Microorganisms,medium and cultivationThe mutant strain A.niger B77(A.niger),an industrial producer of glucoamylase(N 65/1980,National Bank of Industrial Micro-organisms and Cell Cultures,Bulgaria)was used in this study. Spores from established culture(6e7days old)incubated on potato glucose agar slants at30 C were used for the preparation of inocula.The liquid growth medium consisted of(g/L):glucose30 and corn steep liqueur45;pH adjusted to4.8.Cultivation of A.niger was carried out in500e ml Erlenmeyer flasks with75ml growth medium on a rotary shaker at30 C.After 48h of cultivation,the mycelium was harvested byfiltration from the medium,washed twice with distilled water and stored at4 C until use.The dry weight of the free fungal biomass(FC)was determined after drying at85 C for48h.2.3.2.Immobilizing materials and techniquesPoly(vinyl)alcohol(PVA)hydrogel was obtained as describe earlier(Tsekova et al.,2008).The PVA e hydrogel used for immo-bilization was cut into small pieces(4Â4Â4mm in size).They were washed with distilled water and than submerged in500-ml Erlenmeyerflasks containing75ml growth medium(previously autoclaved for20min at120 C).The carrier pieces were inoculated with10ml A.niger spores suspension(1Â106/ml)were cultivated as described above.After48h of incubation,the PVA e immobi-lized biomass of A.niger was harvested from the medium,washed twice with distilled water and stored at4 C until used as a bio-sorbent.The dry weight of PVA e biomass was determined as described for FC.Method described by El e Naggar et al.(2006)was used to prepare Ca alginate beads the spore suspension(10ml,1Â106/ml) was mixed with Na e alginate prior to its stabilization.Beads of approximately4mm diameter were obtained by selecting an appropriate orifice size through which the polymer/spores mixed passed.The cultivation was carried out as described above.After 48h of incubation,the Ca alginate e immobilized biomass of A.niger was harvested from the medium,washed twice with distilled water and stored at4 C until used as a biosorbent.The dry weight of Ca alginate e biomass was determined as described for FC.2.4.Batch biosorption studiesBatch biosorption experiments were carried out in500ml Erlenmeyerflasks,as follows:preweighed biosorbent samples (wet or immobilized biomass)with concentration varying from0.1 to0.5g/l(dry weight)were examined.Each sample was added to 100ml of real wastewater,containing heavy metal ions.Biosorption studies were performed at different initial pH from5.5to7.5using 0.1N NaOH.The mixtures were then agitated at120rpm on a rotary shaker up to30min at25 C.Then the content of theflasks was separated byfiltration using a Whatman N 1filter paper.2.5.Metal uptake(q)Uptake of metal ions was calculated from a metal mass balance yielding:q¼VðC iÀC fÞ(1) where:q is mg metal ions per g dry biosorbent;V is the reaction volume(l),C i and C f are the initial and residual metal concen-trations(mg/l),respectively,and m is the amount of dry bio-sorbent(g).The concentration of the metal ions in thefiltrates was deter-mined using atomic absorption spectrophotometer with an air/ acetyleneflame(model2380;Perkin Elmer,Uberlingen,Germany).Aliquots of wet biomass as well as of immobilized biomass, followed by drying for48h at85 C,were considered as dry biosorbent to calculate the uptake.The efficiency of heavy metal removal was calculated from the amount of metal ions adsorbed on the biosorbent and the amount of metal ions available in the wastewater,as the following equation:%removal¼mg heavy metal ions removedmg heavy metal ions availableÂ100(2)2.6.ReproducibilityAll the experiments were run in triplicates and controls were also run on same pattern without addition of biosorbent.The data shown are average from three separate experiments.Table1Characteristics of the biosorption process for removal of heavy metals from waste-water using free biomass as a sorbent.Performance of biosorbentof A.niger biomassHeavy metals in mixed solution,mg/lCu Zn Ni Fe Pb Cd Mn Initial concentrations mg/l7 1.30.90.20.050.0813 Equilibrium concentrations mg/l 2.8 1.10.460.060.0060.0048.1 Equilibrium time min20151555520 Heavy metal uptake mg/g140.66 1.50.460.150.2516.3 Removal efficiency%6015.448.970889537.7 Free biomass(m)0.3g/l;pH e6.5;number of parallels:(n)3;relative standard deviation(RSD)3e8%.Table2Characteristics of