CN112831540A - 一种奶粉加工中耐热地衣芽孢杆菌的特异培养基及其应用 - Google Patents
一种奶粉加工中耐热地衣芽孢杆菌的特异培养基及其应用 Download PDFInfo
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Abstract
一种奶粉加工中耐热地衣芽孢杆菌的特异培养基及其应用,属于生物技术领域。该特异培养基包括以下组分:蛋白胨6‑12g/L,甘露醇6‑12g/L,牛肉粉0.5‑1.5g/L,氯化钠5‑15g/L,酚红0.01‑0.03g/L,磷酸二氢钾1‑2g/L,氯化钙0.1‑0.5g/L,琼脂15‑25g/L,硫酸亚铁铵0.1‑0.5g/L,Vc 0.1‑1m/L,50%卵黄乳液3‑8ml,多粘菌素B 10000‑20000IU,pH值7.2±0.1。本发明有益效果为:本发明培养基组方简单,操作方便;实验结果便于观察,分析;提高检测效率和准确率;与传统培养基相比,其成本更低,利于推广应用。
Description
技术领域
本发明属于生物技术领域,具体涉及一种奶粉加工中耐热地衣芽孢杆菌的特异培养基及其应用。
背景技术
婴幼儿是一类特殊的群体,婴幼儿配方奶粉作为除母乳之外的主要膳食营养来源,保证奶粉的质量安全尤为重要。在婴幼儿配方奶粉的安全事件中,微生物污染事件出现也较为频繁,由于原料乳中带有耐热类微生物或者加工过程中某些环节控制不到位导致奶粉中含有芽孢杆菌,这样既造成了奶粉质量问题引起安全事故,又给企业生产带来严重损失造成不利影响。我们对南方某品牌婴幼儿配方奶粉加工中耐热微生物进行了分离鉴定,发现主要的芽孢杆菌为地衣芽孢杆菌。
地衣芽孢杆菌地衣芽孢杆菌(学名:Bacillus licheniformis)是土壤中常见的一种兼氧型革兰氏阳性菌嗜热细菌,地衣芽孢杆菌细胞形态和排列呈杆状、单生,地衣芽孢杆菌生命力极强,可在高温、酸性等严重影响自身生命活动的情况下,以内生孢子芽孢的形式来保护自身不受伤害。内生孢子可存活时间长(不消耗自身养分),当环境条件恢复正常时,其可变回活细胞,继续生长繁衍。目前地衣芽孢杆菌大多数用于调整菌群失调,该菌能产生抗活性物质,并具有独特的生物夺氧作用机制,能抑制其它微生物的生长繁殖。虽然地衣芽孢杆菌作为有益菌被广泛使用,但其同时也是一种重要的食源性条件致病菌,给人类健康和养殖业带来了一定的影响。据国内外报道,几乎所有种类食品加工中都曾检测到地衣芽孢杆菌,而且不同的致病菌株可引起不同疾病。产毒菌株可引起人类和动物食物中毒,中毒后还可导致人眼部发炎、心内膜炎、菌血症等,但最常见的是引起腹泻型和呕吐型食物中毒。随着人们对芽孢杆菌类细菌的研究,发现芽孢杆菌自身存在很多安全性问题。经有研究表明,芽孢杆菌能产生耐药性,也产生肠毒素,呕吐毒素等,对动物体、人体产生不良影响。虽然地衣芽孢杆菌长期以来被认为安全的菌种,也有相关报道称其可产生肠毒素。经研究证实,存在毒性与地衣芽孢杆菌中存在毒力基因有关。在奶粉加工中地衣芽孢杆菌是一种典型的能够严重影响奶粉品质和带来食品安全风险的一类有害微生物。
随着关于地衣芽孢杆菌感染病例的报道逐渐增多,越来越多相应的检测方法也被迅速建立。传统检测方法、分子学检测方法、免疫学检测方法都有各自的优缺点,需要根据检测的需要和条件限制进行选择,从而发挥每个检测方法的最大优势。目前已报道的芽胞杆菌属的检测方法有分离培养生化鉴定法、普通 PCR、多重PCR、荧光PCR、LAMP 及基因芯片等,传统的细菌检测方法是通过其生物学特性进行鉴定,主要包括增菌培养、分离纯化、菌落形态观察、革兰氏染色镜检、生化鉴定和生化分型几个步骤。但是分离培养生化鉴定法和多重PCR、荧光PCR、LAMP基因芯片技术操作繁琐,有的检测时间相对较长,有的要求过高。
