CN113186238B - 4,6-α-葡萄糖基转移酶及其在改善馒头品质中的应用 - Google Patents

4,6-α-葡萄糖基转移酶及其在改善馒头品质中的应用 Download PDF

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CN113186238B
CN113186238B CN202110294041.6A CN202110294041A CN113186238B CN 113186238 B CN113186238 B CN 113186238B CN 202110294041 A CN202110294041 A CN 202110294041A CN 113186238 B CN113186238 B CN 113186238B
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glucosyltransferase
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陈晟
吴敬
盛露菲
杨卫康
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Jiangnan University
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Abstract

本发明公开了4,6‑α‑葡萄糖基转移酶及其在改善馒头品质中的应用，属于基因工程技术领域。将来源于发酵乳杆菌(Lactobacillus fermentum)的4,6‑α‑葡萄糖基转移酶(gtf16)通过异源表达的方式制备得到4,6‑α‑葡萄糖基转移酶，将获得的4,6‑α‑葡萄糖基转移酶应用于改性糊精的制备中，所制备的改性糊精具有热量低，分子量大，抗消化酶水解等优势。该方法能耗低，时间短，转化效率高，产物中副产物少，对于工业化制备高分子量改性糊精具有重要的意义。将这种改性糊精添加到馒头中可以延缓淀粉老化，增加馒头的货架期。

Description

4,6-α-葡萄糖基转移酶及其在改善馒头品质中的应用
技术领域
本发明涉及4,6-α-葡萄糖基转移酶及其在改善馒头品质中的应用，属于基因工程技术领域。
背景技术
近年来，随着人类物质生活水平的提高，人们对于健康饮食的也随之增长，低脂低糖这些已经成为人们新的标准，因此这一类符合标准的功能性碳水化合物已成为21世纪健康食品发展的风向标。
抗性糊精是包含α-1,4、α-1,6、α-1,3、α-1,2键的葡萄糖聚合物，由于富含慢消化的α-1,6键及抗消化的α-1,3、α-1,2键，不易被人体内消化酶分解，不易被小肠消化吸收，因此能够促进肠道益生菌的增殖；同时抗性糊精具有性质稳定、水溶性好、持水性高、饱腹感强等特点，因此其作为一种优质膳食纤维在饮料、烘焙、乳制品等行业具有广泛的应用。
目前，抗性糊精的生产方式主要有物理改性、化学改性及生物改性。而物理或化学的生产方式均存在能源消耗大、污染严重、原料利用率低、分离纯化工艺复杂等问题，而生物酶法制备具有绿色、节能、高效、安全等优势，可在弥补物理化学方法的不足之处的同时又提升转化效率，是一种制备抗性糊精的重要发展趋势。
4,6-α-葡萄糖基转移酶可利用淀粉或淀粉衍生物合成富含α-1,6键可溶性葡聚糖，且具有作用底物谱较宽、形成的产物形式多样、抗消化成分及产率均较高等优势。该家族酶的发现为抗性糊精的制备提供了新思路，使其酶法工业化生产成为可能。通过4,6-α-葡萄糖基转移酶作用麦芽糊精得到一种富含α-1-6键的产物，称为改性糊精。
目前4,6-α-葡萄糖基转移酶制备的改性糊精分子量较低，无法满足于对于高分子量改性糊精的需求。
发明内容
针对目前酶法制备得到的改性糊精分子量较低、无法适应某些领域对于高分子量改性糊精的需求的问题，本发明报道了一种采用酶法制备改性糊精的工艺技术，可获得分子量高，低热量不易被人体消化的改性糊精产品，该产品可作为一种新型益生元；它具有的更好的持水性与饱腹感可广泛应用于食品行业，将这种改性糊精添加到馒头中可以延缓淀粉老化，增加馒头的货架期。此制备方法可以为维持和改善人类健康提供有力保障，而且为酶法制备高分子量改性糊精的工业化生产奠定坚实的基础。
本发明提供了一种制备改性糊精的方法，所述方法是以麦芽糊精为底物，以氨基酸序列如SEQ ID NO.1所示的4,6-α-葡萄糖基转移酶为催化剂，制备改性糊精。