the biosorption process for removal of heavy metals from waste-water:PVA e biomass m¼0.3g/l;pH e6.5.Performance of biosorbent Heavy metals in mixed solution,mg/lCu Zn Ni Fe Pb Cd Mn Initial concentrations mg/l7 1.30.90.20.050.0813 Equilibrium concentrations mg/l 2.30.540.460.040.0060.0047.2 Equilibrium time min1510555515 Heavy metal uptake mg/g15.6 2.5 1.50.530.140.2519.3 Removal efficiency%67.158.548.980889544.6 Number of parallels:(n)3;relative standard deviation(RSD)3e8%.K.Tsekova et al./International Biodeterioration&Biodegradation64(2010)447e451 4483.Results and discussion3.1.Sorption of heavy metals from wastewater by freeand immobilized cells of A.niger3.1.1.Sorption of heavy metals from industrial wastewater by free biomass of A.niger (FC)A.niger biomass absorbed Fe 3þ,Pb 2þand Cd 2þions from industrial wastewater more rapidly than other ions (Table 1)within 15e 20min.Experiments indicated that sorption equilibrium reached much faster in case of industrial wastewater sample (up to 20min)in comparison to single ions solution (up to 30min)using same biosorbent (Tsekova et al.,2010a ).These results are impor-tant,as equilibrium time is one of the important parameters for selecting a wastewater treatment system.This may be due to the presence of co e metal ions in the industrial ef fluents as well as to the differences in the heavy metal ions concentrations (Muhammad et al.,2009;Chatterjee et al.,2010).The removal percentages order at equilibrium was:Cd 2þ(95%)>Pb 2þ(88%)>Fe 3þ(70%)>Cu 2þ(60%)>Ni 2þ(48.9%)>Mn 2þ(37.7%)>Zn 2þ(15.4%)3.1.2.Sorption of heavy metals by immobilized biomass of A.nigerThe effect of immobilization of A.niger on PVA e hydrogel as well as on Ca alginate on the removal of heavy metal ions by adsorption was investigated.The metal removal study,illustrated in Tables 2,3showed that their removals were affected by immobilization of A.niger in comparison to the removal ef ficiency by free biomass (Table 1).In general,the both immobilized biosorbents displayed higher bio-sorption capacities for Mn 2þand Cu 2þ,presented in the ef fluent at higher initial concentrations.There was however considerabledifference in total biosorption capability of the test fungal bio-sorbents.Immobilized on Ca alginate cells of A.niger showed highest total biosorption capacity of 71.6mg/g for all heavy metal ions (Table 3),followed by PVA immobilized cells(68.9mg/g,Table 2)and free cells(59.3mg/g j ,Table 1),respectively.Meanwhile,the superior biosorption potential of Ca alginate e immobilized cells (22.6mg/g and 17mg/g)over PVA e immobilized ones (19.3mg/g and 15.6mg/g)was observed in the case of Mn 2þand Cu 2þions,respectively.In general,removal ef ficiency of the test biosorbents,for available metal ions,was observed to follow the sequence in following mode:FC (59.3%)<PVA e biomass (68.9%)<Ca alginate e biomass (71.6%).The both immobilized biosorbents displayed high removal potential for Cd 2,Pb 2þ,Fe 3þ,in comparison to other heavy metal ions from the industrial ef fluent.At the same time the immobilized biomass in Ca alginate exhibited the highest biosorption potency toward Mn 2þand Cu 2þions,that ’s why it was chosen for the following investigations.3.1.3.Effect of pHFig.1shows the effect of pH on the biosorption of different metals by A.niger immobilized biomass.Removal ef ficiency was analyzed over a pH range 5.5e 7.5.The results show that the metal sorption was a function of pH,as the pH increased from 5.5to 6.5,adsorption capacity increased at first for all metals.Maximum adsorption occurs at pH 6.5for Fe 3þ,Cu 2þ,Pb 2þand Cd 2þ,and at pH 7.5for Zn 2þ,Ni2þand Mn 2þ.The maximum removal ef ficiencies (%)for the different metals by Ca alginate e biomass were 96.3%for Cd 2þ(at pH 7.5)>90%for Pb 2þ(at pH 7.5)>80%for Fe 3þ(at pH 6.5)>72.8%for Cu 2þ(at pH 6.5)>61.5%for Mn 2þ(at pH 7.5)>59.7%for Zn 2þ(at pH 7.5)>58.9%for Ni 2þ(at pH 7.5).After pH 7.5the ef ficiency of the metal removal process increases drastically due to the formation of metal hydroxides with their respective metal ions (Zouboulis et al.,2003).This is mostly due to the metal precipitation as hydrox-ides which depend on the pH and ion concentration,but not due to the biosorption (Al e Qodah,2006;AjayKumar et al.,2009).pH value is one of the main factors in biosorption ef ficiency of different biosorbents.The different pH binding