逆流提取机组使用说明书

2011年4月 The Chinese Journal of Process Engineering Apr. 2011收稿日期：2010−12−13，修回日期：2011−03−09基金项目：国家水体污染控制与治理科技重大专项基金资助项目(编号2008ZX07207-003-3)作者简介：高建萍(1987−)，女，山东省潍坊市人，硕士研究生，主要研究方向为蛋白质分离纯化；通讯联系人，张贵锋，E-mail: gfzhang@,王明林，E-mail: mlwang@.多级逆流固液提取技术提取大豆分离蛋白高建萍1,2， 刘 琳1， 张贵锋2， 王明林1， 黄永东2， 刘永东2， 苏志国2(1. 山东农业大学食品科学与工程学院，山东 泰安 271018；2. 中国科学院过程工程研究所生化工程国家重点实验室，北京 100190)摘 要：采用液相色谱和生物质谱技术对豆粕中不同蛋白质在提取过程中的释放行为进行了研究，并将多级逆流固液提取技术用于大豆分离蛋白提取. 结果表明，豆粕中的2S 组分由于分子量低和高水溶性在提取过程中释放速率较快，提取2次总释放量超过80%, 3次提取7S 和11S 组分总释放量约为69%，提高固相和液相间7S 和11S 的浓度梯度或延长提取时间有助于7S 和11S 组分释放. 采用该提取技术可提高提取液中的总蛋白浓度，在相同蛋白质收率下可节水11%，同时较高的蛋白质浓度可增加酸沉过程中蛋白质收率.关键词：大豆分离蛋白；高效液相色谱−质谱联用技术；蛋白质识别；释放；多级逆流固液提取 中图分类号：X522 文献标识码：A 文章编号：1009−606X(2011)02−0312−061 前 言大豆分离蛋白(Soybean Protein Isolate, SPI)是从脱脂豆粕中提取的一种植物蛋白，总蛋白质含量超过90%，良好的保水性、乳化性、吸油性和凝胶性等使其广泛用作食品添加剂和食品原料[1]. 大豆中的蛋白质根据离心过程中的沉降系数可分为2S, 7S (Conglycinin), 11S (Glycinin)和15S 四种组分，所占比例约为9.4%, 34%, 42%, 4.6%[2,3]. 7S 组分中主要是7S 球蛋白(β-Conglycinin)，是由α(70.6 kDa), α′(80.2 kDa), β(48.4 kDa)三种亚基组成的三聚体结构糖蛋白，分子量约为180 kDa ，等电点5.2∼6.2[1,4,5]. 11S 组分中主要是11S 球蛋白，是一种由6个亚基对组成的多聚亚基蛋白，分子量320∼380 kDa ，每个亚基对由1个酸性亚基和1个碱性亚基通过二硫键连接而成，等电点为4.6[6,7]. 7S 和11S 组分的含量是影响SPI 功能特性的关键因素.目前，我国普遍采用的SPI 提取工艺属碱溶酸沉工艺，即豆粕经pH 7.8 (NaOH 调节)的水溶液浸提后离心去除不溶物，提取液经酸沉处理后再进行离心以分离出凝乳，凝乳经水洗、中和后直接进行喷雾干燥，获得SPI 粉[8,9]，提取液去除凝乳后获得的乳清水属高浓度有机废水. 豆粕中蛋白质的提取多采用传统的罐式提取，存在用水量大、7S 和11S 组分提取率低等缺陷. 提取1 t SPI 需用豆粕约2.4 t ，用水量超过29 t ，产生24 t 高浓度乳清废水(COD>20%)；此外，低提取率导致豆渣中7S 和11S 残留量高，亚基解离导致SPI 性能降低. 因此，研究新型SPI 提取工艺以降低用水量并提高7S 和11S 组分收率具有重要的现实意义. 多级逆流固液提取 (Multi-stage countercurrent solid −liquid extraction)技术是固相物料和溶剂运动方向相反、连续定量加入固相物料和溶剂、导出残留物和提取液的连续分离技术[10,11]. 根据提取设备差异，多级逆流固液提取工艺可分为罐组式、连通器式、螺旋式及离心式等多种形式. 在多级逆流固液提取过程中不同部位的溶剂存在浓度梯度，增加了溶剂和固相物料间的浓度差异，从而加快了目标物释放速率并获得较高的提取液浓度[12]. 多级逆流固液提取技术由于溶剂用量少、提取时间短和生产效率高等优点[13]，近年来在食品、中药、天然产物等领域中的应用日益增多.针对SPI 提取过程中存在的用水量大、7S 和11S 提取率低并导致高浓度有机废水产生量大等共性问题，本研究以蛋白质释放行为研究为基础，比较了不同条件下蛋白质的释放速率差异，研究了多级逆流固液提取技术用于提取大豆分离蛋白以降低用水量的可行性.2 材料与方法2.1 材料与试剂脱脂豆粕由大庆日月星公司提供，胰蛋白酶(序列纯)购自美国Promega 公司，三氟乙酸(TFA)和乙腈购自美国Fisher 公司，其他试剂均为市售分析纯. 2.2 仪器高效液相色谱−质谱联用系统(HPLC −MS)由美国Agilent 公司1100液相色谱和美国Thermo Fisher 公司LCQ Deca XP 电喷雾质谱组成，数据采集与处理软件为Xcalibur 1.3，数据分析软件为Biowork 3.1(含Turbosequest), Ultrospec 2000紫外分光光度计(瑞典Pharmacia 公司).2.3 实验方法2.3.1 蛋白质释放过程分析称取100 g 豆粕，加入800 mL pH 7.8的NaOH 溶液，50℃摇床中连续振荡10 min ，快速抽滤，收集滤液；向残余豆粕中加入400 mL pH 7.8的NaOH 溶液，重复上述过程4次. 将收集到的滤液离心(10000 r/min, 5 min)，上清液用HPLC 分离，收集分离出的各色谱峰，酶解后进行液质联用分析以进行蛋白质识别；通过HPLC 各色谱峰面积研究不同蛋白质释放过程.将上述实验获得的蛋白质提取液合并，调节pH 值至4.5，冷却至4~6℃，以10000 r/min 离心5 min ，收集上清液，酶解处理后用液质联用技术进行蛋白质种类分析.2.3.2 多级逆流固液提取实验多级逆流固液提取工艺实验装置系统图见图1. 在提取过程中清水从右向左移动，豆粕相对于提取液从左向右移动，整体上豆粕与提取液运动方向相反，其中S 0为新鲜豆粕，S 1, S 2和S 3分别为提取一、二和三次后的豆粕，W 为清水，提取液E 1为豆粕S 2与清水混合后的提取液，提取液E 2为豆粕S 1与E 1混合后的提取液，提取液E 3为新鲜豆粕S 0与E 2混合后的提取液. 多级逆流固液萃取实验启动时3个提取罐装满水溶液，向第1个罐中加入新鲜豆粕，连续搅拌并离心分离，洗涤后的豆粕依次导入第2和第3个提取罐直至稳态操作；在稳态多级逆流固液萃取过程中，清水W 与提取过2次的豆粕S 2均匀混合，通过旋流分离器分离后产生的豆粕残渣S 3不再使用，产生的提取液为一次蛋白提取溶液E 1；该提取液与提取过1次的豆粕S 1混合均匀，连续离心后的豆粕残渣为2次提取的残渣S 2，产生的提取液为二次蛋白提取溶液E 2；该提取液与新鲜的豆粕S 0均匀混合，连续离心后产生豆粕残渣S 1，产生的提取液为3次蛋白提取溶液E 3.图1 多级逆流固液提取实验装置系统图Fig.1 Schematic diagram of experimental apparatus for multi-stage countercurrent solid −liquid extraction2.4 分析方法 2.4.1 蛋白质含量测定蛋白质含量测定采用福林酚法. 2.4.2 凝胶过滤色谱分析大豆蛋白提取溶液用凝胶过滤色谱法(HPSEC)分析，色谱柱为TSK3000SW[300 mm×7.5 mm (I.D.), 10 µm]，流动相为0.02 mol/L 磷酸缓冲液(pH 7.0)，流速0.4 mL/min ，检测波长280 nm ，进样量10 µL. 2.4.3 HPLC 分析大豆蛋白提取溶液用去离子水稀释后用HPLC 分析，色谱条件：色谱柱Zorbax 300 SB C 18[250 mm×4.6 mm (I.D.), 5 µm]，流动相A 为水(含0.1% TFA)，流动相B 为乙腈(含0.1% TFA)，梯度：0~45 min 5%~60% B ，45~50 min 60%~90% B ，流速0.75 mL/min ，波长280 nm. 2.4.4 酶解方法将上节分离出的色谱峰在100℃下热变性处理10 min ，真空干燥，然后溶于200 µL 0.05 mol/L NH 4HCO 3溶液(pH 8.0)中，加入50 µg 胰蛋白酶[溶于50 µL 0.05mol/L NH 4HCO 3 (pH 8.0)中]，混合均匀后于37℃恒温8 h ，酶解产物直接进行HPLC −MS 分析. 蛋白质提取液经酸沉淀处理后的上清液于100℃下热变性处理10 min ，将pH 值调至8.0后加入胰蛋白酶，酶解条件同上. 2.4.5 HPLC −MS 分析色谱柱为Zorbax SB C 18[150 mm×2.1 mm (I.D.), 5 µm]；流动相A 为水(含0.1% TFA), B 为乙腈(含0.1% TFA)；梯度0~80 min 10%~90% B ，进样量30 µL. 