浙江农业学报 ›› 2021, Vol. 33 ›› Issue (3): 479-489.DOI: 10.3969/j.issn.1004-1524.2021.03.13
收稿日期:
2020-07-26
出版日期:
2021-04-02
发布日期:
2021-03-25
通讯作者:
程亮
作者简介:
, 程亮,E-mail: liangcheng1979@163.com基金资助:
YUE Denggao, CHENG Liang*(), GUO Qingyun
Received:
2020-07-26
Online:
2021-04-02
Published:
2021-03-25
Contact:
CHENG Liang
摘要:
探究出芽短梗霉菌(Aureobasidium pullulans)PA-2脂肽类物质的抑菌活性,并以脂肽类物质的产量为响应值对其发酵条件进行优化,为脂肽类物质的生产与应用奠定理论基础。采用酸沉淀法对脂肽类物质进行粗提,通过原位酸水解-茚三酮显色法对脂肽粗提物进行定性检测,并用琼脂打孔扩散法对其抑菌活性进行测定,通过中心组合设计(central composite design,CCD)构建响应面对其发酵条件进行优化。最终初步判定粗提物为环状脂肽类物质;该物质对金黄色葡萄球菌(Staphylococcus aureus)、酿酒酵母菌(Saccharomyces cerevisiae)、大肠埃希菌(Escherichia coli)、樱桃球腔菌(Mycosphaerella cerasella)和大麦网斑病(Pyrenophora teres)具有明显的抑制作用,抑菌圈分别为3.65、1.95、2.15、1.35、2.18 cm;优化后的最适发酵条件为:接种量6.8%、转速216 r·min -1、温度26 ℃、装液量125 mL、pH 7。在此条件下模型预测的脂肽类物质产量为0.94 g·L -1,实际为0.92 g·L -1,比优化前的产量(0.61 g·L -1)提高了51%。优化后的发酵条件可提高脂肽类物质产量,降低发酵成本,可用于上述有抑制效果的菌株防治。
中图分类号:
岳登高, 程亮, 郭青云. 出芽短梗霉菌PA-2脂肽类物质抑菌活性及其发酵条件优化[J]. 浙江农业学报, 2021, 33(3): 479-489.
YUE Denggao, CHENG Liang, GUO Qingyun. Antibacterial activities and optimization of fermentation conditions of lipopeptides produced by Aureobasidium pullulans PA-2[J]. Acta Agriculturae Zhejiangensis, 2021, 33(3): 479-489.
试验因素 Factor | 试验水平Level | ||||
---|---|---|---|---|---|
-2.378 | -1 | 0 | +1 | +2.378 | |
接种量Inoculation amount (A)/% | 2.24 | 5 | 7 | 9 | 11.76 |
转速Rotation speed(B)/(r·min-1) | 118.66 | 160 | 190 | 220 | 261.34 |
温度Temperature(C)/℃ | 20.24 | 23 | 25 | 27 | 29.80 |
装液量Liquid volume (D)/mL | 31.10 | 100 | 150 | 200 | 268.90 |
初始pH Initial pH(E) | 4.62 | 6 | 7 | 8 | 9.38 |
表1 响应面法-通用旋转中心组合设计因素及水平
Table 1 Response surface method-general rotating center combination design factors and levels
试验因素 Factor | 试验水平Level | ||||
---|---|---|---|---|---|
-2.378 | -1 | 0 | +1 | +2.378 | |
接种量Inoculation amount (A)/% | 2.24 | 5 | 7 | 9 | 11.76 |
转速Rotation speed(B)/(r·min-1) | 118.66 | 160 | 190 | 220 | 261.34 |
温度Temperature(C)/℃ | 20.24 | 23 | 25 | 27 | 29.80 |
装液量Liquid volume (D)/mL | 31.10 | 100 | 150 | 200 | 268.90 |
初始pH Initial pH(E) | 4.62 | 6 | 7 | 8 | 9.38 |
图2 脂肽类物质对不同菌株的抑制效果 A,金黄色葡萄球菌;B,酿酒酵母;C,大肠埃希菌;D,白色念珠菌;E,樱桃球腔菌;F,大麦网斑病;CK-,阴性对照;CK+,阳性对照。
Fig.2 Inhibitory effect of crude lipopeptide on different strains A, Staphylococcus aureus; B, Saccharomyces cerevisiae; C, Escherichia coli; D, Candida albicans; E, Mycosphaerella cerasella; F, Pyrenophora teres; CK-, Negative control; CK+, Control.