发明内容
针对现有技术存在的问题,本发明的目的在于设计提供一种奶粉加工中耐热地衣芽孢杆菌的特异培养基及其应用的技术方案。
本发明通过以下技术手段解决上述技术问题:
所述的一种奶粉加工中耐热地衣芽孢杆菌的特异培养基,其特征在于包括以下组分:蛋白胨6-12g/L,甘露醇6-12g/L,牛肉粉0.5-1.5g/L,氯化钠5-15g/L,酚红 0.01-0.03g/L,磷酸二氢钾1-2g/L,氯化钙0.1-0.5g/L,琼脂15-25g/L,硫酸亚铁铵0.1-0.5g/L,Vc 0.1-1m/L,50%卵黄乳液3-8ml,多粘菌素B 10000-20000IU,pH值7.2±0.1。
所述的一种奶粉加工中耐热地衣芽孢杆菌的特异培养基,其特征在于包括以下组分:蛋白胨8-10g/L,甘露醇8-10g/L,牛肉粉0.8-1.2g/L,氯化钠8-12g/L,酚红 0.01-0.03g/L,磷酸二氢钾1-2g/L,氯化钙0.2-0.4g/L,琼脂18-22g/L,硫酸亚铁铵0.2-0.4g/L,Vc 0.2-0.81m/L,50%卵黄乳液4-6ml,多粘菌素B 12000-18000IU,pH值7.2±0.1。
所述的特异培养基在奶粉加工中快速检测耐热地衣芽孢杆菌的应用。
利用所述的特异培养基快速检测耐热地衣芽孢杆菌的方法,其特征在于包括以下步骤:
取适量奶粉在60-80℃加热20-30min,冷却后涂布于特异培养基中培养,培养后平板上生长的紫红色小落,且菌落周围出现黄色透明圈,即可确定该奶粉中含有耐热地衣芽孢杆菌。
所述的方法,其特征在于培养条件为:50-60℃培养箱中培养18-24小时。
所述的方法,其特征在于对耐热地衣芽孢杆菌进行验证鉴定,提取该菌的DNA,以提取得到的DNA为模板,采用特异性引物进行PCR扩增,所述的特异性引物F1的核苷酸序列如SEQ ID No.1所示,特异性引物F2的核苷酸序列如SEQ ID No.2所示,若扩增产物电泳显示为400bp大小条带,即可明确确定该菌为地衣芽孢杆菌菌株。
所述的方法,其特征在于PCR反应体系为:10×含20mmol/L Mg2+的Buffer 2μL、2.5mmol/L的 dNTPs1.5μL、0.25μmol/L 的引物F1和0.25μmol/L 的引物F2各1μL、5U/μL的Taq DNA聚合酶0.5μL、DNA模板2μL、双或三蒸水12μL。
所述的方法,其特征在于PCR程序为:变性温度95℃、3分钟;后35个循环为94℃、30秒;56℃、30秒;72℃、70秒;延伸温度72℃、10分钟。
本发明有益效果为:本发明培养基组方简单,操作方便;实验结果便于观察,分析;提高检测效率和准确率;与传统培养基相比,其成本更低,利于推广应用。
具体实施方式
以下将结合具体实施例对本发明进行详细说明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件,或者按照制造厂商所建议的条件。
实施例1:一种奶粉加工中耐热地衣芽孢杆菌的特异培养基
该培养基包括以下组分:蛋白胨10g/L,甘露醇10g/L,牛肉粉1.2g/L,氯化钠10g/L,酚红 0.02g/L,磷酸二氢钾1.5g/L,氯化钙0.3g/L,琼脂20g/L,硫酸亚铁铵0.3g/L,Vc0.5m/L,50%卵黄乳液5ml,多粘菌素B 15000IU,pH值7.2。
将蛋白胨,甘露醇,牛肉粉,氯化钠,酚红,磷酸二氢钾,氯化钙,琼脂和Vc称量放入1000ml烧杯中,先用600ml水溶解,定容至1000ml,调节pH为7.2,高压蒸汽121℃灭菌20分钟,灭菌完成后取出,冷却至40-50℃,加入过滤除菌的硫酸亚铁铵,卵黄乳液及多粘菌素B,摇匀后倾入无菌平皿,平板凝固后放置做鉴定菌株备用。