质谱条件：离子源喷雾电压4.5 kV ，毛细管温度300℃，扫描范围m /z 300~2000. 精确质量数扫描和二级质谱扫描(MS/MS)均为数据依赖型扫描，MS/MS 碰撞能量为35%. 质谱数据用Turbosequest 软件进行检索，数据库为从Swiss-Prot 下载的包括大豆中已发现的全部蛋白质的氨基酸序列.3 结果与讨论3.1 豆粕提取液中蛋白质种类识别采用HPLC对豆粕提取液及其后续处理样品进行S 0. Fresh soybean mealS 1, S 2, S 3. Soybean meal after extraction byone, two and three timesW. Fresh waterE 1. Supernatant from the mixture of W with S 2E 2. Supernatant from the mixture of E 1 with S 1E 3. Supernatant from the mixture of E 2 with S 01. Extraction container 2. Pump3. Cyclone separator分析. 图2是豆粕提取液、经酸沉处理乳清废水和沉淀物重新溶解后的HPLC 图谱，可见豆粕提取液中不同种类成分的保留时间分布范围较宽，乳清废水中主要成分集中在23 min 以前，而沉淀物的保留时间主要集中在24 min 以后. 大豆中蛋白质主要是2S, 7S, 11S 和15S 组分，其中2S 中主要是蛋白酶抑制剂类及细胞色素C, 7S 中主要是球蛋白和少量的血球凝集素、脂肪氧化酶及β-淀粉酶，11S 中主要是球蛋白. 大豆中蛋白质分子量范围较宽，许多蛋白质以多聚亚基形式存在. 这些多亚基蛋白在提取过程中可能发生解离或聚集，且不同蛋白质组分及其亚基的等电点、分子量和亲疏水性不同.图2 豆粕提取液、乳清废水和沉淀物的HPLC 分析图谱 Fig.2 HPLC chromatograms of extraction solutions of soybeanmeal, supernatant and precipitation precipitate收集图2中不同保留时间的色谱峰，酶解产物用HPLC −MS 进行分析，质谱数据用Turbosequest 软件进行数据库检索以识别每个色谱峰中的蛋白质种类. 图3(a)是提取的上清液中保留时间为35.4 min 的色谱峰经胰蛋白酶处理后的总离子流图，表明酶解产物存在离子m /z 575.6；精确质量数扫描图谱表明离子m /z 575.6带双电荷[图3(c)]，该离子对应的多肽在图3(a)中的保留时间为15.1 min ，数据库检索表明该离子对应的多肽序列为VFDGELQEGR ，存在于11S 球蛋白G1 (A1aBx)亚基序列中，表明图2(a)保留时间为35.4 min 的色谱峰中存在11S 球蛋白. 确定了大豆中主要蛋白质在色谱图中的保留时间，其中2S 组分蛋白质主要分布于20 min 以前的色谱峰；保留时间为23 min 的色谱峰中检测出11S 球蛋白的碱性亚基和2S 中的Kunitz-trypsin Inhibitor (KTI)蛋白；保留时间为24.3 min 的色谱峰中主要是7S 的α亚基和11S 的A5A4B3亚基等；保留时间为24~36 min 的是完整的7S 和11S 球蛋白及发生部分解离的7S 和11S 球蛋白.图3 图2中保留时间为35.4 min 的色谱峰酶解产物的色谱−质谱分析Fig.3 HPLC −MS analysis of the digested chromatographic peakin 35.4 min of retaining time [(a) total ion, (b) peak in 15.1 min of retaining time, (c) zoom scan, m /z 575.3 and (d) MS/MS spectrum, m /z 575.3]反相色谱主要根据样品极性进行分离，但样品分子量超过一定范围时其保留时间随分子量增加而延长. 从整体上分析，大豆内丰度较高的4类蛋白质组分中2S 组分的分子量较小，较易分散于水溶液中，在色谱图中的整体保留时间较短. 7S 和11S 中的球蛋白均属多亚基蛋白质，图2中有些色谱峰中只检测出了7S 和11S 球蛋白的部分亚基，表明在提取过程中存在多亚基球蛋白的亚基解离. 24 min 之后的色谱峰中可检测出7S 和11S 的全部亚基，表明这些色谱峰中主要是完整的7S 和11S 球蛋白. 文献[14]报道15S 组分较难溶于溶液而残留于粕渣中.3.2 大豆中蛋白质的释放行为为比较豆粕中不同组分的释放行为，重点以2S 组分及7S 和11S 球蛋白为指标，考察了不同提取条件下提取液中各组分相对含量的变化. 图4是不同提取次数所得豆粕提取液的HPLC 图谱. 从图可以看出，随提取次数增加，上清液中2S 组分(保留时间为14.98~20 min)含量逐渐降低，保留时间为23 min 的色谱峰中存在11S 球蛋白的碱性亚基和2S 中的KTI ，该色谱峰面积随提取次数增加逐渐降低，HPLC −MS/MS 分析结果表明，提取2次后该色谱峰中主要是11S 球蛋白的碱性亚基，而2S 中的KTI 含量较低，表明2S 组分具有较快的释放速率. 含7S 和11S 球蛋白的各色谱峰(保留时间为24.67, 29.7和36.2 min)面积变化趋势表明，随提取次数逐渐增加，上清液中7S 和11S 球蛋白所占比例逐渐增加. 用HPSEC 对不同提取次数的上清液进行了分析，图5表明，随提取次数增加，提取液中高分子量组分增加，与HPLC −MS 和反相色谱分析结果一致.05010005010001020304005010035.3929.6223.0720.7014.623.90(a) Soybean meal (b) SupernatantR e l a t i v e a b s o r b a n c e 36.1424.3122.9720.0613.7010.535.2635.3929.6424.8020.0713.6110.534.22(c) PrecipitationprecipitateTime (min)01020304050050100150600800100012001400050100572574576578580501002004006008001000050100(a)45.5043.0139.0937.6527.8823.0520.1911.196.01R e l a t i v e a b u n d a n c eTime (min)(b)1149.6955.5697.0667.5575.6(c)578.7576.7575.7575.3573.0571.3(d)904.3903.3788.4602.2525.9489.2361.1246.8m /z图4 提取一次、三次和五次的豆粕提取液HPLC 图谱 图5 提取一次、二次和三次的豆粕提取液HPSEC 图谱Fig.4 HPLC chromatograms of protein solutions extracted Fig.5 HPSEC chromatograms of protein solutions extractedonce, thrice and five times from soybean meal once, twice and thrice from soybean meal以二级质谱中检测出的目标多肽对应的一级质谱峰面积计算不同种类蛋白质在提取过程中的动态变化. 图6是以提取液中KTI 、亚基A5A4B3和α亚基的多肽峰面积代表的2S, 7S 和11S 组分在提取过程中的相对释放量随提取次数的变化. 2S 组分在第1次提取时的相对释放量高达55%左右，提取2次时相对释放量超过80%，分子量较高的7S 和11S 组分的相对释放量较2S 组分低，4次提取总释放量不足90%. 可见，7S 和11S 组分由于释放速率低于2S 组分，其提取过程需更长的时间；此外，在提取过程中增加豆粕与提取液之间7S 和11S 的浓度梯度有利于提高其释放速率，释放出的高分子量组分及时移出有助于增加豆粕与提取液之间的浓度梯度并提高豆粕中7S 和11S 组分的释放量.图6 大豆蛋白质提取过程中2S 组分中KTI, 11S 组分中的A5A4B3亚基和7S 球蛋白中的α亚基释放量动态变化 Fig.6 Relative release rate of KTI of 2S fraction, A5A4B3 subunitof 11S fraction and α subunit of β-conglycinin of 7S fraction in extraction of proteins from soybean meal3.3 多级逆流固液提取利用多级逆流固液提取法提取了大豆分离蛋白，研究了不同固液比下豆粕中蛋白质释放过程、总蛋白释放量、提取液中不同蛋白质相对含量及所制大豆分离蛋白的分子量范围.