CN111171348B - 一种抑制南美白对虾腐败菌的含虾青素生物抗菌复合膜的制备方法 - Google Patents
一种抑制南美白对虾腐败菌的含虾青素生物抗菌复合膜的制备方法 Download PDFInfo
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Abstract
本发明涉及食品包装材料领域,公开了一种抑制南美白对虾腐败菌的含虾青素生物抗菌复合膜的制备方法,本发明以明胶和壳聚糖作为复合膜基质,在此基础上加入具有抗菌作用的ε‑聚赖氨酸和从南美白对虾壳中提取的具有高抗氧化性的虾青素,制备出生物抗菌复合膜。该抗菌膜能有效地抑制南美白对虾腐败菌的增殖,且本发明还偶然发现了添加虾青素不仅能够为复合膜带来抗氧化性,并且在特定含量下还可显著改善壳聚糖‑明胶基膜的物理性能。此外,该复合膜的制备方法简便、易操作、可用于扩大化生产。
Description
技术领域
本发明涉及食品包装材料领域,尤其涉及一种抑制南美白对虾腐败菌的含虾青素生物抗菌复合膜的制备方法。
背景技术
食品腐败通常是由微生物污染引起的,尤其是对于含水量高、蛋白质含量高的食品来说,如果保鲜措施不够理想,极易发生腐败。为了消除腐败菌,延长食品的保质期,在食品贮藏过程中人们已经开发了多种保鲜方法。其中,在薄膜聚合物基质中加入活性剂作为包装材料被认为是一种有效的食品保鲜方法。
明胶作为一种天然蛋白质,由胶原蛋白部分水解而成,具有食用性和生物可降解性,在高分子薄膜中得到广泛应用。壳聚糖是一种天然的多糖,由几丁质脱乙酰而得,具有良好的抗菌性和成膜能力,也是一种广泛应用于食品包装的生物材料。然而,由于明胶膜和壳聚糖膜自身的一些缺陷,如:纯明胶膜亲水性很强且抗氧化性能低,纯壳聚糖膜柔韧性和伸展性差等,限制了明胶膜和壳聚糖膜在抗菌包装中广泛应用。
与化学或酶改性相比,聚合物共混可以提高纯组分的物理性能,并且由于其无毒和低成本的优点,通常被认为是一种有效的策略。据文献报道,壳聚糖与明胶的结合已显示出在获得具有新特性的生物复合材料方面的优势,例如用于组织工程的新型双层壳聚糖明胶支架,具有抗氧化和抗菌性能的明胶-壳聚糖共混可食性膜,改善了机械和物理性能。
此外,近年来,将天然生物活性物质掺入可生物降解的包装材料中引起了人们的极大关注,因为天然添加剂的加入可以减少食品中合成添加剂的加入量,从而降低潜在的健康风险。ε-聚赖氨酸是一种天然的阳离子多肽,由25-35个均匀的L-赖氨酸组成,具有广谱抗菌性和生物降解性,被食品和药物管理局认定为安全的(GRAS),可用于煮饭或寿司饭。虾青素是一种天然抗氧化剂,具有超单重氧猝灭活性。然而,目前还未有研究报道虾青素作在壳聚糖明胶基膜中的应用。
因此,有必要在上述基础上不断地进行更为深入的研究,以期获得一种兼具高抗菌性、高抗氧化性及高物理性能的复合膜材料。
发明内容
为了解决上述技术问题,本发明提供了一种抑制南美白对虾腐败菌的含虾青素生物抗菌复合膜的制备方法。本发明以明胶和壳聚糖作为复合膜基质,在此基础上加入具有抗菌作用的ε-聚赖氨酸和从南美白对虾壳中提取的具有高抗氧化性的虾青素,制备出生物抗菌复合膜。该抗菌膜能有效地抑制南美白对虾腐败菌的增殖,且本发明还偶然发现了添加虾青素不仅能够为复合膜带来抗氧化性,并且在特定含量下还可显著改善壳聚糖-明胶基膜的物理性能。此外,该复合膜的制备方法简便、易操作、可用于扩大化生产。
