CN104894040B - 食品中沙门氏菌一步法选择性增菌培养基及其制备方法 - Google Patents
食品中沙门氏菌一步法选择性增菌培养基及其制备方法 Download PDFInfo
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Abstract
本发明属于沙门氏菌的选择性增菌培养基,涉及到一种用于富集食品中数量极少且对受损伤细胞具有修复作用的选择性增菌培养基。其特征在于,以质量份数计,其原料配方组成为:胰蛋白胨2.5‑7.5份,鱼粉蛋白胨2.5‑7.5份,牛肉浸膏2‑8份,氯化钠2.5‑7.5份,磷酸氢二钠1‑2份,磷酸二氢钾6‑12份,甘露醇1‑3份,丙酮酸钠5‑15份,阿卡波糖0.2‑1份,胆盐5号1.25‑5份,硫代硫酸钠10‑30份,蒸馏水1000份,pH值为7.2~7.5。
Description
技术领域
本发明属于沙门氏菌的选择性增菌培养基,涉及到一种用于富集食品中数量极少且对受损伤细胞具有修复作用的选择性增菌培养基。
技术背景
沙门氏菌(Salmonella spp.)是肠杆菌科中能引起食源性疾病的重要致病菌。我国细菌性食物中毒事件有70%~80%是由沙门氏菌引起的,沙门氏菌引起的食物中毒常常占据细菌性食物中毒的首位。
不论是标准化学检验协会(AOAC),国际标准化组织(ISO),还是国标GB4789.4-2010中对食品中沙门氏菌的检测都要经过前增菌和增菌两个步骤对样品中沙门氏菌进行富集培养,然后再进行后续的分离、生理生化及血清学鉴定,这通常要耗费大量的时间和精力,不同的增菌方法也都可能会影响检测结果,并且受冷热环境等因素的影响沙门氏菌通常处于亚致死状态,所以经常会导致以增菌失败而漏检。当前的增菌培养基有很多,主要是对多种致病微生物的复合增菌,不同致病菌在统一环境下生长会相互抑制,目前针对食品中单一致病菌的一步增菌培养基很少,所以对食品中特定致病菌增菌培养基的研究具有重要意义。
发明内容
本发明的目的在于提供一种可快速培养食品中数量极少且受亚致死损伤的沙门氏菌增菌的培养基,适用于后续荧光定量PCR的检测。
本发明的目的通过如下技术方案实现:
一种食品中沙门氏菌快速增菌培养基,以质量份数计,其配方组成为:胰蛋白胨2.5-7.5份,鱼粉蛋白胨2.5-7.5份,牛肉浸膏2-8份,氯化钠2.5-7.5份,磷酸氢二钠1-2份,磷酸二氢钾6-12份,甘露醇1-3份,丙酮酸钠5-15份,阿卡波糖0.2-1份,胆盐5号1.25-5份,硫代硫酸钠10-30份,蒸馏水1000份,pH值为7.2~7.5。
本发明的优选配方为,以质量份数计:胰蛋白胨5份,鱼粉蛋白胨5份,牛肉浸膏4份,氯化钠5份,磷酸氢二钠1.5份,磷酸二氢钾9份,甘露醇2份,丙酮酸钠8份,阿卡波糖0.3份,胆盐5号2.5份,硫代硫酸钠25份,蒸馏水1000份,pH值为7.2。
本发明的又一个目的是提供沙门氏菌增菌培养基制备方法,它是由以下步骤制成:
(1)按照比例将胰蛋白胨,鱼粉蛋白胨,牛肉浸膏,氯化钠,磷酸氢二钠,十二水合磷酸二氢钾,甘露醇,丙酮酸钠,阿卡波糖,胆盐5号,硫代硫酸钠加至蒸馏水中搅拌加热溶解。
(2)冷却至室温后,用1 mol/L NaOH调节pH为7.2~7.5。
(3)121℃高压灭菌15 min。
本发明以BPW,LB,NB,TSB为基础培养基,选择其中合适的组分进行混配,得到基础培养基。蛋白胨,鱼粉蛋白胨,牛肉浸膏为细菌生长提供必需的碳源,氮源,无机盐,生长因子等营养成分。磷酸氢二钠和磷酸二氢钾作为缓冲试剂可以保持培养基中pH保持稳定。再选择甘露醇,丙酮酸钠作为生长促进剂,其中丙酮酸钠具有修复受损伤细菌及促进沙门氏菌生长的作用,阿卡波糖,胆盐5号,硫代硫酸钠作为杂菌生长抑制剂。
相对于现有技术,本发明具有以下优点:
本发明能融合食品中沙门氏菌检测前增菌及选择性增菌两个步骤,将增菌时间缩短到12-16 h,并且沙门氏菌在增殖的同时其他微生物受到一定程度的抑制。
本发明按照配方和制备方法配制的增菌培养基可直接做沙门氏菌的分离培养及鉴定,也可直接用于后续荧光定量PCR的检测。
具体实施方式
为更好理解本发明,下面结合实施例对本发明做进一步地详细说明,但是本发明要求保护的范围并不局限于实施例表示的范围
实施例1
称取胰蛋白胨 2.5 g,鱼粉蛋白胨 7.5 g,牛肉浸膏2 g,氯化钠7.5 g,磷酸氢二钠 1 g,磷酸二氢钾 12 g,甘露醇1 g,丙酮酸钠 15 g,阿卡波糖 0.2 g,胆盐5号 5 g,硫代硫酸钠 10 g,蒸馏水1000 mL。
将胰蛋白胨、鱼粉蛋白胨、牛肉浸膏、氯化钠、磷酸氢二钠、磷酸二氢钾、甘露醇、丙酮酸钠、阿卡波糖、3号胆盐、硫代硫酸钠、蒸馏水搅拌溶解,用1 mol/L NaOH调pH至7.2,121℃高压灭菌15 min,常温下保存。
将菌体浓度为109 CFU/L的沙门氏菌培养液稀释106倍,然后取1 mL接入99 mL制备好的用于沙门氏菌增菌培养基中,使培养基中沙门氏菌菌体浓度为101 CFU/L,在37℃,200r/min摇床培养12和16 h,取增菌培养后的增菌液50uL进行适当梯度稀释后涂布到TTB选择性培养基平板培养24-48 h后进行菌落计数。结果见表1.