图7是固液比为1:10 g/mL 和单级停留时间为6 min 条件下不同提取级数蛋白提取液的HPLC 和HPSEC 图谱. 由于2S 等低分子量蛋白经2次提取后释放较完全，豆粕S 2中残留的蛋白质主要是7S 和11S 蛋白，E 1曲线表明保留时间为29 min 的组分所占比例较高，因此提取液E 1中7S 和11S 组分所占比例较高；HPSEC 图谱中保留时间为13.43 min 的色谱峰强度较高也表明提取液E 1中主要是分子量较高的蛋白质[图7(b)]，该结果与7S 和11S 组分释放行为研究结果(图4)一致. S 1与E 1充分混合后S 1中的7S 和11S 组分继续释放，提取液E 2中7S 和11S 组分绝对含量增加. 同时，图7中提取液E 2中低分子量的组分含量也有所升高，原因在于第1次提取过程中未完全释放的2S 组分由于溶出速率高于7S 和11S 组分，相对比例逐渐增加. 在多级逆流固液提取过程中，不同提取级数中2S, 11S 和7S 组分的相对释放量的动态变化见图8，与图7结果一致.在多级逆流固液提取过程中，随提取次数增加豆粕中蛋白组分由左向右逐渐被释放，由于大豆蛋白中不同蛋白组分释放行为的差异，7S 和11S 组分从E 3到E 1的相对比例逐渐增加，从E 1到E 3逐级提取后7S 和11S 绝对含量逐渐升高. 实验比较了基于多级逆流法提取和传统提取过程获得的蛋白质溶液中7S 和11S 所占比例，结果表明多级逆流固液提取法获得的蛋白质溶液中7S 和11S 所占比例提高了8%. 可见在多级逆流固液提取过程中豆粕S 0经1次提取后仍有大量未完全释放的7S 和11S 组分，在第2次和第3次提取时释放量较大，从而提高了蛋白提取液中7S 和11S 组分的比例，这对增加SPI 中高分子量组分相对含量并改善SPI 功能特性十分关键.5010005010001020304005010036.0729.5419.9622.9919.365.34(a) OnceR e l a t i v e a b s o r b a n c e35.3429.5723.0220.0214.573.85(b) Thrice 41.0634.2629.5823.0020.0313.694.24(c) Five times Time (min)0501000501000102030405005010035.3124.9113.49(a) OnceR e l a t i v e a b s o r b a n c e 29.9435.3528.7616.6413.45(b) Twice46.4128.7519.1513.40(c) ThriceTime (min)1234530405060708090100R e l a t i v e r e l e a s e r a t e (%)Extraction times图7 多级逆流固液提取过程中不同提取级数豆粕提取液的HPLC 图谱和HPSEC 图谱Fig.7 HPLC and HPSEC chromatograms of protein solutions during multi-stage countercurrent solid −liquid extraction图8 多级逆流固液提取过程中不同提取级数的提取液中2S组分中KTI 和11S 组分中A5A4B3亚基、7S 球蛋白中 的α亚基释放量的动态变化Fig.8 Relative release rate of KTI of 2S fraction, A5A4B3subunit of 11S fraction and α subunit of β-conglycinin of 7S fraction in three extraction stages during multi-stage countercurrent solid −liquid extraction实验采用福林酚法测定了多级逆流固液提取大豆分离蛋白提取液中的蛋白质浓度，进行3次多级逆流平行实验. 豆粕中蛋白质含量为55%，多级逆流固液提取工艺中大豆分离蛋白的提取率为77.6%，而目前工业上每生产1 t 大豆蛋白粉需2.4 t 豆粕，大豆蛋白粉中的蛋白质含量按90%计算，则原料豆粕中蛋白质的收率不足70%. 大豆中2S, 7S, 11S 和15S 四种组分的含量分别为9.4%, 34%, 42%和4.6%，经过提取后15S 中主要蛋白质存在于豆渣中，但仍有少量溶出；2S 组分尽管平均分子量较低且易溶解，但部分蛋白质的等电点接近 4.5，因此，酸沉阶段会有部分2S 组分与7S 和11S 共同沉淀. 蛋白质等电点沉淀过程主要与溶液pH 有关，但蛋白质浓度会影响沉淀过程的收率，本实验中蛋白质收率提高的主要原因是提取液的浓度较高. 多级逆流固液提取技术是一种有效提高大豆分离蛋白提取率的方法.目前工业上使用的大豆分离蛋白提取过程属错流提取工艺，第1次提取时豆粕与水的比例为1:8 g/mL ，离心后向残余豆粕中加入清水，加入量与第1次提取时豆粕用量的比例为4:1 mL/g ，整体上豆粕与水的比例为1:12 g/mL. 由于错流提取工艺用水量较高，提取液中蛋白质浓度与用水量近似呈反比，7S 和11S 组分的浓度降低后导致其在酸沉阶段沉淀不完全，乳清废水中存在一定量的7S 和11S 球蛋白及其亚基，增加了后续水处理过程负荷，同时也降低了大豆分离蛋白的收率. 本研究采用多级逆流固液提取过程中的固液比为1:8 g/mL ，降低了用水量. 结果表明提取液中7S 和11S 组分的总释放率可达75%左右，在酸沉淀阶段7S 和11S 组分的收率提高了3%，降低了乳清废水中7S 和11S 组分的总量，同时降低用水量后乳清废水中2S 组分浓度比错流提取工艺有所增加.比较了固液比分别为1:7, 1:8和1:9 g/mL 条件下多级逆流固液提取工艺中大豆中蛋白质的释放量及7S 和11S 组分的相对含量变化，结果(表1)表明，固液比为1:8和1:9 g/mL 条件下大豆分离蛋白的相对释放量比固液比为1:7 g/mL 条件下分别提高了12.9%和20.7%，其中7S 和11S 组分在提取物中的总含量分别提高了14.6%和20.8%. 在提取过程中固液比越高，多级逆流固液提表1 不同固液比条件下大豆分离蛋白的相对释放量及7S 和11S 组分的相对含量Table 1 Relative release rate of SPI and content of 7Sand 11S fractions in extraction process with different ratios of solid to liquidRatio of solid to liquid(g/mL) Relative release rateof SPI (%) Relative content of 7S and 11S fractions (%)1:7 1:8 1:971.3 84.2 92.069.2 83.8 90.005010005010001020304005010036.1728.9622.9320.055.42E 1(a) HPLCR e l a t i v e a b s o r b a n c e 36.6128.9822.9620.0513.565.44E 236.1428.8322.8719.985.39E 3Time (min)501000501000102030405005010035.3228.7516.6313.43(b) HPSECE 1R e l a t i v e a b s o r b a n c e 35.5428.8021.6516.7313.50E 238.8835.5029.5521.6313.52E 3Time (min)20406080100123R e l a t i v e r e l e a s e r a t e (%)Extraction solution取工艺对离心分离过程要求越高，在工业上会增加动力消耗. 同时，固液比过高将导致7S和11S组分浓度过高，也会影响其释放行为. 与传统的蛋白提取工艺相比，固液比为1:10 g/mL条件下用水量节省了11%，同时由于提取液中较高的7S和11S浓度，在酸沉阶段7S和11S的收率较传统工艺中的酸沉阶段提高了3%.4 结 论(1)以脱脂豆粕为原料，利用碱溶酸沉法提取大豆分离蛋白，利用高效液相色谱−质谱联用系统对大豆分离蛋白提取液中的蛋白质种类进行了分析，识别出提取液中2S, 7S和11S等主要成分，而分子量较大的15S组分则残留于豆粕残渣中.(2)在蛋白质组分识别的基础上，对不同蛋白质组分在提取过程中的释放行为进行了研究. 结果表明，2S组分具有相对较快的释放速率，而7S和11S组分的释放速率相对较低.(3)利用多级逆流固液提取技术提取大豆分离蛋白可有效提高7S和11S组分的相对释放量，且可使大豆分离蛋白的提取率比传统提取方法提高8%.