CN111838308B - 一种可食性果蔬纳米涂膜保鲜剂及其制备方法和应用 - Google Patents
一种可食性果蔬纳米涂膜保鲜剂及其制备方法和应用 Download PDFInfo
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- A—HUMAN NECESSITIES
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Abstract
本发明公开了一种可食性果蔬纳米涂膜保鲜剂,其组成按质量百分数计如下:疏水性纳米植物糖原的Pickering乳液40‑60wt%;抗菌肽EnterolysinA0.3‑0.7wt%;肉桂醛1‑3wt%;壳聚糖4‑6wt%;山梨醇2‑4wt%;其余为去离子水。实验证明,本发明制备的疏水性纳米植物糖原的Pickering乳液不仅能延长抗菌成分的药效,还能够提高抗菌成分的抑菌性,使涂膜保鲜剂的防腐效果大大增加,延缓果蔬的腐败变质。抗菌肽Enterolysin、肉桂醛作为抗菌剂被Pickering乳液包埋联合使用时具有协同作用而无拮抗作用,可以增加抑菌效果,增强食品防腐剂的稳定性。
Description
技术领域
本发明属于材料技术领域,具体涉及一种可食性果蔬纳米涂膜保鲜剂及其制备方法和应用。
背景技术
涂膜保鲜技术作为一种低毒性、环保、方便的保鲜方式在果蔬保鲜上的应用越来越较广泛,其发展前景十分广阔。涂膜保鲜技术主要是使用成膜性较好的大分子物质和抗菌成分配制成一定浓度的溶液,在果蔬表面进行喷洒或涂抹,形成一种具有保护作用的可食用的薄膜,有效地减弱采后果实的呼吸强度,抑制细菌的生长和水分流失,保持其色泽、新鲜度和饱满度,延缓果蔬组织衰老和腐败。同时,由于薄膜将果实与外界环境分隔,使得果实与外界环境的接触大大减少,从而降低了果实的氧化和环境中有害微生物对果实的侵染,进而达到保鲜和抗菌的目的。
目前,可食性涂膜保鲜材料比较多样,但现有的涂膜保鲜技术也存在一定的不足,比较突出的就是膜强度和膜韧性不够,抗菌性能不强且不持久。水果在贮藏过程中,由于搬运、相互摩擦、光照等作用,非常容易破坏水果表面的膜材料的完整性,膜中的抗菌成分也会慢慢失效,最终使膜性能下降,保鲜效果降低。
发明内容
本发明目的在于针对现有技术的不足,提供一种力学性能、抗菌效果良好且能够食用的果蔬涂膜保鲜剂及其制备方法和应用。
为达到上述目的,采用技术方案如下:
一种可食性果蔬纳米涂膜保鲜剂,包含疏水性纳米植物糖原的Pickering乳液、抗菌肽Enterolysin A、肉桂醛、壳聚糖、山梨醇;其组成按质量百分数计如下:
疏水性纳米植物糖原的Pickering乳液40-60wt%;抗菌肽Enterolysin A0.3-0.7wt%;肉桂醛1-3wt%;壳聚糖4-6wt%;山梨醇2-4wt%;其余为去离子水。
按上述方案,所述疏水性纳米植物糖原的Pickering乳液按以下方式制备而来:
将疏水性纳米植物糖原、中链甘油三酯与去离子水混合,控制pH7.0以下,18000rpm以上高速均质4分钟,即得疏水性纳米植物糖原的Pickering乳液;其中疏水性纳米植物糖原、中链甘油三酯与去离子水的质量比为2.5:25:22.5。
按上述方案,所述疏水性纳米植物糖原按以下方式制备而来:
将20wt%的辛烯基琥珀酸酐/异丙醇溶液加入到30wt%水溶性纳米植物糖原悬浮液中,使水溶性纳米植物糖原悬浮液浓度稀释到10wt%;
用3wt%的NaOH水溶液调节pH到8.5,常温充分反应2h后,用2.5M盐酸调节pH为6.5;
在35℃恒温8小时,培养完成后离心取沉淀,用蒸馏水和无水乙醇反复洗涤,得到疏水性纳米植物糖原。
