应用嗜热脂肪芽胞杆菌检测原料奶中抗生素残留的研究
摘要
本文主要应用包埋的嗜热脂肪芽胞杆菌芽胞检测牛奶中的抗生素残留,对营养物质、指示剂添加量、包埋条件以及检测条件等指标进行了研究,以提高此方法的灵敏度与稳定性。然后通过与小样发酵法、国标TTC法、试剂盒(ECLIPSE50)法、试纸条法四种方法作对照,统计阳性率,进一步确认本研究方法的可行性与灵敏度。
本试验研究主要包括以下几个方面的内容:
(一)首先将从普通微生物菌种保藏中心购买的试验菌(ATCC12980=NCTC10339)进行复壮、优化,在胰蛋白胨大豆琼脂培养基上培养,让其在65℃、pH值7.2条件下生长,挑选生长比较快速的菌落。但嗜热脂肪芽胞杆菌只形成针头大小的菌落,为革兰氏阳性菌。
(二)嗜热脂肪芽胞杆菌芽胞的制备。本试验,芽胞悬浮液中的营养菌体可选择两种方法除去,一是用溶菌酶法,二是加热离心法。通过两种方法的对比,并且考虑到成本因素,本文选择加热离心法。斜面菌种培养后,用生理盐水洗下菌苔,振荡,过滤,制成一定浓度的悬浮液进行加热离心制备芽胞。悬浮液70℃加热10 min在有效除去营养菌体碎片的情况下比更高温度、加热更长时间下存活的芽胞数量多,效果最好。
(三)嗜热脂肪芽胞杆菌芽胞包埋:通过对比卡拉胶、结冷胶、瓜尔胶和海藻酸钙四种包埋剂的凝胶强度、扩散性以及操作的简易程度,选择海藻酸钙作为嗜热脂肪芽胞杆菌芽胞的包埋剂。
(四)营养要求的研究,利用正交试验选择的最优营养液配方是:葡萄糖的添加量为1%,蛋白胨为1%。营养液的pH值控制在7.2左右,1.6%的指示剂的添加量为每100 mL营养液加入0.06 mL。
(五)确定检测条件:处理好的牛奶样品检测时,每个检测小管内添加0.2 mL紫色营养液,再加入牛奶样品0.1 mL。在65℃水浴培养150 min。取检样200份,检测时间为135 min,其中173份为阴性,27份为阳性,没有可疑样品,阳性率为13.5%,本研究方法具有可行性。
(六)稳定性试验:将制备好的检测小管在4℃保存1周、2周、3周以及4周,记录其阴性检测时间分别为前三周都是135 min,第四周检测时间开始增加为150 min,但也在检测时间范围内,所以本方法的检测稳定性为一个月。
(七)与小样发酵法、国标(TTC)法、试剂盒法及试纸条法对照试验:制备相同检样各40份,分别采取小样发酵法、TTC法(国标GB5409-85)、试剂盒法(ECLIPSE50)和试纸条法(青霉素类)与本方法进行对照检测其灵敏度。阳性率分别为小样发酵法22.5%,TTC法25%,本方法23.75%,试剂盒法26.25%,试纸条法32.5%。
本研究方法的灵敏度介于小样发酵与试剂盒法之间,灵敏度具有可靠性。小样发酵法的灵敏度较低,国标TTC法可疑样品份数较多且操作比较复杂,菌液浓度难以控制。试剂盒法和试纸条法操作都比较简单,其中试纸条法时间比较短,但它们经过商品化,成本较高,适合于研究试验室应用,不便于在一般的养殖场和收奶站中推广使用。
本课题目的在于根据现有的检测方法对嗜热脂肪芽胞杆菌检测牛奶中抗生素残留的方法加以改进及创新,建立一种简单、快速、灵敏度高、能够普遍推广应用的抗生素残留检测方法。该检测方法通过与其他方法作对照,在检测时间和灵敏度方面都能达到要求,而且成本大大降低,所以本研究方法具有可行性。
This paper studied the detecting antibiotics residual in milk with Bacillus stearothermophilus's gemmas of embeded. I have studing some indexs for increasing the method's sensitivity and stability. These indexs are nutritive substances, indicator's quantities, conditiones of embedded and conditiones of detected ect. By to contrasting with subsample fermentation method, encoding standard (TTC) methed, kit (ECLIPSE50) method and paper method which these four methods, then statistics positive rates and further to confirm the article's experiment method's feasibility and sensitivity.
This empirical studing mainly including below several aspects :
1. First we let the test organism of purchase (ATCC12980=NCTC10339) form CGMCC rejuvenescent, optimize and cultured at the tryptone-soy-agar medium. let they growth in the condition that temperature is 65℃and pH value is 7.2, then choice colonys which growth fast. Bacillus stearothermophilus's colonys only have pinhead-sized. they are Gram-positive bacteria.
