Study on Quantitative Counting Method of Lactic Acid Bacteria Powder
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摘要: 目的：以9株乳杆菌、6株双歧杆菌、3株球菌、1株凝结芽孢杆菌菌粉为研究对象，在国标GB 4789.35-2016的基础上，对稀释倍数、稀释液成分、培养基成分进行比较研究，考察对乳酸菌菌粉计数活菌数的影响。方法：采用稀释平板计数的方法，对不同的乳酸菌菌粉进行活菌计数。结果：初始乳酸菌菌粉样品稀释倍数对最终计数活菌数无明显影响，ISO稀释液对部分乳杆菌、双歧杆菌菌粉的活菌计数结果有显著提高（P<0.05）；双歧杆菌菌粉采用TOS琼脂培养基的计数结果显著优于国标培养基（P<0.05）；凝结芽孢杆菌菌粉采用改良芽孢计数培养基计数结果优于PCA和NA计数培养基。结论：平板计数方法中，稀释液中含有酪蛋白胨能提升双歧杆菌菌粉的活菌计数数量；TOS琼脂培养基更有利于双歧杆菌的增殖培养；改良芽孢计数培养基更有利于凝结芽孢杆菌的芽孢萌发增殖。Abstract: Objective: On the basis of national standard GB 4789.35-2016, a comparative study was conducted on dilution ratio, diluent components and medium components to investigate the influence on the number of viable bacteria when counting lactic acid bacteria powder. Nine strains of Lactobacillus, six strains of Bifidobacterium, three strains of coccus and one strain of Bacillus coagulans were used as research objects. Methods: The method of dilution plate count was used to count the viable bacteria of different lactic acid bacteria powders. Results: According to research findings, the dilution ratio of the initial lactic acid bacteria powder sample had no significant effect on the final count of viable bacteria, and the ISO dilution had significant effects on the viable count results of some Lactobacillus and Bifidobacterium powders (P<0.05); The counting result of Bifidobacterium powder using TOS agar medium was better than that of national standard medium (P<0.05); The counting result of Bacillus coagulans powder using modified spore counting medium was better than PCA and NA counting culture base. Conclusion: The diluent containing casein peptone could increase the number of viable counts of Bifidobacterium powder when plate counting method was used; TOS agar medium was more conducive to the proliferation and culture of Bifidobacterium; The improved spore counting medium was more conducive to the spore germination and proliferation of Bacillus coagulans.
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Key words:
- lactic acid bacteria /
- counting method /
- Bifidobacterium /
- Bacillus coagulans /
- casein peptone
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表 1 梯度稀释液研究
Table 1. Research on serial dilutions