本发明的具体技术方案为:一种抑制南美白对虾腐败菌的含虾青素生物抗菌复合膜的制备方法,包括以下步骤:
1)向明胶中添加水,加热充分溶解,制得8-12(w/v)%明胶水溶液。
2)向明胶水溶液中添加明胶体积2-4(w/v)%的甘油,充分搅拌均匀。
3)然后添加ε-聚赖氨酸溶液和从南美白对虾壳中提取的虾青素,使ε-聚赖氨酸和虾青素在所得溶液中的浓度分别不低于0.13g/L和15μg/mL。
4)将步骤3)所得溶液均质处理,调节pH值为3.5-4.5。
5)向步骤4)所得溶液中添加明胶质量4-8%的壳聚糖粉末,避光下搅拌,至壳聚糖完全溶解,得到生物抗菌复合膜液。
6)然后,将生物抗菌复合膜液平铺到板上,烘干,得到干燥的抑制南美白对虾腐败菌的生物抗菌复合膜。
本发明以明胶和壳聚糖作为复合膜基质,在此基础上加入具有抗菌作用的ε-聚赖氨酸和从南美白对虾壳中提取的具有高抗氧化性的虾青素,制备出生物抗菌复合膜。ε-聚赖氨酸与蛋白质之间可形成静电复合物,同时聚赖氨酸自身又具有出色的抗菌性,并且ε-聚赖氨酸与壳聚糖相互配合后,可显著提高壳聚糖的抗菌活性。而虾青素是一种天然抗氧化剂,因此,本发明复合膜能充分抑制南美白对虾腐败菌生长和体外自由基氧化,可有效地抑制南美白对虾腐败菌的增殖。
此外,壳聚糖的作用除了带来抗菌性外,其作用还在于可提高明胶膜的稳定性(明胶膜在37℃下发生溶胀,不利于保持膜稳定)。甘油的作用添加至膜基质中可以提高膜的柔韧性。
作为优选,步骤1)中,加热温度为40-50℃。
作为优选,步骤3)中,所述ε-聚赖氨酸的浓度不高于0.3g/L。
作为优选,步骤3)中,所述ε-聚赖氨酸的浓度为0.25-0.3g/L。
作为优选,步骤3)中所述虾青素的浓度低于25μg/mL。
作为优选,步骤3)中所述虾青素的浓度为15-20μg/mL。
本发明团队在研究过程中还偶然发现了添加虾青素不仅能够为复合膜带来抗氧化性,并且在特定含量下还可显著改善壳聚糖-明胶基膜的物理性能。一方面,本发明团队发现,虾青素在明胶膜中的添加能够显著降低复合膜的水溶解性。更为重要的另一方面是,本发明团队发现,当将虾青素在明胶-壳聚糖基复合膜中的添加量控制在上述特定范围时,能够显著改善复合膜的水蒸气透过率(表明水蒸汽更容易透过复合膜)。本发明团队进一步通过断面电镜照片中发现,普通的明胶膜断面有规则裂痕,但是密度不高;未添加ε-聚赖氨酸和虾青素的壳聚糖/明胶膜的断面能够观察到有不规则裂痕贯穿整个膜。而本发明含有ε-聚赖氨酸和虾青素的复合膜的断面有形状较为规则的裂痕,并且当虾青素含量控制在特定范围时裂痕密度可远远高于普通明胶,此外可从复合膜的表面原子力显微镜照片中观察到丰富密集的微孔。分析其原因为:①断面规则、密集的裂痕缩短了水分子扩散距离;②膜表面的多孔结构增加了复合膜的比表面积,有利于水分子的通过。
如果虾青素的添加量过少,则效果提升不明显,而如果添加过量,例如当达到25μg/mL时,不仅会大幅提高成本,同时又反而会降低抗氧化效果(清除DPPH自由基),对DPPH自由基清除率显著降低,分析其原因可能是一方面虾青素浓度过大会时聚集,导致自由基清除效果降低,另一方面高浓度的虾青素还会与ε-聚赖氨酸相互作用增强而发生聚集。
作为优选,步骤4)中,均质条件为:10000-15000r/min,5-15s。
作为优选,步骤5)中,所述壳聚糖粉末的脱乙酰度为80.0-95.0%。
