CN111303466A - 一种具有光敏抑菌功能的保鲜薄膜及其制备方法 - Google Patents
一种具有光敏抑菌功能的保鲜薄膜及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种具有光敏抑菌功能的保鲜薄膜及其制备方法,利用β‑环糊精的空腔结构及黄连素的光敏特性,以聚醋酸乙烯酯为基质,添加β‑环糊精来包合黄连素,通过可见光照射,诱导其释放单线态氧、活性自由基,迁移到肉制品表面上而发挥作用。本发明制备具有光敏抑菌效果的薄膜,以此改变黄连素分子振动自由度,调节其荧光效率,克服了黄连素单线态氧在溶液中寿命低的缺点,提高了黄连素单线态氧的产率与其抗菌性能,拓展其在食品活性包装中更广泛的应用。
Description
技术领域
本发明涉及保鲜薄膜材料技术领域,具体地说,涉及一种具有抑菌功能的保鲜薄膜及其制备方法。
背景技术
食品包装的主要目的是保障食品储运、销售等各个环节的卫生与安全,控制流通环节中微生物等造成的食品污染及由此引发的食源性疾病。食品的货架期与食品的特点及储运环境密切相关,最重要的是,其包装系统的质量。现阶段,冷鲜肉保鲜大多采用添加防腐剂或物理包装的方式,防腐剂安全性广受质疑;现有物理包装材料则存在不够环保、保鲜防护作用不够强等问题。
聚醋酸乙烯酯(PVAc)无色无味,透明无毒,具有热塑性、粘着力强、耐稀酸、稀碱等特性,主要用于制涂料漆和用作胶粘剂,还可用作口香糖中糖胶(chicle)的替代品。
环糊精(Cyclodextrin,简称CD)是直链淀粉在由芽孢杆菌产生的环糊精葡萄糖基转移酶作用下生成的一系列环状低聚糖的总称,通常含有6~12个D-吡喃葡萄糖单元。其中研究得较多并且具有重要实际意义的是含有6、7、8个葡萄糖单元的分子,分别称为α-、β-和γ-环糊精。环糊精能有效地增加一些水溶性不良的药物在水中的溶解度和溶解速度,能提高药物的稳定性和生物利用度,具有药物缓释和改善剂型等作用。β-环糊精(β-CD)的分子洞适中,应用范围广,生产成本低,无毒无害无副作用,且能被人体吸收等特性,是食品工业上使用最多的环糊精产品。天然β-环糊精通常包含在口服药物中,由于环糊精具有亲水的外缘及疏水内腔的截锥圆筒腔穴,能够包络各种客体分子,常被用来作为药物的缓释剂,使药物的有效成份包络在其腔穴中,让药物慢慢地释放出来,提高药效。这种包络作用还可以对一些药物起到稳定作用,延长药物有效期,其在医药业上的应用,具有广阔的诱人前景。
黄连素是一种天然的异喹啉生物碱,许多药用植物的根茎中黄连素的含量高达8~9%,黄连素在中医科学中是一种广谱抗菌药物,具有良好抗菌功能,虽然黄连素不是药食同源的物质,不能作为抗菌剂直接添加到食品中,水溶性低和苦味也阻止了它的利用度,限制了其在食品或农产品领域中的应用。但是黄连素同时还有聚集荧光增强的特性,该特性有可能赋予黄连素在固体状态下高的荧光效率,这有利于单线态到三线态的穿越,诱导高产率活性氧的产生,同时,单线态氧的寿命与其所处环境密切相关,在气体环境中,可达到1h以上,而在不同溶液中寿命则要短的多,只有2~1000微秒,因此,空气中长寿命的单线态氧的存在,恰恰为生鲜等肉类产品的包装提供了一个新的高效抗菌方式,特别适合应用于薄膜包装对食品进行光敏杀菌,除此之外,光动力杀菌还能够避免耐药菌株的产生。但是直到现在,黄连素的光催化产生活性氧的研究以及其应用在国际上研究并不多,在食品保鲜包装上的应用更是鲜有报道。
发明内容
为了解决现有技术中存在的问题,克服黄连素常温下溶解性差及单线态氧在溶液中寿命低的缺点,本发明的目的是提供一种具有光敏抑菌功能的食品保鲜薄膜及其制备方法,该薄膜仅少量添加抑菌剂黄连素,即可在光照条件下诱导黄连素释放单线态氧和活性自由基,提高抗菌性能。在保证食品安全的前提下,最大限度的保持冷鲜肉等生鲜食品原有的品质和商品价值。