靶标菌 Target bacteria | 平均抑菌圈直径 Antibacterial circle diameter/cm |
---|---|
金黄色葡萄球菌Staphylococcus aureus | 3.65 |
酿酒酵母菌Saccharomyces cerevisiae | 1.95 |
大肠埃希菌Escherichia coli | 2.15 |
白色念珠菌Candida albicans | 0 |
樱桃球腔菌Mycosphaerella cerasella | 1.35 |
大麦网斑病Pyrenophora teres | 2.18 |
表2 脂肽粗提物对不同细菌的抑菌直径
Table 2 Antibacterial diameter of crude lipopeptide extracts against different bacteria
靶标菌 Target bacteria | 平均抑菌圈直径 Antibacterial circle diameter/cm |
---|---|
金黄色葡萄球菌Staphylococcus aureus | 3.65 |
酿酒酵母菌Saccharomyces cerevisiae | 1.95 |
大肠埃希菌Escherichia coli | 2.15 |
白色念珠菌Candida albicans | 0 |
樱桃球腔菌Mycosphaerella cerasella | 1.35 |
大麦网斑病Pyrenophora teres | 2.18 |
序号 No. | A | B | C | D | E | 脂肽产量 Lipopeptide yield/(g·L-1) | 预测值 Predicted value/(g·L-1) |
---|---|---|---|---|---|---|---|
1 | 0 | -2.38 | 0 | 0 | 0 | 0.32 | 0.31 |
2 | 0 | 2.38 | 0 | 0 | 0 | 0.82 | 0.83 |
3 | 1 | -1 | 1 | 1 | -1 | 0.26 | 0.25 |
4 | 1 | -1 | 1 | 1 | 1 | 0.25 | 0.26 |
5 | 0 | 0 | 0 | 0 | 0 | 0.83 | 0.86 |
6 | 0 | 0 | 0 | 2.38 | 0 | 0.25 | 0.24 |
7 | 0 | 0 | 0 | 0 | 0 | 0.89 | 0.86 |
8 | 0 | 0 | 0 | 0 | 0 | 0.82 | 0.86 |
9 | -1 | -1 | 1 | -1 | 1 | 0.45 | 0.48 |
10 | 1 | -1 | -1 | 1 | -1 | 0.46 | 0.45 |
11 | -1 | 1 | 1 | -1 | -1 | 0.77 | 0.76 |
12 | -1 | -1 | -1 | 1 | 1 | 0.45 | 0.44 |
13 | -1 | -1 | -1 | -1 | 1 | 0.28 | 0.27 |
14 | 1 | 1 | 1 | 1 | 1 | 0.48 | 0.45 |
15 | 1 | 1 | -1 | -1 | 1 | 0.52 | 0.53 |
16 | -1 | 1 | -1 | 1 | -1 | 0.56 | 0.51 |
17 | 1 | 1 | -1 | 1 | -1 | 0.59 | 0.58 |
18 | 0 | 0 | 0 | 0 | 2.38 | 0.36 | 0.34 |
19 | -1 | 1 | 1 | 1 | -1 | 0.50 | 0.52 |
20 | 1 | 1 | 1 | -1 | -1 | 0.78 | 0.75 |
21 | -2.38 | 0 | 0 | 0 | 0 | 0.45 | 0.42 |
22 | -1 | -1 | 1 | 1 | -1 | 0.34 | 0.28 |
23 | -1 | 1 | -1 | 1 | 1 | 0.54 | 0.55 |
24 | -1 | -1 | 1 | 1 | 1 | 0.33 | 0.34 |
25 | -1 | 1 | -1 | -1 | 1 | 0.45 | 0.47 |
26 | 0 | 0 | 0 | -2.38 | 0 | 0.36 | 0.36 |
27 | -1 | -1 | 1 | -1 | -1 | 0.39 | 0.43 |
28 | 1 | -1 | -1 | -1 | 1 | 0.33 | 0.33 |
29 | -1 | 1 | 1 | -1 | 1 | 0.81 | 0.77 |
30 | -1 | 1 | -1 | -1 | -1 | 0.43 | 0.45 |