2. To prepare gemmas of Bacillus stearothermophilus .in this experiment have two methods can to get rid of nutrition thallus from gemmas soliquoid, one is lysozyme method the other is to heat and centrifuge. By the two methods to contrast, and to consider the cost factor, I choose to heat and centrifuge method in the article. After cultured inclined plane strain washed lawn with normal sodium, succussed, filtered, make it to some density gemmas soliquoid, and then heated and centrifuged. gemmas soliquoid in 70℃heated 10 minutes can survival more gemmas , the effective is best than higher temperature and heating more time.
3. Embedded gemmas of Bacillus stearothermophilus :by contrasting the four medium of gel strength, diffusibility, simple and easy level of operating that carrageenan, gellan gum, guar gum, calcium alginate . I choose calcium alginate for gemmas medium of Bacillus stearothermophilus.each detection tubule are to put two or three gelatum beads . sealled up at 0~4℃until used.
4. The optimization medium to use direct cross experiment is glucose 1%, peptone 1%. The pH value of medium is controlling about 7.2, every 100mL medium are added to 0.06mL 1.6% of indicator.
5. Determine the detection of condition: raw milk sample after treated , every detection tubule are to add 0.2 mL purple medium then to put 0.1mL treated milk sample, at 65℃are culturing 2.5 hours.I take detection samples 200 in all , detection time are 135 minutes, negative samples are 173 and positive samples are 27, no doubtful sampl- es , positive rates are 13.5%, so this studiing method to have feasi- bility.
6. Stability experiment: detection tubules were put in to 4℃until one month , every week detect once , record they are negative time , front three weeks detection time are 135 minutes, the fourth week detection time are 150 minutes, but all the time are normal . so this experiment's detection stability is one month.
7. This experiment comparie to subsample fermentation method, encoding standard (TTC) methed, kit (ECLIPSE50) method and paper method: preparation 40 samples ,each sample have five parallel and use the five methods detect which sensitivity. positive rates subsample fermentation method 22.5%, encoding standard (TTC) 25%, this method 23.75%, kit (ECLIPSE50) 26.25%, paper 32.5%.
The studiing method sensitivity between subsample fermentation and kit method ,the sensitivity have reliability. Sensitivity of subsample fermentation method low, no do- ubtful samples too more of TTC method, even operation too complex ,bacterium liqu- id density hard to controlling. Commercialization of kit and paper methods oper- ation are simple but the cost very expensive, they can use in laboratory, not easy to general nursery and milk station.
This paper aims at developing the detection method of antibiotic in milk by Bacillus stearoth- ermophilus based on exisiting detection method, and set up a simple rapid and sens- itive antibiotic detection method which can be applied widely. This method can arrive the detection limit and detection time and the detection cost decrease largely . So the method is available.
本试验研究主要包括以下几个方面的内容:
(一)首先将从普通微生物菌种保藏中心购买的试验菌(ATCC12980=NCTC10339)进行复壮、优化,在胰蛋白胨大豆琼脂培养基上培养,让其在65℃、pH值7.2条件下生长,挑选生长比较快速的菌落。但嗜热脂肪芽胞杆菌只形成针头大小的菌落,为革兰氏阳性菌。
(二)嗜热脂肪芽胞杆菌芽胞的制备。本试验,芽胞悬浮液中的营养菌体可选择两种方法除去,一是用溶菌酶法,二是加热离心法。通过两种方法的对比,并且考虑到成本因素,本文选择加热离心法。斜面菌种培养后,用生理盐水洗下菌苔,振荡,过滤,制成一定浓度的悬浮液进行加热离心制备芽胞。悬浮液70℃加热10 min在有效除去营养菌体碎片的情况下比更高温度、加热更长时间下存活的芽胞数量多,效果最好。
(三)嗜热脂肪芽胞杆菌芽胞包埋:通过对比卡拉胶、结冷胶、瓜尔胶和海藻酸钙四种包埋剂的凝胶强度、扩散性以及操作的简易程度,选择海藻酸钙作为嗜热脂肪芽胞杆菌芽胞的包埋剂。
(四)营养要求的研究,利用正交试验选择的最优营养液配方是:葡萄糖的添加量为1%,蛋白胨为1%。营养液的pH值控制在7.2左右,1.6%的指示剂的添加量为每100 mL营养液加入0.06 mL。
(五)确定检测条件:处理好的牛奶样品检测时,每个检测小管内添加0.2 mL紫色营养液,再加入牛奶样品0.1 mL。在65℃水浴培养150 min。取检样200份,检测时间为135 min,其中173份为阴性,27份为阳性,没有可疑样品,阳性率为13.5%,本研究方法具有可行性。
(六)稳定性试验:将制备好的检测小管在4℃保存1周、2周、3周以及4周,记录其阴性检测时间分别为前三周都是135 min,第四周检测时间开始增加为150 min,但也在检测时间范围内,所以本方法的检测稳定性为一个月。
(七)与小样发酵法、国标(TTC)法、试剂盒法及试纸条法对照试验:制备相同检样各40份,分别采取小样发酵法、TTC法(国标GB5409-85)、试剂盒法(ECLIPSE50)和试纸条法(青霉素类)与本方法进行对照检测其灵敏度。阳性率分别为小样发酵法22.5%,TTC法25%,本方法23.75%,试剂盒法26.25%,试纸条法32.5%。
本研究方法的灵敏度介于小样发酵与试剂盒法之间,灵敏度具有可靠性。小样发酵法的灵敏度较低,国标TTC法可疑样品份数较多且操作比较复杂,菌液浓度难以控制。试剂盒法和试纸条法操作都比较简单,其中试纸条法时间比较短,但它们经过商品化,成本较高,适合于研究试验室应用,不便于在一般的养殖场和收奶站中推广使用。