按上述方案,所述水溶性纳米植物糖原按以下方式制备而来:
称取甜玉米种子,加入去离子水,在4℃下浸泡24h;
浸泡后的玉米种子去水后粉碎,然后加入3倍体积去离子水,4℃下静置24h;
取上清液,并加醋酸调节pH至4.8-5,再置于4℃下静置24h;
离心去除沉淀,得到的上清液煮沸后,再次离心进一步除去蛋白质至无油无沉淀;
所得上清液按体积1:1加入无水乙醇搅拌混合,4℃下静置至沉淀完全,离心取沉淀冷冻干燥,即得水溶性纳米植物糖原。
按上述方案,所述抗菌肽Enterolysin A按以下方式制备而来:
从臭豆腐样品中用DNA试剂盒提取DNA,利用获得的相应引物采用PCR技术扩增Enterolysin A基因,构建克隆载体pGM-T,通过Genome walking的方法获得Enterolysin A的全长基因序列,构建工程菌BL21,通过诱导剂IPTG诱导表达载体pET28a在大肠杆菌BL21中表达,使用制备型高效液相色谱分离纯化Enterolysin A,真空冷冻干燥得到高纯度的抗菌肽Enterolysin A。
按上述方案,所述壳聚糖按以下方式制备而来:
取软化蟹壳与乙醇混合,在80-90℃加热回流0.5-1.5h,冷却抽滤,滤渣干燥得甲壳素;将甲克素与饱和的NaOH乙醇溶液混合,80-90℃加热回流3-5h,冷却、抽滤、水洗、干燥得壳聚糖。
上述可食性果蔬纳米涂膜保鲜剂的制备方法,包括以下步骤:
取壳聚糖和山梨醇溶解到去离子水中,60-80℃匀速搅拌加热8-12min,冷却到室温待用;
向疏水性纳米植物糖原的Pickering乳液加入抗菌肽EnterolysinA和肉桂醛,搅拌均匀待用;
将以上溶液按体积比1:1混合,搅拌均匀,得到可食性果蔬纳米涂膜保鲜剂。
上述可食性果蔬纳米涂膜保鲜剂作为果蔬保鲜膜的应用,包括以下步骤:
将上述果蔬涂膜保鲜剂喷涂至果蔬表面,干燥后在果蔬表面形成保鲜膜。
相对于现有技术,本发明的有益效果如下:
抗菌肽Enterolysin A从中国传统发酵食品臭豆腐中克隆纯化获得,可以抑制乳杆菌、肠杆菌、枯草芽孢杆菌、单增李斯特氏菌、金黄色葡萄球菌属等。肉桂醛作为一种天然安全的抑菌剂也对人体无毒或低毒,对抑制霉菌,抗真菌有很好的效果。实验证明,本发明将抗菌肽Enterolysin A与肉桂醛被Pickering乳液包埋联合使用时,添加的Pickering乳液对于复合使用的抑菌剂具有协同作用而无拮抗作用,可以增加抑菌效果,增强食品抑菌剂的稳定性。
所述疏水性纳米植物糖原的Pickering乳液是良好的乳化剂,能使保鲜剂各成分均一,该乳液中纳米植物糖原会在油水界面形成阻挡层和三维形式堆积网络,将抗菌成分包埋其中,延长药效。实验证明,本发明制备的疏水性纳米植物糖原的Pickering乳液不仅能延长抗菌成分的药效,还能够提高抗菌成分的抑菌性,使涂膜保鲜剂的防腐效果大大增加,延缓果蔬的腐败变质。
壳聚糖有良好的成膜性、抗菌性以及可食用性,其与Pickering乳液中纳米植物糖原交互作用,增强了复合膜的成膜性。实验证明,含有疏水性纳米植物糖原的Pickering乳液所形成膜比其他Pickering乳液制的膜的韧性和强度得到较大改善。
添加山梨醇能够软化膜的刚性结构,从而增加膜的柔韧性、降低膜的阻隔性,使膜具有光泽和富有弹性。
本发明所得涂膜保鲜剂融合了多糖(壳聚糖)、多肽(抗菌肽)和油脂(中链甘油三酯)这些人体最重要的营养素,使膜本身具有很高的营养价值;在食用果蔬时,食用者除了可以补充果蔬的水分和维生素外,还可以同时补充多糖、多肽和油脂等多种营养成分,可以作为一种营养补充剂。
本发明的果蔬涂膜保鲜剂具有水溶性好,具有良好的透湿性和抗拉伸性能,可有效抑制果蔬的呼吸作用,达到果蔬保鲜的目的,并且保鲜剂成分中的壳聚糖从废弃物蟹壳中提取,植物糖原从甜玉米中提取,提取率高达17%,用量较少,成本低廉且具有可食性。
