CN117756953A - 一种小麦麸皮抗冻阿拉伯木聚糖制备方法、结构鉴定与应用 - Google Patents
一种小麦麸皮抗冻阿拉伯木聚糖制备方法、结构鉴定与应用 Download PDFInfo
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Abstract
本发明涉及一种小麦麸皮抗冻阿拉伯木聚糖(AX)制备方法、结构鉴定与应用，属于食品添加剂领域。其特征在于以小麦麸皮为原料，经碱液‑尿素浸提、分级醇沉、冰壳分离、离子交换柱和分子筛凝胶柱层析后得到高纯度抗冻AX，分析了其分子结构与抗冻活性。本发明制备方法简单、绿色安全，可大规模生产，制得抗冻AX活性高，作为添加剂应用于冷冻食品，且本发明对抗冻AX进行了结构鉴定，为深入研究其抗冻的构效关系、作用机制奠定基础。
Description
技术领域
本发明涉及一种小麦麸皮抗冻阿拉伯木聚糖(AX)制备方法、结构鉴定与应用，属于食品添加剂领域。
背景技术
冷冻食品因具有生产标准化、规模化和食用便捷等优点而备受消费者青睐。然而目前我国冷冻食品无论在产量还是品质方面都远不能满足市场的需要，其品质劣化一直是制约其行业发展的重要因素。冰晶生长引起的食品组织结构破坏是导致冷冻食品品质退化的根本原因。冷冻食品常用改良剂如亲水胶体、乳化剂、氧化剂、酶制剂等可在一定程度上提高冷冻食品品质，然而这类添加剂主要通过强化食品组织结构或抑制水分迁移来降低冰晶生长引起的损伤效应，因而其使用存在盲目性。蛋白类和糖类等抗冻物质主要存在于耐寒生物体中，虽可通过有效调控冰晶形成或生长来保护组织结构，但由于其得率低、价格昂贵而未实现大规模生产与应用。因此，探索安全易得且高效的冷冻食品调质剂是实现其货架期延长、推动行业发展的关键。
作为小麦加工副产物，我国麸皮年产量超过2000万吨，但90％以上被用于饲料、酿酒等传统加工，大部分纤维多糖未得到有效利用。麦麸纤维多糖安全性高，成本低廉，是公认的天然膳食纤维。有实验表明，麦麸多糖可有效抑制冰晶生长，具有开发成为冷冻食品抗冻调质剂的潜力。
专利(公开号CN111548428A，公开日2020年8月18日)公开了“一种金针菇抗冻多糖的提取方法”，冷诱导处理的金针菇经粉碎、高温高压水提、离心、碱提、中和、浓缩、干燥后得到金针菇抗冻多糖，具有较高的抗冻活性，但未对金针菇抗冻多糖成分、结构参数进行说明。专利(公开号CN111704677A，公开日2020年9月25日)公开了“一种利用冰壳法分离麦麸抗冻多糖的方法”，以小麦麸皮为原料，经碱液-尿素浸提、醇沉、冰壳分离得到麦麸抗冻多糖，其主要成分为AX，具有较高的热滞活性，然而抗冻多糖中AX纯度不高，且其分子结构与对冰晶生长抑制活性及其构效关系不明。
本发明通过冰壳法和柱层析法对小麦麸皮中的抗冻AX进行分离纯化，对纯化得到的抗冻AX结构进行了鉴定，明确了各多糖组分的结构组成，为探究其抗冻活性机制提供了依据；麦麸抗冻AX对冰晶生长抑制活性高，可作为天然安全的食品调质剂应用于冷冻食品，为冷冻食品货架期延长及麦麸增值利用提供新思路。
发明内容
技术问题
本发明目的在于提供一种小麦麸皮抗冻AX制备方法、结构鉴定与应用，通过冰壳吸附和柱层析从小麦麸皮中分离纯化抗冻AX，分析其分子结构与抗冻活性，为深入研究抗冻AX抗冻构效关系、作用机制奠定基础。
技术方案
本发明的技术方案概述如下：以小麦麸皮为原料，经碱液-尿素浸提、醇沉、冰壳分离、离子交换柱和分子筛凝胶柱层析后得到高纯度抗冻AX，对其分子结构和抗冻活性进行了分析，具体步骤包括：
(1)抗冻AX制备：按公知方法，小麦麸皮经灭酶、碱液-尿素浸提和分级醇沉后得到麦麸多糖粗品，利用冰壳特异性吸附原理从麦麸多糖粗品中富集抗冻多糖组分，经冻干后制得麦麸抗冻多糖；配置浓度为0.5％～2％(w/v)的麦麸抗冻多糖水溶液，用DEAE-52离子交换柱对麦麸抗冻多糖进行一次纯化，用0～0.5M NaCl进行等度洗脱，用苯酚-硫酸法跟踪洗脱曲线收集多糖，浓缩、冷冻干燥；优选0.1M NaCl洗脱，此时得到的多糖含量最高；配置浓度为1％～2％(w/v)的多糖水溶液，以纯水为洗脱液，用Sephadex G-100分子筛凝胶柱进行二次纯化，使用苯酚-硫酸法跟踪洗脱曲线，出现单一对称峰，收集主峰，浓缩、冻干后得到纯化抗冻AX；