pro files for different metal ions are due to the nature of the chemical interactions of metal ions with the biosorbent.Solution pH in fluences surface metal binding sites of the biosorbents and the chemistry of the cell walls,as well as physicochemistry and hydrolysis of the metals.Table 3Characteristics of the biosorption process for removal of heavy metals from waste-water:Ca alginate e biomass m ¼0.3g/l;pH e 6.5.Performance of biosorbentHeavy metals in mixed solution,mg/l CuZnNiFePbCdMn Initial concentrationsmg/l 7 1.30.90.20.050.0813Equilibrium concentrations mg/l 1.90.580.410.040.0050.003 6.2Equilibrium time min 1510555515Heavy metal uptake mg/g 17 2.4 1.60.530.150.3022.6Removal ef ficiency%72.855.454.4809096.252.3Number of parallels:(n)3;relative standard deviation (RSD)3e 8%.102030405060708090100Cu Zn Ni Fe Pb Cd Mnr e m o v a l , %pH - 6.5pH - 7.5Fig.1.Effect of pH on metal ions removal from wastewater using Ca alginate e immobilized cells of Aspergillus niger as a biosorbent.(m 0.3g/l dry weight).Vertical bars show standard error of means of three replicates.K.Tsekova et al./International Biodeterioration &Biodegradation 64(2010)447e 4514493.1.4.Effect of adsorbent weight (g/l)The effect of adsorbent weight (g/l)on the adsorption ef ficiency of the best fungal biosorbent (Ca alginate e immobilized cells)is shown on Fig.2.Adsorption experiments were carried out at different biosorbent doses ranging from 0.1to 0.5g/l in mixed ions solution.It was observed as a general trend that there is an increase of the removal percentage with increase in adsorbent weight from 0.1to 0.3g/l.The maximum removal of the most heavy metal ions was attained at an adsorbent dose of 0.3g/l with no further signi ficant increase in the removal percentage at higher biosorbent concentration tested was observed.In the case of Fe 3þ,Mn 2þand Ni 2þmaximum removal was attained at 0.5g/l of adsorbent weight.Removal ef ficiency increases for Fe 3þfrom 80%till 90%,Mn 2þfrom 52.3%till 61.5%,and for Ni 2þfrom 58.9%till 79%when the sorbent mass increases from 0.3g/l to 0.5g/l.These results are in agreement with previously studies on many other adsorbents (Yu et al.,2001;Dakiky et al.,2002).As the biosorbent mass increases the number of available binding sites or surface area for the heavy metal ions also increased.However,the removal ef ficiency of Pb 2þand Cd 2þretained constant when the biosorbent weight increased.Accord-ing to the previous works,higher biosorbent dose could produce a “screening ”effect on the binding sites,thus resulting in lower heavy metal uptake (Yahaya et al.,2009;Tsekova et al.,2010a,b ).3.1.5.Secondary chemical treatment of the filtrate after biosorptionTo the filtrate obtained after biosorption 5ml 2%sodium diethyldithiocarbamate (NaDDTC)solution and 4g activated char-coal were added,mixed for 10min and filtered through Whatman N 1filter paper.In this second filtrate the concentration of all the examined heavy metals was below the detection limit of the measurement method.Such successful procedure for completely removing of heavy metals from ef fluents has been not reported in the literature yet.4.ConclusionThe ability of A.niger biomass to bind and remove heavy metals,i.e.Cu 2þ,Zn 2þ,Ni 2þ,Pb 2þ,Cd 2þ,Fe 3þ,Mn 2þfrom real wastewater was investigated.To overcome the separation problems of using freely suspended biomass form,as well as,mass loss after regen-eration of the biosorbent,the biomass was immobilized in the polymer matrixes (PVA and Ca alginate gels).Biosorption studies of Ca alginate e A.niger beads have been found to be effective in removing of relatively low concentrations of these seven heavy metals from wastewater.The process was mainly in fluenced by pH and biosorbent dose.At pH 6.5and biosorbent dose of 0.5g/l dry weight the removal ef ficiencies obtained for Ca alginate e biomass beads were:for Cd 2þe 96.2%;for Pb 2þe 90%;for Fe 3þe 90%;for Cu 2þe 73.5%;for Ni 2þe 70.9%for Zn 2þe 60.9%and Mn 2þe 61.5%.The results obtained showed that immobilized biomass of A.niger ,appears as a possible biosorbent to be used for treatment of metal e polluted industrial wastewaters.The secondary chemical treatment with NaDDTC-activated charcoal showed complete removal of all the studied heavy metals.AcknowledgementsThis work was supported by the National Science Fund at the Ministry of Education