附图说明
图1:实施例1水溶性纳米植物糖原透射电镜图。
具体实施方式
以下实施例进一步阐释本发明的技术方案,但不作为对本发明保护范围的限制。
实施例1
水溶性纳米植物糖原悬浮液的制备过程:
称取甜玉米种子,加入去离子水,在4℃下浸泡24h。去除水溶液,用磨粉机粉碎后,按料水比1:3(v/v)加去离子水,4℃下静置24h;取上清液,加醋酸调节pH至4.8-5,再置于4℃下静置24h;3000g离心10分钟去除沉淀,得到上清液在沸水浴中煮沸,再次3000g离心10分钟进一步除去蛋白质至无油无沉淀;将得到的上清液加入无水乙醇搅拌混合,上清液与无水乙醇按体积1:1混合,4℃下静置至沉淀完全,3000g离心后将沉淀物冷冻干燥(温度-55℃,真空度0.026mbar)得到水溶性纳米植物糖原。
本实施例所得水溶性纳米植物糖原透射电镜图如图1所示,将制得的水溶性纳米植物糖原溶解到去离子水中得到浓度30wt%的水溶性纳米植物糖原悬浮液待用。
疏水性纳米植物糖原的制备过程:
辛烯基琥珀酸酐(OSA)在异丙醇中溶解得到浓度20wt%的溶液,加入到浓度30wt%的水溶性纳米植物糖原悬浮液中,使水溶性纳米植物糖原悬浮液浓度稀释到10wt%。用3wt%的NaOH溶液调节pH到8.5,充分反应2h,用2.5M盐酸调节pH为6.5。放在35℃恒温培养箱8小时。培养完成后3000g离心10分钟取沉淀,用蒸馏水和无水乙醇先后洗涤,重复3次,收集疏水性植物糖原纳米颗粒。
疏水性纳米植物糖原的Pickering乳液的制备:
将疏水性植物糖原纳米颗粒与去离子水混,再加入中链甘油三酯至,控制pH7.0下,18000rpm高速均质4分钟,得疏水性纳米植物糖原的Pickering乳液。其中疏水性植物糖原纳米颗粒、中链甘油三酯、去离子水的质量比为2.5:25:22.5。
实施例2
食源性抗菌肽Enterolysin A制备过程:
1、从臭豆腐样品中用DNA试剂盒提取DNA。
2、Enterolysin A基因扩增:利用获得的相应引物采用PCR技术扩增臭豆腐中的Enterolysin A基因。
3、克隆载体构建:PCR产物经琼脂糖电泳割胶纯化后,按照目的片段与载体摩尔比3:1的比例与克隆载体pGM-T在16℃条件下连接过夜。连接结束后将连接产物转入大肠杆菌TOP10感受态细胞中,然后涂布于LB(Amp+)平板(提前1h将16μL50mg/mL IPTG、40μL 20mg/mL X-gal涂布于LB(Amp+)平板上,37℃放置)上培养,构成Enterolysin A基因克隆文库。次日挑取白斑过夜培养,提取重组克隆质粒pGM-T/Enterolysin A(质粒提取试剂盒)。
4、通过Genome walking的方法获得Enterolysin A的全长基因序列:
以DNA为模板,利用Genome Walking技术设计引物进行步移扩增。扩增后的PCR产物用1%琼脂糖凝胶电泳检测,割胶回收,连接pGM-T载体,转化Escherichia coli TOP 10大肠杆菌感受态细胞中,涂布于含有AMP(100μg/mL)的LB平板,37℃过夜,挑白色单菌落抽提阳性重组子质粒DNA,测定插入DNA片段的序列。
5、构建工程菌BL21。
用限制性内切酶EcoRI、XhoI分别双酶切纯化后的相应的PCR产物和pET28a(+)表达载体。割胶回收目的片段和载体片段。按目的片段与载体摩尔比10:1的比例将两者在16℃条件下过夜连接,连接结束后将连接产物转化大肠杆菌BL21感受态细胞中,然后涂布于LB(Kan+)平板上培养。次日挑取单菌落过夜培养,提取重组表达质粒pET28a进行双酶切鉴定,选取阳性质粒进行重组蛋白表达。
