基于Box Behnken响应面法优化口腔模拟环境IgY抑制牙龈卟啉单胞菌增殖

Optimization of IgY inhibiting Porphyromonas gingivalis proliferation in oral simulation environment based on Box Behnken design response surface methodology

摘要:

【目的】利用Box Behnken响应面法研究免疫球蛋白Y (IgY)影响牙龈卟啉单胞菌(Porphyromonas gingivalis)的生长繁殖特性，发现口腔模拟环境中P. gingivalis的响应值与IgY剂量、初始P. gingivalis数量、培养时间的关系。【方法】以P. gingivalis最终的菌落数为评价指标，通过单因素试验研究培养时间、IgY剂量和初始P. gingivalis数量的最佳条件，再依据Box Behnken中心组合原则设计三因素三水平响应面试验进行优化，评价IgY的抑制效果。优化得到的试验条件重复3次，以验证模型的准确性。【结果】单因素试验结果表明，以180 mmol/L的IgY剂量，2×106 CFU/mL的初始P. gingivalis数量，37 °C厌氧培养4 h，口腔模拟环境IgY对P. gingivalis繁殖的影响达到最佳状态。通过Design Expert 8.0软件对回归方程的分析得到各因素最佳条件为：IgY剂量165 mmol/L、初始P. gingivalis数量2×106 CFU/mL、培养时间4.5 h。采用优化实验条件，重复3次试验，P. gingivalis最终菌落数是5.92×105 CFU/mL，与模型预测值5.85×105 CFU/mL相对误差1%，方差分析表明模型P值显著，缺失值P值不显著，理论值与实测值拟合良好。【结论】IgY能显著抑制牙龈卟啉单胞菌的增殖，优化后口腔模拟环境中的P. gingivalis最终菌落数显著降低，回归模型能够预测口腔模拟环境中P. gingivalis的增殖。

Abstract:

[Objective] Box Behnken design response surface methodology was used to optimize the colony count of IgY against Porphyromonas gingivalis in oral simulation environment in order to find the appropriate dose of IgY, initial density of P. gingivalis and culture time. [Methods] Final colony count was used as the evaluation index, on the basis of single factor tests. Box Behnken RSM was used with three factors such as number of colonies at different dose of IgY, initial density of P. gingivalis and culture time in the artificial saliva. The experiment was repeated three times under the optimal conditions to verify the accuracy of the model. [Results] The results of single factor test showed that IgY had the best effect on the reproduction of P. gingivalis by the dose of 180 mmol/L IgY, the initial amount of 2×106 CFU/mL P. gingivalis and the incubation at 37 °C for 4 h. The optimum culture conditions were as follows: dose of IgY was 165 mmol/L, the initial amount of P. gingivalis was 2×106 CFU/mL, culture time was 4.5 h. Under these conditions the final number of colonies was 5.92×105 CFU/mL. Compared with the predicted value of 5.85×105 CFU/mL, the relative error with the theoretical value was 1%. Analysis of Variance showed the P value of the model was significant, and the missing P value was not significant. The theoretical value was in good agreement with the measured value. [Conclusion] The number of colonies of P. gingivalis in the simulated oral environment was significantly decreased, which indicated that the regression model could be a good predictor of the response of P. gingivalis in the oral cavity.