在一种实施方式中，所述方法包括如下步骤
(1)将10％-30％的麦芽糊精糊化，得到麦芽糊精溶液；
(2)待步骤(1)中的麦芽糊精溶液降温至35-45℃后，将普鲁兰酶和氨基酸序列如SEQ ID NO.1所示的4,6-α-葡萄糖基转移酶加入糊化麦芽糊精溶液中进行改性。
在一种实施方式中，编码所述4,6-α-葡萄糖基转移酶的基因的核苷酸序列如SEQID NO.2所示。
在一种实施方式中，所述4,6-α-葡萄糖基转移酶的添加量不低于1000U/g底物。
在一种实施方式中，所述普鲁兰酶的添加量为10-50U/g底物。
在一种实施方式中，所述改性的条件为在40-45℃、pH为4.5-5.5下反应不低于10h。
本发明提供了一种制备馒头的方法，所述方法是利用所述制备改性糊精的方法制备得到的改性糊精制备馒头。
在一种实施方式中，将改性糊精与中筋面粉质量比为(2-10):100，得到混合粉，将混合粉、酵母和水混匀，并恒温醒发，醒发后排气并于蒸锅中继续醒发，醒发结束后用沸水蒸煮，结束后再焖3-6min，制备得到馒头。
在一种实施方式中，混合粉与酵母的质量比为(70-90):1。
在一种实施方式中，恒温醒发为在30-40℃下醒发1-2h，醒发后排气并于蒸锅中继续醒发25-35min，醒发结束后用沸水蒸煮20-30min。
本发明提供了所述制备改性糊精的方法制备得到的改性糊精在制备米面制品、乳制品、饮料、婴幼儿配方食品、肉制品、糖果中的应用。
在一种实施方式中，所述的米面制品包含烘焙制品、馒头、面条、米粉、方便面条、方便米饭。
在一种实施方式中，述乳制品包含酸奶、奶酪、冰淇淋、植物蛋白乳制品。
在一种实施方式中，所述饮料包含运动饮料、低糖饮料、膳食纤维饮料、五谷杂粮饮料。
本发明提供了所述制备改性糊精的方法制备得到的改性糊精在制备发酵食品中的应用。
本发明的有益效果：
(1)本发明利用酶法制备出一种高分子量的改性糊精，性质稳定，不易被消化，持水性强，可增加饱腹感；
(2)相比物理和化学法制备改性糊精，本方法原料利用率高，且获得的改性糊精产物非还原端起具有连续多个α-1,6糖苷键，比市售改性糊精产品中α-1,6糖苷键含量高很多；
(3)反应过程中，制备工艺比较简单，操作容易，尤其是反应条件比较温和，对设备的要求不高；能耗低，操作简单，收率高，成本低。
(4)将制备得到的改性糊精应用于馒头的制备中，可以显著改善馒头的性质，延缓淀粉老化，馒头的硬度，增加馒头的货架期。也可作为食品添加剂，制备其他的乳制品、烘焙食品、糖果和饮料，可有效改善食品的质地和口感，并且有助于降低食品产品的热值或血糖负荷，有助于低GI饮食。可改善糖尿病和肥胖症等代谢疾病。
附图说明
图1为4,6-α-葡萄糖基转移酶最适温度与最适pH；
图2为4,6-α-葡萄糖基转移酶底物特异性；M:G1-G7；1：葡萄糖(G1)；2:麦芽糖(G2)；3:麦芽三糖(G3)；4：麦芽四糖(G4)；5：麦芽五糖(G5)；6：麦芽六糖(G6)；7：麦芽七糖(G7)；8：蔗糖；9：异麦芽二糖；10：异麦芽三糖；11：黑曲霉二糖；12：潘糖。
图3为4,6-α-葡萄糖基转移酶产物核磁共振色谱图。
图4为4,6-α-葡萄糖基转移酶产物分子量图。
图5为4℃冷藏不同天数馒头硬度变化。
图6为4℃冷藏不同天数后复蒸馒头硬度变化。
具体实施方式
实施例中所述的培养基配方如下：
LB培养基(g/L)：蛋白胨10，酵母提取物5，NaCl 10。
TB培养基(g/L)：蛋白胨10，酵母粉24，甘油5，K2HPO4·3H2O 16.43，KH2PO4 2.31。
实施例中所述的普鲁兰酶购自山东隆科特酶制剂有限公司，酶活为2000U/mL。
实施例中所述的Pancreatin from porcine pancreas(8×USP specification)和淀粉葡萄糖苷酶均购自Sigma Aldrich化学有限公司。
薄层色谱法(TLC)分析不同底物下酶反应产物的组成：方法参见Leemhuis H,Dijkman W P,Dobruchowska J M,et al,Structural characterization of linearisomalto-/malto-oligomer products synthesized by the novel GTFB 4,6-α-glucanotransferase enzyme from Lactobacillus reuteri 121.Glycobiology,2012,22(4),517-528.