参考文献：[1] Castro R F, Marina M L, Garfa M C. Perfusion Reversed-phaseHigh-performance Liquid Chromatography/Mass Spectrometry Analysis of Intact Soybean Proteins for the Characterization of Soybean Cultivars [J]. J. Chromatogr. A, 2007, 1170: 34−43.[2] Panthee D R, Kwanyuen P, Sams C E, et al. Quantitative Trait Locifor β-Conglycinin (7S) and Glycinin (11S) Fractions of Soybean Storage Protein [J]. J. Am. Oil Chem. Soc., 2004, 81(11): 1005−1012.[3] Renkema J M S, Lakemond C M M, De Jongh H H J, et al. The Effectof pH on Heat Denaturation and Gel Forming Properties of Soy Proteins [J]. J. Biotechnol., 2000, 79(3): 223−230.[4] Delwiche S R, Pordesimo L O, Panthee D R, et al. Assessing Glycinin(11S) and β-Conglycinin (7S) Fractions of Soybean Storage Protein by Near-infrared Spectroscopy [J]. J. Am. Oil Chem. Soc., 2007, 84(12): 1107−1115.[5] Liu C, Wang H L, Gui Z M, et al. Optimization of Extraction andIsolation for 11S and 7S Globulins of Soybean Seed Storage Protein [J]. Food Chem., 2007, 102(4): 1310−1316.[6] Zarkadas C G, Gagnon C, Poysa V, et al. Protein Quality andIdentification of the Storage Protein Subunits of Tofu and Null Soybean Genotypes, Using Amino Acid Analysis, One- and Two-dimensional Gel Electrophoresis and Tandem Mass Spectrometry [J]. Food Res. Int., 2007, 40(1): 111−128.[7] Yuan Y J, Velev O D, Chen K, et al. Effect of pH and Ca2+-inducedAssociations of Soybean Proteins [J]. J. Agric. Food Chem., 2002, 50(17): 4953−4958.[8] Duranti M, Barbiroli A, Scarafoni A, et al. One-step Purification ofKunitz Soybean Trypsin Inhibitor [J]. Protein Express. Purif., 2003, 30(2): 167−170.[9] 赵西周，陈春佳，张效伟，等. 连续性碱溶酸沉生产大豆分离蛋白特点分析 [J]. 中国油脂, 2002, 5(5): 61−63.[10] Wang Q E, Ma S M, Fu B Q, et al. Development of Multi-stageCountercurrent Extraction Technology for the Extraction of Glycyrrhizic Acid (GA) from Licorice (Glycyrrhiza uralensis Fish) [J]. Biochem. Eng. J., 2004, 21(3): 285−292.[11] 胡小中，温光源，李里特. 多级逆流醇浸法制取大豆浓缩蛋白工艺的研究 [J]. 食品工业科技, 2009, 30(3): 272−275.[12] 王英，崔政伟. 连续动态逆流提取的动态和现状 [J]. 包装与食品机械, 2009, 27(1): 49−53.[13] Li W, Zheng C, Wang J S, et al. Microwave Multi-stageCountercurrent Extraction of Dihydromyricetin from Ampelopsis Grossedentata [J]. Food Technol. Biotechnol., 2007, 45(4): 374−380. [14] 周兵，周瑞宝. 大豆球蛋白的性质 [J]. 西部粮油科技, 1998,23(4): 39−43.Extraction of Soybean Protein Isolate UsingMulti-stage Countercurrent Solid−Liquid Extraction MethodGAO Jian-ping1,2, LIU Lin1, ZHANG Gui-feng2, WANG Ming-lin1, HUANG Yong-dong2, LIU Yong-dong2, SU Zhi-guo2(1. College of Food Science and Engineering, Shandong Agricultural University, Taian, Shandong 271018, China;2. State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, CAS, Beijing 100190, China) Abstract: Release behavior of proteins in soybean meal during their extraction process using high performance liquid chromatography−mass spectrometry was investigated. The possibility for extraction of soybean protein isolate using multi-stage countercurrent solid−liquid extraction (MSCSLE) was also studied. The result indicated that 2S protein showed a higher release rate and more than 80% of total 2S proteins were released after extraction in two times. The total release rate of 7S and 11S proteins reached 69% after three times of extraction, indicating that 7S and 11S fractions had a lower release rate during extraction process. Increasing the concentration gradient between the solid and liquid or extending the extraction time enhanced the release of 7S and 11S fractions. The concentration of total proteins obtained using MSCSLE was higher than that obtained by multi-time extraction, resulting in higher 7S and 11S recovery rate. Under the same recovery rate of soybean protein isolate, 11% of water could be saved. During precipitation, 8% of total 7S and 11S protein could be recovered.Key words: soybean protein isolate; high performance liquid chromatography−mass spectrum; protein identification; release; multi-stage countercurrent solid−liquid extraction。

