CN112481350A - 用于快速检测霉菌和酵母菌总数的复合培养基及其制备方法 - Google Patents
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Abstract
本发明公开一种用于快速检测霉菌和酵母菌总数的复合培养基,该复合培养基每100g蒸馏水中含有:酪蛋白胰酶消化物0.8g‑1.0g、葡萄糖1.0g‑4.0g、磷酸二氢钾0.2g‑0.3g、氯化钠0.05g‑0.2g、酵母浸膏0.4g‑0.8g、孟加拉红0.4g‑0.8g、琼脂1.5g‑3g、氯霉素0.1g‑0.2g、硫酸镁0.2g‑0.4g、氯化锰0.015g‑0.03g、氯化钙0.05g‑0.2g、吐温60 0.1g‑0.25g。采用本发明复合培养基可快速检测各类食品中的霉菌和酵母菌总数,检出率比马铃薯琼脂和孟加拉红琼脂高20%,菌落形态大,易于计数,并且72h菌落基本成型,基本全部生长。
Description
技术领域
本发明属于微生物学检测技术领域,具体涉及一种用于快速检测霉菌和酵母菌总数的复合培养基。
背景技术
霉菌和酵母菌总数是食品样品在一定条件下(如培养基、培养温度和培养时间等)培养后,所得每g(ml)样品中形成的微生物霉菌和酵母菌总数。霉菌和酵母菌广泛存在于自然界,有的霉菌还可用来制作食品,但也有一些霉菌是有害的,会引起食物霉变,会刺激人体消化道,严重可中毒,对肝脏有损害。
目前,对霉菌和酵母菌总数的检测培养基,通常为马铃薯琼脂或孟加拉红琼脂,多数霉菌和酵母菌在该两种琼脂上培养120h才能完全形成肉眼可分辨的全部菌落,耗费时间较长,检测率有待提高。
发明内容
针对现有技术中存在的不足,本发明的目的在于提供一种既经济又能快速检测霉菌和酵母菌总数的复合培养基,可缩短检测时间,提高样品的检测率。
其中,所述的培养基的组成,每100g蒸馏水中含有:酪蛋白胰酶消化物0.8g-1.0g、葡萄糖1.0g-4.0g、磷酸二氢钾0.2g-0.3g、氯化钠0.05g-0.2g、酵母浸膏0.4g-0.8g、孟加拉红0.4g-0.8g、琼脂1.5g-3g、氯霉素0.1g-0.2g、硫酸镁0.2g-0.4g、氯化锰0.015g-0.03g、氯化钙0.05g-0.2g、吐温60 0.1g-0.25g。
优选每100g蒸馏水中含有:酪蛋白胰酶消化物0.9g、葡萄糖3.0g、磷酸二氢钾0.2g、氯化钠0.1g、酵母浸膏0.6g、孟加拉红0.6g、琼脂2g、氯霉素0.1g、硫酸镁0.3g、氯化锰0.02g、氯化钙0.1g、吐温60 0.2g。
配制方法:将复合培养基各组分加入水中,煮沸溶解至澄清溶液,调节pH值为7.0~7.5,115℃高压灭菌10~15min。
检测方法:称取样品25土0.l g,加入无菌均质瓶中,加入无菌稀释液至250ml,均质1min-2min,稀释度为1:10;取上述步骤的稀释1ml,放入灭菌平皿中,倾倒复合培养基溶液,于28℃土1℃的培养箱内培养72h-120h;计数,得霉菌和酵母菌总数的检测结果。
本发明的复合培养基各成分作用原理如下:
酪蛋白胰酶消化物,动物组织的胃酶消化物:提供营养基础;
酵母浸膏:提供维生素和氨基酸;
葡萄糖:提供碳源;
吐温60,硫酸镁,氯化锰,氯化钙:表面活性剂和促生长因子,可作用于微生物细胞膜某受体,使休眠状态的菌落迅速感知外界适宜的生长环境,快速生长,同时降低菌落表面和培养基的表面张力,使营养物质更易吸收;
琼脂粉:凝固剂;
氯霉素:细菌抑制剂。
比较现有常用三种霉菌培养基:沙氏葡萄糖琼脂培养基(SDA)、马铃薯琼脂培养基(PDA)、孟加拉红琼脂培养基。本配方不同于以上3种培养基,以提高培养基检出率为目的,反复试验对相关成分保留并且含量适当增减,再增加一定量的吐温60,硫酸镁,氯化锰,氯化钙几项表面活性剂及促生长因子,改变不同浓度研究对酵母菌生长的影响。研究结果显示,本发明配方克服传统配方的局限性,增加促生长因子,尽可能的将检出率提高到最大化。
本发明的霉菌和酵母菌复合培养基,与已有技术相比具有以下优点:采用本发明复合培养基可快速检测各类食品中的霉菌和酵母菌总数,检出率比马铃薯琼脂和孟加拉红琼脂高20%,菌落形态大,易于计数,并且72h菌落基本成型,基本全部生长。
具体实施方式
