CN109095510A - 四氧化三铁纳米颗粒及其制备方法和在抑制沙门氏菌增殖中的应用 - Google Patents
四氧化三铁纳米颗粒及其制备方法和在抑制沙门氏菌增殖中的应用 Download PDFInfo
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Abstract
本发明公开了一种四氧化三铁纳米颗粒及其制备方法及在抑制沙门氏菌增殖中的应用,将四氧化三铁纳米颗粒添加到饲料中用来抗家禽的沙门氏菌感染。本发明首次将四氧化三铁纳米颗粒应用在抗家禽的沙门氏菌感染;本发明采用在育雏期饲料中添加四氧化三铁纳米颗粒,可以显著降低感染沙门氏菌肉鸡体内的沙门氏菌载菌量,并对肝组织有利好作用,不产生副作用,使用成本较低,使用过程方便,生产过程简单,本发明将为抗沙门氏菌的治疗开启新的途径,也为开发抗沙门氏菌的药物提供了新的研究思路。
Description
技术领域
本发明涉及一种纳米酶的应用,具体涉及一种四氧化三铁纳米颗粒及其制备方法和在抑制沙门氏菌增值中的应用。
背景技术
纳米酶颗粒是近两年新兴的一种纳米材料,具有制备成本低,方法简单且易保存的优点,目前已被证实具有优良的抗菌活性,而且与现有抗生素相比很难诱导细菌产生耐药性,主要是因为纳米材料的杀伤作用是多方面的,细菌难以产生有针对性的抗药性,且纳米酶具有在低剂量使用时即可有效抑制多种细菌的增殖,又不降低细胞本身的活性,具有较高的生物安全性和环境友好性。铁离子作为机体免疫应答反应过程中各种信号物质的结构域,在机体的免疫反应中起到至关重要的作用。四氧化三铁纳米颗粒作为纳米酶的一种,已被证实可在体外条件下对部分革兰氏阴性菌有抑制作用,但其作为饲料添加剂,在动物体内的杀菌作用目前未见报道和证实。
肠炎沙门氏菌(S.Enteritidis)作为重要的人兽共患的肠道致病菌,不仅可以感染家禽引起各种疾病,还能通过污染的禽肉造成人类沙门氏菌感染,引起食物中毒和各种食源性疾病,直接威胁人体的健康。同时,由于沙门氏菌在禽类生产上有垂直传播的特性,其可经由母体直接传给下一代,造成禽场一旦发现有沙门氏菌就很难进行净化的局面。沙门氏菌主要引起禽类的胃肠炎,一般借由粪口途径侵染雏鸡,定殖在消化道后,可不断增值并侵袭体内包括生殖系统在内的各脏器,由此经禽蛋产品传播到人类,引起肠炎和食物中毒。雏鸡由于其免疫系统未发育健全,极易感染环境中存在的沙门氏菌,引发急性感染,造成较高的发病率与死亡率,而存活的感染雏鸡在性成熟以后虽然很难再表现沙门氏菌病症,但其已成为病菌的隐形携带者,在生产上容易被忽视;继而通过生殖道和消化道组织排毒,造成禽蛋污染则可能引发商品代肉鸡的感染和食物源性安全事件的发生。
国内外对沙门氏菌防治工作都非常重视,也研究出了许多种疫苗和净化措施,但沙门氏菌感染在人和动物中仍然时有发生。目前传统防治禽类沙门氏菌的方法有:①使用灭活疫苗或弱毒疫苗等物质进行提前防治;②使用抗生素进行治疗;③对种鸡群进行净化。现有技术中,疫苗的使用只能进行防治,无法在鸡群感染后对其有效治疗,而抗生素的使用则直接导致鸡群的耐药性提高,另外,对种鸡群的沙门氏菌净化代价较高,投入成本较大。由于目前畜牧业提倡无抗生产,目前已全面禁止在畜禽生产中使用抗生素,因此,寻找抗生素替代品的任务刻不容缓。
发明内容
为了解决以上技术问题,本发明提供了一种四氧化三铁纳米颗粒的制备方法与应用,将该四氧化三铁纳米颗粒用作抗沙门氏菌的饲料添加剂。
为实现上述目的,本发明提供了一种四氧化三铁纳米颗粒的制备方法,该方法包括以下步骤:
(1)将三氯化铁溶解于乙烯乙二醇溶液中,后加入醋酸钠,充分搅拌后加到水热反应釜中,通入氮气,在温度160~200℃的条件下反应6~12h生成沉淀产物;
(2)将沉淀产物冷却至室温,机械搅拌后进行超声波分散;
