响应面法优化副猪嗜血杆菌12型H31菌株发酵培养基组分的研究

doi:10.16431/j.cnki.1671-7236.2025.08.032

摘要/Abstract

摘要： 【目的】应用响应面法优化副猪嗜血杆菌(Haemophilus parasuis,Hps)12型H31菌株的发酵培养基组分，以提高活菌数，降低Hps 12型H31菌株发酵生产疫苗的成本，为工业化生产Hps疫苗提供参考。【方法】通过单因素试验考察牛血清、酵母提取物、烟酰胺腺嘌呤二核苷酸(NAD)、硫酸镁(MgSO₄)4种培养基成分对Hps 12型H31菌株活菌数的影响，确定各因素最佳水平范围后，选取合适水平进行BBD(Box-Behnken design)响应面优化设计。通过对牛血清、酵母提取物、NAD、MgSO₄ 4种培养基成分进行不同组合，系统优化培养基中4种组分的添加量。利用二次响应面回归方法建立活菌数与各组分之间的数学模型，并通过回归分析确定各组分对活菌数的影响程度。【结果】单因子优化显示，活菌数较大时发酵培养基中各组分添加量区间范围分别为：牛血清8%～10%、酵母提取物0.4%～0.6%、NAD 0.05%～0.1%、MgSO₄ 0.3～0.5 g/L。在此基础上进一步应用响应面优化，经优化后Hps 12型H31菌株的最佳发酵培养基组分为：牛血清浓度为9.91%，MgSO₄浓度为0.21 g/L，酵母提取物浓度为0.59%,NAD浓度为0.09%。与初始培养基成分相比，该菌株经优化后的活菌数从1.2×10⁸ CFU/mL提高到3.0×10⁹ CFU/mL，提升了24倍。【结论】本试验通过响应面法优化Hps 12型H31菌株发酵培养基，优化后的发酵培养基能提高Hps的活菌数，为工业化生产Hps疫苗提供参考。

关键词: 副猪嗜血杆菌12型H31菌株; 响应面法; 发酵培养基优化

Abstract: 【Objective】 This study aimed to optimize the fermentation medium components of Haemophilus parasuis (Hps) serotype 12 H31 strain by response surface methodology,improve the number of viable bacteria,reduce the cost of vaccine production by fermentation of Haemophilus parasuis serotype 12 H31 strain,and provide reference for industrial production of Hemophilus parasuis vaccine. 【Method】 Employing single-factor experiments,the effects of bovine serum,yeast extract,nicotinamide adenine dinucleotide (NAD) and MgSO₄ on the viable bacteria count of Haemophilus parasuis serotype 12 H31 strain were investigated meticulously.After determining the optimal level ranges,suitable levels were selected for Box-Behnken design (BBD) response surface optimization design.The fermentation conditions were systematically optimized by different combinations of bovine serum,yeast extract,NAD and MgSO₄.The mathematical model between viable count and each component was established by quadratic response surface regression method,and the influence degree of each component on viable count was determined by regression analysis. 【Result】 Single factor optimization revealed that the optimal ranges of the various components added to the fermentation medium were as follows:When the viable count was high,bovine serum was 8%-10%,yeast extract was 0.4%-0.6%,NAD was 0.05%-0.1% and MgSO₄ was 0.3-0.5 g/L.Further application of surface optimization resulted in the determination of the optimal culture medium components for Haemophilus parasuis serotype 12 H31 strain after optimization, bovine serum concentration was 9.91%,MgSO₄ concentration was 0.21 g/L,yeast extract concentration was 0.59%,NAD concentration was 0.09%.Compared with the initial medium components,the viable count of the strain was increased from 1.2×10⁸ to 3.0×10⁹ CFU/mL,which was 24 times higher after optimization. 【Conclusion】 In this experiment,the response surface method was used to optimize the fermentation conditions of Haemophilus parasuis serotype 12 H31 strain.The optimized fermentation medium could improve the number of viable bacteria of Haemophilus parasuis well,and provide a reference for the industrial production of Haemophilus parasuis vaccine.

Key words: Haemophilus parasuis serotype 12 H31 strain; response surface methodology; optimization of fermentation medium

中图分类号:

S852.61

赖小婷, 吴崟沣, 任晓敏, 洪健渠, 严芬. 响应面法优化副猪嗜血杆菌12型H31菌株发酵培养基组分的研究[J]. 中国畜牧兽医, 2025, 52(8): 3838-3846.

LAI Xiaoting, WU Yinfeng, REN Xiaomin, HONG Jianqu, YAN Fen. Optimization of Fermentation Medium Components of Haemophilus parasuis Serotype 12 H31 Strain by Response Surface Methodology[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(8): 3838-3846.