作为优选,步骤6)中,烘干温度为40-50℃。
作为优选,所述生物抗菌复合膜的膜厚为0.4-0.6μm。
与现有技术对比,本发明的有益效果是:
(1)本发明以明胶和壳聚糖作为复合膜基质,在此基础上加入具有抗菌作用的ε-聚赖氨酸和从南美白对虾壳中提取的具有高抗氧化性的虾青素,制备出生物抗菌复合膜。该抗菌膜能有效地抑制南美白对虾腐败菌的增殖,且本发明还偶然发现了添加虾青素不仅能够为复合膜带来抗氧化性,并且在特定含量下还可显著改善壳聚糖-明胶基膜的物理性能(尤其是水蒸气透过率)。
(2)本发明复合膜的制备方法简便、易操作、可用于扩大化生产。
附图说明
图1实施例1中不同浓度ε-聚赖氨酸对南美白对虾腐败菌的抑菌效果图;
图2为实施例1中不同浓度虾青素对DPPH自由基清除效果图;
图3为含虾青素的生物抗菌膜对南美白对虾腐败菌的抑菌效果图;
图4为单纯明胶膜、壳聚糖/明胶膜和复合膜的微观形态观察图;注:(a)-(c)表示膜照片;(d)-(f)扫描电镜观察膜表面;(g)-(i)扫描电镜观察膜断面;(j)-(1)原子力显微镜观察膜表面;
图5为两种复合膜和明胶膜、壳聚糖/明胶膜水蒸汽透过率比较图;
图6为复合膜和明胶膜、壳聚糖/明胶膜对玉米油高温氧化延缓作用对比图。
具体实施方式
下面结合实施例对本发明作进一步的描述。
总实施例
一种抑制南美白对虾腐败菌的含虾青素生物抗菌复合膜的制备方法,包括以下步骤:
1)向明胶中添加水,40-50℃加热充分溶解,制得8-12(w/v)%明胶水溶液。
2)向明胶水溶液中添加明胶体积2-4(w/v)%的甘油,充分搅拌均匀。
3)然后添加ε-聚赖氨酸溶液和从南美白对虾壳中提取的虾青素,使ε-聚赖氨酸和虾青素在所得溶液中的浓度分别不低于0.13g/L和15μg/mL。
4)将步骤3)所得溶液10000-15000r/min均质处理5-15s,调节pH值为3.5-4.5。
5)向步骤4)所得溶液中添加明胶质量4-8%的壳聚糖粉末(脱乙酰度为80.0-95.0%),避光下搅拌,至壳聚糖完全溶解,得到生物抗菌复合膜液。
6)然后,将生物抗菌复合膜液平铺到板上,40-50℃烘干,得到干燥的抑制南美白对虾腐败菌的生物抗菌复合膜(膜厚为0.4-0.6μm)。
作为优选,步骤3)中,所述ε-聚赖氨酸的浓度不高于0.3g/L。所述虾青素的浓度低于25μg/mL,进一步优选,ε-聚赖氨酸的浓度为0.25-0.3g/L。虾青素的浓度为15-20μg/mL。
实施例1
1、ε-聚赖氨酸对南美白对虾腐败菌(混合菌液)的抑菌效果以南美白对虾腐败菌(混合菌)为指示菌,测定了不同浓度ε-聚赖氨酸(终浓度为2.0、1.0、0.5、0.25、0.13g/L)对南美白对虾腐败菌的抑菌效果,结果见图1。图1结果显示ε-聚赖氨酸对南美白对虾腐败菌的抑菌率随着ε-聚赖氨酸浓度的增加并未显著性增大(p>0.05),说明低浓度的ε-聚赖氨酸对南美白对虾腐败菌仍具有非常高的抑菌效果。
2、来源于南美白对虾的虾青素的体外抗氧化性
采用无水乙醇溶剂提取法,从南美白对虾的虾壳中提取出虾青素,用于含虾青素的生物抗菌膜液制备。鉴于虾青素为脂溶性,所以选取脂溶性的DPPH模型来研究提取的虾青素的体外清除DPPH自由基效果。
