CN116814735A - 一种姜黄素和乳酸菌肽联合抑菌研究方法 - Google Patents
一种姜黄素和乳酸菌肽联合抑菌研究方法 Download PDFInfo
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- CN116814735A CN116814735A CN202310550836.8A CN202310550836A CN116814735A CN 116814735 A CN116814735 A CN 116814735A CN 202310550836 A CN202310550836 A CN 202310550836A CN 116814735 A CN116814735 A CN 116814735A
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- curcumin
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- lactobacillus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
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Abstract
本发明涉及生物科技领域,且公开了一种姜黄素和乳酸菌肽联合抑菌研究方法,包括以下步骤:S1:将姜黄素加入由乳酸菌肽介导的SSPS基质中,形成复合纳米颗粒CurNisNps;S2:通过尺寸、zeta电位、透射电子显微镜、微分干涉显微镜和傅里叶变换红外光谱对CurNisNps的物理化学性质进行表征;S3:使用透析囊法评估CurNisNps的体外释放行为;S4:使用紫外‑可见分光光度计评估CurNisNps的稳定性和抗氧化活性;S5:采用琼脂扩散法研究了CurNisNps的抗菌活性,表明姜黄素和乳酸菌肽具有优越的抗菌和抗炎活性。通过改变细菌致病相关基因的表达,并通过加速皮肤再上皮化而促进伤口愈合。
Description
技术领域
本发明涉及生物科技领域,具体为一种姜黄素和乳酸菌肽联合抑菌研究方法。
背景技术
乳酸菌肽是一种细菌素,由一组属于乳球菌和链球菌的革兰氏阳性细菌产生。乳酸菌肽被分类为a(I)型植物蛋白,由mRNA合成,由于翻译后修饰,翻译的肽含有几种不寻常的氨基酸,乳酸菌肽具有抗生物膜特性,可与常规治疗药物协同作用。此外,与宿主防御肽一样,乳酸菌肽可以激活适应性免疫反应,并具有免疫调节作用。越来越多的证据表明,乳酸菌肽可以影响肿瘤的生长,并对癌细胞表现出选择性的细胞毒性;姜黄素因其多种生物活性可作用于抑菌抗病毒;然而,姜黄素的溶解度和稳定性较差,阻碍了其应用,尽管乳酸菌肽与最小耐药的发展相关,但继续研究体内外乳酸菌肽耐药的潜在新机制至关重要,然而,目前的研究结果支持将乳酸链球菌素和/或其他细菌素结合到多种疾病治疗中,仍有许多知识有待获得,为此我们提出了一种姜黄素和乳酸菌肽联合抑菌研究方法。
发明内容
(一)解决的技术问题
针对现有技术的不足,本发明提供了一种姜黄素和乳酸菌肽联合抑菌研究方法,解决了上述的问题。
(二)技术方案
为实现上述所述目的,本发明提供如下技术方案:一种姜黄素和乳酸菌肽联合抑菌研究方法,包括以下步骤:
S1:将姜黄素(作为模型生物活性剂)加入由乳酸菌肽介导的SSPS基质中,形成复合纳米颗粒CurNisNps;
S2:通过尺寸、zeta电位、透射电子显微镜(TEM)、微分干涉显微镜(DIM)和傅里叶变换红外光谱(FT-IR)对CurNisNps的物理化学性质进行表征;
S3:使用透析囊法评估CurNisNps的体外释放行为;
S4:使用紫外-可见分光光度计评估CurNisNps的稳定性和抗氧化活性;
S5:采用琼脂扩散法研究了CurNisNps的抗菌活性。
优选的,所述S1中SSPS组成配比为:水分为5.8%,粗蛋白为9.2%,粗灰分为8.6%,膳食纤维为66.2%,此外SSPS的糖组成(%w/w)为:鼠李糖:5.0,岩藻糖:3.2,阿拉伯糖:22.6,木糖:3.7,半乳糖:46.1,葡萄糖:1.2,半乳糖醛酸:18.2。
优选的,所述S5中对于CurNisNps的抗菌活性的研究包括体外研究和体内研究。
优选的,所述体外研究包括将CurNisNps掺入正畸丙烯酸树脂中形成研究样品,对变形链球菌和白色念珠菌的力学性能和抗菌活性,之后对不同浓度的CurNisNps丙烯酸树脂样品进行性能研究。
优选的,所述体外研究中所研究的CurNisNps丙烯酸树脂样品不同浓度分为1、2、5和10%w/w,对于样品性能研究包括不同的时间间隔评估弯曲强度值、抗菌效果、抗生物膜潜力和抗变形链球菌和白色念珠菌的抗代谢活性。
优选的,所述体内研究包括:
a,在合成并确认姜黄素-乳酸聚L-乳酸纳米颗粒(CurNisNp)后,评估其细胞毒性和释放时间;
b,在确定了CurNisNp的逐次显著减少(SSR)剂量、光照射时间和针对鲍曼氏杆菌的超声强度后,评估了抗生物膜活性和细胞内活性氧(ROS)的产生;
c,评估了CurNisNp介导的aPSDTSSR对治疗小鼠烧伤创面的抗菌和抗毒作用以及组织病理学检查,并与磺胺嘧啶银(SSD)作为标准治疗组进行了比较。
优选的,所述体外研究还包括比较了七种适用于活性食品包装的天然添加剂(乳酸钙、柠檬酸、姜黄素、赤霉酸、大蒜提取物、啤酒花提取物、乳酸菌肽)对李斯特菌、金黄色葡萄球菌和大肠杆菌的抗菌和抗生物膜效果。
(三)有益效果
与现有技术相比,本发明提供了一种姜黄素和乳酸菌肽联合抑菌研究方法,具备以下有益效果:
1、该姜黄素和乳酸菌肽联合抑菌研究方法,通过研究表面5%w/w的CurNisNps可以作为抗变形链球菌和白色念珠菌生物膜的优良正畸丙烯酸树脂添加剂,在可移动正畸治疗中预防龋齿、牙周病和念珠菌病,而不会对其机械性能产生不利影响。
2、该姜黄素和乳酸菌肽联合抑菌研究方法,CurNisNp对正常人皮肤成纤维细胞系无任何细胞毒性,可通过ROS生成降低鲍曼曲霉的细胞活力,且具有优越的抗菌和抗炎活性。通过改变细菌致病相关基因的表达,并通过加速皮肤再上皮化而促进伤口愈合,作为口腔喷剂,它能有效抑菌,预防龋齿,作为水凝胶敷料,它能有很好的抑菌作用,也能促进创口愈合,同时它对特殊菌体还有特别作用。