为了实现本发明目的,本发明的技术方案如下:
第一方面,本发明提供一种具有光敏抑菌功能的保鲜薄膜的制备方法,所述方法包括如下步骤:
(1)利用β-环糊精(β-CD)对黄连素进行包合,得到β-环糊精/黄连素包合物(β-CD/黄连素包合物);
(2)以所述β-环糊精/黄连素包合物(β-CD/黄连素包合物)、柠檬酸酯和聚醋酸乙烯酯(PVAc)为原料,溶解于乙醇溶液中,得到薄膜涂层液;将所述薄膜涂层液均匀涂布于保鲜膜双侧表面,烘干即得。
进一步地,在上述步骤(1)中,所述β-环糊精与所述黄连素的摩尔用量比为1:1。该包合条件下,形成的包合物比游离的黄连素热稳定更高。同时由于包合作用,可有有效减少黄连素分子的非辐射能量损失,增加分子荧光与系间窜越ISC过程,从而提高单线态氧的产率。
更为具体地,步骤(1)包括以下步骤:
S1:将β-环糊精和黄连素溶于去离子水中,将二者按照1:1的摩尔用量比,在80℃、300r/min条件下包合,包合时间为1h;
S2:将S1所得样品置于旋转蒸发仪上,减压,于70~90℃完全蒸干,真空干燥48h,即得β-环糊精/黄连素包合物黄色粉末,避光保存,备用。
进一步地,在上述步骤(2)中,所使用的β-环糊精/黄连素包合物中黄连素的质量相对于所使用的聚醋酸乙烯酯的质量比为1~5%。
更为具体地,步骤(2)包括以下步骤:
s1:将步骤(1)制备的β-环糊精/黄连素包合物溶于10mL去离子水中,得到β-环糊精/黄连素包合物溶液;
s2:将4.5g PVAc、上述β-环糊精/黄连素包合物溶液和200μL柠檬酸酯溶解在75mL95%的乙醇溶液中,70℃、300r/min条件下搅拌直至充分混合,得到薄膜涂层液;
s3:将上述薄膜涂层液均匀涂布在保鲜薄膜双侧表面,涂布厚度为1mm;
s4:静置,待乙醇挥发完全后,重复s3操作,进行第二层涂布;
s5:室温条件下干燥1天,将干燥的薄膜在室温、真空度0.1atm的条件下置于真空干燥箱中烘干。
作为优选,所述保鲜薄膜为PVDC薄膜。
第二方面,本发明提供一种具有光敏抑菌功能的保鲜薄膜,所述保鲜薄膜为含有光敏抑菌涂层的薄膜,所述光敏抑菌涂层中含有由β-环糊精包合的黄连素。
进一步地,所述具有光敏抑菌功能的保鲜薄膜由本发明前述方法制备而成。
本发明涉及到的原料或试剂均为普通市售产品,涉及到的操作如无特殊说明均为本领域常规操作。
在符合本领域常识的基础上,上述各优选条件,可以相互组合,得到具体实施方式。
本发明的有益效果在于:
本发明应用环糊精分子包合技术制备黄连素/β-环糊精包合物,改善黄连素在常用溶剂中的分散性能,通过紫外吸收/荧光发射确定了黄连素/β-环糊精的最佳包合摩尔比,成功将其掺入到聚醋酸乙烯酯的薄膜基质中实现抑菌保鲜,解决了黄连素作为抑菌因子应用于食品包装材料的增溶与稳定分散的技术难点。
本发明通过在膜基质中添加低浓度的黄连素,并避免了抑菌剂常见的直接添加方式,有效防止食品污染。所制备的保鲜薄膜可通过光动力杀菌的方式提升黄连素的杀菌效率,使膜中低浓度的黄连素产生高效的杀菌效果。
本发明通过对大肠杆菌和金黄色葡萄球菌的抑菌实验,证明黄连素/β-环糊精包合物制备成的薄膜抗菌效果优于单纯黄连素薄膜的抑菌效果,并且随着浓度和光照时间的增加,抑菌效果显著增强,尤其对金黄色葡萄球菌的抑菌效果更佳。通过该保鲜薄膜对冷鲜猪肉低温贮藏期间肉品品质的影响研究发现,添加不同浓度黄连素的薄膜和添加黄连素包合物的薄膜冷鲜肉品质差异显著,同时光照时间延长可以显著提高保鲜效果。
总的来说,本发明利用β-环糊精的空腔结构及黄连素的光敏特性性,以聚醋酸乙烯酯为基质,添加β-环糊精来包合黄连素,通过自然光照射,诱导其释放单线态氧、活性自由基,迁移到肉制品上而发挥作用,制备具有光敏抑菌效果的薄膜,以此改变黄连素在水溶液中的分子振动自由度,调节其荧光效率,克服了黄连素常温下溶解性差及单线态氧在溶液中寿命低的缺点,提高了黄连素单线态氧的产率与其抗菌性能,拓展其在食品活性包装中更广泛的应用。