逆流提取原理
逆流提取是一种常用的化工分离技术，其原理是利用两种不相溶的溶剂在一个
装置中进行逆向流动，从而实现对混合物中成分的分离和提取。

逆流提取技术在化工生产中有着广泛的应用，如萃取、脱水、脱气等过程中都可以采用逆流提取技术。

逆流提取的原理可以简单地理解为，混合物在两种不相溶的溶剂中有不同的溶
解度，通过两种溶剂的逆向流动，可以使得混合物中的成分在两种溶剂中分别得到提取。

在逆流提取的过程中，通常会选择一个较为极性的溶剂和一个较为非极性的溶剂，以便更好地实现对混合物中成分的分离。

在实际的逆流提取过程中，通常会采用萃取塔或者萃取槽等装置，通过将混合
物和两种溶剂分别输入到萃取塔中，利用逆向流动的原理，使得混合物中的成分在两种溶剂中得到提取。

在萃取塔中，混合物首先与一个溶剂相接触，然后在逆向流动的过程中与另一个溶剂相接触，通过这样的循环过程，可以实现对混合物中成分的分离和提取。

逆流提取技术的优点在于可以实现对混合物中成分的高效分离，同时可以在一
定程度上减小溶剂的消耗。

此外，逆流提取技术还可以适用于对温度、压力敏感的物质进行提取，具有较高的适用性。

然而，逆流提取技术也存在一些局限性，例如对于粘度较高的物质，逆流提取
技术的效率可能会受到一定的影响；另外，在实际操作中需要考虑流体动力学、传质和传热等多种因素，因此需要对操作条件进行精确控制。