and Science of Republic of Bulgaria (Grant DOO2-185/2008)and Operative Program Human Resources (Grant BG051PO001e 3.3.04/32).ReferencesAjayKumar,A.V.,Darwish,N.A.,Hilal,N.,2009.Study of various parameters in thebiosorption on heavy metals on activated sludge (Special Issue for Environ-ment).World Applied Sciences Journal 5,32e 40.Al e Qodah,Z.,2006.Biosorption of heavy metal ions from aqueous solutions byactivated sludge.Desalination 196,164e 176.Chatterjee,S.K.,Bhattacharjee,I.,Chandra,G.,2010.Biosorption of heavy metalsfrom industrial waste water by Geobacillus thermodenitri ficans .Journal of Hazardous Materials 175,117e 125.Christian,V.,Shrivastava,R.,Shukla,D.,Modi,H.A.,Vyas,B.R.M.,2005.Degradationof xenobiotic compounds by lignin e degrading white e rote fungi:enzy-mology and mechanism involved.Indian Journal of Experimental Biology 43,301e 312.Dakiky,M.,Khamis,M.,Manassra,A.,Mer ’eb,M.,2002.Selective adsorption ofchromium (VI)in industrial wastewater using low e cost abundantly available adsorbents.Advance Environmental Research 6(4),533e 540.El e Naggar,M.Y.,El e Assar,S.A.,Youseff,A.Y.,El e Sersy,N.A.,Beltagy,E.A.,2006.Extracellular b e mannanase production by the immobilization of the locally isolated Aspergillus niger .International Journal of Agriculture &Biology 8(1),57e 62.Gupta,R.,Mukerji,K.G.,2001.Bioremediation:Past,present and future.In:Tewari,R.,Mukerji,K.G.,Gupta,J.K.,Gupta,L.K.(Eds.),Role of microbes in the management of environmental pollution.A.P.H.Publishing Corp,New Delhi,pp.73e 81.Kapoor, A.,Viraraghvan,T.,1995.Fungal biosorption:an alternative treatmentoption for heavy metal bearing wastewater:a review.Bioresource Technology 53,195e 206.Cu Zn Ni Fe Pb Cd Mnr e m o v a l , %m - 0.1g/l m - 0.3g/l m - 0.5g/lFig.2.Effect of sorbent dose on metal ions removal from wastewater using Ca alginate e immobilized cells of Aspergillus niger as a biosorbent.(pH 6.5).Vertical bars show standard error of means of three replicates.K.Tsekova et al./International Biodeterioration &Biodegradation 64(2010)447e 451450Leitão, A.L.,2009.Potential of Penicillium species in the bioremediationfield.International Journal of Environmental Research and Public Health6, 1393e1417.Mata,Y.N.,Blázquez,M.L.,Ballester,A.,González,F.,Munoz,J.A.,2009.Biosorption of cadmium,lead and copper with calcium alginate xerogels and immobilized Fucus vesiculosus.Journal of Hazardous Materials163,555e562. Muhammad,R.,Nadeem,R.,Hanif,M.A.,Ansari,T.M.,Rehman,K.U.,2009.Pb(II) biosorption from hazardous aqueous streams using Gossypium hirsutum (Cotton)waste biomass.Journal of Hazardous Materials161,88e94.Silva,R.M.P.,Rodriguez,A.Á.,De Oca,J.M.G.M.,Moreno,D.C.,2009.Biosorption of chromium,copper,manganese and zinc by Pseudomonas aeruginosa AT18 isolated from a site contaminated with petroleum.Bioresource Technology100, 1533e1538.Tripathi,A.K.,Harsh,N.S.K.,Gupta,N.,2007.Fungal treatment of industrial efflu-ents:a mini e review.Life Science Journal4(2),78e81.Tsekova,K.,Christova,D.,Todorova,D.,Ivanova,S.,2008.Biosorption of ternary mixture of heavy metals by entrapped in PVA e hydrogel biomass of Penicillium cyclopium.Comptes rendus de l`Academie bulgare des Sciences61(9),1175e1180.Tsekova,K.,Todorova,D.,Dencheva,V.,Ganeva,S.,2010a.Biosorption of copper(II)and cadmium(II)from aqueous solutions by free and immobilized biomass ofAspergillus niger.Bioresource Technology101,1727e1731.Tsekova,K.,Christova,D.,Dencheva,V.,Ganeva,S.,2010b.Biosorption of binarymixture of copper and cobalt by free and immobilized biomass of Penicilliumptes rendus de l`Academie bulgare des Sciences63(1),85e90.Volesky,B.,Holan,Z.R.,1995.Biosorption of heavy metals.Biotechnology Progress11(3),235e250.Yahaya,Y.A.,Don,M.M.,Bhatia,S.,2009.Biosorption of copper(II)onto immobilizedcells of Pycnoporus sanguineus from aqueous solution:equilibrium and kineticstudies.Journal of Hazardous Materials161,189e195.Yu,B.,Zhang,Y.,Shukla,S.S.,Dorris,K.L.,2001.The removal of heavy metals fromaqueous solutions by sawdust adsorption:removal of lead and comparison ofits adsorption with copper.Journal of Hazardous Materials84,83e94.Zouboulis,A.I.,Matis,K.A.,Loukidou,M., Sebesta,F.,2003.Metal biosorption by PAN e immobilized fungal biomass in simulated wastewaters.Colloids andSurfaces212,185e195.K.Tsekova et al./International Biodeterioration&Biodegradation64(2010)447e451451。