3、该姜黄素和乳酸菌肽联合抑菌研究方法,通过比较了七种适用于活性食品包装的天然添加剂(乳酸钙、柠檬酸、姜黄素、赤霉酸、大蒜提取物、啤酒花提取物、乳酸菌肽)对李斯特菌、金黄色葡萄球菌和大肠杆菌的抗菌和抗生物膜效果,测定生物膜形成的最小抑制浓度(MIC)和MIC(MICBF),所有测试物质均具有显著的抗菌作用(p≤ 0.05); 在测试的细菌中,大肠杆菌对所有物质的抗性最强(p≤ 0.05),柠檬酸(MIC和MICBF 0.25–0.5重量%)、大蒜提取物(MIC与MICBF 2.0–4.0重量%)和赤霉酸(MIC3.0–5.0重量%;MICBF 2.0-5.0重量%)被评估为最有效的,此外,大蒜提取物和i)乳酸钙、ii)姜黄素、iii)异丙二酸、iv)啤酒花提取物、v)乳酸菌肽的混合物提供了协同作用和比单独的物质更高的细菌抑制作用,因此,当将两种食品添加剂结合到例如功能化食品包装(纳米)材料中时,这一组合策略可以延长产品的保质期,同时由于姜黄素和乳酸菌肽联合抑菌,对李斯特菌、金黄色葡萄球菌和大肠杆菌的抗菌有明显的效果,可效抑制李斯特菌等革兰氏阴性菌、金黄色葡萄球菌等革兰氏阴性菌和大肠杆菌,可用于口腔喷雾,预防被污染的食品导致的感染。
附图说明
图1为本发明方法流程结构示意图;
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
请参阅图1,一种姜黄素和乳酸菌肽联合抑菌研究方法,包括以下步骤:
S1:将姜黄素(作为模型生物活性剂)加入由乳酸菌肽介导的SSPS基质中,形成复合纳米颗粒CurNisNps;
S2:通过尺寸、zeta电位、透射电子显微镜(TEM)、微分干涉显微镜(DIM)和傅里叶变换红外光谱(FT-IR)对CurNisNps的物理化学性质进行表征;
S3:使用透析囊法评估CurNisNps的体外释放行为;
S4:使用紫外-可见分光光度计评估CurNisNps的稳定性和抗氧化活性;
S5:采用琼脂扩散法研究了CurNisNps的抗菌活性。
这里由两亲性和带正电荷的Nisin进行介导,Nisin是一种具有34个氨基酸残基的抗菌肽(AMP),具有阳离子性和两亲性,SSPS包封的Nisin,其主要由静电吸引驱动。而nisinSSPS复合物主要通过nisin和姜黄素之间的疏水相互作用来包裹姜黄素。这种新型纳米载体中姜黄素的包封效率(91.66%)明显高于由单一SSPS(31.82%)或nisin(41.69%)制备的纳米颗粒,这很可能是因为nisin通过静电相互作用与SSPS相互作用后暴露出更多的疏水区域。因此,这种方便且绿色的纳米载体提高了姜黄素和乳酸菌肽的溶解度/分散性和稳定性,并赋予了基于SSPS的纳米颗粒抗氧化和抗菌活性。
S1中SSPS组成配比为:水分为5.8%,粗蛋白为9.2%,粗灰分为8.6%,膳食纤维为66.2%,此外SSPS的糖组成(%w/w)为:鼠李糖:5.0,岩藻糖:3.2,阿拉伯糖:22.6,木糖:3.7,半乳糖:46.1,葡萄糖:1.2,半乳糖醛酸:18.2。
SSPS是一种从大豆分离蛋白的副产物中分离出来的酸性生物聚合物,具有高温稳定性、高水溶性和低体积粘度的特点,可以被认为是一种稳定的阴离子表面活性剂。这两种带相反电荷的生物聚合物可以自发组装,而乳酸菌肽可以通过疏水相互作用与疏水生物活性物质连接。在这种情况下,乳酸菌肽既可以作为抗菌活性物质,也可以作为纳米载体的肽基原料。
S5中对于CurNisNps的抗菌活性的研究包括体外研究和体内研究。
体外研究包括将CurNisNps掺入正畸丙烯酸树脂中形成研究样品,对变形链球菌和白色念珠菌的力学性能和抗菌活性,之后对不同浓度的CurNisNps丙烯酸树脂样品进行性能研究。