总的来说，逆流提取技术作为一种重要的化工分离技术，在化工生产中有着广
泛的应用前景。

通过对逆流提取原理的深入理解和技术改进，可以更好地发挥逆流提取技术在化工生产中的作用，为提高生产效率和产品质量提供有力支持。

逆流提取的原理范文逆流提取是一种常用的物质分离技术，它是通过逆向流动相对于溶剂移动物质的方法，利用相互之间的物理和化学差异来分离目标物质。

1.相互溶解性差异：在逆流提取中，通常通过溶剂与被提取物质之间的溶解度差异来实现分离。

具体而言，溶剂的选择应使目标物质有较高的溶解度，而不被提取物质溶解。

当溶剂与混合物接触时，目标物质将优先溶解在溶剂中，而不被提取物质相对较少地溶解。

通过逆向流动，提取溶剂中的目标物质可以得到分离。

2.组分的分配差异：逆流提取还利用了溶剂与被提取物质之间的分配差异。

当混合物与溶剂接触时，被提取物质会在两相之间通过分配达到平衡。

目标物质有较高的分配系数，即在溶剂和混合物之间分配的比例较高，而不被提取物质的分配系数较低。

通过逆向流动，目标物质会逐渐从混合物中分配到溶剂中，实现分离。

3.物理性质的差异：逆流提取还利用了混合物中组分的物理性质差异。

例如，如果目标物质在混合物中具有较高的挥发性，而不被提取物质的挥发性较低，逆流提取可以通过控制温度和压力来促使目标物质从混合物中蒸发到溶剂中。

反之亦然，如果目标物质的沸点较高，可以通过逆流提取将其从溶剂中蒸发回混合物中，实现分离。

4.化学亲和力：逆流提取还可以利用目标物质与溶剂之间的化学亲和力来实现分离。

如果目标物质与溶剂之间具有特定的相互作用，如氢键、离子相互作用或金属络合等，可以通过控制反应条件来改变这些相互作用的强弱。

这将导致目标物质与混合物中其他成分之间的化学反应，进而实现分离。

总结起来，逆流提取的原理是通过溶剂与被提取物质之间的溶解度差异、分配差异、物理性质差异以及化学亲和力等多种作用机制来实现分离。

逆流提取是一种灵活、高效且广泛应用于各种领域的分离技术，可以用于提取和纯化化学品、药物、天然产物等。

UNPACKINGAfter unpacking unit, inspect carefully for any damage thatmay have occurred during transit. Check for loose, missing, or damaged parts. If any damage is observed, a shipping damage claim must be filed with carrier. Do not use Manual Fluid Extractor if broken, bent, cracked ordamaged parts (including labels) are noted. Any Manual Fluid Extractor that appears damaged in any way, operates abnormally or is missing parts should beremoved from service immediately. If you suspect that the Manual Fluid Extractor was subjected to a shock load (a load that was dropped suddenly, unexpectedly, etc.) immediately discontinue use until it has been checked by a factory authorized service center.Please read and save these instructions. Read through this owner’s manual carefully before using product. Protect yourself and others by observing all safety information, warnings, and cautions. Failure to comply with instructions could result in personal injury and/or damage to product or property. Please retain instructions for future reference.The following safety information is provided as guidelines to help you operate your Manual Fluid Extractor under the safest possible conditions. Any tool or piece of equipment can be potentially dangerous to use when safety or safe handling instructions are not known or not followed. The following safety instructions are to provide the user with the information necessary for safe use and operation. Please read and retain these instructions for the continued safe use of your service system. Failure to follow instructions listed below may result in serious injury. In addition, make certain that anyone that uses the equipment understands and follows these safety instructions as well.24389MANUAL FLUID EXTRACTORThank you very much for choosing an OEMTOOLS Product!For future reference, please complete the owner’s record below:Model: _______________ Purchase Date: _______________ Save the receipt, warranty and these instructions. It is important that you read the entire manual to become familiar with this product before you begin using it. This machine is designed for certain applications only. OEMTOOLS cannot be responsible for issues arising from modification. We strongly recommend this machine is not modified and/or used forany application other than that for which it was designed. If you have any questions relative to a particular application, DO NOT use the machine until you have first contacted OEMTOOLS to determine if it can or should be performed on the product.IMPORTANT INSTRUCTIONSAND SAFETY RULES1.Know your tool. Read this manual carefully. Learnthe tool’s applications and limitations, as well as,potential hazards specific to it.2.Keep work area clean and well lit. Cluttered or darkwork areas invite accidents.3.Keep children away. All children should be keptaway from the work area. Never let a child handlea tool without strict adult supervision.4.Do not operate this tool if under the influenceof alcohol or drugs. Read warning labels onprescriptions to determine if your judgment orreflexes are impaired while taking drugs. If there is any doubt, do not attempt to operate.e safety equipment. Eye protection shouldbe worn at all times when operating this tool.Use ANSI approved safety glasses. Everydayeyeglasses are NOT safety glasses. Dust mask,non-skid safety shoes, hard hat or hearingprotection should be used in appropriateconditions.6.Wear proper apparel. Loose clothing, gloves, neck-ties, rings, bracelets or other jewelry may present a potential hazard when operating this tool. Keep all apparel clear of the tool.7.Don’t overreach. Keep proper footing and balanceat all times when operating this tool.8.Check for damage. Check your tool regularly. Ifpart of the tool is damaged it should be carefullyinspected to make sure that it can perform itsintended function correctly. If in doubt, the partshould be repaired. Refer all servicing to a qualified technician. Consult your dealer for advice.9.Keep away from flammables. Do not attemptto operate this tool near flammable materialsor combustibles. Failure to comply may causeserious injury or death.10.Store idle tools out of the reach of children anduntrained persons. Tools may be dangerous in the hands of untrained users.•Maintain tools with care.•Keep tools dry and clean.•Properly maintained tools are less likely to bind and are easier to control. Do not use a damaged tool. Tag damaged tools “Do not use” until repaired.•Check for misalignment or binding of moving parts, breakage of parts, and any other condition that may affect the tool’s operation.•If damaged, have the tool serviced before using. Many accidents are caused by poorly maintained tools.•Use only accessories that are recommended by the manufacturer for your model. Accessories that may be suitable for one tool may become hazardous when used on another tool.•Tool service must be performed only by qualified repair personnel. Service or maintenance performed by unqualified personnel could result in a risk of injury.•When servicing a tool, use only identical replacement parts. Use of unauthorized parts or failure to follow maintenance instructions may create a risk of injury.•Maintain a safe working environment. Keep the work area well lit. Make sure there is adequate surrounding workspace. Keep the work area free of obstructions, grease, oil, trash, and other debris. Do not use this product in a damp or wet location.•Maintain labels and nameplates on this product. These carry important information. If unreadable or missing, contact OEM for a replacement.•Keep the handle dry, clean, and free from brake fluid,oil, and grease.