CN115160613B - 一种保鲜水凝膜的制备工艺 - Google Patents
一种保鲜水凝膜的制备工艺 Download PDFInfo
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Abstract
本发明公开了一种保鲜水凝膜的制备工艺，包括如下步骤：(1)将羧甲基壳聚糖和菊糖溶于去离子水，搅拌溶解后进行聚合反应，得到羧甲基壳聚糖/菊糖水凝胶；(2)将肠球菌素加入到步骤(1)得到的羧甲基壳聚糖/菊糖水凝胶中进行聚合反应，反应结束后制膜，即得。本发明以羧甲基壳聚糖/菊糖为载体负载不同浓度的肠球菌素，从制备稳定性、抗菌性能、保鲜效果的综合评价，得到了较好的羧甲基壳聚糖/菊糖/肠球菌素水凝胶膜，为食品工业提供新型、有效抗菌包装。
Description
技术领域
本发明属于食品加工技术领域，涉及一种保鲜水凝膜的制备工艺。
背景技术
随着生活水平的提高，人们逐渐追求健康、营养和饮食的多样化，特别是对水果、蔬菜、肉类和水产品等生鲜食品的需求日益增加。新鲜食物的新鲜度是消费者的一个重要指标。生鲜食品在冷藏和销售过程中面临微生物的腐败变质，导致保质期短，并且以被致病菌等污染，如单核增生李斯特菌，因此开发抗菌包装材料是保证新鲜食品质量、卫生品质和延长食品货架期的关键。抗菌包装是将包装材料与抗菌药物相结合，抑制微生物生长，是提高食品卫生质量和延长货架期的一种新兴技术。近年来，利用静电纺丝技术将天然生物来源的抗菌物质加载到食品级载体上制备纳米纤维膜成为研究的热点。静电纺丝技术是指聚合物溶液在高压静电场作用下克服表面张力产生带电射流，并最终固化得到纳米纤维。由于电纺丝纤维结构完整、比表面积大、体积大以及纤维排列的特殊性，纳米纤维和电纺丝技术在食品包装和医疗领域受到青睐。但静电技术操作成本高、制作周期长，给其在实际应用过程中造成一定不利的影响。而水凝胶的盛行，在于其具有生物相容性、操作性好、也是抗菌物质良好载体，常被医学用于载药定点靶向治疗。在食品方面，由于水凝胶具有一定流动性和粘度，使其在食品保鲜应用方面展现强有力的优势。水凝胶在低温放置过程中会形成稳定的胶状体，与低温放置食品所需保鲜材料相吻合。水凝胶对水溶性抗菌剂具有一定负载能力，因此通过聚合接枝反应将抗菌剂负载与水凝胶空间网络结构，将使水凝胶膜呈现出较好的表面抑菌作用。随着水凝胶具有一定亲水性，在保鲜过程中会吸附组织中渗出的汁液，并且水凝胶具有一定粘附性，因而会较好保持肉组织的表观状态。
羧甲基壳聚糖是一种重要的水溶性壳聚糖衍生物，有许多医学功效，如促进创面愈合、止血、抑制瘢痕、镇痛和抑菌作用，另外在化工、环保、保健品方面也有广泛的应用。羧甲基壳聚糖具备良好的生物相容性和生物降解性，在水凝胶和愈合创伤类生物材料中广泛应用，也被广泛应用于组织工程基质材料。此外，羧甲基壳聚糖很容易加工成纳米颗粒，使其更适用于药物递送，生物成像，生物传感器和基因疗法，近年来羧甲基壳聚糖在绿色化学方面也有很多应用。因其独有的生物特性，羧甲基壳聚糖在生物医学和制药领域有广泛的应用。其水溶性特点，单一羧甲基壳聚糖亲水性较高，易在保鲜过程被分解和过度吸收肉中水分。菊糖广泛存在于植物组织中，约有3.6万种植物中含有菊糖，尤其是菊芋、菊苣块根中含有丰富的菊糖。由于菊糖较好保健特性和富含氨基和羟基，因此可为水凝胶膜良好的基材。因而，通过将羧甲基壳聚糖与菊糖进行聚合接枝，不但可增强膜的稳定性，也可降低膜的亲水特性，并且负载对李斯特菌具有较好抑菌效果的肠球菌素，将使水凝胶膜被更好地用于海鲜类保鲜包装。
