纸片法检测饮料中菌落总数的研究
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摘要
本论文对检测纸片的显色体系，营养液配方，制作工艺进行了优化；分析了NaCl，琼脂，乳化剂对纸片质量的影响；将纸片法用于饮料类食品的检测作了研究，以国标法(平板计数法，PCA)作对照，确定了纸片法的精确度和检测时间，分析了纸片法与国标法的符合率，实验结果表明：
1．TTC对大肠菌，乳酸菌，醋酸菌三种试验菌株的生长均有影响，浓度在0.004％时显色情况最好，生成的红色菌落数目最多；浓度在0.004％以下生成的菌落不能充分显色，高于0.005％时细菌生长则受到抑制。将贴有0.004％TTC纸片的平板与未加TTC平扳作对照时，结果显示两方法差异无显著性(t=0.3558，P＞0.05)，因此，确定TTC显色剂使用浓度为0.004％。
2．根据二次最优饱和设计“D-416-A”，设计营养液优化试验方案，第8号与第10号试验结果最优；由回归方程计算得知，当蛋白胨，酵母膏，葡萄糖，磷酸氢二钾编码值为(1，0.9，1，0.65)时预测值最大。对应配方与第8号和第10号试验配方比较接近，经二次优化试验表明，差异不显著(P＞0.05)。确定每100mL营养液中加入蛋白胨，酵母膏，葡萄糖，磷酸氢二钾分别为2.59g，1.05g，0.239g，0.25g。
3．对灭菌时间的研究表明，116℃／20min和121℃／15min培养基都可达到无菌状态，但121℃／15min灭菌条件下菌悬液接种后比116℃／20min生长较好，确定121℃／15min的灭菌条件，
4．经L_9(3~4)正交试验。纸片的浸渍温度，浸渍时间，烘烤温度，烘烤时间对纸片质量都有较大的影响。实验结果表明，当浸渍温度70℃，浸渍时间2min，烘烤温度55℃，烘烤时间1h时纸片得分最高。
5．对培养基中氯化钠(NaCl)的使用量进行了研究，实验结果表明，每100mL营养液中加入0.45g／NaCl时细菌生长呈现最好态势。
6．琼脂对纸片质量也有较大影响，当浓度大于0.1％时，随着琼脂用量增加，纸片得分逐渐下降，而且细菌生长速度也逐渐减慢；当浓度低于0.1％时，细菌生长虽不会受到抑制，但纸片得分偏低。确定每100mL营养液中加入0.1g琼脂。
7．营养液中加入乳化剂后纸片质量有所提高，单甘酯，蔗糖酯，Span-40对纸片质量均有不同程度的影响，当浓度大于0.15mg／mL时，浓度增加对纸片质量不会造成明显的影响；使用0.15mg／mL Span-40纸片得分最高，细菌生长也不会受到影响。
8．对纸片检测特性的研究表明，(1)当培养时间大于15h，菌落数目增加不明显，与国标法培养24h的结果差异不显著(P＞0.05)；(2)对混合菌悬液和三类饮料作10次重复试验，变异系数在0.9％-4.714％之间，表明纸片法具有较高的精确度；(3)以国标法作对照，对363份饮料样品作了检查，纸片法合格316份，合格率87.05％，国标法合格321份，合格率88.42％；两法均合格302份，均不合格46份，符合率为95.87％，两者检出的绝对菌落数比国标法：纸片法为1：0.9342；(4)检测纸片用于方便面酱包，菜包，粉包等食品的检测，与国标法比较获
西南农业大学三00四届硕士学位论文中天摘要
里里里里巴里巴里里里皿口纽里里巴
得的菌落数比值为l:0.7576一1:0.91之间，两者具有不同程度的差异;(5)纸片保质期久180
大。
9.经粗略计算，纸片法和国标法作一次饮料菌落总数检测，成本分别为12.6元和18.3
元人民币，纸片法比国标法经济。
The changing colour system, culture medium formula and making technology of test paper were optimized in this paper, effect of NaCl, agar on quality of test paper were analysed ,and detection time .precision was determined also. Compared with national standard method(PCA) the accuracy, as well as consistency were set by petrifilm method.Findings as follows:
1 .TTC had effect on the test bacteria Coliform bacteria, Lactobacilus lactis, Acetobacter, the optimum changing colour occurred and maximum colonies obtained when concentration of TTC was 0.004%,the growth of colonies couldn't be fully coloured when concentration of TTC is below 0.004% and restrained when higher than 0.005%.Test paper macerated in 0.004% TTC compared with PCA ,the results hadn't remarkable differences(t=0.3558,P>0.05).