31 | -1 | 1 | 1 | 1 | 1 | 0.49 | 0.54 |
32 | 0 | 0 | -2.38 | 0 | 0 | 0.38 | 0.39 |
33 | 1 | -1 | 1 | -1 | -1 | 0.42 | 0.43 |
34 | 1 | 1 | 1 | 1 | -1 | 0.45 | 0.48 |
35 | 1 | -1 | -1 | -1 | -1 | 0.34 | 0.31 |
36 | 0 | 0 | 2.38 | 0 | 0 | 0.52 | 0.51 |
37 | 2.38 | 0 | 0 | 0 | 0 | 0.42 | 0.45 |
38 | 0 | 0 | 0 | 0 | -2.38 | 0.28 | 0.30 |
39 | 1 | -1 | 1 | -1 | 1 | 0.44 | 0.43 |
40 | 0 | 0 | 0 | 0 | 0 | 0.84 | 0.86 |
41 | 1 | -1 | -1 | 1 | 1 | 0.46 | 0.47 |
42 | -1 | -1 | -1 | -1 | -1 | 0.23 | 0.21 |
43 | 0 | 0 | 0 | 0 | 0 | 0.85 | 0.86 |
44 | -1 | -1 | -1 | 1 | -1 | 0.30 | 0.37 |
45 | 1 | 1 | 1 | -1 | 1 | 0.71 | 0.72 |
46 | 1 | 1 | -1 | 1 | 1 | 0.60 | 0.58 |
47 | 0 | 0 | 0 | 0 | 0 | 0.86 | 0.86 |
48 | 0 | 0 | 0 | 0 | 0 | 0.90 | 0.86 |
49 | 0 | 0 | 0 | 0 | 0 | 0.91 | 0.86 |
50 | 1 | 1 | -1 | -1 | -1 | 0.55 | 0.55 |
表3 中心组合试验结果及预测
Table 3 Center combination experiment results and prediction
序号 No. | A | B | C | D | E | 脂肽产量 Lipopeptide yield/(g·L-1) | 预测值 Predicted value/(g·L-1) |
---|---|---|---|---|---|---|---|
1 | 0 | -2.38 | 0 | 0 | 0 | 0.32 | 0.31 |
2 | 0 | 2.38 | 0 | 0 | 0 | 0.82 | 0.83 |
3 | 1 | -1 | 1 | 1 | -1 | 0.26 | 0.25 |
4 | 1 | -1 | 1 | 1 | 1 | 0.25 | 0.26 |
5 | 0 | 0 | 0 | 0 | 0 | 0.83 | 0.86 |
6 | 0 | 0 | 0 | 2.38 | 0 | 0.25 | 0.24 |
7 | 0 | 0 | 0 | 0 | 0 | 0.89 | 0.86 |
8 | 0 | 0 | 0 | 0 | 0 | 0.82 | 0.86 |
9 | -1 | -1 | 1 | -1 | 1 | 0.45 | 0.48 |
10 | 1 | -1 | -1 | 1 | -1 | 0.46 | 0.45 |
11 | -1 | 1 | 1 | -1 | -1 | 0.77 | 0.76 |
12 | -1 | -1 | -1 | 1 | 1 | 0.45 | 0.44 |
13 | -1 | -1 | -1 | -1 | 1 | 0.28 | 0.27 |
14 | 1 | 1 | 1 | 1 | 1 | 0.48 | 0.45 |
15 | 1 | 1 | -1 | -1 | 1 | 0.52 | 0.53 |
16 | -1 | 1 | -1 | 1 | -1 | 0.56 | 0.51 |
17 | 1 | 1 | -1 | 1 | -1 | 0.59 | 0.58 |
18 | 0 | 0 | 0 | 0 | 2.38 | 0.36 | 0.34 |
19 | -1 | 1 | 1 | 1 | -1 | 0.50 | 0.52 |
20 | 1 | 1 | 1 | -1 | -1 | 0.78 | 0.75 |
21 | -2.38 | 0 | 0 | 0 | 0 | 0.45 | 0.42 |
22 | -1 | -1 | 1 | 1 | -1 | 0.34 | 0.28 |
23 | -1 | 1 | -1 | 1 | 1 | 0.54 | 0.55 |
24 | -1 | -1 | 1 | 1 | 1 | 0.33 | 0.34 |