实施例1：异源表达发酵乳杆菌(Lactobacillus fermentum)来源的4,6-α-葡萄糖基转移酶(Gtf16)
以带gtf16全长基因(核苷酸序列如SEQ ID NO.2所示)的质粒为模板，设计引物，经PCR后得到目的基因4,6-α-葡萄糖基转移酶基因(gtf16)，构建重组质粒pBSMuL3-gtf16。具体步骤如下：
PCR引物：
D-F：CCATGGGGAAACTGCTGAAAACCCTG(SEQ ID NO.3)；
D-R：TTCGAATAGTAGAAGTTATAAGCGTATTATT(SEQ ID NO.4)。
PCR体系为：20μM引物D-F和D-R各0.5μL，dNTP Mix 4μL，5xPS Buffer 10μL，2.5U/μL的PrimeStar聚合酶0.5μL，模板0.5μL，加双蒸水补齐50μL。
PCR条件：94℃预变性4min；98℃变性10s，55℃退火10s，72℃延伸3.5min，30个循环。
将PCR产物进行胶回收、与克隆载体pMD18T于16℃连接过夜，转化E.coli JM109，涂布在含有氨苄(100μg/mL)抗性的LB平板，37℃培养10-12h，挑取转化子，提取重组质粒并双酶切验证，然后对验证正确的重组质粒测定DNA序列，阳性克隆子即pMD18T-gtf16-JM109。酶切回收目的基因，将其与表达载体pBSMuL3进行双酶切，并于16℃连接过夜，转化E.coli JM109，涂布含有卡那霉素(100μg/mL)抗性的LB平板，37℃培养10-12h，挑取转化子，提取重组质粒并双酶切验证，并对验证正确的重组质粒测定DNA序列，阳性克隆子即pBSMuL3-gtf16。
将重组质粒pBSMuL3-gtf16转化到枯草芽孢杆菌WS11感受态中，得到基因工程菌枯草芽孢杆菌WS11/pBSMuL3-gtf16，涂布含卡那霉素(100μg/mL)抗性的LB平板上，经37℃培养10-12h。挑选单菌落至含卡那霉素(100μg/mL)抗性的10mL液体LB培养基中，37℃培养8h，将测序验证正确的菌株于保存甘油管，贮存于-80℃冰箱。
实施例2：摇瓶发酵产酶
将实施例1中得到的重组菌株接种于LB培养基中，在37℃下培养8h后，以5％接种量转接至50mLTB发酵培养基中，先于37℃、200rpm恒温条件下扩大培养1-2h，在菌体OD600增长至0.5-1.0时加入异丙基-β-D-硫代吡喃半乳糖苷(IPTG)(终浓度为0.08mmol/L)，最终置于25℃、200rpm下进行24h的摇瓶发酵。发酵结束后，将发酵液经高压匀浆破碎后离心，上清即为重组菌所产的4,6-α-葡萄糖基转移酶酶液。
实施例3：4,6-α-葡萄糖基转移酶酶活测定
1.酶活测定
用碘法测定实施例3中发酵所得Gtf16的总酶活。碘试剂：用适当的蒸馏水将2.6gI2和26g KI溶解，并将其转移到100mL容量瓶中定容，4℃保存，使用前用蒸馏水以1:260的比例稀释。
反应时，在40℃下，以直链淀粉(0.125％，w/v，即1.25g/L)为底物，在0.2mol/LNa2HPO4-0.1mol/L柠檬酸缓冲液(pH 5.0)中测定酶活力。反应时，取200μL的底物于1.5mL离心管中，40℃温浴10min。加入200μl的Gtf16酶液40℃反应10min，反应结束后取200μl反应液加入到3800μL的碘显色液中显示5min，分光光度计测定660nm下的吸光度。空白用缓冲液代替酶液，对照取200μl的缓冲液加入到3800ul的碘显色液中显示5min。并在OD660下测吸光值。以五个不同浓度的标准品(0.1、0.2、0.3、0.4、0.5mg/mL)制作直链淀粉的校准曲线。一个相对酶活单位定义为:单位时间吸光值下降一个百分点为一个酶活单位。
酶活(U/mL)＝(100*D(稀释倍数)*(A对照-A实验))/(10min*0.1ml*(A对照-A空白)
2.最适pH和最适温度测定
在pH 7.0下测定Gtf16最适温度，范围为30℃-55℃；在最适温度下测定最适pH，范围为3.0-8.0(反应缓冲液为磷酸-柠檬酸)。
