杨树桑黄多糖提取条件优化及其抑菌活性研究

doi:10.13560/j.cnki.biotech.bull.1985.2024-0677

摘要/Abstract

摘要：

【目的】 优化杨树桑黄多糖（Sanghuongporus vaninii polysaccharides, SVP）提取工艺，探讨SVP的抑菌机制，为天然抑菌剂的开发提供参考。【方法】 在单因素实验的基础上，利用响应面分析法优化多糖水提醇沉法提取工艺，提高多糖提取率；通过最小抑菌浓度、生长曲线和酶活性等的测定，研究SVP对大肠杆菌和金黄色葡萄球菌的抑菌活性。【结果】 在浸提温度86℃、时间1.6 h、料液比1∶24 g/mL时，多糖的提取率为4.07%；多糖对大肠杆菌和金黄色葡萄球菌的最小抑菌浓度分别为2.000 mg/mL和1.500 mg/mL。与阴性对照组相比，大肠杆菌和金黄色葡萄球菌的胞外碱性磷酸酶（alkaline phosphatase, AKP）活性分别升高3.7倍（P<0.05）和2.6倍（P<0.05）、β-半乳糖苷酶的活性分别升高6.2％（P<0.01）和7.3％（P<0.01）、ΔOD _260nm分别增加了60％（P<0.01）和1.2倍（P<0.01）、胞内Na⁺/K⁺-ATPase的酶活性分别下降28.9％（P<0.001）和34.8％（P<0.001）、Ca²⁺-ATPase的酶活性分别下降43.2％（P<0.001）和35.6％（P<0.001）、ATP酶的活性分别降低了20.1％（P<0.001）和34.6％（P<0.001）、苹果酸脱氢酶（malate dehydrogenase, MDH）活性分别降低了23.5％（P<0.001）和28.7％（P<0.0001）、琥珀酸脱氢酶（succinate dehydrogenase, SDH）活性分别降低了17.9％（P<0.01）和13.8％（P<0.01）。【结论】 响应面优化后，SVP的实际提取率比预测提取率高5.71%，同时SVP对大肠杆菌和金黄色葡萄球菌有较强的抑制作用，作用机制可能与破坏细菌细胞膜、抑制能量代谢酶等的活性相关。

关键词: 真菌, 杨树桑黄多糖, 响应面, 抑菌活性

Abstract:

【Objective】 The aim is to optimize the extraction process of Sanghuongporus vaninii polysaccharides（SVP）, explore the antibacterial mechanism of SVP, and provide reference for the development of natural antimicrobial agents. 【Method】 Based on single factor experiments and response surface analysis, we optimized the process of extracting polysaccharides by water extraction and alcohol precipitation, and improve the polysaccharides extraction yield. By measuring the minimum inhibitory concentration, growth curve, and enzyme activities, we studied the antibacterial activity of SVP against Escherichia coli and Staphylococcus aureus. 【Result】 When the extraction temperature is 86℃, the time is 1.6 h, and the ratio of material to liquid is 1:24 g/mL, the extraction yield of polysaccharides is 4.07%. The minimum inhibitory concentrations of polysaccharides against E. coli and S. aureus are 2.000 mg/mL and 1.500 mg/mL, respectively. Compared with the negative control group, the extracellular alkaline phosphatase（AKP）activity of E. coli and S. aureus increased by 3.7 times（P < 0.05）and 2.6 times（P < 0.05）, the enzyme activity of β-galactosidase increased by 6.2%（P < 0.01）and 7.3%（P < 0.01）, the ΔOD _260nm increased by 60%（P < 0.01）and 1.2 times（P < 0.01）, the intracellular Na⁺/K⁺-ATPase activity decreased by 28.9%（P < 0.001）and 34.8%（P < 0.001）, the Ca²⁺-ATPase activity decreased by 43.2%（P < 0.001）and 35.6%（P < 0.001）, the ATPase activity decreased by 20.1%（P < 0.001）and 34.6%（P < 0.001）, the malate dehydrogenase（MDH）activity decreased by 23.5%（P < 0.001）and 28.7%（P < 0.001）, and the succinate dehydrogenase（SDH）activity decreased by 17.9%（P < 0.01）and 13.8%（P < 0.01）, respectively. 【Conclusion】 After response surface optimization, the actual extraction yield of SVP is 5.71% higher than the predicted extraction yield, and SVP has a strong inhibitory effect on E. coli and S. aureus. Its mechanism of action may be related to the destruction of bacterial cell membranes, inhibition of energy metabolism enzymes, etc.

Key words: fungus, Sanghuongporus vaninii polysaccharides, response surface, antibacterial activity

叶卓妍, 周家淇, 林丹媛, 孙赫男, 王艳珍. 杨树桑黄多糖提取条件优化及其抑菌活性研究[J]. 生物技术通报, 2024, 40(11): 58-67.

YE Zhuo-yan, ZHOU Jia-qi, LIN Dan-yuan, SUN He-nan, WANG Yan-zhen. Study on the Optimization of Extraction Conditions and Antibacterial Activities of Polysaccharides from Sanghuongporus vaninii[J]. Biotechnology Bulletin, 2024, 40(11): 58-67.