实施例2:一种奶粉加工中耐热地衣芽孢杆菌的特异培养基
该培养基包括以下组分:蛋白胨6g/L,甘露醇6g/L,牛肉粉0.5g/L,氯化钠5g/L,酚红 0.01g/L,磷酸二氢钾1g/L,氯化钙0.1g/L,琼脂15g/L,硫酸亚铁铵0.1g/L,Vc 0.1m/L,50%卵黄乳液3ml,多粘菌素B 10000IU,pH值7.2±0.1。
实施例3:一种奶粉加工中耐热地衣芽孢杆菌的特异培养基
蛋白胨12g/L,甘露醇12g/L,牛肉粉1.5g/L,氯化钠15g/L,酚红 0.03g/L,磷酸二氢钾2g/L,氯化钙0.5g/L,琼脂25g/L,硫酸亚铁铵0.5g/L,Vc 1m/L,50%卵黄乳液8ml,多粘菌素B 20000IU,pH值7.2±0.1。
实施例4:地衣芽孢杆菌显色培养
将活化后的地衣芽孢杆菌、大肠杆菌和金黄色葡萄球菌分别涂布于上实施例1得到的特异培养基中,55℃培养18h。
结果:
菌种类别 | 生长状况 | 菌落颜色 |
地衣芽孢杆菌 | +++ | 紫红色菌落(蓝色透明圈) |
金黄色葡萄球菌 | ﹣ | 无 |
大肠杆菌 | - | 无 |
由此可见,在特异培养基上地衣芽孢杆菌培养后平板上生长的紫红色小落,且菌落周围出现黄色透明圈。
同样,采用实施例2和3得到的特异培养基进行实施例4相同的显色培养,同样,地衣芽孢杆菌培养后在平板上生长的紫红色小落,且菌落周围出现黄色透明圈。
实施例5:验证鉴定
将实施例4得到的待鉴定菌株接入盛有70ml上述培养基的150ml三角烧瓶中,在50-60℃、150转/分钟的恒温摇床中培养20小时,按照下列方法提取该菌的DNA:
(1)1.5 mL培养液,12000rpm离心1分钟,收集细菌菌体。
(2)加584μL TE悬浮沉淀,并加30.7μL 10% SDS,10μL溶菌酶,混匀,37℃保温1小时,95℃水浴保温5分钟。
(3)加92.3μL 5mol/L NaCl,92.3μL CTAB/NaCl溶液,混匀,65℃保温20分钟。
(4)用等体积氯仿:异戊醇(24:1)抽提,11000rpm离心10分钟,将上清液移至干净离心管。
(5)用等体积氯仿:异戊醇(24:1)抽提,静置5分钟左右,11000rpm离心10分钟,上清液移至干净离心管。
(6)加一倍体积无水乙醇,颠倒混合,-20℃放置10分钟,12000rpm离心8分钟,沉淀DNA。
(7)去液体,加70%乙醇漂洗2次,离心5~6秒,干燥后加30μL双蒸水,震荡混匀,离心5~6秒,加入DNA保存液10ul作为DNA模板保存液,4℃保存。
(8)根据地衣芽孢杆菌16sRNA设计特异性引物F1和F2,特异性引物F1 5′-GAGGAAGGTGGGGATGACGT-3′(如SEQ ID No.1所示)和F2 5′-GCCAAGGCATCCACC-3′(如SEQ IDNo.2所示),在下列条件下进行PCR扩增:待检测菌株DNA: 10×Buffer(含20mmol/L Mg2+)2μL、dNTPs(2.5mmol/L)1.5μL、上F1和F2引物(0.25μmol/L)各1μL、Taq DNA聚合酶(5U/μL)0.5μL、DNA模板2μL、双或三蒸水12μL,扩增条件控制为变性温度95℃、3分钟;后35个循环为94℃、30秒;56℃、30秒;72℃、70秒;延伸温度72℃、10分钟。
(9)用微量移液枪小心将PCR反应后的样品液加入琼脂糖胶板样品槽中,总体积不可超过样品槽容量。每加完一个样品要更换枪头,以防止互相污染,注意上样时要小心操作,避免损坏凝胶或将样品槽底部凝胶刺穿;加完样后,放入电泳槽中,立即接通电源,控制电压保持在60~80V,电流在40mA以上。当溴酚蓝条带移动到距凝胶前约2cm时,停止电泳;电泳完毕后,将凝胶移入0.5μg/mL的EB溶液中,室温下染色20~25分钟;用特异性引物扩增后电泳检测400bp大小,即可鉴定该菌为地衣芽孢杆菌菌株。