参考文献

[1] ZHOU M,GUO Y,ZHAO J,et al.Identification and characterization of novel immunogenic outer membrane proteins of Haemophilus parasuis serovar 5[J].Vaccine,2009,27(38):5271-5277.
[2] 王显勇.副猪嗜血杆菌病防控技术要点[J].今日畜牧兽医,2024,40(5):23-25. WANG X Y.Prevention and control technology of Haemophilus parasuis disease[J].Today Animal Husbandry and Veterinary Medicine,2024,40(5):23-25.(in Chinese)
[3] 肖月,吴庆鹏,龚旺,等.副猪嗜血杆菌病研究进展[J].江西畜牧兽医杂志,2024,1:46-50. XIAO Y,WU Q P,GONG W,et al.Research progress on Haemophilus parasuis disease[J].Jiangxi Journal of Animal Husbandry & Veterinary Medicine,2024,1:46-50.(in Chinese)
[4] 张小兰.副猪嗜血杆菌4型H25株、5型H45株及12型H31株的发酵工艺研究[J].福建畜牧兽医,2024,46(4):49-54. ZHANG X L.Study on the fermentation process of Haemophilus parasuis type 4 H25,type 5 H45,and type 12 H31 strains[J]. Fujian Animal Husbandry and Veterinary Medicine,2024,46(4):49-54.(in Chinese)
[5] 肖敏,刘建,卢昌,等.副猪嗜血杆菌的分离鉴定与灭活疫苗的初步研制[J].动物医学进展,2022,43(8):35-40. XIAO M,LIU J,LU C,et al.Isolation and identification of Haemophilus parasuis and preliminary development of inactivated vaccine[J].Progress in Veterinary Medicine,2022,43(8):35-40.(in Chinese)
[6] 戴璐,张千,万佳佳,等.1株血清10型副猪嗜血杆菌的分离鉴定与生物学特性分析[J].中国畜牧兽医,2024,51(4):1671-1685. DAI L,ZHANG Q,WAN J J,et al.Isolation,identification,and analysis of biological characteristics of a serotype 10 Haemophilus parasuis strain[J].China Animal Husbandry & Veterinary Medicine,2024,51(4):1671-1685.(in Chinese)
[7] 苗森,牛得权,张阳阳,等.基于响应面分析的高抗原活性猪链球菌2型疫苗培养基的研制与效果评价研究[J].饲料研究,2024,47(1):86-92. MIAO S,NIU D Q,ZHANG Y Y,et al.Development and efficacy evaluation of high antigenicity Streptococcus suis type 2 vaccine culture medium based on response surface analysis[J].Feed Research,2024,47(1):86-92.(in Chinese)
[8] 邱文彬,于江,张玉玉,等.副猪嗜血杆菌流行病学调查及两株分离株耐药性和致病性分析[J].家畜生态学报,2020,41(2):64-71. QIU W B,YU J,ZHANG Y Y,et al.Epidemiological investigation of Haemophilus parasuis and the drug resistance and pathogenicity analysis of two isolates[J].Journal of Domestic Animal Ecology,2020,41(2):64-71.(in Chinese)
[9] 刘建忠,熊亚红,翁丽萍,等.生物过程的优化[J].中山大学学报(自然科学版),2002,S1:132-137. LIU J Z,XIONG Y H,WENG L P,et al.Bioprocess opimization[J].Acta Scientiarum Naturalium Universitatis Sunyatseni,2002,S1:132-137.(in Chinese)
[10] SIGAL I A,WHYNE C M.Mesh morphing and response surface analysis:Quantifying sensitivity of vertebral mechanical behavior[J].Annals of Biomedical Engineering,2010,38(1):41-56.
[11] LAU H L,WONG F W F,RAHMAN R N Z R A,et al.Optimization of fermentation medium components by response surface methodology (RSM) and artificial neural network hybrid with genetic algorithm (ANN-GA) for lipase production by Burkholderia cenocepacia ST8 using used automotive engine oil as substrate[J].Biocatalysis and Agricultural Biotechnology,2023,50:102696.
[12] 宋鹏,李理想,赵彪,等.基于MTT比色和响应面法优化侧孢短芽孢杆菌最大活菌数培养条件[J].食品与机械,2024,40(2):28-35. SONG P,LI L X,ZHAO B,et al.Optimization of cultivation conditions for maximum live bacterial count of Bacillus laterosporus based on MTT colorimetry and response surface methodology[J].Food & Machinery,2024,40(2):28-35.(in Chinese)
[13] 张阳阳,万强,牛得权,等.基于响应面分析的高抗原活性猪水肿病大肠杆菌疫苗培养基的研制及效果评价[J].微生物学通报,2023,50(9):4141-4153. ZHANG Y Y,WANG Q,NIU D Q,et al.Development and evaluation of a vaccine medium for highly antigenic porcine edema disease E.coli based on response surface methodology[J].Microbiology China,2023,50(9):4141-4153.(in Chinese)