本课题目的在于根据现有的检测方法对嗜热脂肪芽胞杆菌检测牛奶中抗生素残留的方法加以改进及创新,建立一种简单、快速、灵敏度高、能够普遍推广应用的抗生素残留检测方法。该检测方法通过与其他方法作对照,在检测时间和灵敏度方面都能达到要求,而且成本大大降低,所以本研究方法具有可行性。
This paper studied the detecting antibiotics residual in milk with Bacillus stearothermophilus's gemmas of embeded. I have studing some indexs for increasing the method's sensitivity and stability. These indexs are nutritive substances, indicator's quantities, conditiones of embedded and conditiones of detected ect. By to contrasting with subsample fermentation method, encoding standard (TTC) methed, kit (ECLIPSE50) method and paper method which these four methods, then statistics positive rates and further to confirm the article's experiment method's feasibility and sensitivity.
This empirical studing mainly including below several aspects :
1. First we let the test organism of purchase (ATCC12980=NCTC10339) form CGMCC rejuvenescent, optimize and cultured at the tryptone-soy-agar medium. let they growth in the condition that temperature is 65℃and pH value is 7.2, then choice colonys which growth fast. Bacillus stearothermophilus's colonys only have pinhead-sized. they are Gram-positive bacteria.
2. To prepare gemmas of Bacillus stearothermophilus .in this experiment have two methods can to get rid of nutrition thallus from gemmas soliquoid, one is lysozyme method the other is to heat and centrifuge. By the two methods to contrast, and to consider the cost factor, I choose to heat and centrifuge method in the article. After cultured inclined plane strain washed lawn with normal sodium, succussed, filtered, make it to some density gemmas soliquoid, and then heated and centrifuged. gemmas soliquoid in 70℃heated 10 minutes can survival more gemmas , the effective is best than higher temperature and heating more time.
3. Embedded gemmas of Bacillus stearothermophilus :by contrasting the four medium of gel strength, diffusibility, simple and easy level of operating that carrageenan, gellan gum, guar gum, calcium alginate . I choose calcium alginate for gemmas medium of Bacillus stearothermophilus.each detection tubule are to put two or three gelatum beads . sealled up at 0~4℃until used.
4. The optimization medium to use direct cross experiment is glucose 1%, peptone 1%. The pH value of medium is controlling about 7.2, every 100mL medium are added to 0.06mL 1.6% of indicator.
5. Determine the detection of condition: raw milk sample after treated , every detection tubule are to add 0.2 mL purple medium then to put 0.1mL treated milk sample, at 65℃are culturing 2.5 hours.I take detection samples 200 in all , detection time are 135 minutes, negative samples are 173 and positive samples are 27, no doubtful sampl- es , positive rates are 13.5%, so this studiing method to have feasi- bility.
6. Stability experiment: detection tubules were put in to 4℃until one month , every week detect once , record they are negative time , front three weeks detection time are 135 minutes, the fourth week detection time are 150 minutes, but all the time are normal . so this experiment's detection stability is one month.
7. This experiment comparie to subsample fermentation method, encoding standard (TTC) methed, kit (ECLIPSE50) method and paper method: preparation 40 samples ,each sample have five parallel and use the five methods detect which sensitivity. positive rates subsample fermentation method 22.5%, encoding standard (TTC) 25%, this method 23.75%, kit (ECLIPSE50) 26.25%, paper 32.5%.
The studiing method sensitivity between subsample fermentation and kit method ,the sensitivity have reliability. Sensitivity of subsample fermentation method low, no do- ubtful samples too more of TTC method, even operation too complex ,bacterium liqu- id density hard to controlling. Commercialization of kit and paper methods oper- ation are simple but the cost very expensive, they can use in laboratory, not easy to general nursery and milk station.
This paper aims at developing the detection method of antibiotic in milk by Bacillus stearoth- ermophilus based on exisiting detection method, and set up a simple rapid and sens- itive antibiotic detection method which can be applied widely. This method can arrive the detection limit and detection time and the detection cost decrease largely . So the method is available.
引文
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