[Preparation of Lactobacillus paracei TK1501 postbiotic and its inhibitory effect against Helicobacter pylori infection in mice]

Abstract

Objective: To prepare a postbiotic using soybean fermentation product of Lactobacillus paracasei TK1501 and evaluate its inhibitory effect against Helicobacter pylori (Hp) infection in mice.

Methods: L. paracasei TK1501 was cultured for 32 h at 37 ℃ in an anaerobic condition for solid substrate fermentation with a solid to water ratio of 1:1.5 in the substrate and an inoculation density of 5×10⁷ CFU/mL. The postbiotic was isolated and purified using macroporous resin XAD-16N adsorption, cation exchange chromatography and HPLC, and its stability and antibacterial activity were assessed. The inhibitory effect of this postbiotic against Hp infection was evaluated in a mouse model with gastric mucosal Hp infection, which were treated with the postbiotic via gavage for 4 weeks at the dose of 0.02 or 0.1 mL. Serum levels of TNF-α and IL-1β of the mice were analyzed after the treatments, and gastric tissues of the mice were collected for HE staining.

Results: L. paracasei TK1501 postbiotic could be easily degraded by protease and had good thermal stability and tolerance to exposures to acid, base, and organic solvents. In the in vitro experiment, the postbiotic showed strong inhibitory effects in bacterial cultures of Staphylococcus aureus, Hp and other common pathogenic bacteria without obviously affecting the resident bacteria in the digestive tract. In the mouse models, treatment with the postbiotic at the dose of 0.1 mL significantly alleviated Hp infection and lowered the serum levels of TNF-α and IL-1β of the mice.

Conclusion: L. paracasei TK1501 postbiotic has strong inhibitory effects on Hp and Staphylococcus aureus but not on normal intestinal flora in mice.

Keywords: Helicobacter pylori infection; L. paracasei TK1501; isolation.

图1

各种蛋白酶处理后的抑菌圈分析实验 Fig.1 Changes in antibacterial activity of the postbiotic after treatment with different proteases

图2

大孔吸附树脂流出液各梯度洗脱液抑菌圈 Fig.2 Bacterial ring for each gradient eluate of the effluent of macroporous adsorbent resin. A1-A3: Effluent of supernatant after large pore adsorption chromatography column; A4-A6: 10%, 20% and 40% ethanol eluent; A7-A9: 60%, 80%, and 100% ethanol eluent.

图3

阳离子交换柱各梯度洗脱液抑菌圈 Fig.3 Bacterial inhibition by each gradient eluate of cation exchange columns. B1: Eluent of 20 mmol/L NaOAc+0.2 mol/L NaCl; B2: Eluent of 20 mmol/L NaOAc+0.5 mol/L NaCl; B3: Eluent of 20 mmol/L NaOAc +1.0 mol/L NaCl.

图4

HPLC分析色谱图 Fig.4 HPLC analysis of the ethanol eluents of the macroporous adsorbent resin and cation exchange resin. A: 20% ethanol elution chromatogram. B: 20 mmol/L sodium acetate+0.2 mol/L sodium chloride elution chromatogram of cation exchange resin.

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L. paracasei TK1501后生元热稳定性试验 Fig.5 Thermal stability test L. paracasei TK1501 postbiotic. C1: Blank control group; C2-C6: Postbiotics heated at 60-100 ℃ for 10 min.

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L. paracasei TK1501后生元酸碱耐受性试验 Fig.6 Acid and base tolerance test L. paracasei TK1501 postbiotic. D1-D7: Postbiotics at pH3-9 (D3 is the blank control).

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不同组别小鼠胃部炎症因子表达水平变化 Fig.7 Changes of serum levels of gastric inflammatory factors in different groups of mice. **P<0.01 vs blank group; ^## P<0.01 vs model group.

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不同组别小鼠胃组织切片HE染色分析 Fig. 8 HE staining of gastric tissues in different groups of mice. A: blank group; B: Hp infection model group; C: L. paracasei TK1501 postbiotic control group; D: L. paracasei TK1501 postbiotic low dose group; E: L. paracasei TK1501 postbiotic high dose group.