图/表 10

图1 不同条件对SVP提取率的影响 A：浸提温度；B：浸提时间；C：料液比

Fig. 1 Effects of different conditions on the extraction yield of SVP A: Extraction temperature. B: Extraction time. C: Material-liquid ratio

表1 响应面三因素三水平试验设计

Table 1 Response surface three factor and three level experimental design

因素Factor	水平Level
因素Factor	-1	0	1
A温度/℃	70	80	90
B时间/h	1	2	3
C料液比/（g·mL^-1）	1∶10	1∶20	1∶30

表2 响应面试验的方案及提取率

Table 2 Box-Behnken design

序号 No.	水平Level			提取率Extraction yield/%
序号 No.	A/℃	B/h	C/（g·mL^-1）	实验Experiment	预测Prediction
1	90	2	1∶10	2.66	2.21
2	80	3	1∶10	2.77	2.49
3	90	2	1∶30	3.39	2.87
4	80	2	1∶20	4.12	3.08
5	90	1	1∶20	2.46	2.42
6	70	2	1∶10	2.37	2.72
7	70	3	1∶20	2.90	3.15
8	80	2	1∶20	4.25	3.33
9	80	2	1∶20	4.03	1.94
10	80	1	1∶30	2.78	2.75
11	80	1	1∶10	2.04	2.80
12	70	2	1∶30	3.20	3.23
13	90	3	1∶20	3.12	4.15
14	70	1	1∶20	2.17	4.15
15	80	2	1∶20	4.00	4.15
16	80	2	1∶20	4.36	4.15
17	80	3	1∶30	3.14	4.15

表3 回归方程方差分析

Table 3 Results of analysis of variance（ANOVA）for a fitted regression equation

方差来源Source	平方和Sum of squares	自由度df	均方Square	F值F value	P值P value	显著性Significance
模型	8.91	9	0.99	54.14	< 0.0001	***
A	0.12	1	0.12	6.62	0.0369	*
B	0.77	1	0.77	41.99	0.0003	***
C	0.89	1	0.89	48.82	0.0002	***
AB	0.00	1	0.00	0.08	0.7896
AC	0.00	1	0.00	0.19	0.6784
BC	0.04	1	0.04	1.93	0.2071
A²	1.68	1	1.68	91.91	< 0.0001	***
B²	3.09	1	3.09	169.11	< 0.0001	***
C²	1.59	1	1.59	87.10	< 0.0001	***
残差	0.13	7	0.02
失拟项	0.04	3	0.01	0.54	0.6798
纯误差	0.09	4	0.02	-	-
总误差	9.04	16	-	-	-

图2 多糖提取响应面图和等高线图 A：浸提时间和温度对多糖提取率的影响；B：料液比和浸提温度对多糖提取率的影响；C：料液比和浸提时间对多糖提取率的影响

Fig. 2 Response surface map and contour map of polysaccharides extraction A: The influence of extraction time and temperature on the extraction yield of polysaccharides. B: The influence of material-liquid ratio and extraction temperature on the extraction yield of polysaccharides. C: The influence of material-liquid ratio and extraction time on the extraction yield of polysaccharides

表4 SVP对大肠杆菌、金黄色葡萄球菌的MIC

Table 4 MIC of SVP against E. coli and S. aureus

菌种Strain	最小抑菌浓度MIC/（mg·mL^-1）
E. coli	2.000
S. aureus	1.500

图3 SVP对生长曲线的影响 A:大肠杆菌生长曲线；B:金黄色葡萄球菌生长曲线。***P<0.001 vs. 阴性对照组。

Fig. 3 Effects of SVP on the growth curve A:Growth curve of E. coli. B :Growth curve of S. aureus. ***P<0.001 vs. control group.

图4 SVP对大肠杆菌和金黄色葡萄球菌细胞壁和细胞膜酶活性的影响 A: AKP酶活性；B：β-半乳糖苷酶活性；C：Na+/K+-ATPase活性；D：Ca2+-ATPase活性。*P<0.05, **P<0.01, ***P<0.001 vs. 阴性对照组

Fig. 4 Effects of SVP on cell wall and membrane enzyme activities of E. coli and S. aureus A: AKP enzyme activity. B β-Galactosidase activity. C: Na+/K+-ATPase activity. D: Ca2+-ATPase activity. *P<0.05, **P<0.01, ***P<0.001 vs. control group

图5 SVP对大肠杆菌和金黄色葡萄球菌核酸泄露的影响 **P<0.01 vs. 阴性对照组

Fig. 5 Effects of SVP on the leakage of nucleic acid of E. coli and S. aureus **P<0.01 vs. control group

图6 SVP对大肠杆菌和金黄色葡萄球菌能量代谢酶活性的影响 A: ATPase活性；B: MDH酶活性；C: SDH酶活性。**P<0.01,***P<0.001 vs. 阴性对照组

Fig. 6 Effects of SVP on energy metabolism enzyme activities of E. coli and S. aureus A: ATPase activity; B MDH enzyme activity; C: SDH enzyme activity. **P<0.01, ***P<0.001 vs. control group

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