以下通过具体实施例对本发明进行进一步解释说明,其中,酪蛋白胰酶消化物购自于上海一研生物科技有限公司。
实施例1
取酪蛋白胰酶消化物0.9g、葡萄糖3.0g、磷酸二氢钾0.2g、氯化钠0.1g、酵母浸膏0.6g、孟加拉红0.6g、琼脂2g、氯霉素0.1g、硫酸镁0.3g、氯化锰0.02g、氯化钙0.1g、吐温600.2g。将上述组分加入100g蒸馏水中,煮沸溶解至澄清溶液,调节pH值为7.0,高压灭菌115℃,15min后备用。
实施例2
酪蛋白胰酶消化物0.8g、葡萄糖4.0g、磷酸二氢钾0.2g、氯化钠0.2g、酵母浸膏0.4g、孟加拉红0.4g、琼脂3g、氯霉素0.1g、硫酸镁0.2g、氯化锰0.03g、氯化钙0.05g、吐温600.25g。将上述组分加入100g蒸馏水中,煮沸溶解至澄清溶液,调节pH值为7.0,高压灭菌115℃,15min后备用。
实施例3
酪蛋白胰酶消化物1.0g、葡萄糖1.0g、磷酸二氢钾0.3g、氯化钠0.05g、酵母浸膏0.8g、孟加拉红0.8g、琼脂1.5g、氯霉素0.2g、硫酸镁0.4g、氯化锰0.015g、氯化钙0.2g、吐温60 0.1g。将上述组分加入100g蒸馏水中,煮沸溶解至澄清溶液,调节pH值为7.0,高压灭菌115℃,15min后备用。
实验论证:
1菌种试验用菌株的传代次数不得超过5代,白色念珠菌(Candida albicans)[CMCC(F)98 001],黑曲霉(Aspergillus niger)[CMCC(F)98 003]
2菌液制备
2.1接种白色念珠菌的新鲜培养物至沙氏葡萄糖液体培养基中或沙氏葡萄糖琼脂培养基上,培养温度20—25℃,培养时间2-3天。上述培养物用pH7.0无菌氯化钠-蛋白胨缓冲液或0.9%无菌氯化钠溶液制成每1ml含菌数为20CFU~200CFU。
2.2接种黑曲霉的新鲜培养物至沙氏葡萄糖琼脂斜面培养基上,培养温度20—25℃,培养5—7天,加入3—5ml含0.05%(ml/ml)聚山梨酯80的pH7.0无菌氯化钠-蛋白胨缓冲液或0.9%无菌氯化钠溶液,将孢子洗脱。然后,采用适宜的方法吸出孢子悬液至无菌试管内,用含0.05%(ml/ml)聚山梨酯80的pH7.0无菌氯化钠-蛋白胨缓冲液或0.9%无菌氯化钠溶液制成每1ml含孢子数为20CFU~200CFU的黑曲霉孢子悬液。
2.3菌液制备后若在室温下放置,应在2小时内使用;若保存在2—8℃,可在24小时内使用。黑曲霉孢子悬液可保存在2—8℃,在验证过的贮存期内使用。
3.质量检查
3.1定量
3.1.1接种
选择10-6稀释度的工作菌悬液0.1mL,均匀涂布接种于马铃薯琼脂培养基,孟加拉红琼脂培养基以及本发明实施例1培养基。每一稀释度接种两个平板。可使用倾注法进行接种,并按标准规定的培养条件28℃±1℃,5d培养平板。
3.1.2计数
统计三种培养基的培养时间以及菌落数目。
培养基 | 培养时间(h) | 霉菌数目(CFU) | 酵母菌数目(CFU) |
平板计数培养基 | 120 | 56 | 78 |
孟加拉红培养基 | 120 | 56 | 72 |
实施例1培养基 | 72 | 68 | 88 |
3.1.3结论
通过本发明与市售培养基霉菌和酵母菌检测结果比对后,实验结果表明本发明检测结果在72h内检出率比马铃薯琼脂和孟加拉红琼脂高20%(在允许误差范围内),菌落形态也较大。
Claims (2)
1.一种用于快速检测霉菌和酵母菌总数的复合培养基,其特征在于该复合培养基每100g蒸馏水中含有:酪蛋白胰酶消化物0.8g-1.0g、葡萄糖1.0g-4.0g、磷酸二氢钾0.2g-0.3g、氯化钠0.05g-0.2g、酵母浸膏0.4g-0.8g、孟加拉红0.4g-0.8g、琼脂1.5g-3g、氯霉素0.1g-0.2g、硫酸镁0.2g-0.4g、氯化锰0.015g-0.03g、氯化钙0.05g-0.2g、吐温60 0.1g-0.25g。
2.一种权利要求1所述的用于快速检测霉菌和酵母菌总数的复合培养基的制备方法,其特征在于该方法包括以下步骤:将复合培养基各组分加入水中,煮沸溶解至澄清溶液,调节pH值为7.0~7.5,115℃高压灭菌10~15min。
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