(2)抗冻AX结构鉴定：配置1％～2％(w/v)抗冻AX水溶液，用配备示差折光检测器的分子排阻高效液相色谱进行分析，以普鲁兰多糖为标品计算其分子量；抗冻AX经酸解、衍生化后，通过反相高效液相色谱测定其单糖组成；抗冻AX经甲基化、水解、还原和乙酰化后进行气相-质谱联用检测；抗冻AX溶于D2O中，进行1H、COSY、NOESY、HSQC和HMBC核磁共振(NMR)分析；
(3)抗冻AX活性分析：用20％～40％(w/v)的蔗糖溶液配置0.1％～1.0％(w/v)的抗冻AX水溶液，取5～10μL的置于载玻片间制备成厚度约为5～10μm的样品膜；将载玻片置于温度可控的冷台上，以20℃/min的冷却速率从室温冷却至-50℃，再以10℃/min上升至-8℃，用偏光显微镜观察抗冻AX溶液在-8℃下形成冰晶颗粒在120min内的动态变化，通过图像分析定量分析冰晶尺寸，确定抗冻AX对冰晶生长抑制活性。
本发明人为深度开发小麦加工副产物麸皮资源，通过大量实验研究得到本发明：在成功纯化得到抗冻AX基础上，对其分子结构和抗冻活性进行了系统研究。抗冻AX主要由阿拉伯糖和木糖组成，阿拉伯糖与木糖之比为1.0～1.2，分子量范围为180～720kDa，以[→4)-β-D-Xylp-(1→]为主链，支链主要为Araf，连接方式以T-Araf为主。抗冻AX活性较高，对-8℃冰晶生长尺寸抑制效应为15％～72％。
有益效果
与现有技术相比，本发明具有以下优点：
(1)通过冰壳法和柱层析法对小麦麸皮中的抗冻AX进行分离纯化，其操作简单、绿色安全、适于生产；
(2)对纯化得到的抗冻AX结构进行了鉴定，明确了各多糖组分的结构组成，为探究其抗冻活性机制提供了依据；
(3)麦麸抗冻AX对冰晶生长抑制活性高，可作为天然安全的调质剂应用于冷冻食品，为冷冻食品货架期延长及麦麸增值利用提供新思路。
附图说明
图1为抗冻AX代表性分子排阻色谱图
图2为抗冻AX代表性单糖组成色谱图
图3为抗冻AX甲基化分析结果
图4为抗冻AX的1H NMR图谱
图5为抗冻AX的COSY NMR图谱
图6为抗冻AX的NOESY NMR图谱
图7为抗冻AX的HSQC NMR图谱
图8为抗冻AX的HMBC NMR图谱
图9为抗冻AX对冰晶生长的抑制效果
具体实施方式
实施例1：抗冻AX的制备方法
麦麸抗冻多糖提取：小麦麸皮经过除杂、高温灭酶、粉碎过筛后，与浓度为1.5MNaOH和2.0M尿素混合溶液混合，麦麸与NaOH和尿素溶液按质量体积比1∶20混合，于25℃下充分搅拌8h，反应后5000rpm下离心10min后取上清液；在上清液中逐步加入无水乙醇溶液进行梯度乙醇分级沉淀，先将体系乙醇浓度调为20％，静置得到沉淀，再调至30％，依次连续获取其他乙醇浓度下的沉淀(40％，50％，60％，70％)，在室温条件下透析48h；将麦麸粗多糖溶液冻干得到麦麸粗多糖；将圆底烧瓶倒入其容积1/5的水，设置圆底烧瓶转速为80rpm，在-40℃冰浴中浸泡120s形成均匀冰壳；多余的水倒出后，再次将圆底烧瓶浸入冰浴中，直至圆底烧瓶内的冰层形成裂缝。将形成冰壳的圆底烧瓶中浸入-2.5℃的冰浴中，倒入其体积1/2、质量体积浓度为2.0％的麦麸粗多糖水溶液，在转速为50rpm的条件下浸泡60min；将圆底烧瓶中的冰层在25～35℃下融化，得到麦麸抗冻多糖。
麦麸抗冻AX纯化：配置浓度为2％(w/v)的麦麸抗冻多糖水溶液，上样于DEAE-52离子交换柱，用0.1M NaCl进行等度洗脱，洗脱过程中使用苯酚-硫酸法跟踪洗脱曲线，根据洗脱曲线分别收集多糖部分，浓缩冻干；配置浓度为2％(w/v)的上述多糖水溶液，上样于Sephadex G-100柱，用水进行洗脱，使用苯酚-硫酸法跟踪洗脱曲线，出现一个单一对称峰，收集主峰，浓缩冻干，得抗冻AX。