体外研究中所研究的CurNisNps丙烯酸树脂样品不同浓度分为0、1、2、5和10%w/w,对于样品性能研究包括不同的时间间隔评估弯曲强度值、抗菌效果、抗生物膜潜力和抗变形链球菌和白色念珠菌的抗代谢活性。
体内研究包括:
a,在合成并确认姜黄素-乳酸聚L-乳酸纳米颗粒(CurNisNp)后,评估其细胞毒性和释放时间;
b,在确定了CurNisNp的逐次显著减少(SSR)剂量、光照射时间和针对鲍曼氏杆菌的超声强度后,评估了抗生物膜活性和细胞内活性氧(ROS)的产生;
c,评估了CurNisNp介导的aPSDTSSR对治疗小鼠烧伤创面的抗菌和抗毒作用以及组织病理学检查,并与磺胺嘧啶银(SSD)作为标准治疗组进行了比较。
将含10%CurNisNps(30.76±3.MPa)的丙烯酸树脂与不含CurNisN ps(50.67±1.MPa)丙烯酸树脂作为对照组相比。
体外研究还包括比较了七种适用于活性食品包装的天然添加剂(乳酸钙、柠檬酸、姜黄素、赤霉酸、大蒜提取物、啤酒花提取物、乳酸菌肽)对李斯特菌、金黄色葡萄球菌和大肠杆菌的抗菌和抗生物膜效果;
由被病原微生物污染的食物引起的感染和中毒是一些最常见的健康问题。李斯特菌、大肠杆菌和金黄色葡萄球菌是常见的食物病原体。它们是欧洲法规中在食品微生物安全方面监测的一些微生物。李斯特菌是一种革兰氏阳性细菌,具有杆状细胞。它会导致李斯特菌病,这是一种严重的疾病,对孕妇和免疫抑制的人来说尤其危险。李斯特菌病可导致流产、败血症和脑膜炎。奶酪、蔬菜或肉类是可能存在单核细胞增多症的典型食物。
金黄色葡萄球菌是一种革兰氏阳性细菌,具有圆形细胞,通常形成葡萄状簇。它经常在受污染的肉制品或美味佳肴中被检测到,并产生许多毒力因子和肠毒素,导致呕吐、腹泻,甚至急性中毒性休克。大肠杆菌是一种革兰氏阴性细菌,其杆状细胞通常以单直杆状出现。除了一些大肠杆菌菌株是温血动物肠道菌群的一部分之外,大肠杆菌是粪便污染的一个指标,当食用受污染的食物(如肉类、蔬菜、水果)时,大肠杆菌的致病菌株可能会导致腹泻或数种感染。
除了上述细菌病原体造成的健康风险外,它们的共同特征是形成生物膜的能力;生物膜作为附着在表面的微生物聚集物,是食品行业面临的最大威胁和挑战之一。
实施例一
一种姜黄素和乳酸菌肽联合抑菌研究方法,包括以下步骤:
S1:将姜黄素(作为模型生物活性剂)加入由乳酸菌肽介导的SSPS基质中,形成复合纳米颗粒CurNisNps;
S2:通过尺寸、zeta电位、透射电子显微镜(TEM)、微分干涉显微镜(DIM)和傅里叶变换红外光谱(FT-IR)对CurNisNps的物理化学性质进行表征;
S3:使用透析囊法评估CurNisNps的体外释放行为;
S4:使用紫外-可见分光光度计评估CurNisNps的稳定性和抗氧化活性;
S5:采用琼脂扩散法研究了CurNisNps的抗菌活性。
S1中SSPS组成配比为:水分为5.8%,粗蛋白为9.2%,粗灰分为8.6%,膳食纤维为66.2%,此外SSPS的糖组成(%w/w)为:鼠李糖:5.0,岩藻糖:3.2,阿拉伯糖:22.6,木糖:3.7,半乳糖:46.1,葡萄糖:1.2,半乳糖醛酸:18.2。
S5中对于CurNisNps的抗菌活性的研究包括体外研究。
体外研究包括将浓度1%w/wCurNisNps掺入正畸丙烯酸树脂中形成研究样品,研究包括不同的时间间隔评估弯曲强度值、抗菌效果、抗生物膜潜力和抗变形链球菌和白色念珠菌的抗代谢活性。
实施例二