酶的固定化技术及其应用曾鸿雁(西南科技大学,四川,绵阳)摘要:随着工业生物技术和酶工程的不断发展，酶在各个领域的广泛应用，对酶的要求也越来越严格。

本文针对目前酶工程技术之一酶的固定化，对酶的固定化技术及其展望做一综述。

关键词：酶，固定化，技术Immobilization of Enzyme And its Applications Abstract：with the continuous development of biotechnology industrial and enzyme engineering , enzyme are widely used in various fields and the requirements to enzymes also become more and more stringent . This article is to review the enzyme immobilization, which is one of the current enzyme engineering technologiesKey words: enzyme, immobilization, technology一、引言酶是一类具有生物催化性质的高分子物质，其催化性具有专一性强、催化效率高和作用脚尖温和等特点。

但是在实际工业生产中，由于实际环境因素，应用酶的过程出现了一些不足之处：①酶的催化效率不高。

人们在使用酶的过程中，往往要求酶的催化效率要足够高，以加快反应速度，提高劳动生产率，然而实际上很多酶的催化效率不够高而难于满足人们的使用要求。

②酶的稳定性较差。

大多数酶稳定性较差，在高温、强酸、强碱和重金属离子等外界因素的影响下，都容易变形失活。

③酶的一次性使用。

酶一般是在溶液中与底物反应，这样酶在反应系统中，与底物和产物混合在一起，反应结束后，即使酶仍有很高的活力，也难于回收利用。

乳糖酶的固定化及低乳糖奶褐变抑制技术的研究的开题报告一、研究背景和意义乳糖酶是一种能够分解乳糖的酶类，在乳制品工业中有着广泛的应用。

然而，部分人群由于乳糖不耐受或过敏等原因，不能食用含有较高浓度乳糖的乳制品，这对于乳制品的生产和销售造成了一定的限制。

因此，乳糖酶的固定化及低乳糖奶褐变抑制技术的研究具有重要的现实意义。

二、研究内容和目标本研究的主要内容包括乳糖酶的固定化及低乳糖奶褐变抑制技术。

具体目标如下：1. 选择合适的固定化载体，建立乳糖酶的固定化工艺。

2. 筛选合适的工艺参数，优化乳糖酶固定化过程。

3. 研究低乳糖奶褐变的抑制效果及机理。

4. 确定最佳的低乳糖奶制备工艺，实现乳糖浓度降至符合过敏人群需求的水平。

三、研究方法和技术路线本研究主要采用以下方法和技术路线：1. 首先，选择适宜的固定化载体进行固定化研究，并通过表面形态和化学组成的表征分析其性质。

2. 深入研究不同的试验条件对固定化效果的影响，优化固定化工艺并确定最优条件。

3. 通过模拟不同的加工与贮存条件下的褐变过程，研究不同条件下低乳糖奶的抑制效果及机理。

4. 在实验条件下设计出不同浓度的低糖乳制备工艺，定量评估低乳糖奶的品质，并根据结果对制备工艺进行优化。

四、研究预期结果和意义本研究预期可以得出以下研究成果：1. 确定一种高效的乳糖酶固定化载体，建立固定化工艺优化条件，为工业化应用提供技术基础。

2. 研究低乳糖奶褐变的抑制效果及机制，为乳制品工业提供低乳糖产品的制备和贮存技术的指导。

3. 设计出符合过敏人群需求的低糖乳制备工艺，并定量评估低乳糖奶的品质，对于推广为新的高附加值乳制品有着积极的意义。

黑曲霉产乳糖酶酶学性质研究牛奶是最接近完善的营养食品，它包含了人类生长和维持健康的一切营养成分，具有丰富的蛋白质氨基酸和维生素，是天然钙质的极好来源。

乳糖是奶类中特有的糖类，也是重要的营养成分之一，该物经小肠粘膜上皮细胞绒毛远端刷状缘上的乳糖酶水解成为半乳糖和葡萄糖后，才能被小肠吸收利用。

乳糖酶学名半乳糖苷半乳糖水解酶，又名半乳糖苷酶，是一种白色粉末，无味道，无嗅，溶解后是一种浅棕色液体，乳糖酶通过特定条件水解半乳糖苷键将乳糖水解成葡萄糖和半乳糖，易被肠道吸收。