从表1可知,经过12-16 h的增菌,沙门氏菌的菌体浓度由10 CFU/mL增至108 CFU/mL~109 CFU/mL,满足后期PCR检测需要。
实施例 2
称取胰蛋白胨 7.5 g,鱼粉蛋白胨 2.5 g,牛肉浸膏 8 g,氯化钠 2.5 g,磷酸氢二钠 2 g,磷酸二氢钾 6 g,甘露醇 3 g,丙酮酸钠 5 g,阿卡波糖 1 g,胆盐5号 1.25 g,硫代硫酸钠 30 g,蒸馏水1000份。
将胰蛋白胨、鱼粉蛋白胨、牛肉浸膏、氯化钠、磷酸氢二钠、磷酸二氢钾、甘露醇、丙酮酸钠、阿卡波糖、3号胆盐、硫代硫酸钠、蒸馏水搅拌溶解,1 mol/L NaOH调pH至7.5,121℃高压灭菌15 min,常温下保存。
将菌体浓度为109 CFU/L的沙门氏菌培养液稀释106倍,然后取1 mL接入99 mL制备好的用于沙门氏菌增菌培养基中,使培养基中沙门氏菌菌体浓度为101 CFU/L,在37℃,200r/min摇床培养12和16 h,取增菌培养后的增菌液50uL进行适当梯度稀释后涂布到TTB琼脂平板培养24-48 h后进行菌落计数。结果见表2.
从表1可知,经过12-16 h的增菌,沙门氏菌的菌体浓度由10 CFU/mL增至108 CFU/mL~109 CFU/mL,满足后期PCR检测需要。
实施例3
称取胰蛋白胨 5 g,鱼粉蛋白胨 5 g,牛肉浸膏 4 g,氯化钠 5 g,磷酸氢二钠1.5 g,磷酸二氢钾 9 g,甘露醇 2 g,丙酮酸钠 10 g,阿卡波糖 0.6 g,3号胆盐 2.5 g,硫代硫酸钠 25 g,蒸馏水1000 mL。
将胰蛋白胨、鱼粉蛋白胨、牛肉浸膏、氯化钠、磷酸氢二钠、磷酸二氢钾、甘露醇、丙酮酸钠、阿卡波糖、3号胆盐、硫代硫酸钠、蒸馏水搅拌溶解, 1mol/L NaOH调pH至7.3,121℃高压灭菌15 min,常温下保存。
将菌体浓度为109 CFU/L的沙门氏菌培养液稀释106倍,然后取1 mL接入99 mL制备好的用于沙门氏菌增菌培养基中,使培养基中沙门氏菌菌体浓度101 CFU/L,在37℃,200 r/min摇床培养12和16 h,取增菌培养后的增菌液50uL进行适当梯度稀释后涂布到TTB琼脂平板培养24-48 h后进行菌落计数。结果见表3
Claims (1)
1.用于食品中沙门氏菌一步法选择性增菌培养基,其特征在于,以质量份数计,其原料配方组成为:胰蛋白胨2.5-7.5份,鱼粉蛋白胨2.5-7.5份,牛肉浸膏2-8份,氯化钠2.5-7.5份,磷酸氢二钠1-2份,磷酸二氢钾6-12份,甘露醇1-3份,丙酮酸钠5-15份,阿卡波糖0.2-1份,胆盐5号1.25-5份,硫代硫酸钠10-30份,蒸馏水1000份,pH值为7.2~7.5;以质量份数计;用于沙门氏菌一步法选择性增菌培养基的制备方法,包括如下步骤:
(1)将胰蛋白胨,鱼粉蛋白胨,牛肉浸膏,氯化钠,磷酸氢二钠,磷酸二氢钾,甘露醇,丙酮酸钠,阿卡波糖,胆盐5号,硫代硫酸钠加入到蒸馏水中,微加热助溶;
(2)冷却至室温后,用1mol/L NaOH调节pH为7.2~7.5;
(3)121℃高压灭菌15min。
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