将提取的虾青素用20%乙醇稀释,分别得到浓度为5、7、9、11、13、15、20和25μg/mL的虾青素溶液,上述虾青素溶液中分别加入ε-聚赖氨酸(选择终浓度为0.25g/L),用于研究不同浓度虾青素和ε-聚赖氨酸复合后的抗氧化性,结果见图2。从图2可知:虾青素浓度为5~9μg/mL时,DPPH自由基的清除在70%左右。当虾青素浓度为15μg/mL时,对DPPH自由基的清除率可达到90%以上。继续提升虾青素浓度至20μg/mL,发现对DPPH自由基清除效果提升不够明显,而当虾青素浓度提升为为25μg/mL时,发现对DPPH自由基清除率降到55%左右,说明虾青素浓度过大不利于进一步提高DPPH自由基清除效果,推测与高浓度虾青素易在ε-聚赖氨酸中发生聚集有关。因此,确定生物抗菌膜液中虾青素的浓度为15-20μg/mL比较适宜。
3、含虾青素的生物抗菌膜对南美白对虾腐败菌的抑菌效果
将南美白对虾腐败菌用肉汤培养基培养至对数期,取50μL涂布到无菌营养琼脂上,然后分别将单独明胶膜、添加壳聚糖的明胶膜(记作:壳聚糖/明胶膜)和添加ε-聚赖氨酸、虾青素、壳聚糖的明胶膜(记作:复合膜,虾青素15μg/mL,ε-聚赖氨酸0.25g/L)剪成1.7cm大小膜片,贴在已涂菌的营养琼脂上,在37℃恒温培养箱中培养24h,观查是否有抑菌圈形成。此外,还测定了不同膜对南美白对虾腐败菌的抑菌率。结果见图3。图3结果显示:未调节pH条件下制备的明胶膜(pH 6.0)对南美白对虾腐败菌的抑菌率低于10%,而用5%柠檬酸调节明胶液pH至4.0(用于溶解壳聚糖)后,明胶膜本身具有较强的抑菌效果,与柠檬酸自身抑菌作用有关。明胶膜在37℃下发生溶胀(表现为抑菌圈向外扩大),说明单独的明胶膜在37℃会发生溶解,不利于保持膜稳定。在明胶中添加壳聚糖可以提高膜的稳定性(表现为膜未发生溶胀),在壳聚糖和明胶基质中加入ε-聚赖氨酸和虾青素制备的复合膜对实验腐败菌的抑菌率可达到100%,且未发生明显溶胀现象。
4、含虾青素的生物抗菌膜的微观形态
采用扫描电镜、原子力显微镜分别观察单纯明胶膜、壳聚糖/明胶膜和复合膜的表面、断面微观形态,结果见图4。从图4可以看出复合膜为淡红色,与明胶膜比较,复合膜的表面较为平整,断面有形状较为规则裂痕。未添加ε-聚赖氨酸和虾青素的壳聚糖/明胶膜表面紧密,光滑,断面能够观察到有不规则裂痕贯穿整个膜。
5、含虾青素的生物抗菌膜的理化性质分析
分别测定明胶膜、壳聚糖/明胶膜和复合膜的含水量、水溶解性、膜厚度和水蒸汽透过率,结果见表1和图5。
表1明胶膜、壳聚糖/明胶膜和复合膜的性能比较
注:表中数据用平均值±标准差表示(n=3),同行小写字母不同表示差异显著(p<0.05)。
表1结果表明:与明胶膜相比较,添加ε-聚赖氨酸和虾青素的复合膜的溶解性降低,说明疏水性增强。从图5中可知,复合膜(虾青素15μg/mL,ε-聚赖氨酸0.25g/L)的水蒸汽透过率显著高于明胶膜,而当虾青素浓度为20μg/mL时,水蒸汽透过率进一步显著高于壳聚糖/明胶膜。而当虾青素浓度高于25μg/mL时,不仅对DPPH自由基清除率急剧降低(见图2结果),而且制成膜的表面连续性也急剧降低,无实用价值,因此未对其进行进一步的含水量、溶解性和膜厚度等检测。
综上所述,复合膜中虾青素适宜添加浓度范围为15-20μg/mL,并且如果要使复合膜的水蒸汽透过率达到最佳,浓度范围为20μg/mL左右
6、含虾青素的生物抗菌膜的抗氧化效果