大量研究表明哺乳动物的乳糖酶活性随年龄的增长，具有典型的生理性降低，成人乳糖酶下降的不可逆受基因控制。

[1] 假如乳糖不被吸收，它将被排放到肠中，被肠道微生物发酵产酸、产气，从而导致胃肠功能失调，并造成有价值的蛋白质和矿物质的损失，如铁、锌、钙质的丢失，这与小儿佝偻病和成年人的骨质疏松症都有关系，专家们的有关调查研究结果显示：乳糖酶缺乏患者比正常情况下的人的骨骼结构差，而更为严重的是，如果体内缺乏乳糖酶半乳糖使得乳糖将无法被水解成为单糖（葡萄糖和半乳糖），而半乳糖供人体吸收、利用，能促进脑苷和粘多糖的合成从而起到促进幼儿智力发育的作用。

乳糖吸收障碍不仅伴有乳糖不耐的征兆，而且还伴有食欲下降，导致饮食量减少、腹泻，也导致一定数量已被消化的食物的丧失，这些消化的食物本能够被吸收却没有被吸收，包括有价值的蛋白、微生物、矿物质和一些牛奶中的糖类、使得牛奶的利用率极低、也浪费了牛奶这种宝贵的饮食资源。

乳糖不耐症在世界范围内是一种多发疾病，易发人群有以下几种情况：（1）成年人主要是由于遗传原因，体内乳糖酶从出生一年后开始衰减。

（2）是先天性疾病。

婴儿在刚出生时肠道内就缺乏乳糖酶活性，这将导致严重的肠胃系统失调。

（3）由于早产而造成婴儿肠道低乳糖酶活性。

（4）蛋白质热值吸收障碍严重的儿童，其乳糖酶活性在一个时期内会暂时消失。

乳糖不耐症发生的人种间差别：据悉，70%的成年黑人、10-15%的成年白人、95%的亚洲人受乳糖不耐症困扰。

天然产物研究与开发N at Prod Res Dev 2010,22:2092214,231文章编号:100126880(2010)022*******收稿日期:2009204223 接受日期:2009209224基金项目:湘潭市科技重点资助项目ZD20081027;教育部/大学生创新实验计划0项目081053013*通讯作者Te:l 862731258298175;E 2m ai:l hyzeng @xtu 复合诱变选育高产脂肪酶黑曲霉菌株及其固定化酶性质孟 娟,曾虹燕*,张艳梅,夏 葵,刘 贵湘潭大学化工学院生物技术研究所,湘潭411105摘 要:通过硫酸二乙酯(DES)和微波复合诱变,获得遗传性状稳定的高产脂肪酶黑曲霉突变菌株C M 2,酶活达174.93U /mL 。

对菌株C M 2培养条件的优化,以橄榄油和(N H 4)2S O 4为最佳碳、氮源,在28e 、p H 7.5的条件下,发酵CM2菌株68h ,脂肪酶活为180.52U /mL 。

大孔树脂固定化脂肪酶在35~55e 和p H 7.5~9.5之间有很好的稳定性。

游离酶和固定化酶的表观失活活化能分别为52.6842kJ/m ol 和30.8391kJ /mo,l 固定化酶对温度的敏感度降低,耐受性增强。

在微水相中脂肪酶催化22辛醇和乙酸乙烯酯不对称酯交换反应中,(S )2乙酸辛酯的对映选择性高(游离酶e .e .S85.7%;固定化酶e .e .S87.7%),显示了该固定化酶在22辛醇的手性拆分方面具有良好的应用前景。