发明内容
本发明所要解决的技术问题是针对现有技术的不足，提供一种保鲜水凝膜的制备工艺，以羧甲基壳聚糖/菊糖为载体负载肠球菌素，制备得到保鲜效果好、抗菌性能强的羧甲基壳聚糖/菊糖/肠球菌素水凝胶膜，为食品工业提供新型、有效抗菌包装。
为了解决上述技术问题，本发明公开了一种保鲜水凝膜的制备工艺，包括如下步骤：
(1)将羧甲基壳聚糖和菊糖溶于去离子水，搅拌溶解后进行聚合反应，得到羧甲基壳聚糖/菊糖水凝胶；
(2)将肠球菌素加入到步骤(1)得到的羧甲基壳聚糖/菊糖水凝胶中进行聚合反应，反应结束后制膜，即得。
具体地，步骤(1)中，所述的羧甲基壳聚糖(CMC)与去离子水的质量比为4％～6％：1，优选为4％：1；所述的菊糖(Inulin)与去离子水的质量比为6％～8％：1，优选为8％：1；所述的羧甲基壳聚糖和菊糖的总质量与去离子水的质量比为12％：1。
具体地，步骤(1)中，所述的搅拌溶解，搅拌溶解温度为室温，搅拌溶解时间为4h。
具体地，所述的聚合反应，反应温度为45℃，反应时间为1h。
具体地，步骤(2)中，所述的肠球菌素用量为羧甲基壳聚糖与菊糖总质量的1.0％～1.5％，优选为1.5％。
具体地，步骤(2)中，所述的制膜，制膜温度为45℃。
上述的制备工艺制备得到的水凝膜也在本发明的保护范围之内。
所述的水凝膜在食品保鲜中的应用也在本发明的保护范围之内。
所述的食品为畜类、蛋类、禽类、水产类、乳制品类、蔬菜类食品中的任意一种或几种的组合。
优选地，所述的食品为鱼。
有益效果：
本项发明以羧甲基壳聚糖/菊糖为载体，首先筛选两者最佳复配比例，并以最佳复配比例的水凝胶为载体负载不同浓度的肠球菌素，从制备稳定性、抗菌性能、保鲜效果的综合评价，得到了较好的羧甲基壳聚糖/菊糖/肠球菌素水凝胶膜，为食品工业提供新型、有效抗菌包装。
附图说明
下面结合附图和具体实施方式对本发明做更进一步的具体说明，本发明的上述和/或其他方面的优点将会变得更加清楚。
图1CMC与菊糖共聚后水凝胶溶液的流变及红外光谱；其中，图1a为水凝胶溶液流变学；图1b为对应水凝胶溶液；图1c为共聚后水凝胶溶液的红外光谱。
图2冷冻干燥后水凝胶结构；其中，图2a～图2e，分别为2％CMC+10％Inulin、4％CMC+8％Inulin、6％CMC+6％Inulin、8％CMC+4％Inulin、10％CMC+2％Inulin。
图3负载肠球菌素水凝胶膜结构；其中，图3a～图3e，分别为4％CMC+8％Inulin(Control)、4％CMC+8％Inulin+0.012、4％CMC+8％Inulin+0.024、4％CMC+8％Inulin+0.036、4％CMC+8％Inulin+0.048水凝胶膜。
图4负载肠球菌素水凝胶膜红外光谱。
图5负载肠球菌素水凝胶膜XRD、DSC、DTG及TG表征；其中图5a～图5d分别为XRD、DSC、DTG及TG表征。
图6负载肠球菌素水凝胶膜溶胀率及水接触角；其中图6a为水凝胶膜溶胀率，图6b～图6f分别为对应溶胀后水凝胶膜，图6g为各组水接触角。