10−1试管 10−2试管 10−3试管 10−4试管 10−5试管 10−6试管 10−7试管 10−8试管 10−9试管 测试1 国标稀释液 国标稀释液 国标稀释液 国标稀释液 国标稀释液 国标稀释液 国标稀释液 国标稀释液 国标稀释液 测试2 国标稀释液 国标稀释液 国标稀释液 国标稀释液 国标稀释液 国标稀释液 国标稀释液 ISO稀释液 ISO稀释液 测试3 国标稀释液 国标稀释液 国标稀释液 国标稀释液 ISO稀释液 ISO稀释液 ISO稀释液 ISO稀释液 ISO稀释液 测试4 国标稀释液 国标稀释液 ISO稀释液 ISO稀释液 ISO稀释液 ISO稀释液 ISO稀释液 ISO稀释液 ISO稀释液 测试5 ISO稀释液 ISO稀释液 ISO稀释液 ISO稀释液 ISO稀释液 ISO稀释液 ISO稀释液 ISO稀释液 ISO稀释液 
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表 2 不同益生菌菌粉稀释倍数计数结果（×1011 CFU/g）
Table 2. Counting results of dilution times of different probiotic bacteria powder (×1011 CFU/g)
分类 菌株 2 g→38 mL 10 g→90 mL 25 g→225 mL（国标）
乳杆菌嗜酸乳杆菌LA85 2.4±0.1 2.3±0.2 2.4±0.1 植物乳杆菌Lp90 5.4±0.1 5.3±0.1 5.4±0.1 鼠李糖乳杆菌LRa05 4.4±0.2 4.5±0.1 4.5±0.1 干酪乳杆菌LC89 5.4±0.1 5.5±0.2 5.5±0.2 副干酪乳杆菌LC86 5.3±0.1 5.4±0.1 5.4±0.1 唾液乳杆菌LS97 2.3±0.1 2.2±0.1 2.3±0.2 罗伊氏乳杆菌LR08 2.3±0.2 2.2±0.1 2.3±0.1 保加利亚乳杆菌LB42 1.1±0.2 1.2±0.1 1.2±0.2 格氏乳杆菌LG08 3.4±0.1 3.4±0.1 3.3±0.1
双歧
杆菌乳双歧杆菌BLa80 5.4±0.2 5.6±0.1 5.5±0.1 长双歧杆菌BL21 3.4±0.1 3.5±0.2 3.4±0.2 短双歧杆菌BBr60 5.4±0.1 5.6±0.1 5.5±0.1 青春双歧杆菌BAC30 1.3±0.2 1.2±0.2 1.2±0.2 婴儿双歧杆菌BI45 1.4±0.2 1.4±0.2 1.3±0.1 两歧双歧杆菌BBi32 5.4±0.1 5.5±0.2 5.4±0.2
球菌嗜热链球菌ST81 1.2±0.1 1.3±0.2 1.2±0.1 乳酸片球菌PA53 4.6±0.1 4.7±0.2 4.6±0.1 戊糖片球菌PP06 3.5±0.1 3.5±0.1 3.4±0.2 芽孢杆菌 凝结芽孢杆菌BC99 1.2±0.1 1.3±0.2 1.2±0.1
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表 3 不同稀释液计数对活菌数的影响 （×1011 CFU/g）
Table 3. Effect of different diluent counts on the number of viable bacteria (×1011 CFU/g)
分类 菌株 国标稀释液 ISO稀释液 改良稀释液一 改良稀释液二 改良稀释液三
乳杆菌嗜酸乳杆菌LA85 2.3±0.2 3.2±0.1* 2.4±0.2 2.5±0.2 2.2±0.2 植物乳杆菌Lp90 5.3±0.1 5.8±0.2 5.4±0.2 5.4±0.2 5.5±0.2 鼠李糖乳杆菌LRa05 4.3±0.2 4.6±0.1 4.5±0.2 4.4±0.2 4.3±0.1 干酪乳杆菌LC89 5.4±0.2 5.7±0.2 5.5±0.2 5.4±0.1 5.7±0.1 副干酪乳杆菌LC86 5.3±0.2 5.5±0.1 5.4±0.2 5.3±0.2 5.5±0.1 唾液乳杆菌LS97 2.3±0.2 3.5±0.2* 2.8±0.1 2.7±0.1 2.4±0.2 罗伊氏乳杆菌LR08 2.1±0.2 2.5±0.2 2.3±0.1 2.3±0.2 2.4±0.2 保加利亚乳杆菌LB42 1.1±0.2 1.3±0.2 1.2±0.1 1.2±0.1 1.1±0.2 格氏乳杆菌LG08 3.2±0.1 3.5±0.2 3.3±0.2 3.4±0.1 3.3±0.2
双歧
杆菌乳双歧杆菌BLa80 5.3±0.2 6.7±0.1* 5.6±0.2 5.7±0.2 5.4±0.1 长双歧杆菌BL21 3.2±0.2 4.1±0.2* 3.5±0.1 3.5±0.1 3.2±0.2 短双歧杆菌BBr60 5.4±0.1 6.7±0.2* 5.8±0.2 5.8±0.1 5.3±0.1 青春双歧杆菌BAC30 1.3±0.2 1.9±0.1* 1.2±0.2 1.3±0.2 1.3±0.2 婴儿双歧杆菌BI45 1.2±0.2 1.9±0.1* 1.2±0.1 1.3±0.1 1.2±0.1 两歧双歧杆菌BBi32 5.3±0.2 6.9±0.2* 5.8±0.1 5.5±0.2 5.2±0.1