附图说明
图1为本发明实验例1中1×10-5mol/L黄连素水溶液(a)与亚甲基蓝(b)在可见光照射下吸收光谱随时间的变化;
图2为本发明实验例1中1×10-5mol/L黄连素水溶液室温条件不同时间的吸收光谱;
图3为本发明实验例1中不同照射时间下各物质单线态氧的释放情况;
图4为本发明实验例1中黄连素与β-环糊精不同摩尔比下的吸收和发射光谱图;
图5为本发明实验例1中黄连素薄膜和黄连素/β-环糊精包合物薄膜对大肠杆菌的抑制率;
图6为本发明实验例1中黄连素薄膜和黄连素/β-环糊精包合物薄膜对金黄色葡萄球菌的抑制率;
图7为本发明实验例1中不同黄连素/β-环糊精包合物添加量的薄膜光照与暗光条件下样品菌落总数的变化;
图8为本发明实验例1中不同黄连素/β-环糊精包合物添加量的薄膜光照与暗光条件下样品TVB-N值的变化;
图9为本发明实验例1中不同黄连素/β-环糊精包合物添加量的薄膜光照与暗光条件下样品pH值的变化。
具体实施方式
下面将结合实施例对本发明的优选实施方式进行详细说明。需要理解的是以下实施例的给出仅是为了起到说明的目的,并不是用于对本发明的范围进行限制。本领域的技术人员在不背离本发明的宗旨和精神的情况下,可以对本发明进行各种修改和替换。
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1
本实施例用于说明本发明所述具有光敏抑菌功能的保鲜薄膜的制备方法。
具体步骤如下:
一、制备β-CD/黄连素包合物
S1:将0.045g黄连素和0.15gβ-环糊精溶于去离子水中(二者摩尔用量比为1:1),在80℃、300r/min条件下包合,包合时间为1h;
S2:将S1所得样品置于旋转蒸发仪上,减压,于70~90℃完全蒸干,真空干燥48h,即得β-环糊精/黄连素包合物黄色粉末,避光保存,备用。
二、制备具有光敏抑菌功能的保鲜薄膜
s1:将步骤(1)制备的β-环糊精/黄连素包合物溶于10mL去离子水中,得到β-环糊精/黄连素包合物溶液;
s2:将4.5g PVAc、上述β-环糊精/黄连素包合物溶液和200μL柠檬酸酯溶解在75mL95%的乙醇溶液中,70℃、300r/min条件下搅拌直至充分混合,得到薄膜涂层液,且黄连素相对于PVAc的用量为1%(w/w);
s3:裁取略短于涂布机面板的PVDC保鲜膜置于涂布机上,将涂布机速度设定为20,校准刮刀高度为0,然后设定高度为1mm,将s2中所述薄膜涂层液倒入PVDC保鲜膜的一侧,当刮刀与溶液接触后开始涂布;
s4:静置3-4h后待乙醇挥发完全后重新以新膜的厚度校准为零后重复上s3步骤进行第二层涂抹;
s5:25℃条件下干燥1天,将干燥的薄膜在室温和真空度0.1atm的条件下置于真空干燥箱中24h烘干,即得具有光敏抑菌功能的保鲜薄膜,该薄膜用代号Bbr1/β-CD1示。
实施例2
本实施例与实施例1的区别在于:将0.045g黄连素的替换为0.225g,将0.15gβ-环糊精替换为0.76g。
所得薄膜涂层液中,黄连素相对于PVAc的用量为5%(w/w),制得的薄膜用代号Bbr5/β-CD5表示。
对比例1
本对比例与实施例1的区别在于:在制备光敏抑菌薄膜的步骤中,用0.045g的黄连素替代β-环糊精/黄连素包合物,制备薄膜涂层液,所得薄膜用代号Bbr1/β-CD0表示。
对比例2
本对比例与实施例2的区别在于:在制备光敏抑菌薄膜的步骤中,用0.225g的黄连素替代β-环糊精/黄连素包合物,制备薄膜涂层液,所得薄膜用代号Bbr5/β-CD0表示。
对比例3
本对比例与实施例1的区别在于:在薄膜制备的步骤中,黄连素、β-环糊精的添加量均为0%,制备对照组薄膜涂层液,该薄膜用代号Bbr0/β-CD0表示。
实验例1
本实验例用于验证实施例1~2和对比例1~3的抑菌效果。为直观比较起见,将实施例1~2和对比例1~3的涂层组分通过表1体现:
表1.各个涂层的加料比例表
1、黄连素稳定性检测
配置1×10-5mol/L的黄连素溶液,置于强度为60mW/cm2的可见光灯下辐照0min、30min、60min、90min后,分析不同光照时间下黄连素作为光敏剂的稳定性,以同浓度的亚甲基蓝做对照。
在市售的光敏剂中,亚甲基蓝(MB)被认为是非常有效的光敏抗菌治疗药物。MB的最大吸附峰在650nm,甚至当它暴露在弱光下也能表现出相当优异的抗菌活性,对几种微生物包括革兰氏阳性菌和革兰氏阴性菌都有非常好的抗菌效果。相比于MB,黄连素的生理毒性更小,图1分析可知,黄连素在扫描范围波长内有两个非常明显的吸收峰,分别为348nm和420nm,且随着照射时间的延长,黄连素吸收峰并没有明显变化。
图2则显示,放置6d后的黄连素,其吸收波峰也没有明显的幅动,而图1中亚甲基蓝随着照射时间的延长,吸收峰呈有规律的下降趋势。可见不同照射时间下,黄连素在水溶液中的光稳定性明显优于亚甲基蓝。
2、黄连素光敏化释放的单线态氧检测
新型单线态氧荧光探针(Singlet Oxygen Sensor Green reagent,SOSG)是一种高强灵敏度、选择性好的单线态氧荧光小分子探针,不会同羟基自由基、过氧化氢和超氧阴离子等活性氧发生反应。SOSG用作化学发光探针,可用于评估黄连素光敏活性。
亚甲基蓝是目前商业品中能非常高效产生单线态氧和自由基的化学物质,本发明中以其作为参照物,评价黄连素单线态氧产率。图3可知,SOSG在可见光光照下,光谱变化不大,可见光不能有效改变SOSG的分子结构从而改变其分子荧光。但是黄连素,黄连素/β-环糊精包合物,以及亚甲基蓝在可见光光照下都明显产生了光生-单线态氧,而且黄连素/β-环糊精包合物单线态氧的产率高于黄连素,这可能是由于环糊精限制了黄连素的分子振动,降低了非辐射的能量损耗,增强分子荧光与ISC过程,从而增加了黄连素向三线态氧的能量转移所导致。
3、黄连素/β-环糊精包合物摩尔比的确定
配置黄连素/β-环糊精摩尔配比为10:0,9:1,8:2,7:3,6:4,5:5,4:6,3:7,2:8,1:9,0:10的水溶液,置于80℃恒温水浴锅中,300r/min搅拌至完全溶解。25℃条件下,使用420nm的激发波长记录荧光发射光谱以检测包合物的最佳配比。
图4为5×10-5mol/L黄连素水溶液与β-环糊精相互作用而产生的紫外吸收和荧光发射的光谱变化。在发射谱图中,谱线随着摩尔比的减小而发生蓝移现象,这主要是由于黄连素穿进β-环糊精中,降低了共轭体系之间相互作用所导致。在发射谱图中,在555nm处出现峰值,相较Blank样品发射峰蓝移了5nm。摩尔比大于5:5时,谱线发生了一定程度的蓝移,但小于5:5时,无论吸收谱图还是发射谱图都没有再产生蓝移的现象,证明了黄连素与β-环糊精之间的穿合定量关系是1:1。
4、薄膜的抑菌实验
首先将大肠杆菌和金黄色葡萄球菌进行活化,于37℃培养24h。取所述的实施例1~2及对比例1~3所制备的薄膜,裁剪为2cm2的正方形,放置在24孔板中,将活化好的大肠杆菌和金黄色葡萄球菌取100μL菌悬液涂到薄膜样品上,黑暗或以60mW/cm2强度的可见光辐照20min、光照40min后,每个样品吸取梯度稀释后的100μL在琼脂平板上涂布,37℃孵育12h,进行菌落计数,每组2个平行。
通过图5和图6可以看出,对比例3的薄膜对大肠杆菌和金黄色葡萄球菌几乎无抑制作用。实施例1~2添加了黄连素和黄连素/β-环糊精包合物的薄膜对大肠杆菌和金黄色葡萄球菌都起到了较强的抑制效果,并且随着黄连素和黄连素/β-环糊精包合物的浓度增大和照射时间的增加,抑制效果都得到了增强。并且,实施例1与实施例2中添加黄连素/β-环糊精包合物制备的薄膜较对比例1和对比例2单纯添加黄连素制备的薄膜,对大肠杆菌和金黄色葡萄球菌的抑菌性能更佳。