6、通过诱导剂IPTG诱导表达载体pET28a在大肠杆菌BL21中表达。
(1)从-77℃甘油管中活化菌种BL21、划LB平板,37℃培养箱中培养。
(2)挑取单菌落转接于含有5mLLB加入相应抗生素的液体培养基中,于37℃,200rpm摇床中培养过夜。
(3)将过夜培养菌接种到含有50mL LB液体培养基的250mL三角瓶中(按1%接种比例),200rpm,20℃培养8h左右,测OD600nm到O.6左右时加入诱导剂IPTG(终浓度为0.8mM),20℃,200rpm诱导培养20h。
(4)将诱导培养好的转化菌BL21于4℃,10000g离心10min,得菌体。
7、Enterolysin A的分离纯化
使用截留孔径为1kd和3kd的超滤离心管对上述纯化蛋白洗脱液进行脱盐处理;使用制备型高效液相色谱(High performance liquid chro-matography,HPLC)对蛋白进行高度纯化,收集洗脱峰,旋转蒸发除去有机溶剂,真空冷冻干燥得到高纯度抗菌肽Enterolysin A。
实施例3
可食性果蔬纳米涂膜保鲜剂的制备方法:
原料按质量百分数计:疏水性纳米植物糖原的Pickering乳液50wt%;抗菌肽Enterolysin A0.5wt%;肉桂醛2wt%;壳聚糖5wt%;山梨醇3wt%;去离子水39.5wt%。
取成膜剂壳聚糖和增塑剂山梨醇溶解到去离子水中,70℃加热10分钟,加热期间不断轻微匀速搅拌,防止气泡产生和壳聚糖变性,加热完后冷却到室温待用。
取Pickering乳液加入抗菌肽Enterolysin A和肉桂醛,搅拌均匀待用。
将以上两者溶液1:1混合,搅拌均匀即得到可食性纳米果蔬涂膜保鲜剂。
取本实施例所得可食性果蔬纳米涂膜保鲜剂20mL添加到直径为11.8cm培养皿中平铺均匀,室温(25℃)干燥24小时,在相对湿度为60%,温度为25℃的条件下晾干48小时,揭膜。
对比例:使用改性淀粉Pickering乳液代替本实施例疏水性纳米植物糖原的Pickering乳液,其余成分和步骤不变,所述改性淀粉Pickering乳液是利用辛烯基琥珀酸酐(OSA)疏水改性淀粉作为乳化剂与20%(V/V)正十四烷作为油相制备O/W稳定Pickering乳液。
经测试,本实施例和对比例膜性能参数如表1所示。可见从水溶性、膜强度和膜伸长率对比可知,含有疏水性纳米植物糖原的Pickering乳液的膜性能优于含改性淀粉Pickering乳液的膜性能。
表1不同Pickering乳液涂膜保鲜剂膜性能参数
实施例4
Enterolysin A和肉桂醛在Pickering乳液中最低抑菌浓度(MIC)的测定
实施方法:Enterolysin A和肉桂醛对细菌的MIC的测定采用标准肉汤微量稀释法药敏试验方法。制备不同质量浓度的单一抗菌剂,参照GB2760—2014《食品安全国家标准食品添加剂使用标准》中食品添加剂最大允许范围,分别制备不同质量浓度的Enterolysin A(0μg/mL、4μg/mL、8μg/mL、16μg/mL、32μg/mL、50μg/mL、100μg/mL、150μg/mL)、肉桂醛(0μg/mL、15μg/mL、30μg/mL、60μg/mL、100μg/mL、150μg/mL、250μg/mL、500μg/mL)、被Pickering乳液包埋的EnterolysinA(0μg/mL、4μg/mL、8μg/mL、16μg/mL、32μg/mL、50μg/mL、100μg/mL、150μg/mL)、被Pickering乳液包埋的肉桂醛(0μg/mL、15μg/mL、30μg/mL、60μg/mL、100μg/mL、150μg/mL、250μg/mL、500μg/mL)的MH培养基。向每组培养基接入相同浓度和体积的金黄色葡萄球菌,使细菌在恒温培养箱37℃、180r/min条件下培养20h,每组实验进行三次平行实验。