25 | -1 | 1 | -1 | -1 | 1 | 0.45 | 0.47 |
26 | 0 | 0 | 0 | -2.38 | 0 | 0.36 | 0.36 |
27 | -1 | -1 | 1 | -1 | -1 | 0.39 | 0.43 |
28 | 1 | -1 | -1 | -1 | 1 | 0.33 | 0.33 |
29 | -1 | 1 | 1 | -1 | 1 | 0.81 | 0.77 |
30 | -1 | 1 | -1 | -1 | -1 | 0.43 | 0.45 |
31 | -1 | 1 | 1 | 1 | 1 | 0.49 | 0.54 |
32 | 0 | 0 | -2.38 | 0 | 0 | 0.38 | 0.39 |
33 | 1 | -1 | 1 | -1 | -1 | 0.42 | 0.43 |
34 | 1 | 1 | 1 | 1 | -1 | 0.45 | 0.48 |
35 | 1 | -1 | -1 | -1 | -1 | 0.34 | 0.31 |
36 | 0 | 0 | 2.38 | 0 | 0 | 0.52 | 0.51 |
37 | 2.38 | 0 | 0 | 0 | 0 | 0.42 | 0.45 |
38 | 0 | 0 | 0 | 0 | -2.38 | 0.28 | 0.30 |
39 | 1 | -1 | 1 | -1 | 1 | 0.44 | 0.43 |
40 | 0 | 0 | 0 | 0 | 0 | 0.84 | 0.86 |
41 | 1 | -1 | -1 | 1 | 1 | 0.46 | 0.47 |
42 | -1 | -1 | -1 | -1 | -1 | 0.23 | 0.21 |
43 | 0 | 0 | 0 | 0 | 0 | 0.85 | 0.86 |
44 | -1 | -1 | -1 | 1 | -1 | 0.30 | 0.37 |
45 | 1 | 1 | 1 | -1 | 1 | 0.71 | 0.72 |
46 | 1 | 1 | -1 | 1 | 1 | 0.60 | 0.58 |
47 | 0 | 0 | 0 | 0 | 0 | 0.86 | 0.86 |
48 | 0 | 0 | 0 | 0 | 0 | 0.90 | 0.86 |
49 | 0 | 0 | 0 | 0 | 0 | 0.91 | 0.86 |
50 | 1 | 1 | -1 | -1 | -1 | 0.55 | 0.55 |
方差来源 Soruces of variation | 平方和 Sum of squares | 自由度 Degree of freedom | 方差 Variance | F值 F value | P值 P value | 显著性 Significance |
---|---|---|---|---|---|---|
回归模型Regression model | 2.06 | 20 | 0.10 | 89.28 | <0.000 1 | ** |
A | 1.426×10-3 | 1 | 1.426×10-3 | 1.23 | 0.275 6 | |
B | 0.51 | 1 | 0.51 | 439.53 | <0.000 1 | ** |
C | 0.029 | 1 | 0.029 | 24.76 | <0.000 1 | ** |
D | 0.028 | 1 | 0.028 | 24.26 | <0.000 1 | ** |
E | 3.887×10-3 | 1 | 3.887×10-3 | 3.37 | 0.076 9 | |
AB | 1.125×10-4 | 1 | 1.125×10-4 | 0.097 | 0.757 2 | |
AC | 0.025 | 1 | 0.025 | 21.92 | <0.000 1 | ** |
AD | 1.800×10-3 | 1 | 1.800×10-3 | 1.56 | 0.221 9 | |
AE | 3.613×10-3 | 1 | 3.613×10-3 | 3.13 | 0.087 5 | |
BC | 0.016 | 1 | 0.016 | 14.03 | 0.000 8 | ** |
BD | 0.019 | 1 | 0.019 | 16.46 | 0.000 3 | ** |
BE | 2.450×10-3 | 1 | 2.450×10-3 | 2.12 | 0.156 0 | |
CD | 0.20 | 1 | 0.20 | 169.10 | <0.000 1 | ** |
CE | 4.500×10-4 | 1 | 4.500×10-4 | 0.39 | 0.537 4 | |