关键词:黑曲霉;脂肪酶;复合诱变;固定化;酶拆分中图分类号:Q814.2;Q939;TQ645文献标识码:AC o m positeM u ta ti on Breed i ng of As pergill u s n i ger Stra i n w ithH i gh L i pase A cti vity and I mm ob ilized L i pase P roper tiesMENG Juan,Z ENG H ong 2yan *,Z HANG Yan 2me,i X I A Ku,i LI U GuiB iotechnolo gy R es ea rch Institute ,College of Che m ica l Eng i neering,Xiang tan Un i versit y ,Xiang t a n 411105,Ch i na Abstr a ct :The As perg illus ni g erC M 2w it h h i gh li pase acti vity (174.93U /mL)was o bta i ned by co m bini ngm icro wave ir 2rad i atio n with DES .The hered i ta ry character of t he strai n C M 2was stab le .The culture cond iti ons of the stra i n C M 2were opti m ized .The li pase acti vity was 180.52U /mL when t he stra i n was cu ltured i n the m ed i u m at 28e and p H 7.5for 68h ,whe re olive oil and (N H 4)2SO 4we re used as the carbo n and n itro gen so urces ,respecti ve l y .Co mpar i ng with the free li pase ,the i m mob ili zed lipase adsorbed on the m acroporous resi n possessed hig her stab ilit y at 35~55eand p H 7.5~9.5.The apparen t i nacti vatio n energy of free and i m m o b ili zed li pase was 52.6842and 30.8391kJ/m o,l respectively .The result sho wed that i m m o b ilized lipase was m ore tolerant and i nsensiti ve to te m pe rature .The transester ifi catio n of (R,S )222oc tanol w ith vi nyl ace tate asyrn m e tricall y cata l yzed by the free and i m m obilized li pases i n m icroaqueous phase .The enantiose l ectivit y of (S )2oc t yl aceta te was h i gh ,and e .e .S85.7%for the free lipase and e .e .S87.7%f orthe i m m o b ilized li pase .The results sho wed tha t t he li pase fro m As pergillus ni ger strain had a goo d appli catio n potential inthe enzy m a tic resol utio n of (R,S )222oc tano 1.K ey word s :As perg illus ni ger ;li pase ;m utati on ;i m mob iliza ti on ;resol utio n of 22octanol脂肪酶(EC 3.1.1.3)作为一类非水相生物催化剂在有机合成中具有重要的应用价值。

固定化黑曲霉发酵玉米糖液生产柠檬酸的研究3周剑平1　龚伟中1,2　王治业1　杨　晖1,3　麻和平1 (甘肃省科学院生物研究所　兰州730000)1 (中国科学院兰州化学物理研究所　兰州730000)2(兰州大学生命科学学院　兰州73000)3摘要:利用PVA复合凝胶包埋黑曲霉的孢子及菌丝体。

固定后的细胞经过一系列预培养,用于发酵玉米生产柠檬酸。

试验确定的固定化细胞摇瓶发酵生产柠檬酸最适条件为:玉米糖液浓度10Bx,培养温度35℃,摇瓶转速250r/min。

经此条件发酵64h,柠檬酸产率最高达到96g/L,通常稳定在90g/L左右。

同时对柠檬酸连续批次发酵生产进行了初步研究,固定化黑曲霉可连续使用8批次以上,其柠檬酸产量稳定在86～92g/L之间,这为柠檬酸连续发酵提供了有利的保证,并探讨了有关的工艺技术条件。

关键词:固定化,黑曲霉,玉米,柠檬酸发酵中图分类号:Q93 文献标识码:A 文章编号:025322654(2001)0520029204S TUDI ES ON CI TRI C ACID PRODU CTION FROM CORN SACCHA RI F YIN GL IQU ID B Y I M MOBIL I ZED ASPERGILLUS NIGERZHOU Jian2Ping1　GONG Wei2Zhong1,2　WANG Zhi2Y e1　Y ANG Hui1,3　MA He2Ping1(Institute o f Biology,Gansu Academy o f Sciences,Lanzhou730000)1(Institute o f Chemistry and Physics,Chinese Academy o f Sciences,Lanzhou730000)2(College o f life science,Lanzhou University,Lanzhou73000)3Abst ract:The immobilized cells of Aspergillus niger with complex PVA were used to produce citric acid by fermen2tating corn saccharifying liquid.The suitable conditions for fermentation in shaking flask were determined as follows:corn saccharifying liquid concentration10Bx,temperature35℃and rotatory speed of shaking flask250r/min.Thecitric acid yield reached96g/L maximally and usually was90g/L after64h fermentation under the suitable conditions.The continuous fermentation of immobilized cell was studied primarily.The experimental result showed that immobi2lized Aspergillus niger can be used continuous for more than eight times and citric acid yield was86～92g/L.Key wor ds:Immobilized cell,Aspergillus niger,Corn,Citric acid Fermentation柠檬酸是一种重要的有机酸,在食品、化工、医药、建筑、印染、皮革、洗涤剂等行业有广泛的用途[1,2]。