球菌嗜热链球菌ST81 1.1±0.1 1.3±0.1 1.2±0.2 1.2±0.1 1.2±0.2 乳酸片球菌PA53 4.6±0.1 4.5±0.1 4.8±0.2 4.6±0.1 4.8±0.2 戊糖片球菌PP06 3.5±0.2 3.4±0.2 3.6±0.1 3.5±0.2 3.6±0.1 芽孢
杆菌凝结芽孢杆菌BC99 1.1±0.1 1.3±0.1 1.2±0.2 1.2±0.1 1.1±0.1 注：“*”表示与国标方法比较有显著性差异（P<0.05）；“**”表示与国标方法比较有极显著性差异（P<0.01）；表4、表6同。
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表 4 不同稀释液计数结果（×1011 CFU/g）
Table 4. Counting results of different dilutions (×1011 CFU/g)
菌株 测试1 测试2 测试3 测试4 测试5 嗜酸乳杆菌LA85 2.3±0.2 2.4±0.2 2.6±0.2 2.8±0.2* 3.2±0.1* 唾液乳杆菌LS97 2.3±0.2 2.5±0.2 2.7±0.3 3.0±0.2* 3.5±0.2* 乳双歧杆菌BLa80 5.3±0.2 5.4±0.2 5.6±0.2 5.8±0.3 6.7±0.1* 长双歧杆菌BL21 3.1±0.2 3.3±0.1 3.4±0.2 3.6±0.2 4.1±0.2* 短双歧杆菌BBr60 5.5±0.1 5.6±0.2 5.8±0.2 6.1±0.1 6.7±0.2* 青春双歧杆菌BAC30 1.2±0.2 1.3±0.1 1.4±0.2 1.5±0.2 1.8±0.1* 婴儿双歧杆菌BI45 1.1±0.2 1.2±0.1 1.3±0.3 1.4±0.2 1.6±0.3* 两歧双歧杆菌BBi32 5.4±0.2 5.5±0.2 5.8±0.2 6.0±0.2* 6.8±0.3*
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表 5 不同培养基对乳杆菌计数活菌数的影响（×1011 CFU/g）
Table 5. Effects of different mediums on Lactobacillus counting viable count (×1011 CFU/g)
菌株 MRS琼脂培养基（国标） 酸化MRS Agar培养基 嗜酸乳杆菌LA85 2.3±0.2 2.3±0.1 植物乳杆菌Lp90 5.3±0.1 5.4±0.1 鼠李糖乳杆菌LRa05 4.3±0.2 4.4±0.1 干酪乳杆菌LC89 5.4±0.2 5.5±0.1 副干酪乳杆菌LC86 5.3±0.2 5.4±0.1 唾液乳杆菌LS97 2.3±0.2 2.4±0.1 罗伊氏乳杆菌LR08 2.2±0.1 2.1±0.2 保加利亚乳杆菌LB42 1.1±0.2 1.2±0.1 格氏乳杆菌LG08 3.2±0.2 3.3±0.1
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表 6 不同培养基对双歧杆菌计数活菌数的影响（×1011 CFU/g）
Table 6. Effects of different mediums on Bifidobacterium counting viable count (×1011 CFU/g)
菌株 MRS琼脂培养基+锂盐
（国标）TOS琼脂培养基 乳双歧杆菌BLa80 5.2±0.1 5.9±0.1* 长双歧杆菌BL21 3.1±0.2 5.5±0.1* 短双歧杆菌BBr60 5.4±0.1 7.5±0.2* 青春双歧杆菌BAC30 1.4±0.2 2.6±0.1* 婴儿双歧杆菌BI45 1.2±0.1 2.1±0.2* 两歧双歧杆菌BBi32 5.3±0.1 7.5±0.1*
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表 7 不同培养基对球菌计数活菌数的影响（×1011 CFU/g）
Table 7. Effects of different mediums on the counting of viable cocci (×1011 CFU/g)
菌株 MC琼脂培养基（国标） M17培养基 嗜热链球菌ST81 1.2±0.2 1.3±0.2 乳酸片球菌PA53 4.8±0.2 4.9±0.2 戊糖片球菌PP06 3.6±0.1 3.7±0.1
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表 8 不同培养基对凝结芽孢杆菌BC99计数活菌数的影响（×1011 CFU/g）
Table 8. Effects of different mediums on counting viable cells of Bacillus coagulans BC99 (×1011 CFU/g)
菌株 NA营养琼脂
培养基PCA
培养基改良芽孢计数
培养基凝结芽孢杆菌BC99 1.1±0.2 1.2±0.1 1.8±0.1* 注：“*”表示有显著性差异（P<0.05）。
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