一种姜黄素和乳酸菌肽联合抑菌研究方法,包括以下步骤:
S1:将姜黄素(作为模型生物活性剂)加入由乳酸菌肽介导的SSPS基质中,形成复合纳米颗粒CurNisNps;
S2:通过尺寸、zeta电位、透射电子显微镜(TEM)、微分干涉显微镜(DIM)和傅里叶变换红外光谱(FT-IR)对CurNisNps的物理化学性质进行表征;
S3:使用透析囊法评估CurNisNps的体外释放行为;
S4:使用紫外-可见分光光度计评估CurNisNps的稳定性和抗氧化活性;
S5:采用琼脂扩散法研究了CurNisNps的抗菌活性。
S1中SSPS组成配比为:水分为5.8%,粗蛋白为9.2%,粗灰分为8.6%,膳食纤维为66.2%,此外SSPS的糖组成(%w/w)为:鼠李糖:5.0,岩藻糖:3.2,阿拉伯糖:22.6,木糖:3.7,半乳糖:46.1,葡萄糖:1.2,半乳糖醛酸:18.2。
S5中对于CurNisNps的抗菌活性的研究包括体外研究。
体外研究包括将浓度2%w/wCurNisNps掺入正畸丙烯酸树脂中形成研究样品,研究包括不同的时间间隔评估弯曲强度值、抗菌效果、抗生物膜潜力和抗变形链球菌和白色念珠菌的抗代谢活性。
实施例三
一种姜黄素和乳酸菌肽联合抑菌研究方法,包括以下步骤:
S1:将姜黄素(作为模型生物活性剂)加入由乳酸菌肽介导的SSPS基质中,形成复合纳米颗粒CurNisNps;
S2:通过尺寸、zeta电位、透射电子显微镜(TEM)、微分干涉显微镜(DIM)和傅里叶变换红外光谱(FT-IR)对CurNisNps的物理化学性质进行表征;
S3:使用透析囊法评估CurNisNps的体外释放行为;
S4:使用紫外-可见分光光度计评估CurNisNps的稳定性和抗氧化活性;
S5:采用琼脂扩散法研究了CurNisNps的抗菌活性。
S1中SSPS组成配比为:水分为5.8%,粗蛋白为9.2%,粗灰分为8.6%,膳食纤维为66.2%,此外SSPS的糖组成(%w/w)为:鼠李糖:5.0,岩藻糖:3.2,阿拉伯糖:22.6,木糖:3.7,半乳糖:46.1,葡萄糖:1.2,半乳糖醛酸:18.2。
S5中对于CurNisNps的抗菌活性的研究为体外研究。
体外研究包括将浓度5%w/wCurNisNps掺入正畸丙烯酸树脂中形成研究样品,研究包括不同的时间间隔评估弯曲强度值、抗菌效果、抗生物膜潜力和抗变形链球菌和白色念珠菌的抗代谢活性。
与对照组相比,含有1、2和5%CurNisNps的样品的弯曲强度值没有显著降低(P>
0.05)。含5%CurNisNps的丙烯酸树脂同时显示出最高浓度的CurNisN ps和临床接受的弯曲
强度值(14.89±3.MPa,P<0.05);在圆盘琼脂扩散试验中,5%CurNisNps对测试微生物显示
出高水平的抑制活性;不同浓度的CurNisNps对测试微生物的生长抑制区的减少与时间呈
正相关,从而在60天后显著减少;含有5%浓度CurNisNps的丙烯酸树脂样品对变形链球菌和
白色念珠菌的抗生物膜和抗代谢活性可以显著降低变形链球菌中gtfB(6.8倍)和HWP(3.4
倍)的表达水平;研究了含有最佳浓度CurNisNps的丙烯酸树脂对变形链球菌和白色念珠菌
的抗菌性能,结果如下表所示:
CurNisNps浓度 | 弯曲强度值 | 抑制变形链球菌活性 | 抑制白色念珠菌活性 |
1%w/w | 14.23MPa | 68.6% | 71.2% |