5、冷鲜肉的储藏保鲜实验
超市购买新鲜猪肉,于无菌环境中处理成25g的小块,样品以实施例1与实施例2及对比例3所述薄膜单独密封包装,将包装好的样品于白光下照射20min(每10min翻一次面),对照组样品不进行可见光辐照处理。处理后的样品有序放置在冰箱中并于4℃下储存。隔天随机抽取样品,25℃环境条件下适应30min后检测样品的菌落总数、pH值和挥发性盐基氮。
(1)菌落总数(APC)
菌落总数(APC)参照GB 4789.2—2016的方法进行测定。
(2)pH值
通过直接插入样品中测量猪肉样品的内部pH,用pH计在不同的位置测5次。使用前,在室温下使用缓冲溶液对pH计进行校准。
(3)挥发性盐基氮(TVB-N)
挥发性碱性氮(TVB-N)含量是评估肉类新鲜度的一种理化指标。TVB-N测定参照GB5009.44-2016半微量定氮法测定。
冷鲜猪肉的菌落总数、挥发性碱基氮、pH值是衡量其新鲜程度的重要指标。图7~9数据显示,随着储存时间的延长,菌落总数、挥发性碱基氮、pH值均呈现增长趋势。实施例2的薄膜包装的样品在光照的协同作用下,各指标增长最为缓慢,样品品质最佳,实施例1中包合物浓度降低协同光照的效果仍然显著优于对比例2高浓度黄连素薄膜的保鲜效果,可见黄连素经β-环糊精包合后可以更好地发挥其光敏抗菌的功效,保鲜效果与包合物浓度和光照呈正相关。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
Claims (9)
1.一种具有光敏抑菌功能的保鲜薄膜的制备方法,其特征在于,所述方法包括如下步骤:
(1)利用β-环糊精对黄连素进行包合,得到β-环糊精/黄连素包合物;
(2)以所述β-环糊精/黄连素包合物、柠檬酸酯和聚醋酸乙烯酯为原料,溶解于乙醇溶液中,得到薄膜涂层液;将所述薄膜涂层液均匀涂布于保鲜膜双侧表面,烘干,即得具有光敏抑菌功能的保鲜薄膜。
2.根据权利要求1所述的制备方法,其特征在于,步骤(1)中,所述β-环糊精与所述黄连素的摩尔用量比为1:1。
3.根据权利要求2所述的制备方法,其特征在于,步骤(1)包括以下步骤:
S1:将β-环糊精和黄连素溶于去离子水中,在80℃、300r/min条件下包合,包合时间为1h;
S2:将S1所得样品置于旋转蒸发仪上,减压,于70~90℃完全蒸干,真空干燥48h,得到β-环糊精/黄连素包合物黄色粉末。
4.根据权利要求1~3任一项所述的制备方法,其特征在于,步骤(2)中,所使用的β-环糊精/黄连素包合物中黄连素的质量相对于所使用的聚醋酸乙烯酯的质量比为1~5%。
5.根据权利要求4所述的制备方法,其特征在于,步骤(2)包括以下步骤:
s1:将步骤(1)制备的β-环糊精/黄连素包合物溶于10mL去离子水中,得到β-环糊精/黄连素包合物溶液;
s2:将4.5g PVAc、上述β-环糊精/黄连素包合物溶液和200μL柠檬酸酯溶解在75mL95%的乙醇溶液中,70℃、300r/min条件下搅拌直至充分混合,得到薄膜涂层液;
s3:将上述薄膜涂层液均匀涂布在保鲜薄膜双侧表面,涂布厚度为1mm;
s4:静置,待乙醇挥发完全后,重复s3操作,进行第二层涂布;
s5:室温条件下干燥1天,将干燥的薄膜在室温、真空度0.1atm的条件下置于真空干燥箱中烘干。
6.根据权利要求5所述的制备方法,其特征在于,s3中,所述保鲜薄膜为PVDC薄膜。
7.一种具有光敏抑菌功能的保鲜薄膜,其特征在于,所述保鲜薄膜为含有光敏抑菌涂层的保鲜薄膜。
8.根据权利要求7所述的具有光敏抑菌功能的保鲜薄膜,其特征在于,所述光敏抑菌涂层中含有由β-环糊精包合的黄连素。
9.根据权利要求7或8所述的具有光敏抑菌功能的保鲜薄膜,其特征在于,由权利要求1~6任一项所述的制备方法制备而成。
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