乳糖酶的应用及固定化乳糖酶是一类重要的酶，也称作β-d-乳糖苷酶或乳糖水解酶。

它可以从乳糖分解为糖原和水，是在食品加工、医药、饲料工业及其他工业方面广泛应用的重要技术催化剂。

一、乳糖酶的分类乳糖酶的分类主要参照结构特征，Casaubón在1998年首先把乳糖酶分为β-析氢乳糖酶（β-D-galactohydrolase）和α-析氢乳糖酶（α-D-galactohydrolase）两类。

Β-析氢乳糖酶又分I型和II型，α-析氢乳糖酶又可分为α-1、α-2、α-3、α-4四类。

随后众多研究者又对乳糖酶的分类作了进一步的改进，Sebastian et al（1996）把所有乳糖酶分为四大类：A、B、C和D四类。

二、乳糖酶的功能乳糖酶的功能是将β–链上的乳糖水解成甘油和水，这种水解作用可以改变各种特定的有机物的性质，使其更容易消化和吸收，也可以改变食品的口感。

乳糖酶还可以用于抑制蛋白质合成酶的作用，这是因为它能够切断蛋白质负责传输信号的活性端，从而阻止蛋白质的整合和合成。

三、乳糖酶的应用1、在食品加工中：乳糖酶可以用来制作乳糖、乳酸、乳清粉等，也可以用来制作无乳或低乳的乳制品；2、在医药中：乳糖酶可以作为一种辅助治疗的药物，具有改善消化障碍和营养障碍的作用；3、在饲料中：乳糖酶可以改善家畜和家禽的消化能力，促进动物体内有机物的吸收和利用；4、在生物质能源利用中：乳糖酶可以实现木质素等有机物的分解，有助于将可再生能源转化为清洁能源。

四、乳糖酶的固定化固定化乳糖酶是将自由的乳糖酶结合到功能分子表面上，以获得更好的稳定性，更安全的使用性和足够的活性，使其更容易应用于各种生产实际。

研究表明，聚乙烯醇质量可以提供足够的固定能力，不仅可以有效抑制乳糖酶的蒸发，而且可以大大提高乳糖酶的固定活性，这有助于提高乳糖酶的稳定性和分离性。

目前，国内外对乳糖酶的固定化研究仍在不断深入和完善。

乳糖酶的固定化及低乳糖奶褐变抑制技术的研究分类号密级学号:丝箜单位代码:§?一名微雇业太学学位论文乳糖酶的固定化及低乳糖奶褐变抑制技术的研究‘生:研究何僮培指导教师:王壶越教援申请学位门类级别:奎堂亟±业专名研方向:萤差塞量直芒晶品厘究在学院:所麴塑型撞堂暄答辩委员会主席≯襄二七年六月摘要本课题比较研究了乳糖酶的固定化方法,确定了乳糖酶的固定化参数,并对几种商业用乳糖酶生产低乳糖牛奶时的用量,最佳水解条件和工艺参数进行了比较研究。

以为褐变程度的指示物,研究不同灭菌条件下低乳糖奶褐变及褐变抑制剂的抗褐变效果,建立了生产低乳糖奶的技术体系,为低乳糖奶乳糖水解率%生产提供技术支持。

研究表明:选用海藻酸钠一明胶包埋法固定化乳糖酶,其最适条件为:海藻酸钠浓度.%,明胶浓度.%,乳糖酶的浓度.%,由此得到的固定化酶的酶活回收率可达.%,但该固定化酶的机械强度不理想。

选用海藻酸钠一卡拉胶协同固定化乳糖酶,其最优条件为:海藻酸钠浓度.%,卡拉胶浓度.%,乳糖酶的浓度.%,由此得到的固定化酶的酶活回收率可达.%,凝胶的稳定性重复使用次数可以达到次。

该固定化酶的最适温度为℃,较游离酶没有改变;最适为.,较游离酶升高了.个单位;耐热性和耐受性变宽,这些特性有利于固定化酶在鲜牛奶中的直接使用。

当热处理温度高于℃时, 乳糖酶的水解能力会明显下降。

牛乳的杀菌条件可以使它们失活。

对酶而言, .~.具有较高的酶解水平:酶用量为.%时,℃作用小时或酶用量为.%时,~℃作用小时,乳糖的水解率达到%以上。

采用对进行检测,其中高温。

,低乳糖奶中一含量为. /,分别是普通牛奶与添加复合褐变抑制剂后的低乳糖牛奶的.倍和.倍。

低乳糖奶中埘含量受乳糖水解率、灭菌温度和贮藏条件的影响。

采用合适水解率如%,灭菌后,迅速冷却并低温贮存,能较好地控制和抑制褐变的程度。

抑制荆、、和配合使用时,对的抑制率达%以上。

低乳糖牛奶中的残留量为.//,符合国家标准中食品添加剂残留量的要求。