在上述条件下，测得Gtf16酶最适pH为5.0，最适温度分别为40℃(见图1)。
实施例4：4,6-α-葡萄糖基转移酶底物特异性研究
分别用25mmol/L的葡萄糖、麦芽低聚糖(DP 2-7)、蔗糖、异麦芽二糖、异麦芽三糖、黑曲霉二糖及潘糖作为底物研究该酶的底物特异性。将过量的Gtf16与上述各底物在37℃下孵育10h后，煮沸10min使酶失活终止反应。所有反应均在pH5.0的0.2mol/L Na2HPO4/0.1mol/L柠檬酸缓冲液中进行，通过薄层色谱法(TLC)分析每种底物下酶反应产物的组成。
薄层色谱的流动相按照正丁醇:乙酸:水(2:1:1，v/v/v)的配比混合制成；显色剂：在纯乙醇中加入5％的浓硫酸。将混合的标准品(G1-G7)和20-30μg的反应混合物分别点样在TLC板上，将板在流动相中展开8-10h，当流动相靠近TLC薄板顶部时将其取出，待其自然干燥后再放于流动相中进行二次展开以便G1-G7完全分离。待TLC板完全干燥后放于显色剂溶液中并使点样面与显色剂充分接触，之后取出TLC板并在95℃的烘箱中放置20min以观察显色结果。
在上述条件下检测到Gtf16不能利用单独的异麦芽二糖、异麦芽三糖、蔗糖、黑曲霉二糖、潘糖、葡萄糖、麦芽二糖底物，当底物聚合度为麦芽三糖及以上时，Gtf16能够进行转苷反应(见图2)。
实施例5：4,6-α-葡萄糖基转移酶产物特异性研究
将过量的发酵乳杆菌(Lactobacillus fermentum)Gtf16与直链淀粉(0.6％w/v)在pH5.0、40℃条件下温浴24h，之后煮沸10min灭酶，离心(12000rpm，15min)取上清冷冻干燥即得酶转化产物。
1.产物分子量测定
采用高效凝胶过滤色谱法(HPGFC)来确定产物的分子量。使用配备有2414示差折光检测器的Waters 1525高性能液相色谱系统进行HPGFC分析，并用Empower3工作站记录和处理色谱数据。使用Ultrahydrogel TM Linear(300mm×7.8mm，内径×2)凝胶过滤柱进行分离，柱温为45℃，以0.9ml/min的流速注入50μL的样品，0.1M NaNO3作为流动相。从
Figure BDA0002983634460000061
购买的Dextran标准溶液(180Da，2700Da，9750Da，36800Da和135350Da)用作标准溶液。
在上述条件下检测到Gtf16产物的平均分子量为23793Da(见图4)。
2.NMR光谱分析
将上述冷冻干燥产物称取40mg，加入500μL含有0.03％三甲基硅烷磷酸酯(TMSP0.03％)的重水(D2O)完全溶解样品。在AVANCE III 400MHz数字NMR光谱仪(BrukerBiospin International AG)上于60℃检测样品的一维核磁共振(NMR)氢谱。其中0.03％TMSP是作为校准化学位移(δ)的标准品。最后通过在δ5.36和δ4.97处的信号峰面积的积分来估算α-1,4和α-1,6键的占比。
在上述条件下检测到Gtf16产物中α-1,6键的百分比为75％(见图3)。
实施例6：4,6-α-葡萄糖基转移酶在高分子量改性糊精制备中的应用
(1)在麦芽糊精中加入pH5.0的缓冲液将其调成20％(200g/L)的溶液并加热(90℃、10-20min)完成糊化，等待温度降至40℃后，加入10U/g底物的普鲁兰酶和1000U/g底物的4,6-α-葡萄糖基转移酶Gtf16，在恒温水浴摇床中40℃反应12h；
(2)升温至100℃灭酶，将反应产物8000rpm离心30min取上清，0.45μm滤膜过滤后喷雾干燥，最终得到乳白色粉末成品。
实施例7：高分子量改性糊精的抗消化实验
(1)将实施例6中获得的高分子量改性糊精产品配制成浓度为25g/L的溶液；
(2)消化酶的制备：将2g猪胰腺胰酶(Sigma Aldrich)置于24mL蒸馏水中充分振荡10min，1 500rpm离心15min，将20mL上清液转移至50ml离心管中，然后加入0.4mL的葡萄糖苷酶和3.6mL蒸馏水充分混合；