图7负载肠球菌素水凝胶膜抑菌及对三文鱼保鲜效果；其中，图7a为LM菌显色培养结果，图7b为三文鱼中LM菌结果；图7c、图7d分别经肠球菌素处理前后共聚焦电镜图；图7e为各组水凝膜对LM抑菌效果；图7f为使用水凝胶膜对三文鱼进行包装保鲜；图7g为负载肠球菌素水凝胶膜包装后贮藏期LM菌数变化，图7h为水凝胶膜包装下贮藏期挥发性盐基氮(TVB-N)变化。
具体实施方式
本发明实施例使用的试验材料如下所示：
试验菌株：单核细胞增生李斯特菌(Listeria monocytogenes，L.monocytogenes,LM)为目标菌，由江苏省农业科学院加工所畜禽研究室从新鲜鸡肉中分离鉴定并提供。
主要化学试剂：李斯特菌显色培养基(BHI)，购于青岛海博生物技术有限公司；羧甲基壳聚糖(Carboxymethyl chitosan，CMC)及菊糖(Inulin)，购于上海源叶生化科技有限公司；肠球菌素A(Enterocin A)，购于国肽生物科技有限公司。
主要仪器设备：EXSTAR series TG/DTA7200热重分析仪，日本SII NanoTechnology Inc公司；Paar Physica MCR 301流变仪，奥地利安东帕公司；PE(Ultra ViewVOX)转盘式激光共聚焦显微镜，美国铂金埃尔默股份有限公司；EVO-LS10扫描电子显微镜，德国卡尔蔡司股份公司；Nicolet iS50傅里叶变换红外光谱仪，美国赛默飞世尔有限公司；差示扫描量热仪，美国TA有限公司；D2 PHASER X射线衍射仪，美国布鲁克公司。
实施例1
以20mL去离子水为溶剂，加入一定质量比的CMC和Inulin于室温下搅拌4h，待体系内固体部分完全溶解后置于45℃水浴锅中进行聚合反应，反应时间为1h。以水质量参照，以添加的CMC和Inulin质量比例不同，分别设为2％CMC+10％Inulin、4％CMC+8％Inulin、6％CMC+6％Inulin、8％CMC+4％Inulin、10％CMC+2％Inulin，形成不同质量配比的羧甲基壳聚糖/菊糖水凝胶。
经过结构表征，确认以4％CMC+8％Inulin为最佳的配比的水凝胶作为载体(具体数据见“实施例3”下“1水凝胶表征”)，随后在总溶液中添加0.012g、0.024g、0.036g、0.048g肠球菌素，并经45℃加热聚合1h后，将水凝胶置于平皿中，放置与45℃干燥箱中进行制膜，根据负载不同浓度的肠球菌素，将膜命名为4％CMC+8％Inulin+0.012、4％CMC+8％Inulin+0.024、4％CMC+8％Inulin+0.036、4％CMC+8％Inulin+0.048；同时以未负载肠球菌素为对照组。
实施例2：实验测定方法
1、水凝胶膜表征
水凝胶流变性能的表征：采用Paar Physica MCR 301应力控制流变仪对水凝胶溶液在静态剪切作用下的流变性能进行表征，平板直径为20mm，测试温度控制在(25±5)℃。
红外光谱测试：波长范围为中红外(4000～600cm-1)，以4cm-1的光谱分辨率对每个光谱累积扫描32次。
差示扫描量热：采用差示扫描量热仪(美国TA)测定不同样品的DSC参数，4mg样品放入有盖的铝样品架中在氮气气氛下以10℃/min的加热速率升温到250℃。
X射线衍射(XRD)：使用D2 PHASER X射线衍射分析仪对负载不同浓度紫苏醛电纺膜进行分析，其中电压为20kV，电流5mA，扫描速率4°/min，步长0.02°，扫描范围5°～40°。