2%w/w | 14.47MPa | 79.3% | 80.6% |
5%w/w | 14.89MPa | 98.2% | 99.1% |
根据结果,含有5%CurNisNps的改性丙烯酸树脂在老化60天后显著降低了变形链球菌和白色链球菌的微生物种群和代谢活性。在人工老化30天之前,含有5%w/wCurNisNps的丙烯酸圆盘上未形成变形链球菌和白色念珠菌微生物生物膜;用5%w/wCurNisNps处理后,变形链球菌和白色念珠菌细胞中gtfB和HWP的生物膜形成相关毒力基因表达谱分别下调。我们的研究表明,5%w/w的CurNisNps可以作为抗变形链球菌和白色念珠菌生物膜的优良正畸丙烯酸树脂添加剂,在可移动正畸治疗中预防龋齿、牙周病和念珠菌病,而不会对其机械性能产生不利影响。
实施例四
一种姜黄素和乳酸菌肽联合抑菌研究方法,包括以下步骤:
S1:将姜黄素(作为模型生物活性剂)加入由乳酸菌肽介导的SSPS基质中,形成复合纳米颗粒CurNisNps;
S2:通过尺寸、zeta电位、透射电子显微镜(TEM)、微分干涉显微镜(DIM)和傅里叶变换红外光谱(FT-IR)对CurNisNps的物理化学性质进行表征;
S3:使用透析囊法评估CurNisNps的体外释放行为;
S4:使用紫外-可见分光光度计评估CurNisNps的稳定性和抗氧化活性;
S5:采用琼脂扩散法研究了CurNisNps的抗菌活性。
S1中SSPS组成配比为:水分为5.8%,粗蛋白为9.2%,粗灰分为8.6%,膳食纤维为66.2%,此外SSPS的糖组成(%w/w)为:鼠李糖:5.0,岩藻糖:3.2,阿拉伯糖:22.6,木糖:3.7,半乳糖:46.1,葡萄糖:1.2,半乳糖醛酸:18.2。
S5中对于CurNisNps的抗菌活性的研究包括体内研究。
体内研究包括:
a,在合成并确认姜黄素-乳酸聚L-乳酸纳米颗粒(CurNisNp)后,评估其细胞毒性和释放时间;
b,在确定了CurNisNp的逐次显著减少(SSR)剂量、光照射时间和针对鲍曼氏杆菌的超声强度后,评估了抗生物膜活性和细胞内活性氧(ROS)的产生;
c,评估了CurNisNp介导的aPSDTSSR对治疗小鼠烧伤创面的抗菌和抗毒作用以及组织病理学检查,并与磺胺嘧啶银(SSD)作为标准治疗组进行了比较。
结果表明,非细胞毒性CurNisNp具有均匀的表面和球形囊泡,持续释放至第14天。通过增加CurNisNp的浓度、光照射时间和超声强度,鲍曼曲霉细胞活力的剂量依赖性降低得以实现。在小鼠中,生物膜生长、基因表达的变化以及通过加速皮肤再上皮化促进伤口愈合的时间依赖性减少。aPSDTSSR组和SSD组之间不仅在抗菌和抗毒活性方面没有显著差异,而且aPSDTSR组的伤口愈合和再上皮化比SSD组更有效。
本体外和体内研究表明,CurNisNp-aPSDT对正常人皮肤成纤维细胞系无任何细胞毒性,可通过ROS生成降低鲍曼曲霉的细胞活力。
通过改变细菌致病相关基因的表达,并通过加速皮肤再上皮化而促进伤口愈合。此外,结果表明,CurNisNp-aPSDT可能是治疗烧伤伤口感染的一种有前途的补充策略。
实施例五
一种姜黄素和乳酸菌肽联合抑菌研究方法,包括以下步骤:
S1:将姜黄素(作为模型生物活性剂)加入由乳酸菌肽介导的SSPS基质中,形成复合纳米颗粒CurNisNps;