DE | 1.125×10-4 | 1 | 1.125×10-4 | 0.097 | 0.757 2 | |
A2 | 0.32 | 1 | 0.32 | 275.38 | <0.000 1 | ** |
B2 | 0.15 | 1 | 0.15 | 129.08 | <0.000 1 | ** |
C2 | 0.30 | 1 | 0.30 | 256.42 | <0.000 1 | ** |
D2 | 0.54 | 1 | 0.54 | 468.04 | <0.000 1 | ** |
E2 | 0.51 | 1 | 0.51 | 443.22 | <0.000 1 | ** |
残差Residual | 0.033 | 29 | 1.155×10-3 | |||
失拟项Lack of fit | 0.026 | 22 | 1.161×10-3 | 1.02 | 0.528 6 | |
纯误差Pure error | 7.950×10-3 | 7 | 1.136×10-3 | |||
合计Cor total | 2.10 | 49 |
表4 拟合回归方程的方差分析结果
Table 4 ANOVA results of fitted regression equations
方差来源 Soruces of variation | 平方和 Sum of squares | 自由度 Degree of freedom | 方差 Variance | F值 F value | P值 P value | 显著性 Significance |
---|---|---|---|---|---|---|
回归模型Regression model | 2.06 | 20 | 0.10 | 89.28 | <0.000 1 | ** |
A | 1.426×10-3 | 1 | 1.426×10-3 | 1.23 | 0.275 6 | |
B | 0.51 | 1 | 0.51 | 439.53 | <0.000 1 | ** |
C | 0.029 | 1 | 0.029 | 24.76 | <0.000 1 | ** |
D | 0.028 | 1 | 0.028 | 24.26 | <0.000 1 | ** |
E | 3.887×10-3 | 1 | 3.887×10-3 | 3.37 | 0.076 9 | |
AB | 1.125×10-4 | 1 | 1.125×10-4 | 0.097 | 0.757 2 | |
AC | 0.025 | 1 | 0.025 | 21.92 | <0.000 1 | ** |
AD | 1.800×10-3 | 1 | 1.800×10-3 | 1.56 | 0.221 9 | |
AE | 3.613×10-3 | 1 | 3.613×10-3 | 3.13 | 0.087 5 | |
BC | 0.016 | 1 | 0.016 | 14.03 | 0.000 8 | ** |
BD | 0.019 | 1 | 0.019 | 16.46 | 0.000 3 | ** |
BE | 2.450×10-3 | 1 | 2.450×10-3 | 2.12 | 0.156 0 | |
CD | 0.20 | 1 | 0.20 | 169.10 | <0.000 1 | ** |
CE | 4.500×10-4 | 1 | 4.500×10-4 | 0.39 | 0.537 4 | |
DE | 1.125×10-4 | 1 | 1.125×10-4 | 0.097 | 0.757 2 | |
A2 | 0.32 | 1 | 0.32 | 275.38 | <0.000 1 | ** |
B2 | 0.15 | 1 | 0.15 | 129.08 | <0.000 1 | ** |
C2 | 0.30 | 1 | 0.30 | 256.42 | <0.000 1 | ** |
D2 | 0.54 | 1 | 0.54 | 468.04 | <0.000 1 | ** |
E2 | 0.51 | 1 | 0.51 | 443.22 | <0.000 1 | ** |
残差Residual | 0.033 | 29 | 1.155×10-3 | |||
失拟项Lack of fit | 0.026 | 22 | 1.161×10-3 | 1.02 | 0.528 6 | |
纯误差Pure error | 7.950×10-3 | 7 | 1.136×10-3 | |||
合计Cor total | 2.10 | 49 |
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