2.Scheme of culture medium was designed by D-416-A,we got optimum desired results by tropical equation, when the code value of peptone, yeast exrtact,glucose,K2HPO4 were 1, 0.9, 1, 0.65, formula of NO.8 and NO. 10 had a little difference compared with optimum formula, they hadn't remarkable difference (P>0.05) by the double experiment .So the main composition of 100mL culture medium was peptone 2.59g,yeast extract 1.05g, glucose 0.239g, K2HPO4 0.35g .
3.when studying on sterilization time we found that there was no bacteria left in culture medium in condition of 116℃/20min or 121℃/15min,but the colonies growth better in culture medium sterilized at 121℃/15min than at 116℃/20min.
4.By L9(34) orthogonal experiment maceration temperature and time, drying temperature and time all had much effect on quality of test paper. Experiments showed that the test paper got the highest mark when macerated at 70℃/2min,dryed at 55℃/1 h.
5.The usage of NaCl had been studied in this paper, the colonies grew best when adding NaCl 0.45g to 100mL culture medium.
6.Agar had effect on quality of test paper, the mark of test paper descended and the colonies grew more slowly when concentration of agar was higher than 0.1%,moreover the mark of test paper was not high though the growth of colonies were normal when concentration of agar is less than 0.1 %.So the optimum concentration of agar in culture medium was 0.1 %.
7.The quality of test paper improved when direactive glyceride, sucrose ester, Span-40 were added into macerating culture medium,different kinds of emulsion had different effect on quality of test paper.when concentration were higher than 0.15mg/mL there weren't remarkable effect on the quality. Test paper got the highest mark when concentration of Span-40 was 0.15mg/mL and the growth of bacteria weren't restrained.
8.According to studying on character of test paper, (1) quantity of colonies didn't increase remarkably when cultivated more than 15h and there was no remarkable differences(P>0.05) compared with national standard method,(2) mixed bacteria and three kinds of beverages were tested, results showed that repeatability was from 0.9% to 4.714%,(3)compared with national standard method 363 beverage samples were tested,316 samples were qualified by test paper method and percent of pass was 87.05%,321 samples were qualified by national standard method and percent of pass was 88.40%,302 samples were qualified and 46 weren't qualified by two methods and consistency was 95.87%,absolute ratio of colonies was 1:0.9342.(4)vacuum dried vegetable, flavour powder and sauce in instant noodles were tested by test paper, absolute ratio of colonies obtained is from 0.7576 to 0.91,the results showed differences compared with national standard method.,(5)guarantee period of test paper is 180 days.
9.Roughly estimating the cost of test paper method and national standard method were 12.6 ?and 18.3
￥ separately in a single test.
1．TTC对大肠菌，乳酸菌，醋酸菌三种试验菌株的生长均有影响，浓度在0.004％时显色情况最好，生成的红色菌落数目最多；浓度在0.004％以下生成的菌落不能充分显色，高于0.005％时细菌生长则受到抑制。将贴有0.004％TTC纸片的平板与未加TTC平扳作对照时，结果显示两方法差异无显著性(t=0.3558，P＞0.05)，因此，确定TTC显色剂使用浓度为0.004％。
2．根据二次最优饱和设计“D-416-A”，设计营养液优化试验方案，第8号与第10号试验结果最优；由回归方程计算得知，当蛋白胨，酵母膏，葡萄糖，磷酸氢二钾编码值为(1，0.9，1，0.65)时预测值最大。对应配方与第8号和第10号试验配方比较接近，经二次优化试验表明，差异不显著(P＞0.05)。确定每100mL营养液中加入蛋白胨，酵母膏，葡萄糖，磷酸氢二钾分别为2.59g，1.05g，0.239g，0.25g。
3．对灭菌时间的研究表明，116℃／20min和121℃／15min培养基都可达到无菌状态，但121℃／15min灭菌条件下菌悬液接种后比116℃／20min生长较好，确定121℃／15min的灭菌条件，
4．经L_9(3~4)正交试验。纸片的浸渍温度，浸渍时间，烘烤温度，烘烤时间对纸片质量都有较大的影响。实验结果表明，当浸渍温度70℃，浸渍时间2min，烘烤温度55℃，烘烤时间1h时纸片得分最高。
5．对培养基中氯化钠(NaCl)的使用量进行了研究，实验结果表明，每100mL营养液中加入0.45g／NaCl时细菌生长呈现最好态势。
6．琼脂对纸片质量也有较大影响，当浓度大于0.1％时，随着琼脂用量增加，纸片得分逐渐下降，而且细菌生长速度也逐渐减慢；当浓度低于0.1％时，细菌生长虽不会受到抑制，但纸片得分偏低。确定每100mL营养液中加入0.1g琼脂。
7．营养液中加入乳化剂后纸片质量有所提高，单甘酯，蔗糖酯，Span-40对纸片质量均有不同程度的影响，当浓度大于0.15mg／mL时，浓度增加对纸片质量不会造成明显的影响；使用0.15mg／mL Span-40纸片得分最高，细菌生长也不会受到影响。
8．对纸片检测特性的研究表明，(1)当培养时间大于15h，菌落数目增加不明显，与国标法培养24h的结果差异不显著(P＞0.05)；(2)对混合菌悬液和三类饮料作10次重复试验，变异系数在0.9％-4.714％之间，表明纸片法具有较高的精确度；(3)以国标法作对照，对363份饮料样品作了检查，纸片法合格316份，合格率87.05％，国标法合格321份，合格率88.42％；两法均合格302份，均不合格46份，符合率为95.87％，两者检出的绝对菌落数比国标法：纸片法为1：0.9342；(4)检测纸片用于方便面酱包，菜包，粉包等食品的检测，与国标法比较获