•Before use, read and understand all warnings, safety precautions, and instructions as outlined in the vehicle manufacturer’s service manual. I t is beyond the scope of this manual to properly describe the correct procedure and test data for each vehicle.•Always perform vehicle service in a properly ventilated area. Never run an engine without proper ventilation for its exhaust. Stop work and take necessary steps to improve ventilation in the work area if you develop momentary eye, nose, or throat irritation as this indicates inadequate ventilation.•Engine parts that are in motion and unexpected movement of a vehicle can injure or kill. When working near moving engine parts, wear snug fit clothing and keep hands and fingers away from moving parts. Keep hoses and tools clear of moving parts. Always stay clear of moving engine parts. Hoses and tools can be thrown through the air if not kept clear of moving engine parts. The unexpected movement of a vehicle can injure or kill. When working on vehicles always set the parking brake or block the wheels.•Be alert for hot engine parts to avoid accidental burns.•If you drove your car recently, fluids could be very hot. Allow at least 2 hours before you handle any fluids. Oil and Coolant burns are very dangerous.•Avoid accidental fire and/or explosion. Do not smoke near engine fuel and battery components.•Never remove the cap from the radiator or expansion tank while the engine is at operating temperature.•Always allow the engine to cool before removing the radiator cap or expansion tank cap. The cooling system is under pressure. Failure to allow the engine to cool before attempting to remove the cap could result in serious injuries.•The warnings, precautions, and instructions discussed in this manual cannot cover all possible conditions and situations that may occur. The operator must understand that common sense and caution are factors which cannot be built into this product, but must be supplied by the operator.•For safety purposes and the prevention of damage to expensive components it is advised that the user have an understanding of basic automotive repair and a working knowledge of automotive systems.•We believe the information contained herein to be reliable. However, general technical information is given by us without charge and the user shall employsuch information at his own discretion and risk. We assume no responsibility for results or damages incurred from the use of such information in whole or in part. Always refer to specific instructions and technical information supplied by vehicle manufacturer.•The manufacturer declines any and all responsibility for damage to vehicles or components if said damage is the result of unskillful handling by the operator or of failure to observe the basic safety rules set forth in the instruction manual.•Used oil, antifreeze, brake fluid and transmission fluid contains chemical compounds that can be harmful to humans and other animals. When managed properly, used oil may again be of beneficial use. Used oil may be blended and recycled as a heating or industrial fuel and also may be re-refined and made into new lubricants. Persons who perform maintenance on their own vehicles are responsible for managing the used fluids in a manner that is protective of human health and the environment and to follow all local laws and regulations concerning their disposal. DISPOSALAt the end of the useful life of the Manual Fluid Extractor, dispose of the components according to all state, federal, and local regulationsPURPOSEThe 24389 Manual Fluid Extractor is designed for the extraction of all types of engine, transmission and lubricating oils from cars, motorcycles, marine engines and industrial machinery. Also suitable for low viscosity fluid such as water or coolantReservoir Capacity 5.5L (1.45 Gallons) PRODUCT SPECIFICATIONS24389-1Tube1 24389-2Suciton Tube1 24389-3Dipstick Tube1 24389-4Pump Handle1 24389-5Reservoir1 24389-6Float Assembly(Not Shown)124389-7Base124389-8Vacuum ReleasePlug1NOTENot all components of the Manual Fluid Extractor are replacement items, but are illustrated as a convenient reference for location and position in the assembly sequence.(1)MainSuctionTube (2)Suciton Tube(3)Dipstick Tube(5)Reservoir(7)Base (4)(8)VacuumReleasePlugAlways wear safety glasses and gloves!OPERATING INSTRUCTIONS• This equipment is intended only for professional use by personnel trained in performing the service functions for which it is has been designed• This equipment is designed for servicing a variety of vehicles in a safe, convenient manner. However, differences in vehicle makes and models may make it impossible to use this equipment as it is intended. Do not attempt to force the use of this equipment on an application for which it is not designed to perform.• The procedures documented in this manual are to serve as guidelines for the use of this equipment.• In addition to these guidelines, always follow the manufacturer’s recommended procedures when servicing each unique vehicle.• The use of this equipment is simple and straightforward if you follow the instructions. However, always keep in mind that you are working with a system that may be under pressure, with fluid that is just waiting to be expelled. When operating this equipment, use common sense, and always stop to think before disconnecting a hose or other component.DO NOT USE THIS EQUIPMENT WITH GASOLINE OR OTHER FLAMMABLE LIQUIDS OR WITHFLUIDS AT TEMPERATURES ABOVE 175° FAHRENHEIT (80° CELSIUS). KEEP AWAY FROM OPEN FLAMES OR EXCESSIVE HEATEXTRACTING MOTOR OIL THROUGH THE DIPSTICK TUBE1. Park vehicle on level ground. Ensure the transmission of the vehicle is in “neutral” or “park” position andapply the parking brake.2. Start the engine. Allow the engine to idle until it has reached normal operating temperature. Turn the engineoff.3. Insert the appropriate diameter dipstick tube into the dipstick fill hole until it reaches the bottom of oil pan.Note: Do not bend the suction tube during the inserting procedure.4. Connect the main suction tube to the dipstick tube.5. Insert the O-Ringed plug of the main suction tube into the reservoir adapter and then insert the adapter intothe top of the reservoir. Ensure the tube connections are tight to prevent leakage.6. Extract the used motor oil by pumping the evacuator handle several times to create a vacuum. Once the fluidbegins to flow into the reservoir, continue to operate the pump until all the