Safety Assessment of Two New Lactobacillus Plantarum Strains Isolated from Traditional Fermented Food of Northeast China
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Abstract: This study aimed to evaluate the safety of previously isolated Lactobacillus strains (L. Plantarum L12 and L20) by in vitro and in vivo methods. Antibiotic sensitivity, bile salt hydrolysis activity, D-lactic acid content, and hemolytic activity were analyzed in vitro. Then, a short-term oral toxicity study was conducted in mice, which received 0.2 mL (109 CFU/mL) of experimental bacterial solutions orally for 30 days. The results showed that both strains had good antioxidant activity, high sensitivity to antibiotics, high bile salt hydrolase activity, low D-lactic acid content, and gamma hemolysis. Intragastric administration had no adverse effects on growth, behavior, food intake, organ weight, or histopathological analysis of the treated mice. Compared with control mice, the serum malondialdehyde concentration and liver glutathione content of probiotic-treated mice were statistically different (P<0.05). These results suggest that strains L12 and L20 are nonpathogenic and likely to be safe for human consumption.
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Keywords:
- probiotics /
- safety /
- oral toxicity /
- Lactobacillus plantarum /
- mice
摘要: 针对已分离的植物乳杆菌L12和L20通过体外和体内两种方法进行安全性评价。通过体外实验测定乳酸水解活性抗生素敏感性，胆盐水解酶活性，D-乳酸含量，γ溶血等指标；体内实验是对小鼠进行短期口服毒性的研究，口服0.2 mL（109 CFU/mL）的实验性细菌溶液30 d。结果表明，体外实验中，两株菌株均具有良好的抗氧化活性，对抗生素敏感性高，胆盐水解酶活性高，D-乳酸含量低，γ溶血；体内实验中灌胃给药对治疗小鼠的生长、行为、摄食量、器官重量或组织病理学分析均无不良影响。与对照组相比，益生菌组小鼠血清MDA浓度和肝脏GSH含量差异有统计学意义(P<0.05)。这些结果表明，菌株L12和L20是非致病性的，人直接食用是安全的。 -
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Figure 3. Photomicrographs of liver paraffin section of mice (a) control (b) L12, and (c) L20
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Figure 4. Serum MDA concentration (a) and total Liver GSH concentration (b) in control and probiotic-treated mice
Note: Values are means with standard deviations shown by vertical bars (n=4). Bars with a different lowercase letter (s) in the same treatments indicate significant differences at P<0.05 among the treatments.
Table 1 Antibiotic susceptibility of Lactobacillus strains L12 and L20
Antibiotic L12 L20 Diameters (mm) Sensitivity Diameters (mm) Sensitivity Rifampin 22±0.2g S 31±0.3b S Vancomycin 15±0.1j I 8.3±0.03j R Kanamycin 7±0.5ll R 27±0.4d S Ceftazidime 30±0.1c S 14±0.05i I Sulphamethoxazole/Trimethoprim 30±0.02c S 17±0.2h I Erythromycin 32±0.04b S 27±0.03d S Ampicillin 24±0.07f S 26±0.05e S Penicillin G 33±0.0a S 31±0.2b S Streptomycin 26±0.03e S 21±0.1g S Gentamicin 29±0.04d S 29±0.03c S Tetracycline 16±0.04i I 32±0.1a S Cefotaxime 2.9±0.05m R 23±0.2f S Ciprofloxacin 20±0.2h S 2.3±0.03k R Norfloxacin 14±0.01k S 1.6±0.6l R Note: The different lowercase letters in the table indicate a significant difference (P<0.05);R=is low -sensitive or resistant, S=is highly sensitive, I=is moderately sensitive. 
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Table 2 Test results of D-lactic acid content and bile salt hydrolase activity of strains
Strain Bile salt hydrolase activity D-lactic acid content Absorbance values Cholecystolytic enzyme (μmol/L) Absorbance values D-lactic acid concentration (μmol/L) L12 43.51±0.25a 227.32±1.89b 4.99±0.25a 18.87±15.36b L20 59.78±0.38b 245.92±2.0a 6.47±0.38a 21.53±20.25a Control 0.11±0.01b 0.62±2.20c Note: The different lowercase letters in the table indicate a significant difference (P<0.05).
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Table 3 Treatment-related sign of toxicity during the 30-days study period
Organ system Observed & inspected items Performance after gavage Central nervous system Behaviour No abnormal calls, no abnormal posture and repeated scratching Neuromuscular system Action Normal movement, no movement disorder Autonomic nervous system Pupil size Normal, no reduction or expansion Secretion No salivation, no tears Respiratory system Nostrils No flow Respiration rate Normal breathing Digestive system Belly type Abdominal type is normal Fecal shape and color Stool formation, dark yellow Urogenital system Perineum No secretions Mammary gland No swelling Skin and coat Color, tension Normal, no sagging, no rash Completeness No hair removal, no erect Eye Eyelid No upper eyelid droop Eyeball No protrusion, no corneal opacity, no congestion of the conjunctiva Transparency Not turbid
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Table 4 Organ relative weights of control and probiotic-treated mice
Strain Concentration (CFU/mL) Gavage dose (mL) Visceral index (organ-body ratio) Spleen Heart Liver Kidney L12 109 0.2 0.008±0.00028 0.008±0.00005 0.05±0.0022 0.06±0.001 L20 109 0.2 0.009±0.0003 0.008±0.00025 0.06±0.004 0.06±0.003 Control 109 0.2 0.009±0.0005 0.008±0.00022 0.06±0.003 0.06±0.0013 Note: There is no significant difference between the experimental group and the control group (P>0.05).
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