Study on the Optimization of Extraction Conditions and Antibacterial Activities of Polysaccharides from Sanghuongporus vaninii

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

Abstract

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

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.

Figures/Tables 10

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

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

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

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	-	-	-

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

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

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.

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

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

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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