扫描电子显微分析(SEM)：水凝胶膜前夜进行冻干后与水凝胶膜样品表面进行喷金(25mA，40s)处理，扫描时加速电压为10kV。
热重分析：在氮气氛围下(气体流量15mL/min)，从室温条件下以10℃/min的升温速率逐渐加热至600℃，利用EXSTAR series TG/DTA7200热重分析仪测定不同样品的热重变化参数。
静态水接触角测试：样品剪裁成2cm×2cm的尺寸大小并将其固定在载玻片上，调节液滴大小稳定5s后读数。
2、水凝胶膜抑菌及保鲜试验
共聚焦电镜观察抑菌：取对数期(37℃培养5～7h)菌液7mL以6000r/min离心10min，去上清用0.85％生理盐水洗涤2～3次，加入7mL灭菌的生理盐水，震荡混匀后分别吸取1mL加入7个灭菌的2mL离心管中，每个离心管与加入25mg的抑菌膜对应编号，室温下处理4h后将菌液吸取到10mL无菌离心管，多次吸取1mL无菌生理盐水进行润洗原管和电纺膜上附着的菌液，润洗后液体再次收集到上述10mL无菌离心管。操作完毕，配平离心，收集菌泥、加入1mL无菌生理盐水制取混匀液，同时移取到1.5mL离心管，再次润洗2～3次之后的菌泥加入1mL 2.5％戊二醛水溶液在4℃固定12h，接下来置于锇酸溶液中再次固定，使用乙醇梯度脱水后对样品镀金，处理好的菌液置于激光共聚焦扫描电镜显下观察抑菌情况。
抑菌圈测定：分别将LM在无菌条件下涂布于李斯特菌显色固体培养基平板表面，待涂布菌液稍干后，将不同浓度的0.8mm直径抑菌膜置于平板的对应位置，置于37℃培养24h后用游标卡尺测定抑菌圈直径。
LM菌总的活菌数量分析(Total viable bacteria，TVC)：在不同采样时间，将10g切碎的三文鱼样品与90mL灭菌生理盐水在无菌袋中混合。将上述混合溶液用磁力搅拌30min，用10倍稀释法对TVC进行分析。选择三种合适的十进制稀释剂，在李斯特菌显色培养基平板上计数。每组样本有3个重复。
挥发性盐基氮(Totalvolatilebasicnitrogen，TVB-N)：切碎的三文鱼样品(5g)加入含25mL蒸馏水的50mL离心管中，均质。匀浆静置30min后过滤，按照GB 5009.228-2016标准用微扩散法测定滤液中TVB-N含量。
实施例3：结果与分析
1、水凝胶表征
对实施例1制备得到的不同比例的羧甲基壳聚糖/菊糖水凝胶进行表征，以CMC与菊糖基质经共聚交联结合形成水凝胶溶液，如图1a所示，溶液粘度随CMC添加比例的增加而增加。2％CMC+10％Inulin水凝胶溶液平均粘度为19.78mPa·s，CMC添加比例增加至4％时，溶液粘度增加近6倍；当CMC所占比例超过6％时，溶液粘度急剧增加。如图1b所示，随着CMC浓度提高，溶液颜色逐渐加深，这对形成的水凝胶膜用于保鲜肉制品的色泽产生一定影响。因此，合适的浓度有助于降低水凝胶膜对肉制品色泽的二次影响。
经对各组聚合后水凝胶溶液进行红外表征(图1c)，4％CMC+8％Inulin水凝胶溶液在2800～3200、1000cm-1产生C-C、C-H、C＝O等共价键合作用。结合水凝胶溶液的粘度。因此，选择4％CMC+8％Inulin水凝胶溶液有助于形成较好的水凝膜，且粘度值有利于保持组织的完整性和对肉制品产生较低色泽影响。
2、未负载肠球菌素水凝胶膜液形态表征
