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
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摘要
【目的】利用Box Behnken响应面法研究免疫球蛋白Y(Ig Y)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Ig Y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、Ig Y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Ig Y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Ig Y剂量,2×10~6 CFU/m L的初始P.gingivalis数量,37°C厌氧培养4 h,口腔模拟环境Ig Y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:Ig Y剂量165 mmol/L、初始P.gingivalis数量2×10~6 CFU/m L、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5.92×10~5 CFU/m L,与模型预测值5.85×10~5 CFU/m L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】Ig Y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。
[Objective]Box Behnken design response surface methodology was used to optimize the colony count of Ig Y against Porphyromonas gingivalis in oral simulation environment in order to find the appropriate dose of Ig Y,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 Ig Y,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 Ig Y had the best effect on the reproduction of P.gingivalis by the dose of 180 mmol/L Ig Y,the initial amount of 2×10~6 CFU/m L P.gingivalis and the incubation at 37°C for 4 h.The optimum culture conditions were as follows:dose of Ig Y was 165 mmol/L,the initial amount of P.gingivalis was 2×10~6 CFU/m L,culture time was 4.5 h.Under these conditions the final number of colonies was 5.92×10~5 CFU/m L.Compared with the predicted value of 5.85×10~5 CFU/m L,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.
[Objective]Box Behnken design response surface methodology was used to optimize the colony count of Ig Y against Porphyromonas gingivalis in oral simulation environment in order to find the appropriate dose of Ig Y,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 Ig Y,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 Ig Y had the best effect on the reproduction of P.gingivalis by the dose of 180 mmol/L Ig Y,the initial amount of 2×10~6 CFU/m L P.gingivalis and the incubation at 37°C for 4 h.The optimum culture conditions were as follows:dose of Ig Y was 165 mmol/L,the initial amount of P.gingivalis was 2×10~6 CFU/m L,culture time was 4.5 h.Under these conditions the final number of colonies was 5.92×10~5 CFU/m L.Compared with the predicted value of 5.85×10~5 CFU/m L,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.
引文
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[14]Zhou Y,Wu WH,Su TW,et al.Use of response surface methodology on the production of active compound MFS from Fusarium solani and its activity on HMG-Co A reductase[J].Microbiology China,2014,41(8):1516-1524(in Chinese)周喻,吴文惠,苏同伟,等.响应面法优化海洋镰刀腐皮菌产出HMG-Co A还原酶抑制剂的培养条件[J].微生物学通报,2014,41(8):1516-1524
[15]Bostanci N,Belibasakis GN.Porphyromonas gingivalis:an invasive and evasive opportunistic oral pathogen[J].FEMS Microbiology Letters,2012,333(1):1-9
[16]Hu Y.The effect of oral malodor system on identifying halitosis-correlated bacteria[D].Chengdu:Master’s Thesis of Sichuan University,2004(in Chinese)胡赟.口臭研究模型对致臭菌确定的探讨[D].成都:四川大学硕士学位论文,2004
[17]Li XY,Liu H,Xu YP,et al.Chicken egg yolk antibody(Ig Y)controls Solobacterium moorei under in vitro and in vivo conditions[J].Applied Biochemistry and Biotechnology,2012,168(6):1448-1458
[18]Taylor AI,Gould HJ,Sutton BJ,et al.Avian Ig Y binds to a monocyte receptor with Ig G-like kinetics despite an Ig E-like structure[J].The Journal of Biological Chemistry,2008,283(24):16384-16390
[19]Rahman S,van Nguyen S,Icatlo FC Jr,et al.Oral passive Ig Y-based immunotherapeutics[J].Human Vaccines&Immunotherapeutics,2013,9(5):1039-1048
[20]Liu PF,Huang IF,Shu CW,et al.Halitosis vaccines targeting Fom A,a biofilm-bridging Protein of Fusobacteria nucleatum[J].Current Molecular Medicine,2013,13(8):1358-1367
[2]Carlander D,Kollberg H,Wej?ker P,et al.Peroral immunotheraphy with yolk antibodies for the prevention and treatment of enteric infections[J].Immunologic Research,2000,21(1):1-6