采用高温加速油脂氧化法,测定玉米油添加复合膜(虾青素15μg/mL,ε-聚赖氨酸0.25g/L)后在60℃下的油脂氧化程度(用丙二醛生成量计),结果见图6。图6结果表明:玉米油在无任何添加膜的情况下,氧化速度随着保温时间的延长而急剧地增加。与对照相比较,玉米油中添加明胶膜和壳聚糖/明胶膜可一定程度上减缓玉米油在高温下的氧化程度。但是,与复合膜相比较,当保温时间为59h时,复合膜处理组的丙二醛含量低于5nmol/mL,为所有处理组最低,说明含虾青素的生物抗菌膜能有效地延缓油脂氧化进程。
综上,本发明制备的含虾青素的生物抗菌液及抗菌膜不仅能有效地抑制南美白对虾腐败菌的生长繁殖,而且具有很好的抗氧化性和膜性能。
需要说明的是,上述实施例中复合膜的制备方法为:
1)向明胶中添加水,45℃加热充分溶解,制得10(w/v)%明胶水溶液。
2)向明胶水溶液中添加明胶体积3(w/v)%的甘油,充分搅拌均匀。
3)然后添加ε-聚赖氨酸溶液和从南美白对虾壳中提取的虾青素。
4)将步骤3)所得溶液12000r/min均质处理10s,调节pH值为4。
5)向步骤4)所得溶液中添加明胶质量6%的壳聚糖粉末(脱乙酰度为80.0-95.0%),避光下搅拌,至壳聚糖完全溶解,得到生物抗菌复合膜液。
6)然后,将生物抗菌复合膜液平铺到板上,45℃烘干,得到干燥的抑制南美白对虾腐败菌的生物抗菌复合膜。
本发明中所用原料、设备,若无特别说明,均为本领域的常用原料、设备;本发明中所用方法,若无特别说明,均为本领域的常规方法。
以上所述,仅是本发明的较佳实施例,并非对本发明作任何限制,凡是根据本发明技术实质对以上实施例所作的任何简单修改、变更以及等效变换,均仍属于本发明技术方案的保护范围。
Claims (8)
1.一种抑制南美白对虾腐败菌的含虾青素生物抗菌复合膜的制备方法,其特征在于包括以下步骤:
1)向明胶中添加水,加热充分溶解,制得8-12(w/v)%明胶水溶液;
2)向明胶水溶液中添加明胶体积2-4(w/v)%的甘油,充分搅拌均匀;
3)然后添加ε-聚赖氨酸溶液和从南美白对虾壳中提取的虾青素,使ε-聚赖氨酸在所得溶液中的浓度不低于0.13g/L,虾青素在所得溶液中的浓度为15-20 μg/mL;
4)将步骤3)所得溶液均质处理,调节pH值为3.5-4.5;
5)向步骤4)所得溶液中添加明胶质量4-8%的壳聚糖粉末,避光下搅拌,至壳聚糖完全溶解,得到生物抗菌复合膜液;
6)然后,将生物抗菌复合膜液平铺到板上,烘干,得到干燥的抑制南美白对虾腐败菌的生物抗菌复合膜。
2.如权利要求1所述的制备方法,其特征在于,步骤1)中,加热温度为40-50℃。
3.如权利要求1所述的制备方法,其特征在于,步骤3)中,所述ε-聚赖氨酸的浓度不高于0.3g/L。
4.如权利要求3所述的制备方法,其特征在于,步骤3)中,所述ε-聚赖氨酸的浓度为0.25-0.3g/L。
5.如权利要求1所述的制备方法,其特征在于,步骤4)中,均质条件为:10000-15000r/min,5-15s。
6.如权利要求1所述的制备方法,其特征在于,步骤5)中,所述壳聚糖粉末的脱乙酰度为80.0-95.0%。
7.如权利要求1所述的制备方法,其特征在于,步骤6)中,烘干温度为40-50℃。
8.如权利要求1所述的制备方法,其特征在于,所述生物抗菌复合膜的膜厚为0.4-0.6μm。
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