(3)取4mL酶转化产物在37℃下温浴10min，加入750μl的消化酶，在0min、20min和120min分别取样1mL，100℃灭活10min。灭活样品通过12000rpm离心10min后用葡萄糖试剂盒测葡萄糖含量。
各含量占比计算公式如下(其中D为稀释倍数)：
RDS％＝(D1 OD505-20min-D2OD505-0min)/21.23*100，
SDS％＝(D3 OD505-120min-D3OD505-20min)/21.23*100，
RS％＝100-RDS-SDS；
其中RDS表示0-20min内被消化物质的含量百分比，SDS表示20-120min内被消化物质的含量百分比，RS表示120min内不能被消化的物质含量百分比，
经检测，制备的高分子改性糊精在其非还原端起具有连续多个α-1,6糖苷键，抗性(RS)含量可达到72％，在胃肠道中很少被消化。
实施例8：高分子量改性糊精应用于馒头制作
称取3、5、7、10％(w.w-1，改性糊精和中筋面粉的质量比)的改性糊精-中筋面粉混合粉各400g，酵母5g，加水量按照各组面粉实测吸水率的80％添加，将粉、酵母和水加入搅拌机中，用3档搅拌约15min，取下面团将其置于干净的盆中，在35℃恒温培养箱中醒发1h，对其进行排气，再将面团分成质量相同的若干份，揉搓成型置于预热的蒸锅中继续醒发约30min，在沸水中蒸25min，关掉电磁炉，继续焖5min后将馒头取出置于容器内。
对上述实验制作的馒头在室温下冷却1h后将其切成约2cm厚度的馒头片，取中间馒头芯部分用质构仪测定馒头的各项指标。质构仪设置参数如下：P/25，测试前速度1mm·s-1，测试速度时速度1mm·s-1，测试后速度1mm·s-1，形变程度50％，两次压缩间隔时间5s，触发力5g。重复测试3次，各数值采用3次试验的平均值。
将馒头置于4℃冷藏3天，5天，7天，同时冷藏后的馒头在100℃下复蒸10min，测得的各质构参数如表1～表4、图5和图6所示，添加改性糊精的馒头的硬度明显低于对照组，馒头的品质明显得到改善，也延长了馒头的储存时间。
表1改性糊精对馒头质构性质的影响-第1天
Figure BDA0002983634460000071
表2改性糊精对馒头质构性质的影响4℃-3天
Figure BDA0002983634460000072
Figure BDA0002983634460000081
表3改性糊精对馒头质构性质的影响4℃-5天
Figure BDA0002983634460000082
表4改性糊精对馒头质构性质的影响4℃-7天
Figure BDA0002983634460000083
Figure BDA0002983634460000091
虽然本发明己以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
SEQUENCE LISTING
<110> 江南大学
<120> 4,6-α-葡萄糖基转移酶及其在改善馒头品质中的应用
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tattatgatg cgatcaccac cctgatgaaa gcacgtaaac agtttgttag cggtggtcag 1440
accatgacca aactgagcga taacctgatt gcaagcgtgc gttatggtaa aggtgttgca 1500
aatgcaaata gcgaaggcac cgatagcctg agccgtacca gtggtatggc agttatcgtt 1560
ggtaataatc cgcagatggc agaacagaca attagtatca atatgggccg tgcgcacgct 1620
aatgaacagt atcgtaatct gctggatacc accgatgatg gtctgaccta taacgcggat 1680
ggcgttgaaa atccggaaac cctgaccacc gatgatagcg gctatctgaa agttaccgtt 1740