实施结果:如表1所示,根据标准肉汤微量稀释法药敏试验方法计算得到的MIC值显示,Enterolysin A和肉桂醛对供试菌株表现出不同的抑菌活性,Enterolysin A对金黄色葡萄球菌的MIC值为0.08mg/mL,肉桂醛对金黄色葡萄球菌的的MIC值为0.25mg/mL,被Pickering乳液包埋的Enterolysin A对金黄色葡萄球菌的MIC值为0.068mg/mL,被Pickering乳液包埋的肉桂醛对金黄色葡萄球菌的MIC值为0.24mg/mL。
结论分析:可见肉桂醛对金黄色葡萄球菌的MIC值大于Enterolysin A对金黄色葡萄球菌的MIC值,未被Pickering乳液包埋的Enterolysin A和肉桂醛对金黄色葡萄球菌的MIC值大于被Pickering乳液包埋的Enterolysin A和肉桂醛对金黄色葡萄球菌的MIC值。说明本发明所用Pickering乳液对食品防腐有促进的作用,两者的效果是协同的。
实施例5
实施方法:肉汤稀释棋盘分析法采用96孔微滴度板的棋盘结构,用肉汤微量稀释法测定不同浓度的Enterolysin A、不同浓度的肉桂醛、被Pickering乳液包埋不同浓度的Enterolysin A和被Pickering乳液包埋的不同浓度的肉桂醛对金黄色葡萄球菌的抑制作用,确定Enterolysin A、肉桂醛以及被Pickering乳液包埋的Enterolysin A和被Pickering乳液包埋的肉桂醛的MIC。
Enterolysin A和肉桂醛的MIC采用标准肉汤微稀释敏感度试验方法测定。具体方法为:在MH培养基中,将细菌稀释至最终浓度约为1000CFU/mL左右的菌液。再在2块96孔微滴板的单个井中加入50μL的食品抑菌剂稀释液和50μL制备的1000CFU/mL左右的菌液。1块在恒温培养箱中37℃下放置24小时过夜培养待测,另1块在恒温培养箱中37℃下放置48小时培养待测。
为评价每种组合的抑菌效果,计算了A(Enterolysin A)、B(肉桂醛)两种药物组合的部分抑菌浓度指数(FICI)与A(或B)单剂的MIC之比。该指数计算如下:
FICI=FICIA+FICIB=(CACOMA/MICA)+(CBCOMB/MICB)
其中FICIA为A药物的抑菌浓度指数;FICIB为B药物的抑菌浓度指数;CACOMA为A与B药物联合时达到最低抑菌浓度情况下,A药物的浓度;CBCOMB为A与B药物联合时达到MIC情况下,B药物的浓度;MICA为A单剂的MIC;MICB为B单剂的MIC。
FICI的解释如下:当FICI值<0.5时表示协同作用,当FICI值>4时表示拮抗作用。
实施结果:如表2所示,可得出未被Pickering乳液包埋的Enterolysin A和肉桂醛对金黄色葡萄球菌相互作用的FICI值为0.275,由于当FICI小于0.5表现为协同作用,表明Enterolysin A与肉桂醛具有协同作用而无拮抗作用。当Enterolysin A与肉桂醛被Pickering乳液包埋时,对金黄色葡萄球菌相互作用的FICI值在0.24,由于当FICI小于0.5表现为协同作用,表明被Pickering乳液包埋的Enterolysin A与肉桂醛具有协同作用而无拮抗作用。由表3可知,培养48小时,没有被Pickering乳液包埋的抑菌剂MIC值增加较快,但是被Pickering乳液包埋的抑菌剂MIC值变化不明显。
可见Enterolysin A与肉桂醛被Pickering乳液包埋联合使用时具有协同作用而无拮抗作用,可以增加抑菌效果,同时可以延长抗菌时间,增强了食品抑菌剂的稳定性。