对实施例1制备得到的不同比例的羧甲基壳聚糖/菊糖水凝胶进行表征，通过扫描电子显微镜(SEM)对水凝胶的形貌进行了进一步研究，如图2所示，所有水凝胶都呈现不规则多孔网络，赋予水凝胶足够的养分渗透性。其中，4％CMC+8％Inulin(图2b)和6％CMC+6％Inulin(图2c)凝胶网络中不规则孔隙均匀分布，冻干凝胶SEM图像中直径(5～25μm)小于直径(10～50μm)。此外，作为保鲜膜的水凝胶能够吸收过多的组织渗出液并保持微环境的湿润是至关重要的。
3、负载肠球菌素水凝胶膜形态表征
对实施例1制备得到的负载不同浓度的肠球菌素的水凝胶膜进行表征，如图3所示。随着肠球菌素负载量的增加，水凝胶膜表面逐渐呈现有凸起和和不规则的地方，说明4％CMC+8％Inulin水凝胶溶液对肠球菌素的负载量有一定承受限制(图3a)。纵观负载肠球菌素水凝胶膜表观，4％CMC+8％Inulin+0.024表面较平整(图3c)，4％CMC+8％Inulin+0.036表明稍有凸起(图3d)，而4％CMC+8％Inulin+0.048水凝胶膜表观完成呈现皱折、不规则(图3e)，表观形态对膜的应用具有重要的影响作用。
4、负载肠球菌素水凝胶膜红外表征
由图4可知，负载肠球菌素改变了CMC+Inulin水凝胶间键合作用。3250cm-1处C-C/C-H伸缩振动强度，4％CMC+8％Inulin+0.024组与Control组相近，4％CMC+8％Inulin+0.036次之；在1500-1700cm-1处，相比对照组，负载肠球菌素水凝胶膜产生新峰键合带，且此处4％CMC+8％Inulin+0.036组C-O、C-C或C＝O键伸缩振动最强，且4％CMC+8％Inulin+0.024对1000cm-1峰带强度减弱程度较小，说明4％CMC+8％Inulin+0.024、4％CMC+8％Inulin+0.036组中肠球菌素与CMC、Inulin产生较好的键合作用，肠球菌素较好地负载与水凝胶键合网络结构。
5、负载肠球菌素水凝胶膜热稳定性表征
图5a表明，负载肠球菌素减弱了水凝胶膜结晶度，4％CMC+8％Inulin+0.024、4％CMC+8％Inulin+0.036两组膜峰在22℃处强度相近，且高于4％CMC+8％Inulin+0.012、4％CMC+8％Inulin+0.048两组。图5b表明，添加适当浓度肠球菌素有助于提高膜溶解温度；同样，DTG溶变曲线也表明(图5c)，4％CMC+8％Inulin+0.012、4％CMC+8％Inulin+0.024、4％CMC+8％Inulin+0.036三组添加的肠球菌提高了膜的热稳定性；TG曲线表明(图5d)，肠球菌素的热稳定性好于水凝胶基质。
6、负载肠球菌素水凝胶膜溶胀率及接触角表征
如图6所示，水凝胶膜溶胀率随着负载肠球菌素浓度的不同呈现显著差异。图6a、图6b～6f结果表明，负载较低浓度肠球菌素的4％CMC+8％Inulin+0.012溶胀率最高，4％CMC+8％Inulin+0.024、4％CMC+8％Inulin+0.036两组溶胀率显著较低；图6g中膜的水接触角也表明4％CMC+8％Inulin+0.024、4％CMC+8％Inulin+0.036两组有较低亲水性。
7、水凝胶膜抑菌及保鲜试验
