Fermentation optimization of Penicillium citrinum PA-33 strain by response surface method
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摘要:目的
优化桔青霉Penicillium citrinum PA-33发酵培养基和发酵条件，以提高桔青霉PA-33的抗菌活性。
方法采用单因素试验确定桔青霉PA-33发酵所需最适基础培养基、碳氮源和无机盐，并利用响应面法设计确定最适发酵培养基配方；在发酵条件单因素试验基础上，采用三元二次通用旋转组合设计和频率分析法优化其最适发酵条件组合。
结果经优化后，最佳发酵培养基配方为马铃薯汁液219.91 g·L−1、甘露醇34.11 g·L−1、黄豆粉6.25 g·L−1；最适发酵条件为装液量50 mL、接种量3.5% (φ)、发酵温度28 ℃，摇床转速150 r·min−1、发酵时间12 d。优化后发酵液对大肠埃希菌Escherichia coli的抑菌圈直径为28.99 mm，较优化前抑菌圈直径 (18.73 mm) 增加了10.26 mm。
结论采用响应面法、三元二次通用旋转组合设计和频率分析法优化发酵工艺，显著提高了桔青霉PA-33发酵液的抗菌活性，为该菌株的抗菌活性物质的分离以及工业化生产提供依据。
Abstract:ObjectiveTo optimize the medium composition and fermentation conditions of Penicillium citrinum PA-33 to improve its antibacterial activity.
MethodSingle factor experiments were used to determine the optimum basic medium, carbon sources, nitrogen sources and inorganic salts. The optimum formulation of fermentation medium was determined by response surface methodology. On the basis of single factor tests of fermentation conditions, the optimal combination of fermentation conditions was optimized by ternary quadratic rotation unitized design and frequency analysis method.
ResultThe optimal medium composition were: Potato juice 219.91 g·L−1, mannitol 34.11 g·L−1 and soybean powder 6.25 g·L−1. The optimal fermentation conditions were: Liquid volume 50 mL, inoculation concentration 3.5% (φ), culture temperature 28 ℃, rotation speed 150 r·min−1 and fermentation for 12 d. The inhibition zone diameter of fermentation broth after optimization on Escherichia coli reached 28.99 mm, increased by 10.26 mm compared with 18.73 mm under the original conditions.
ConclusionResponse surface methodology, ternary quadratic rotation unitized design and frequency analysis method significantly enhance the antibacterial activity of fermentation broth of Penicillium citrinum PA-33, and this study provides a basis for isolation of antibacterial active substances and industrial production of this strain.
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图 1 不同培养基成分对发酵液抗菌活性的影响
B图中横坐标的数字1、2、3、4、5、6、7、8、9和10分别代表葡萄糖、蔗糖、淀粉、玉米粉、乳糖、果糖、糊精、甘油、甘露醇和麦芽糖；C图中横坐标的数字1、2、3、4、5、6、7、8、9、10和11分别代表对照、蛋白胨、酵母浸粉、牛肉膏、胰蛋白胨、黄豆粉、尿素、KNO3、NH4NO3、NH4Cl和陈皮；各图中柱子上方的不同小写字母表示差异显著(P<0.05, Duncan’s法)
Figure 1. Effects of different medium ingredients on the antibacterial activities of fermentation broth
In figure B，number 1，2，3，4，5，6，7，8，9 and 10 represent glucose, sucrose, starch, corn flour, lactose, fructose, dextrin, glycerin, mannitol and maltose, respectively; In figure B，number 1，2，3，4，5，6，7，8，9, 10 and 11 represent control, peptone, yeast extract, beef extract, tryptone, soybean powder, urea, KNO3, NH4NO3, NH4Cl and bran, respectively; In each figure, different lowercase letters on the bars indicate significant difference (P<0.05, Duncan’s test)
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图 2 菌株PA-33抗菌活性的响应曲面图
A1、A2和A3为响应面3D图；B1、B2和B3为等高线图
Figure 2. Response surface plots of antibacterial activity of strain PA-33
A1, A2, and A3: 3D maps of response surfaces; B1, B2, and B3: Contour maps
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图 3 不同发酵条件对菌株PA-33抑菌活性的影响
各图中, 柱子上方的不同小写字母表示差异显著(P<0.05, Duncan’s法)
Figure 3. Effect of different dermentation condition on antimicrobial activity of strain PA-33
In each figure, different lowercase letters on the bars indicate significant difference (P<0.05, Duncan’s test)
表 1 不同质量浓度的培养基成分对菌株PA-33抗菌活性的影响1)
Table 1 Effects of different concentrations of medium components on antibacterial activities of fermentation broth