西南农业大学三00四届硕士学位论文中天摘要
里里里里巴里巴里里里皿口纽里里巴
得的菌落数比值为l:0.7576一1:0.91之间，两者具有不同程度的差异;(5)纸片保质期久180
大。
9.经粗略计算，纸片法和国标法作一次饮料菌落总数检测，成本分别为12.6元和18.3
元人民币，纸片法比国标法经济。
The changing colour system, culture medium formula and making technology of test paper were optimized in this paper, effect of NaCl, agar on quality of test paper were analysed ,and detection time .precision was determined also. Compared with national standard method(PCA) the accuracy, as well as consistency were set by petrifilm method.Findings as follows:
1 .TTC had effect on the test bacteria Coliform bacteria, Lactobacilus lactis, Acetobacter, the optimum changing colour occurred and maximum colonies obtained when concentration of TTC was 0.004%,the growth of colonies couldn't be fully coloured when concentration of TTC is below 0.004% and restrained when higher than 0.005%.Test paper macerated in 0.004% TTC compared with PCA ,the results hadn't remarkable differences(t=0.3558,P>0.05).
2.Scheme of culture medium was designed by D-416-A,we got optimum desired results by tropical equation, when the code value of peptone, yeast exrtact,glucose,K2HPO4 were 1, 0.9, 1, 0.65, formula of NO.8 and NO. 10 had a little difference compared with optimum formula, they hadn't remarkable difference (P>0.05) by the double experiment .So the main composition of 100mL culture medium was peptone 2.59g,yeast extract 1.05g, glucose 0.239g, K2HPO4 0.35g .
3.when studying on sterilization time we found that there was no bacteria left in culture medium in condition of 116℃/20min or 121℃/15min,but the colonies growth better in culture medium sterilized at 121℃/15min than at 116℃/20min.
4.By L9(34) orthogonal experiment maceration temperature and time, drying temperature and time all had much effect on quality of test paper. Experiments showed that the test paper got the highest mark when macerated at 70℃/2min,dryed at 55℃/1 h.
5.The usage of NaCl had been studied in this paper, the colonies grew best when adding NaCl 0.45g to 100mL culture medium.
6.Agar had effect on quality of test paper, the mark of test paper descended and the colonies grew more slowly when concentration of agar was higher than 0.1%,moreover the mark of test paper was not high though the growth of colonies were normal when concentration of agar is less than 0.1 %.So the optimum concentration of agar in culture medium was 0.1 %.
7.The quality of test paper improved when direactive glyceride, sucrose ester, Span-40 were added into macerating culture medium,different kinds of emulsion had different effect on quality of test paper.when concentration were higher than 0.15mg/mL there weren't remarkable effect on the quality. Test paper got the highest mark when concentration of Span-40 was 0.15mg/mL and the growth of bacteria weren't restrained.
8.According to studying on character of test paper, (1) quantity of colonies didn't increase remarkably when cultivated more than 15h and there was no remarkable differences(P>0.05) compared with national standard method,(2) mixed bacteria and three kinds of beverages were tested, results showed that repeatability was from 0.9% to 4.714%,(3)compared with national standard method 363 beverage samples were tested,316 samples were qualified by test paper method and percent of pass was 87.05%,321 samples were qualified by national standard method and percent of pass was 88.40%,302 samples were qualified and 46 weren't qualified by two methods and consistency was 95.87%,absolute ratio of colonies was 1:0.9342.(4)vacuum dried vegetable, flavour powder and sauce in instant noodles were tested by test paper, absolute ratio of colonies obtained is from 0.7576 to 0.91,the results showed differences compared with national standard method.,(5)guarantee period of test paper is 180 days.
9.Roughly estimating the cost of test paper method and national standard method were 12.6 ?and 18.3
￥ separately in a single test.
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