(3)将分散后的产物用蒸馏水及乙醇交叉洗涤,真空干燥后,得到四氧化三铁纳米颗粒。
进一步的,步骤(1)中,三氯化铁与乙烯乙二醇溶液的质量体积比为0.8-0.85:40-50,三氯化铁与醋酸钠的质量比为0.8-0.85:3.5-4。
进一步的,步骤(2)中,超声波的频率为40khz,超声波分散的时间为4-6min。
进一步的,步骤(3)中,交叉洗涤至上清液的pH为6.8-7.2。
本发明还提供了上述任一项的制备方法制得的四氧化三铁纳米颗粒。
其中,四氧化三铁纳米颗粒的平均直径为200-300nm。
本发明还提供了所述的四氧化三铁纳米颗粒的应用,将四氧化三铁纳米颗粒用作抗沙门氏菌的饲料添加剂。
优选的,四氧化三铁纳米颗粒在饲料中的质量添加量为0.005-0.015%。
本发明的有益效果为:本发明通过水热法合成特定粒径大小的四氧化铁,首次将四氧化三铁纳米颗粒应用于家禽的抗菌感染;本发明采用在育雏饲料中添加四氧化三铁纳米颗粒,可以显著降低家禽的沙门氏菌感染,且四氧化三铁纳米颗粒的添加量较低,对降低肝脏的病理变化有利好作用,同时不对其产生副作用,使用成本较低,使用过程方便,生产过程简单,本发明方法将为抗沙门氏菌的治疗开启新的途径,也为开发抗沙门氏菌的药物提供了新的研究思路。
附图说明
图1为本发明实施例2得到的四氧化三铁纳米颗粒的扫描电镜图;
图2为本发明中四氧化三铁纳米颗粒催化H2O2的酶动力学反应图;
图3为本发明中四氧化三铁纳米颗粒催化TMB的酶动力学反应图;
图4为本发明处理组中雏鸡肝脏的载菌量结果;
图5为本发明对照组中雏鸡肝脏的病理切片的电镜图;
图6为本发明添加组1中雏鸡肝脏的病理切片的电镜图。
具体实施方式
下面结合附图和实施例对本发明进行详细地解释说明。
实施例1
一种四氧化三铁纳米颗粒的制备方法包括以下步骤:
(1)将0.8g四氧化三铁溶解在40mL的乙烯乙二醇溶液(质量分数为60%)中,后加入3.5g醋酸钠,充分搅拌后加到水热反应釜中,通入氮气,在温度160℃的条件下反应12h生成沉淀产物;
(2)将沉淀产物冷却至室温,机械搅拌后进行超声波分散,超声波的频率为40khz,超声波分散的时间为4min;
(3)将分散后的产物用蒸馏水及乙醇交叉洗涤,洗涤至上清液的pH为6.8,真空干燥后,得到四氧化三铁纳米颗粒,四氧化三铁纳米颗粒的平均直径为250nm。
实施例2
一种四氧化三铁纳米颗粒的制备方法包括以下步骤:
(1)将0.82g四氧化三铁溶解在45mL的乙烯乙二醇溶液(质量分数为60%)中,后加入3.6g醋酸钠,充分搅拌后加到水热反应釜中,通入氮气,在温度180℃的条件下反应8h生成沉淀产物;
(2)将沉淀产物冷却至室温,机械搅拌后进行超声波分散,超声波的频率为40khz,超声波分散的时间为5min;
(3)将分散后的产物用蒸馏水及乙醇交叉洗涤,洗涤至上清液的pH为7.0,真空干燥后,得到四氧化三铁纳米颗粒,四氧化三铁纳米颗粒的平均直径为200nm。
实施例3
一种四氧化三铁纳米颗粒的制备方法包括以下步骤:
(1)将0.85g四氧化三铁溶解在50mL的乙烯乙二醇溶液(质量分数为60%)中,后加入4.0g醋酸钠,充分搅拌后加到水热反应釜中,通入氮气,在温度200℃的条件下反应6h生成沉淀产物;
(2)将沉淀产物冷却至室温,机械搅拌后进行超声波分散,超声波的频率为40khz,超声波分散的时间为6min;
(3)将分散后的产物用蒸馏水及乙醇交叉洗涤,洗涤至上清液的pH为6.8,真空干燥后,得到四氧化三铁纳米颗粒,四氧化三铁纳米颗粒的平均直径为300nm。
实施例2得到的四氧化三铁纳米颗粒的扫描电镜图如图1所示,由图1可知,四氧化三铁纳米颗粒的粒径为200nm,颗粒均匀,粒径越小的四氧化三铁纳米颗粒在相同质量下具有更高的比表面积,因此表现出的类过氧化物酶活性越高。将实施例2得到的四氧化三铁纳米颗粒进行后续试验。