ρ(马铃薯)/(g·L−1) Patato content 抑菌圈直径/mm Inhibition zone diameter ρ(甘露醇)/(g·L−1) Mannitol content 抑菌圈直径/mm Inhibition zone diameter ρ(黄豆粉)/((g·L−1) Soybean powder content 抑菌圈直径/mm Inhibition zone diameter 100 22.93±0.52ab 20 22.03±0.12c 5.0 23.83±0.69c 150 23.13±0.25ab 25 23.33±0.09b 7.5 27.93±0.19a 200 23.47±0.25a 30 26.60±0.83a 10.0 25.77±0.31b 250 22.53±0.19b 35 25.50±0.08a 12.5 25.13±0.09b 300 21.80±0.16c 40 23.43±0.87b 15.0 22.43±0.17d 1) 同列数据后的不同小写字母表示差异显著 (P<0.05，Duncan’s 法 )
1) Different lowercase letters in the same column indicate significant difference (P<0.05，Duncan’s test)
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表 2 Box-Behnken试验方差分析
Table 2 Analysis of variance for Box-Behnken experiment
来源1) Source SS DF MS F P2) 模型 Model 76.25 9 8.47 22.24 0.000 2** A 1.07 1 1.07 2.80 0.138 3 B 15.62 1 15.62 41.01 0.000 4** C 14.36 1 14.36 37.70 0.000 5** AB 0.07 1 0.07 0.17 0.691 9 AC 9.52 1 9.52 24.98 0.001 6** BC 0.01 1 0.01 0.02 0.894 4 A2 14.56 1 14.56 38.22 0.000 5** B2 12.31 1 12.31 32.30 0.000 7** C2 5.19 1 5.19 13.61 0.007 8** 残差
Residual2.67 7 0.38 失拟
Lack of fit1.96 3 0.65 3.71 0.118 9 误差
Error0.71 4 0.18 总变异
Total variation78.92 16 1) A、B 和 C 分别表示马铃薯、甘露醇和黄豆粉；2) “**” 表示模型具有极显著影响 (P<0.01)
1) A, B and C represent potato, mannitol and soybean powder, respectively; 2) “**” indicates the model is highly significant(P<0.01)
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表 3 三元二次通用旋转组合试验设计及结果1)
Table 3 Design and results for quadratic general rotary composite
试验号
Test No.θ/℃ 接种量(φ)/ %
Inoculation concentration装液量/mL
Liquid volume抑菌圈直径/mm
Inhibition zone diameter1 30(1) 5(1) 60(1) 28.30 2 30(1) 5(1) 40(−1) 28.37 3 30(1) 2(−1) 60(1) 27.80 4 30(1) 2(−1) 40(−1) 27.93 5 26(−1) 5(1) 60(1) 28.73 6 26(−1) 5(1) 40(−1) 28.87 7 26(−1) 2(−1) 60(1) 28.40 8 26(−1) 2(−1) 40(−1) 28.57 9 24.6(−1.682) 3.5(0) 50(0) 26.88 10 31.4(1.682) 3.5(0) 50(0) 27.00 11 28(0) 0.977(−1.682) 50(0) 28.80 12 28(0) 6(1.682) 50(0) 29.23 13 28(0) 3.5(0) 33.2(−1.682) 28.53 14 28(0) 3.5(0) 66.8(1.682) 28.87 15 28(0) 3.5(0) 50(0) 28.80 16 28(0) 3.5(0) 50(0) 28.93 17 28(0) 3.5(0) 50(0) 29.20 18 28(0) 3.5(0) 50(0) 29.13 17 28(0) 3.5(0) 50(0) 29.00 18 28(0) 3.5(0) 50(0) 28.90 1) 括号中的数值为编码值
1) Numbers in the brackets are coded values
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表 4 三元二次通用旋转组合试验结果方差分析
Table 4 Analysis of variance for results from quadratic general rotary united design
来源 Source SS DF MS PCC F P X1 0.283 6 1 0.283 6 −0.515 4 3.617 2 0.086 4 X2 0.385 1 1 0.385 1 0.573 9 4.910 4 0.051 0 X3 0.000 3 1 0.000 3 0.018 9 0.003 6 0.953 5 X12 6.406 3 1 6.406 3 −0.943 9 81.696 5 0.000 1 X22 0.064 5 1 0.064 5 0.275 6 0.822 2 0.385 9 X32 0.028 5 1 0.028 5 −0.187 3 0.363 6 0.559 9 X1X2 0.012 0 1 0.012 0 0.122 8 0.153 2 0.703 7 X1X3 0.001 5 1 0.001 5 0.043 9 0.019 3 0.892 3 X2X3 0.001 0 1 0.001 0 0.035 9 0.012 9 0.911 8 回归 Regression 7.349 1 9 0.816 6 10.413 2 0.000 8 残差 Residual 0.784 2 10 0.078 4 失拟 Lack of fit 0.541 6 5 0.108 3 2.233 2 0.130 9 误差 Error 0.242 5 5 0.048 5 总和 Total 8.133 2 19 1) X1、X2 和 X3 分别表示温度、接种量和装液量；PCC 为偏相关系数
1) X1，X2 andX3 represent temperature，inoculation concentration and liquid volume, respectively; PCC is patial correlation coefficient
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表 5 各变量取值频率分布
Table 5 Frequency distribution of variable value
水平
Levelθ 接种量 Inoculation concentration 装液量 Liquid volume 次数 Times 频率 Frequency 次数 Times 频率 Frequency 次数 Times 频率 Frequency −1.682 0 0.000 5 0.143 7 0.200 −1.000 10 0.286 5 0.143 7 0.200 0.000 25 0.714 5 0.143 7 0.200 1.000 0 0.000 10 0.286 7 0.200 1.682 0 0.000 10 0.286 7 0.200
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