S2:通过尺寸、zeta电位、透射电子显微镜(TEM)、微分干涉显微镜(DIM)和傅里叶变换红外光谱(FT-IR)对CurNisNps的物理化学性质进行表征;
S3:使用透析囊法评估CurNisNps的体外释放行为;
S4:使用紫外-可见分光光度计评估CurNisNps的稳定性和抗氧化活性;
S5:采用琼脂扩散法研究了CurNisNps的抗菌活性。
S1中SSPS组成配比为:水分为5.8%,粗蛋白为9.2%,粗灰分为8.6%,膳食纤维为66.2%,此外SSPS的糖组成(%w/w)为:鼠李糖:5.0,岩藻糖:3.2,阿拉伯糖:22.6,木糖:3.7,半乳糖:46.1,葡萄糖:1.2,半乳糖醛酸:18.2。
S5中对于CurNisNps的抗菌活性的研究包括体外研究。
体外研究还包括比较了七种适用于活性食品包装的天然添加剂(乳酸钙、柠檬酸、姜黄素、赤霉酸、大蒜提取物、啤酒花提取物、乳酸菌肽)对李斯特菌、金黄色葡萄球菌和大肠杆菌的抗菌和抗生物膜效果,具体包括以下步骤:
d,从捷克微生物收集中心获得细菌菌株和培养条件革兰氏阳性细菌单核细胞增生李斯特菌、单核细胞增殖李斯特菌、金黄色葡萄球菌和金黄色葡萄杆菌,和革兰氏阴性菌大肠杆菌和大肠杆菌;
e,将细菌储存在胰蛋白胨大豆肉汤中,并在冰箱中添加25%浓度的甘油中,且温度为-80◦C,为了重新培养,将细菌培养物接种到5ml无菌TSB中,并孵育24小时,孵育温度为37◦C;
f,对抗菌物质苯甲酸、山梨酸,乳酸钙,柠檬酸,姜黄素、赤霉酸,大蒜提取物,啤酒花提取物和乳酸菌肽进行了测试,对上述物质根据其产量分为三组:天然(姜黄素、大蒜提取物、啤酒花提取物、乳酸菌肽)、天然(乳酸钙、柠檬酸、赤霉酸)和合成(苯甲酸、山梨酸),合成的用作对照,测试了以下浓度(wt%)的物质(在无菌蒸馏水中稀释;在姜黄素中,将pH调节至8.3–8.5以增加其水溶性):10.0、7.5、5.0、4.0、3.0、2.5、2.0、1.5、1.25、1.0、0.75、0.5、0.25、0.13、0.07;
g,混合物为了测试所选物质(大蒜提取物、赤霉酸、钙、乳酸盐、姜黄素、乳酸菌肽、啤酒花提取物)的相互作用,制备了十种混合物,将最有效浓度的物质以1:1和1:2的比例混合,所有混合物均含有大蒜提取物(浓度与确定的模式值相对应)作为主要成分。
h,根据最小抑制浓度和最小生物膜形成抑制浓度将单个细菌悬浮液调节至0的光密度,将McFarland在Mueller Hinton Broth中的100μl加入无菌96孔微量滴定板中,并与100μl所需浓度的物质混合,单独接种是阳性对照,培养 24小时,前后温度保持37◦C,分光光度法测量620nm处的吸光度,培养前/培养后测量值之间的差异被认为是细菌细胞活力(浮游细胞在物质存在下生长的能力)的指标,测定MIC值,即隔夜培养后抑制可见细菌生长的物质的最低浓度(A620nm<0.01)。
测定并比较了选定的天然和天然物质及其混合物对食品病原体的抗菌和抗生物膜效果。在MIC和MICBF中,它们显著抑制了革兰氏阳性菌(高达98.7%)和大肠杆菌菌株(高达94.3%),此外,大蒜提取物成功地与规定浓度的其他选定物质结合;大蒜提取物与i)赤霉酸、ii)啤酒花提取物和iii)姜黄素的混合物被评估为最强的混合物。所有测试的混合物对革兰氏阳性细菌都非常有效,并且比单独的物质对大肠杆菌菌株更有效。了解抗菌混合物的协同作用对于食品工业应用具有重要价值;因此,当将两种食品添加剂结合到例如功能化食品包装(纳米)材料中时,这一组合策略可以延长产品的保质期,同时由于姜黄素和乳酸菌肽联合抑菌,对李斯特菌、金黄色葡萄球菌和大肠杆菌的抗菌有明显的效果,可效抑制李斯特菌等革兰氏阴性菌、金黄色葡萄球菌等革兰氏阴性菌和大肠杆菌,可用于口腔喷雾,预防被污染的食品导致的感染。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。