实施例6:应用
取适量需要检测的奶粉在60-80℃加热20-30min,冷却后涂布于特异培养基中培养,若培养后平板上生长的紫红色小落,且菌落周围出现黄色透明圈,即可确定该奶粉中含有耐热地衣芽孢杆菌。
再对平板上得到的菌落进行如实施例5相同的验证鉴定,若扩增产物电泳显示为400bp大小条带,即可明确确定该菌为地衣芽孢杆菌菌株。
序列表
<110> 贝因美(杭州)食品研究院有限公司
浙江科技学院
<120> 一种奶粉加工中耐热地衣芽孢杆菌的特异培养基及其应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> 引物(primer)
<400> 1
gaggaaggtg gggatgacgt 20
<210> 2
<211> 15
<212> DNA
<213> 引物(primer)
<400> 2
gccaaggcat ccacc 15
Claims (8)
1.一种奶粉加工中耐热地衣芽孢杆菌的特异培养基,其特征在于包括以下组分:蛋白胨6-12g/L,甘露醇6-12g/L,牛肉粉0.5-1.5g/L,氯化钠5-15g/L,酚红 0.01-0.03g/L,磷酸二氢钾1-2g/L,氯化钙0.1-0.5g/L,琼脂15-25g/L,硫酸亚铁铵0.1-0.5g/L,Vc 0.1-1m/L,50%卵黄乳液3-8ml,多粘菌素B 10000-20000IU,pH值7.2±0.1。
2.如权利要求1所述的一种奶粉加工中耐热地衣芽孢杆菌的特异培养基,其特征在于包括以下组分:蛋白胨8-10g/L,甘露醇8-10g/L,牛肉粉0.8-1.2g/L,氯化钠8-12g/L,酚红0.01-0.03g/L,磷酸二氢钾1-2g/L,氯化钙0.2-0.4g/L,琼脂18-22g/L,硫酸亚铁铵0.2-0.4g/L,Vc 0.2-0.81m/L,50%卵黄乳液4-6ml,多粘菌素B 12000-18000IU,pH值7.2±0.1。
3.如权利要求1或2所述的特异培养基在奶粉加工中快速检测耐热地衣芽孢杆菌的应用。
4.利用权利要求1或2所述的特异培养基快速检测耐热地衣芽孢杆菌的方法,其特征在于包括以下步骤:
取适量奶粉在60-80℃加热20-30min,冷却后涂布于特异培养基中培养,培养后平板上生长的紫红色小落,且菌落周围出现黄色透明圈,即可确定该奶粉中含有耐热地衣芽孢杆菌。
5.如权利要求4所述的方法,其特征在于培养条件为:50-60℃培养箱中培养18-24小时。
6.如权利要求4所述的方法,其特征在于对耐热地衣芽孢杆菌进行验证鉴定,提取该菌的DNA,以提取得到的DNA为模板,采用特异性引物进行PCR扩增,所述的特异性引物F1的核苷酸序列如SEQ ID No.1所示,特异性引物F2的核苷酸序列如SEQ ID No.2所示,若扩增产物电泳显示为400bp大小条带,即可明确确定该菌为地衣芽孢杆菌菌株。
7.如权利要求6所述的方法,其特征在于PCR反应体系为:10×含20mmol/L Mg2+的Buffer 2μL、2.5mmol/L的 dNTPs1.5μL、0.25μmol/L 的引物F1和0.25μmol/L 的引物F2各1μL、5U/μL的 Taq DNA聚合酶0.5μL、DNA模板2μL、双或三蒸水12μL。
8.如权利要求6所述的方法,其特征在于PCR程序为:变性温度95℃、3分钟;后35个循环为94℃、30秒;56℃、30秒;72℃、70秒;延伸温度72℃、10分钟。
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