使用四氧化三铁纳米颗粒催化双氧水(H2O2)与3,3’,5,5’-四甲基对二氨基联苯(TMB)发生颜色反应,同时增加H2O2或TMB的浓度,测定酶动力学反应参数,结果如图2-3所示。由图2-3的结果可知,四氧化三铁纳米颗粒的表面电荷丰富,容易吸附带正电的TMB,造成较高的亲和力。四氧化三铁纳米颗粒表现出的类过氧化物酶活性可提高机体内的氧化应激活性,提高机体对沙门氏菌增殖的抵抗能力。
将四氧化三铁纳米颗粒颗粒均匀地与日粮核心料充分混合,然后与日粮原料(如表1所示)充分混合,得到含量为0.005%的四氧化三铁纳米颗粒添加的饲料,装袋后避光保存于干燥处待用。
表1 SPF鸡育雏期饲料配方
试验设计:
试验选用0日龄海兰褐蛋鸡180只,平均分成三组,每组60只,试验周期为9天,从0日龄至9日龄,按照四氧化三铁纳米颗粒颗粒的添加分为对照组及四氧化三铁纳米颗粒添加组1和添加组2,对照组在整个试验期饲喂基础饲粮,育雏期添加组1在育雏期饲喂添加含量为0.005%四氧化三铁纳米颗粒饲料,育雏期添加组2在育雏期饲喂添加含量为0.015%四氧化三铁纳米颗粒饲料。
7日龄对所有雏鸡进行沙门氏菌攻毒,且于攻毒后12小时取雏鸡肝脏、脾脏、盲肠食糜及血液中的沙门氏菌含量进行载菌量检测和病理切片检测,且于攻毒后12、24和48小时取雏鸡肝脏进行载菌量检测和病理切片检测。
本发明对照组、添加组1、添加组2中的雏鸡肝脏、肝脏、肝脏及血液中的沙门氏菌含量(LOG CFU/g)的测定结果如表2所示。
表2
在12、24和48时对对照组、添加组1、添加组2中的雏鸡的肝脏切片进行载菌量检测,检测结果如图4所示。病理切片检测结果如图5-6所示:48h对照组肝脏组织出现较多空泡,组织松散,添加组1的雏鸡肝脏组织空泡较少,肝实质清晰。由以上结果可知,本发明采用的四氧化三铁纳米颗粒可以显著降低感染雏鸡的沙门氏菌增殖,降低沙门氏菌对雏鸡肝脏的侵蚀。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明实质内容上所作的任何修改、等同替换和简单改进等,均应包含在本发明的保护范围之内。
Claims (9)
1.一种四氧化三铁纳米颗粒的制备方法,其特征在于,该方法包括以下步骤:
(1)将三氯化铁溶解于乙烯乙二醇溶液中,后加入醋酸钠,充分搅拌后加到水热反应釜中,通入氮气,在温度160~200℃的条件下反应6~12h生成沉淀产物;
(2)将沉淀产物冷却至室温,机械搅拌后进行超声波分散;
(3)将分散后的产物用蒸馏水及乙醇交叉洗涤,真空干燥后,得到四氧化三铁纳米颗粒。
2.根据权利要求1所述的四氧化三铁纳米颗粒的制备方法,其特征在于,步骤(1)中,三氯化铁与乙烯乙二醇溶液的质量体积比为0.8-0.85:40-50,三氯化铁与醋酸钠的质量比为0.8-0.85:3.5-4。
3.根据权利要求1所述的四氧化三铁纳米颗粒的制备方法,其特征在于,步骤(2)中,超声波的频率为40khz,超声波分散的时间为4-6min。
4.根据权利要求1所述的四氧化三铁纳米颗粒的制备方法,其特征在于,步骤(3)中,交叉洗涤至上清液的pH为6.8-7.2。
5.权利要求1-4任一项所述的制备方法制得的四氧化三铁纳米颗粒。
6.根据权利要求5所述的四氧化三铁纳米颗粒,其特征在于,四氧化三铁纳米颗粒的平均直径为200-300nm。
7.权利要求5或6所述的四氧化三铁纳米颗粒在制备抗沙门氏菌饲料中的应用。
8.根据权利要求7所述的四氧化三铁纳米颗粒的应用,其特征在于,四氧化三铁纳米颗粒在饲料中的质量添加量为0.005-0.015%。
9.权利要求5或6所述的四氧化三铁纳米颗粒在制备抗沙门氏菌药物中的应用。
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