aaaggctata gcaaccctta tgtgagcggc tatctgggtg tttgggtgcc ggttgtgagt 1800
ggtaatcagg atgtgacaac caatgcagca accgttagtg cggatagtaa taaaattttt 1860
gaaagcaacg cggcactgga ttcacacatg atttatgaag atttttccct gtatcagccg 1920
gaaccaacct caaccgaaaa ccatgcctat aatattattg cacagaatgc agaactgttt 1980
aataacctgg gtattacgga tttttggatg gccccggcat atacctctgc aagcatgagc 2040
cgctataatg aaggctattc agttgccgat cgctataatc tgggtacaaa tgcaaatcca 2100
accaaatatg gcagcggtga agaactggca aatgcaatcg ctgccctgca tagcgcaggt 2160
ctgaaagtgc aggaagatat tgtgatgaat cagatgatcg gttttagcgg tcaggaagca 2220
gtgaccgtta cacgtaccaa tgcacgcggt atgcagattt atgttaatgg taaaacatac 2280
gctaaccaga tttattttgc gtataccacc ggcggtggta atggccagga aacctatggt 2340
ggcaaatatc tgagcgaact gcaaagtaaa tatccggatc tgtttacaac ccgctttatt 2400
agcaccggcg tggcgccgga ccctacaacc catattacca aatggagcgc aaaatatgaa 2460
aacggcacca gcctgcaaaa cattggcatc ggtctggcag ttaaactgcc taatggcgat 2520
tatgcctatc tgaatgattc caataataaa gcatttaaca ccaccctgcc ggaaaccatg 2580
agcagcgcag attattatgc gaatattgaa gatgat 2616
<210> 3
<211> 26
<212> DNA
<213> 人工序列
<400> 3
ccatggggaa actgctgaaa accctg 26
<210> 4
<211> 31
<212> DNA
<213> 人工序列
<400> 4
ttcgaatagt agaagttata agcgtattat t 31

Claims (4)

1.一种制备馒头的方法，其特征在于，利用改性糊精制备馒头；所述改性糊精的制备方法为：
（1）将10%-30%的麦芽糊精糊化，得到麦芽糊精溶液；
（2）待步骤（1）中的麦芽糊精溶液降温至35-45℃后，将普鲁兰酶和氨基酸序列如SEQID NO.1所示的4,6-α-葡萄糖基转移酶加入糊化麦芽糊精溶液中进行改性；
所述4,6-α-葡萄糖基转移酶的添加量不低于1000U/g底物；所述普鲁兰酶的添加量为10-50 U/g底物；所述改性的条件为在40-45℃、pH为4.5-5.5下反应不低于10 h。
2.根据权利要求1所述的方法，其特征在于，将改性糊精与中筋面粉质量比为(2-10):100，得到混合粉，将混合粉、酵母和水混匀，并恒温醒发，醒发后排气并于蒸锅中继续醒发，醒发结束后用沸水蒸煮，结束后再焖3-6 min，制备得到馒头。
3.根据权利要求2所述的方法，其特征在于，混合粉与酵母的质量比为(70-90):1。
4.根据权利要求2所述的方法，其特征在于，恒温醒发为在30-40℃下醒发1-2 h，醒发后排气并于蒸锅中继续醒发25-35 min，醒发结束后用沸水蒸煮20-30 min。
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