实施例2：抗冻AX的结构鉴定
分子量测定：称取5mg麦麸抗冻AX溶于5mL纯水中，过0.45μm滤膜。采用配备Agilent GPC(PL aquagel-OH Mixed-H 8μm，7.5×300mm)的Agilent高效液相色谱系统分析分子量，以普鲁兰多糖为标品。流动相为100mM硝酸钠，流速为1mL/min，进样量为100μL，柱温保持在30℃，检测器为RID-10A示差折光检测器和G1314A紫外检测器。结果表明，抗冻AX是分子量分布均一的纯化多糖，分子量集在316kDa(图1)。
单糖组成分析：取5mg抗冻AX加入5mL 2M的三氟乙酸中，100℃条件下水解2h。取100μL多糖水解液，加入100μL 0.5M的1-苯基-3-甲基-5-吡唑啉酮(PMP)甲醇溶液进行衍生化，氯仿萃取后过0.45μm滤膜后进行液相分析。色谱条件：ZORBAX Eclipse XDB-C18柱(4.6mm×250mm，5μm)流动相：A：磷酸盐缓冲液(pH6.9)：乙腈(85∶15，v/v)；B：磷酸盐缓冲液(pH6.9)：乙腈(60∶40，v/v)；梯度洗脱比例在0min至10min，B相由8％上升至16％，再在10min至50min内由16％上升至40％。同样地，单糖标品用相同方法衍生化后进行分析。单糖组成结果如图3所示，结果表明，抗冻AX为主要由阿拉伯糖(Ara)、木糖(Xyl)、甘露糖(Man)、半乳糖(Gal)、葡萄糖(Glc)组成，含量分别为48.63％、47.75％、1.19％、1.40％和1.03％，阿拉伯糖与木糖之比为1.02。
甲基化分析：称取10mg抗冻AX溶解于1mL蒸馏水，之后加入同体积碳二亚胺(100mg/mL)反应2h。反应结束后加入1mL2M咪唑，之后将上述溶液分为两等份，一份加入1mL的NaBH4溶液反应3h，另一份加入1mL的NaBD4溶液(30mg/mL)反应3h，加入100μL乙酸终止反应。冻干后加入0.5mL DMSO，45℃下超声处理4h，然后在85℃的水浴锅中水浴1h。水浴结束后加入1mg NaOH，反应30min后加入50μL碘甲烷溶液反应1h。反应结束后加入1mL蒸馏水和2mL二氯甲烷，涡旋混匀后离心，弃水相，此过程重复3次。将样品溶液蒸干后加入100μL 2MTFA，121℃反应90min，30℃下蒸干后加入50mL 2M氨水和50μL 1M NaBD4，混匀后反应2.5h，加入20μL冰醋酸终止整个反应，再用氮气吹干多糖样品。吹干后加入250μL乙酸酐，混匀后在水浴锅(100℃)中反应2.5h。最后加入1mL蒸馏水静置10min后加入500μL二氯甲烷，将上述溶液混匀后再离心，重复用蒸馏水水洗3次，最后取下层二氯甲烷相，取有机相过0.22μm滤膜后用气相色谱进行分析。色谱条件为色谱柱：HP-5MS色谱柱(30m×0.32mm×0.25μm)；进样口温度：250℃；升温程序：140℃，保持1min；2℃/min，上升至220℃，保持1min，10℃/min，上升至250℃，保持2min。进样量：1μL。流动相：氦气。流速：1mL/min。图5为所得色谱图，如表1所示抗冻AX由12种甲基多糖组成，包括五种阿拉伯残基，三种木糖残基及少量的半乳糖残基、甘露糖残基和葡萄糖残基，其中1，4-二乙酰基-2，3，5-三甲基阿拉伯糖醇含量最高，相对摩尔占比为30.37％，是由末端Araf残基产生的，其次是1，4，5-三乙酰基-2，3二甲基木糖醇，由4-连接的木糖残基产生，其中1，4，5-三乙酰基-2，3，6-三甲基葡萄糖醇含量较高，占比为7.59％，这是由4-连接的葡萄糖残基产生，1，5-二乙酰基-2，3，4-三甲基木糖醇含量占比为6.93％，是由末端Xylp残基产生。
表1甲基化分析抗冻AX含有的单糖残基的主要连接方式