fluid has been drained from the engine crankcase.Note: When oil in the tank reaches the maximum level, the vacuum will release to prevent overflowing.7. Remove the main suction hose from the reservoir adapter; pour the used oil from the reservoir into a suitablecontainer, then dispose of the fluid in an appropriate mannerNOTEIf no vacuum can be created after emptying the Reservoir, the user may need to remove the Red Vacuum Release Plug below the Pump Handle and reinstall it. This will release vacuum inside the Pump and restore operation.EXTRACTING TRANSMISSION FLUID THROUGH THE DIPSTICK TUBEIn some applications, this may require jacking or lifting the vehicle. Use appropriate safety stands to avoid serious or fatal injury.1.Operate the vehicle to warm the transmission fluid to a normal operating temperature.Caution: Do not attempt to extract fluids at temperatures greater than 175° Fahrenheit (80° Celsius).2.Properly park the vehicle on level ground and turn the engine off.3.Remove the transmission fluid dipstick.4.Insert the dipstick tube into the dipstick fill hole until it reaches the bottom of the transmission pan.5.Connect the main suction tube to the dipstick tube.6.Insert the O-Ringed plug of the main suction tube into the reservoir adapter and then insert the adapter intothe top of the reservoir. Ensure the tube connections are tight to prevent leakage.7.Extract the used transmission fluid by pumping the evacuator handle several times to create a vacuum.Once the fluid begins to flow into the reservoir, continue to operate the pump until all the fluid has been drained from the transmission pan.8.Remove the main suction hose from the reservoir adapter; pour the used transmission fluid from thereservoir into a suitable container, then dispose of the fluid in an appropriate manner.NOTEIf no vacuum can be created after emptying the Reservoir, the user may need to remove the Red Vacuum Release Plug below the Pump Handle and reinstall it. This will release vacuum inside the Pump and restore operation.EXTRACTING COOLANT FROM A RADIATOR OR EXPANSION TANKNever remove the cap from the radiator or expansion tank while the engine is at operating temperature. Always allow the engine to cool before removing the radiator cap or expansion tank cap. The cooling system is under pressure. Failure to allow the engine to cool before attempting to remove the cap could result in serious injuries.1.Properly park the vehicle on level ground and turn the engine off.2.Allow engine to cool completely. Caution: Do not attempt to extract fluids at temperatures greater than 175°Fahrenheit (80° Celsius).3.Remove the radiator or expansion tank cap.4.Insert the suction tube into the radiator or expansion tank until it reaches the bottom.5.Connect the main suction tube to the suction tube.6.Insert the O-Ringed adapter into the pour spout on the top of the reservoir. Ensure the tube connections aretight to prevent leak.7.Extract the used coolant by pumping the evacuator handle several times to create a vacuum. Once thecoolant begins to flow into the reservoir, continue to operate the pump until all the coolant has been drained from the radiator or expansion tank.8.Remove the reservoir adapter from the reservoir; pour the used coolant from the reservoir into a suitablecontainer, then dispose of the coolant in an appropriate manner.NOTEIf no vacuum can be created after emptying the Reservoir, the user may need to remove the Red Vacuum Release Plug below the Pump Handle and reinstall it. This will release vacuum inside the Pump and restore operation.EXTRACTING BRAKE FLUID FROM THE MASTER CYLINDER1.Properly park the vehicle on level ground and turn the engine off.2.Clean the exterior of the master cylinder and master cylinder cap to prevent dirt from entering the mastercylinder when the cap is removed.3.Remove the cap from the master cylinder reservoir.4.Insert the suction tube into the master cylinder reservoir.5.Connect the main suction tube to the suction tube.6.Insert the O-Ringed adapter into the pour spout on the top of the reservoir. Ensure the tube connections aretight to prevent leak.7.Extract the used brake fluid by pumping the evacuator handle several times to create a vacuum. Once thefluid begins to flow into the reservoir, continue to operate the pump until all the fluid has been drained from the master cylinder.8.Remove the reservoir adapter from the reservoir; pour the used brake fluid from the reservoir into a suitablecontainer, then dispose of the fluid in an appropriate manner.NOTEIf no vacuum can be created after emptying the Reservoir, the user may need to remove the Red Vacuum Release Plug below the Pump Handle and reinstall it. This will release vacuum inside the Pump and restore operation.EXTRACTING PO WER STEERING FLUID FRO M THE PO WER STEERING FLUID RESERVOIR1.Properly park the vehicle on level ground and turn the engine off.2.Clean the exterior of the power steering fluid reservoir to prevent dirt from entering the reservoir when thecap is removed.3.Remove the cap from the power steering fluid reservoir.4.Insert the suction tube into the master cylinder reservoir.5.Connect the main suction tube to the suction tube.6.Insert the O-Ringed adapter into the pour spout on the top of the reservoir. Ensure the tube connections aretight to prevent leak.7.Extract the used power steering fluid by pumping the evacuator handle several times to create a vacuum.Once the fluid begins to flow into the reservoir, continue to operate the pump until all the fluid has beendrained from the power steering fluid reservoir.8.Remove the reservoir adapter from the evacuator reservoir; pour the used power steering fluid from thereservoir into a suitable container, then dispose of the fluid in an appropriate manner.NOTEIf no vacuum can be created after emptying the Reservoir, the user may need to remove the Red Vacuum Release Plug below the Pump Handle and reinstall it. This will release vacuum inside the Pump and restore operation.MAINTENANCE1.Always store the Manual Fluid Extractor in a well-protected area where it will not be exposed to inclementweather, corrosive vapors, abrasive dust, or any other harmful elements.2.Keep the Manual Fluid Extractor clean for better and safer performance.3.Inspect Reservoir, Hoses, Caps, Gaskets, Seals, Gauge and Adapters periodically, and if damaged, replacethem.4.Rinse the evacuator reservoir, pump, adapter, and tubes with clean solvent or engine degreaser, and allowthem to dry thoroughly.。