表2不同处理抑菌剂培养24小时的MIC
表3不同处理抑菌剂培养48小时的MIC
实施例6
挑选色泽和成熟度一致的葡萄洗净,消毒30分钟,在室温(25℃)晾干,将其分成两组,1组不涂膜,作为对照组;另一组在实施例3制备的可食性果蔬纳米涂膜保鲜剂中浸渍1分钟后在室温(25℃)晾干。将两组均储藏在4℃冷藏箱,对照组的贮藏期在20d左右,本发明可食性果蔬纳米涂膜保鲜剂可使其延长到35天的储藏期。
Claims (4)
1.一种可食性果蔬纳米涂膜保鲜剂,其特征在于组成按质量百分数计如下:
疏水性纳米植物糖原的Pickering乳液40-60wt%;抗菌肽Enterolysin A0.3-0.7wt%;肉桂醛1-3wt%;壳聚糖4-6wt%;山梨醇2-4wt%;其余为去离子水;
所述疏水性纳米植物糖原的Pickering乳液按以下方式制备而来:
将疏水性纳米植物糖原、中链甘油三酯与去离子水混合,控制pH7.0以下,18000rpm以上高速均质4分钟,即得疏水性纳米植物糖原的Pickering乳液;其中疏水性纳米植物糖原、中链甘油三酯与去离子水的质量比为2.5:25:22.5;
所述疏水性纳米植物糖原按以下方式制备而来:
将20wt%的辛烯基琥珀酸酐/异丙醇溶液加入到30wt%水溶性纳米植物糖原悬浮液中,使水溶性纳米植物糖原悬浮液浓度稀释到10wt%;
用3wt%的NaOH水溶液调节pH到8.5,常温充分反应2h后,用2.5M盐酸调节pH为6.5;
在35℃恒温8小时,培养完成后离心取沉淀,用蒸馏水和无水乙醇反复洗涤,得到疏水性纳米植物糖原;
所述水溶性纳米植物糖原按以下方式制备而来:
称取甜玉米种子,加入去离子水,在4℃下浸泡24h;
浸泡后的玉米种子去水后粉碎,然后加入3倍体积去离子水,4℃下静置24h;
取上清液,并加醋酸调节pH至4.8-5,再置于4℃下静置24h;
离心去除沉淀,得到的上清液煮沸后,再次离心进一步除去蛋白质至无油无沉淀;
所得上清液按体积1:1加入无水乙醇搅拌混合,4℃下静置至沉淀完全,离心取沉淀冷冻干燥,即得水溶性纳米植物糖原;
所述抗菌肽Enterolysin A按以下方式制备而来:
从臭豆腐样品中用DNA试剂盒提取DNA,利用获得的相应引物采用PCR技术扩增Enterolysin A基因,构建克隆载体pGM-T,通过Genome walking的方法获得Enterolysin A的全长基因序列,构建工程菌BL21,通过诱导剂IPTG诱导表达载体pET28a在大肠杆菌BL21中表达,使用制备型高效液相色谱分离纯化Enterolysin A,真空冷冻干燥得到高纯度的抗菌肽Enterolysin A。
2.如权利要求1所述可食性果蔬纳米涂膜保鲜剂,其特征在于所述壳聚糖按以下方式制备而来:
取软化蟹壳与乙醇混合,在80-90℃加热回流0.5-1.5h,冷却抽滤,滤渣干燥得甲壳素;将甲壳素与饱和的NaOH乙醇溶液混合,80-90℃加热回流3-5h,冷却、抽滤、水洗、干燥得壳聚糖。
3.权利要求1-2任一项所述可食性果蔬纳米涂膜保鲜剂的制备方法,包括以下步骤:
取壳聚糖和山梨醇溶解到去离子水中,60-80℃匀速搅拌加热8-12min,冷却到室温待用;
向疏水性纳米植物糖原的Pickering乳液加入抗菌肽Enterolysin A和肉桂醛,搅拌均匀待用;
将以上溶液按体积比1:1混合,搅拌均匀,得到可食性果蔬纳米涂膜保鲜剂。
4.权利要求1-2任一项所述可食性果蔬纳米涂膜保鲜剂作为果蔬保鲜膜的应用,包括以下步骤:
将上述可食性果蔬纳米涂膜保鲜剂 喷涂至果蔬表面,干燥后在果蔬表面形成保鲜膜。
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