注：相对摩尔量＝峰面积/分子量；相对摩尔比(％)＝相对摩尔量/各组分相对摩尔量总和
核磁共振分析：抗冻AX溶于D2O中，样品30mg与99.9％的D2O交换三次，在60℃下搅拌3h后4℃条件下静置过夜，充分水合后的样品溶液冷冻干燥除去溶剂，然后溶解在0.5mLD2O中6h。样品的1D和2D核磁共振波谱记录在Bruker DRX-600核磁共振波谱仪上。在293K下进行了1H、13C、DQF-COSY、TOCSY、HSQC和HMBC实验。将HOD信号固定在δ4.80ppm处，记录1HNMR谱。以丙酮(HPLC级)为内标，固定甲基碳信号为δ30.24ppm，记录13C NMR谱。(1H-1H相关谱(COSY)、1H-1H全相关谱(TOCSY)、1H-12C杂核单量子相干谱(HSQC)和1H-13C杂核多键相关谱(HMBC)获得了2D-NMR数据，其中化学位移(δ)以ppm表示。图4-8为抗冻AX的核磁共振图谱，结果表明抗冻AX以[→4)-β-D-Xylp-(1→]为主链，支链主要是Araf，连接方式以T-Araf为主，另外也存在半乳糖以[→4)-Galp-(1→]和[→3)-Galp-(1→]两种方式连接。表2为抗冻AX的1H和13C化学位移分类表。
表2抗冻AX的核磁共振图谱分析结果
实施例3：抗冻AX的活性分析
抗冻AX活性分析：用20％的蔗糖溶液配置0.5％的抗冻AX水溶液，取10μL的置于玻片间制备成厚度约为10μm的样品膜。以20℃/min的冷却速率从室温冷却至-50℃。然后再以10℃/min上升至-8℃，用偏光显微镜观察-8℃下形成冰晶尺寸在120min内的动态变化，通过图像分析定量分析冰晶尺寸，以此反映抗冻AX对冰晶生长抑制活性。如图9所示，抗冻AX可明显抑制冰晶生长，在10、60、120min时对冰晶尺寸降低效应分别为19.73％、53.73％和71.86％。
以上详细说明了本发明的实施方式，但这只是为了便于理解而举的实例，不应被视为是对本发明范围的限制。同样，任何所属技术领域的技术人员均可根据本发明的技术方案及其较佳实施例的描述，做出各种可能的等同改变和替换，但所有这些改变和替换都应属于本发明的权利要求的保护范围。
Claims (2)
1.一种小麦麸皮抗冻阿拉伯木聚糖(AX)制备方法、结构鉴定与应用，其特征在于，以小麦麸皮为原料，经碱液-尿素浸提、分级醇沉、冰壳分离、离子交换柱和分子筛凝胶柱层析，得到高纯度抗冻AX，对其分子结构和抗冻活性进行了分析，具体包括以下步骤：
(1)抗冻AX制备：按公知方法，小麦麸皮经灭酶、碱液-尿素浸提、分级醇沉、冰壳分离后制得麦麸抗冻多糖；配置浓度为0.5％～2％(w/v)的麦麸抗冻多糖水溶液，用离子交换柱对麦麸抗冻多糖进行一次纯化，用0～0.5M NaCl进行等度洗脱，用苯酚-硫酸法跟踪洗脱曲线收集多糖，浓缩、冷冻干燥；配置浓度为1％～2％(w/v)的多糖水溶液，以纯水为洗脱液，用分子筛凝胶柱进行二次纯化，浓缩、冻干后得到纯化的抗冻AX；
(2)抗冻AX结构鉴定：配置1％～2％(w/v)抗冻AX水溶液，用分子排阻高效液相色谱测定其分子量；抗冻AX经酸解、衍生化后，通过反相高效液相色谱测定其单糖组成；抗冻AX经甲基化、水解、还原和乙酰化后进行气相-质谱联用检测；抗冻AX溶于D2O中，进行核磁共振分析；
(3)抗冻AX活性分析：用20％～40％(w/v)的蔗糖溶液配置0.1％～1.0％(w/v)的抗冻AX水溶液，用配备冷冻台的偏光显微镜观察抗冻AX溶液在-8℃下形成冰晶尺寸随时间的动态变化，计算抗冻AX对冰晶生长抑制活性。
2.根据权利要求1所述的一种小麦麸皮抗冻AX制备方法、结构鉴定与应用，其特征在于，抗冻AX的分子量范围为180～720kDa，阿拉伯糖与木糖之比为1.0～1.2对-8℃冰晶生长尺寸抑制效应为15％～72％，作为添加剂应用于冷冻食品。
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