[3]Jin WC,Yamada K,Ikami M,et al.Application of Ig Y to sandwich enzyme-linked immunosorbent assays,lateral flow devices,and immunopillar chips for detecting staphylococcal enterotoxins in milk and dairy products[J].Journal of Microbiological Methods,2013,92(3):323-331
[4]Lee EN,Sunwoo HH,Menninen K,et al.In vitro studies of chicken egg yolk antibody(Ig Y)against Salmonella enteritidis and Salmonella typhimurium[J].Poultry Science,2002,81(5):632-641
[5]Sui JX.Study on the antibacterial activity and mechanism of chicken egg yolk antibodies against Listeria monocytogens[D].Qingdao:Doctoral Dissertation of Ocean University of China,2010(in Chinese)隋建新.单增李斯特菌鸡卵黄抗体的抑菌性能及其机理研究[D].青岛:中国海洋大学博士学位论文,2010
[6]Sun XY,Xu Y,Wang C,et al.Effects of egg yolk-derived immunoglobulin(Ig Y)against Porphyromonas gingivalis induced experimental periodontitis in rats[J].Chinese Journal of Conservative Dentistry,2011,21(7):380-385(in Chinese)孙晓瑜,徐燕,王姹,等.被动免疫制剂抗Pg-Ig Y对大鼠实验性牙周炎的治疗[J].牙体牙髓牙周病学杂志,2011,21(7):380-385
[7]Li Y,Xu YP,Wang LL,et al.Application of Ig Y as a nutraceutical in animal feed and disease control[J].Heilongjiang Animal Science and Veterinary Medicine,2016(5):190-193,287(in Chinese)李媛,徐永平,王丽丽,等.药效营养品Ig Y在动物饲料与疾病防治中的应用[J].黑龙江畜牧兽医,2016(5):190-193,287
[8]Hatta H,Tsuda K,Ozeki M,et al.Passive immunization against dental plaque formation in humans:effect of a mouth rinse containing egg yolk antibodies(Ig Y)specific to Streptococcus mutans[J].Caries Research,1997,31(4):268-274
[9]Yokoyama K,Sugano N,Shimada T,et al.Effects of egg yolk antibody against Porphyromonas gingivalis gingipains in periodontitis patients[J].Journal of Oral Science,2007,49(3):201-206
[10]Zhang W,Feng XP,Tao DY,et al.Clinical study on the effect of anti-gingivitis Ig Y toothpaste in control of gingivitis and dental plaque[J].Shanghai Journal of Stomatology,2016,25(4):453-455(in Chinese)张玮,冯希平,陶丹英,等.抗龈炎Ig Y牙膏对牙龈炎、菌斑控制的评价[J].上海口腔医学,2016,25(4):453-455
[11]Shellis RP.A synthetic saliva for cultural studies of dental plaque[J].Archives of Oral Biology,1978,23(6):485-489
[12]Saunders KA,Greenman J,Mc Kenzie C.Ecological effects of triclosan and triclosan monophosphate on defined mixed cultures of oral species grown in continuous culture[J].Journal of Antimicrobial Chemotherapy,2000,45(4):447-452
[13]Hu Y,Hu DY,Zheng LL,et al.Establishment of malodor model and its effects on identifying the halitosis-related bacteria[J].West China Journal of Stomatology,2007,25(3):226-229(in Chinese)胡赟,胡德渝,郑雷蕾,等.口臭研究模型的建立及其对口腔致臭菌的确定[J].华西口腔医学杂志,2007,25(3):226-229
[14]Zhou Y,Wu WH,Su TW,et al.Use of response surface methodology on the production of active compound MFS from Fusarium solani and its activity on HMG-Co A reductase[J].Microbiology China,2014,41(8):1516-1524(in Chinese)周喻,吴文惠,苏同伟,等.响应面法优化海洋镰刀腐皮菌产出HMG-Co A还原酶抑制剂的培养条件[J].微生物学通报,2014,41(8):1516-1524
[15]Bostanci N,Belibasakis GN.Porphyromonas gingivalis:an invasive and evasive opportunistic oral pathogen[J].FEMS Microbiology Letters,2012,333(1):1-9
[16]Hu Y.The effect of oral malodor system on identifying halitosis-correlated bacteria[D].Chengdu:Master’s Thesis of Sichuan University,2004(in Chinese)胡赟.口臭研究模型对致臭菌确定的探讨[D].成都:四川大学硕士学位论文,2004
[17]Li XY,Liu H,Xu YP,et al.Chicken egg yolk antibody(Ig Y)controls Solobacterium moorei under in vitro and in vivo conditions[J].Applied Biochemistry and Biotechnology,2012,168(6):1448-1458
[18]Taylor AI,Gould HJ,Sutton BJ,et al.Avian Ig Y binds to a monocyte receptor with Ig G-like kinetics despite an Ig E-like structure[J].The Journal of Biological Chemistry,2008,283(24):16384-16390
[19]Rahman S,van Nguyen S,Icatlo FC Jr,et al.Oral passive Ig Y-based immunotherapeutics[J].Human Vaccines&Immunotherapeutics,2013,9(5):1039-1048
[20]Liu PF,Huang IF,Shu CW,et al.Halitosis vaccines targeting Fom A,a biofilm-bridging Protein of Fusobacteria nucleatum[J].Current Molecular Medicine,2013,13(8):1358-1367