[14] 马林丽,徐国强,张秋萍,等.优化培养基组分提高猪链球菌2型NT15B菌株活菌数的研究[J].中国畜牧兽医,2024,51(7):2776-2786. MA L L,XU G Q,ZHANG Q P,et al.Study on optimizing the composition of the culture medium to increase the viable cell count of Streptococcus suis type 2 NT15B strain[J].China Animal Husbandry & Veterinary Medicine,2024,51(7):2776-2786.(in Chinese)
[15] SRIVASTAVA R,BARUAH J J A,MICROBIOLOGY E.Culture conditions for production of thermostable amylase by Bacillus stearothermophilus[J].Applied and Environmental Microbiology,1986,52(1):179-184.
[16] 张丽梅.解朊金黄杆菌蛋白质谷氨酰胺酶发酵培养基成分筛选和组合优化研究[D].南京:南京农业大学,2022. ZHANG L M.Study on component screening and combination optimization of protein glutaminase fermentation medium for Lactobacillus plantarum[D].Nanjing:Nanjing Agricultural University,2022.(in Chinese)
[17] 张豆,李月影,申硕,等.新生牛血清快速筛选方法的建立[J].中国生物制品学杂志,2022,35(2):200-204. ZHANG D,LI Y Y,SHEN S,et al.Establishment of a rapid screening method for newborn bovine serum[J].Chinese Journal of Biological Products,2022,35(2):200-204.(in Chinese)
[18] 李世明,胡燕杰,张丽.动物疫苗生产用的新生牛血清管理策略[J].国外畜牧学(猪与禽),2022,42(4):80-85. LI S M,HU Y J,ZHANG L.Management strategy for newborn bovine serum used in animal vaccine production[J].Foreign Journal of Animal Husbandry (Pigs and Poultry),2022,42(4):80-85.(in Chinese)
[19] 康碧静,马芳芳,马春英,等.不同批次新生牛血清对MDCK细胞培养效果的研究[J].福建畜牧兽医,2023,45(1):1-5. KANG B J,MA F F,MA C Y,et al.Study on the effect of different batches of newborn bovine serum on MDCK cell culture[J].Fujian Animal Husbandry and Veterinary Medicine,2023,45(1):1-5.(in Chinese)
[20] 徐涛,金磊,夏鹏志.生物细胞内NAD水平分析技术的研究进展[J].分析测试学报,2024,43(7):1076-1085. XU T,JIN L,XIA P Z.Research progress on NAD level analysis technology in biological cells[J].Journal of Analytical Testing,2024,43(7):1076-1085.(in Chinese)
[21] 林丽苗,周庆丰,余国莲,等.副猪嗜血杆菌培养条件优化[J].现代畜牧兽医,2013,7:56-58. LIN L M,ZHOU Q F,YU G L,et al.Optimization of culture conditions for Haemophilus parasuis[J].Modern Animal Husbandry and Veterinary Medicine,2013,7:56-58.(in Chinese)
[22] 赖志,黄锡琴,龚建培,等.猪繁殖与呼吸综合征活疫苗(CH-1R株)工厂化生产培养条件的研究[J].中国预防兽医学报,2010,32(8):615-618. LAI Z,HUANG X Q,GONG J P,et al.Study on the factory production and cultivation conditions of porcine reproductive and respiratory syndrome live vaccine (CH-1R strain)[J].Chinese Journal of Preventive Veterinary Medicine,2010,32(8):615-618.(in Chinese)
[23] 王擎.两种鱼类细菌疫苗的培养基及发酵工艺优化[D].上海:华东理工大学,2021. WANG Q.Optimization of culture media and fermentation process for two fish bacterial vaccines[D].Shanghai:East China University of Science and Technology,2021.(in Chinese)
[24] 孙承文,江小燕,赖迎迢,等.一株维氏气单胞菌发酵培养基优化及其制备疫苗免疫效力比较[J].微生物学杂志,2022,42(4):48-54. SUN C W,JIANG X Y,LAI Y T,et al.Optimization of fermentation medium for a strain of Pseudomonas aeruginosa and comparison of immune efficacy in vaccine preparation[J].Journal of Microbiology,2022,42(4):48-54.(in Chinese)
[25] 杨帅.生长抑素和皮质抑素双表达DNA疫苗培养基优化及免疫水牛犊牛的促生长效果[D].武汉:华中农业大学,2021. YANG S.Optimization of culture medium for somatostatin and corticosteroid dual expression DNA vaccine and growth promoting effect of immune buffalo calves[D].Wuhan:Huazhong Agricultural University,2021.(in Chinese)