Claims (7)
1.一种姜黄素和乳酸菌肽联合抑菌研究方法,其特征在于:包括以下步骤:
S1:将姜黄素(作为模型生物活性剂)加入由乳酸菌肽介导的SSPS基质中,形成复合纳米颗粒CurNisNps;
S2:通过尺寸、zeta电位、透射电子显微镜(TEM)、微分干涉显微镜(DIM)和傅里叶变换红外光谱(FT-IR)对CurNisNps的物理化学性质进行表征;
S3:使用透析囊法评估CurNisNps的体外释放行为;
S4:使用紫外-可见分光光度计评估CurNisNps的稳定性和抗氧化活性;
S5:采用琼脂扩散法研究了CurNisNps的抗菌活性。
2.根据权利要求1所述的一种姜黄素和乳酸菌肽联合抑菌研究方法,其特征在于:所述S1中SSPS组成配比为:水分为5.8%,粗蛋白为9.2%,粗灰分为8.6%,膳食纤维为66.2%,此外SSPS的糖组成(%w/w)为:鼠李糖:5.0,岩藻糖:3.2,阿拉伯糖:22.6,木糖:3.7,半乳糖:46.1,葡萄糖:1.2,半乳糖醛酸:18.2。
3.根据权利要求1所述的一种姜黄素和乳酸菌肽联合抑菌研究方法,其特征在于:所述S5中对于CurNisNps的抗菌活性的研究包括体外研究和体内研究。
4.根据权利要求3所述的一种姜黄素和乳酸菌肽联合抑菌研究方法,其特征在于:所述体外研究包括将CurNisNps掺入正畸丙烯酸树脂中形成研究样品,对变形链球菌和白色念珠菌的力学性能和抗菌活性,之后对不同浓度的CurNisNps丙烯酸树脂样品进行性能研究。
5.根据权利要求4所述的一种姜黄素和乳酸菌肽联合抑菌研究方法,其特征在于:所述体外研究中所研究的CurNisNps丙烯酸树脂样品不同浓度分为1、2、5和10%w/w,对于样品性能研究包括不同的时间间隔评估弯曲强度值、抗菌效果、抗生物膜潜力和抗变形链球菌和白色念珠菌的抗代谢活性。
6.根据权利要求3所述的一种姜黄素和乳酸菌肽联合抑菌研究方法,其特征在于:所述体内研究包括:
a,在合成并确认姜黄素-乳酸聚L-乳酸纳米颗粒(CurNisNp)后,评估其细胞毒性和释放时间;
b,在确定了CurNisNp的逐次显著减少(SSR)剂量、光照射时间和针对鲍曼氏杆菌的超声强度后,评估了抗生物膜活性和细胞内活性氧(ROS)的产生;
c,评估了CurNisNp介导的aPSDTSSR对治疗小鼠烧伤创面的抗菌和抗毒作用以及组织病理学检查,并与磺胺嘧啶银(SSD)作为标准治疗组进行了比较。
7.根据权利要求3所述的一种姜黄素和乳酸菌肽联合抑菌研究方法,其特征在于:所述体内研究包括:所述体外研究还包括比较了七种适用于活性食品包装的天然添加剂(乳酸钙、柠檬酸、姜黄素、赤霉酸、大蒜提取物、啤酒花提取物、乳酸菌肽)对李斯特菌、金黄色葡萄球菌和大肠杆菌的抗菌和抗生物膜效果。
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