图7a为李斯特菌显色培养基培养菌后的显色状态，并测得三文鱼中存在单核增生李斯特。单核增生李斯特菌在环境中无处不在，在绝大多数食品中都能找到李斯特菌。肉类、蛋类、禽类、海产品、乳制品、蔬菜等都已被证实是李斯特菌的感染源。李斯特菌中毒严重的可引起血液和脑组织感染，很多国家都已经采取措施来控制食品中的李斯特菌，并制定了相应的标准。
如图7b～图7d所示，LM菌与0.024g/mL肠球菌素共培养后，李斯特菌全部被致死，说明肠球菌素对LM菌具有较好的抑制作用。如图7e所示，随着肠球菌素负载浓度的增加，水凝胶膜对LM抑菌直径呈现增加趋势，而4％CMC+8％Inulin+0.048组抑菌圈直径低于4％CMC+8％Inulin+0.036组，说明高浓度肠球菌素不能与CMC+Inulin水凝胶较好相容。因此，结合膜的表征及抑菌效果，本文选用4％CMC+8％Inulin+0.036水凝胶膜进行三文鱼保鲜试验，以未负载肠球菌素的Control组为对照，以浸泡于104LM菌液后的三文鱼为试验对象，贮藏期为8d(图7f)，通过分析贮藏过程中三文鱼中LM菌数及TVB-N变化。结果表明，起始两组膜对LM菌产生一定抑制作用，且负载肠球菌素后增强抑菌作用。相比贮藏过程，4％CMC+8％Inulin+0.036组LM数量始终低于对照组(图7g)。通过TVB-N结果也可知，4％CMC+8％Inulin+0.036组TVB-N值增加缓慢，说明4％CMC+8％Inulin+0.036水凝胶膜可有效抑制三文鱼中腐败微生物的繁殖(图7h)。因此，4％CMC+8％Inulin+0.036水凝胶膜是一种潜在的可应用于海鲜类保鲜的水凝胶保鲜膜。
本发明提供了一种保鲜水凝膜的制备工艺的思路及方法，具体实现该技术方案的方法和途径很多，以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。本实施例中未明确的各组成部分均可用现有技术加以实现。
Claims (9)
1.一种保鲜水凝膜的制备工艺，其特征在于，包括如下步骤：
（1）将羧甲基壳聚糖和菊糖溶于去离子水，搅拌溶解后进行聚合反应，得到羧甲基壳聚糖/菊糖水凝胶；
（2）将肠球菌素加入到步骤（1）得到的羧甲基壳聚糖/菊糖水凝胶中进行聚合反应，反应结束后制膜，即得；
所述的聚合反应，反应温度为45oC，反应时间为1 h。
2.根据权利要求1所述的水凝膜的制备工艺，其特征在于，步骤（1）中，所述的羧甲基壳聚糖与去离子水的质量比为4%~6%：1；所述的菊糖与去离子水的质量比为6%~8%：1；所述的羧甲基壳聚糖和菊糖的总质量与去离子水的质量比为12%：1。
3.根据权利要求1所述的水凝膜的制备工艺，其特征在于，步骤（1）中，所述的搅拌溶解，搅拌溶解温度为室温，搅拌溶解时间为4 h。
4.根据权利要求1所述的水凝膜的制备工艺，其特征在于，步骤（2）中，所述的肠球菌素用量为羧甲基壳聚糖与菊糖总质量的1.0%~1.5%。
5.根据权利要求1所述的水凝膜的制备工艺，其特征在于，步骤（2）中，所述的制膜，制膜温度为45oC。
6.权利要求1~5任意一项所述的制备工艺制备得到的水凝膜。
7.权利要求6所述的水凝膜在食品保鲜中的应用。
8.根据权利要求7所述的应用，其特征在于，所述的食品为畜类、蛋类、禽类、水产类、乳制品类、蔬菜类食品中的任意一种或几种的组合。
9.根据权利要求8所述的应用，其特征在于，所述的食品为鱼。
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