栽培条件对松杉灵芝多糖含量的影响及其抗氧化活性分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0921

生物技术通报 ›› 2024, Vol. 40 ›› Issue (11): 47-57.doi: 10.13560/j.cnki.biotech.bull.1985.2024-0921

• 菌物功效及作用机制专题（专题主编：王迪教授） • 上一篇下一篇

栽培条件对松杉灵芝多糖含量的影响及其抗氧化活性分析

张鑫¹^,²(), 邬柯奕², 祝嗣臣³, 李玉²^,⁴(), 李兰洲²()

1.沈阳农业大学园艺学院，沈阳 110866
2.吉林农业大学食药用菌教育部工程研究中心，长春 130118
3.黑龙江坤健农业股份有限公司，齐齐哈尔 161200
4.中国科学院天津工业生物技术研究所，天津 300308

收稿日期:2024-09-24 出版日期:2024-11-26 发布日期:2024-12-19
通讯作者: 李玉，男，博士，教授，研究方向：菌类资源发掘、保育及其工程化和产业化；E-mail: liyu@jlau.edu.cn；
李兰洲，男，博士，讲师，研究方向：菌物活性成分提取及功效；E-mail: lilanzhou@jlau.edu.cn
作者简介:张鑫，女，博士，研究方向：食药用菌多糖及功效；E-mail: 2021200116@stu.syau.edu.cn
基金资助:
国家食用菌产业技术体系(CARS-20-17);天津市合成生物技术创新能力提升行动项目(TSBICIP-CXRC-006)

Effect of Cultivation Conditions on the Content of Polysaccharide in Ganoderma tsugae and its Antioxidant Activity

ZHANG Xin¹^,²(), WU Ke-yi², ZHU Si-chen³, LI Yu²^,⁴(), LI Lan-zhou²()

1. College of Horticulture, Shenyang Agricultural University, Shenyang 110866
2. Engineering Research Center of Chinese Ministry of Education for Edible and Medicinal Fungi, Jilin Agricultural University, Changchun 130118
3. Heilongjiang Kunjian Agriculture Co. Ltd.，Qiqihaer 161200
4. National Center of Technology Innovation for Synthetic Biology, Tianjin 300308

Received:2024-09-24 Published:2024-11-26 Online:2024-12-19

摘要/Abstract

摘要：

【目的】 优化松杉灵芝栽培和多糖提取条件，并分析其体外抗氧化活性，明确松杉灵芝多糖活性。【方法】 以松杉灵芝的农艺性状、多糖含量为指标，优化松杉灵芝的栽培条件同时分析其体外抗氧化活性，进一步采用响应面设计法优化松杉灵芝多糖提取的方案，考察松杉灵芝粗多糖（GTP）对H₂O₂诱导的HepG2细胞的保护作用。【结果】 松杉灵芝的最优栽培条件为遮阴率60%，温度30℃，湿度80%，CO₂浓度0.03%时，子实体质量和多糖含量均最高。松杉灵芝水提物表现出了良好的体外抗氧化活性，具有清除自由基和抗超氧阴离子作用。最佳提取方案为提取温度82℃、提取时间5.6 h、液料比56 mL/g时，得率为1.90%；GTP通过调控氧化应激相关蛋白的表达，保护H₂O₂诱导的HepG2细胞免受氧化损伤。【结论】 明确了松杉灵芝的栽培条件及其多糖提取工艺，并证实其具有体外抗氧化活性，其对H₂O₂诱导的HepG2保护作用与氧化应激有关。

关键词: 松杉灵芝, 农艺性状, 多糖, 提取工艺, 抗氧化活性

Abstract:

【Objective】 The aim of this study is to optimize the cultivation and polysaccharide extraction conditions of Ganoderma tsugae, analyze its antioxidant activity in vitro, and clarify the polysaccharide activity of G. tsugae. 【Method】 The agronomic traits and sugar content of G. tsugae were used as indicators to optimize the cultivation conditions and analyze its antioxidant activity in vitro. Furthermore, response surface design was used to optimize the extraction of G. tsugae crude polysaccharide（GTP）and to investigate its protective effect on H₂O₂ induced HepG2 cells. 【Result】 The fruiting body mass and polysaccharide content are the highest, while the optimal cultivating conditions for G. tsugae is shading rate of 60%, temperature of 30℃, humidity of 80%, and CO₂ concentration of 0.03%. Water extracts from G. tsugae also present promising antioxidant activity in vitro, with the ability to scavenge free radicals and resist superoxide anions. The optimal extraction method for sugar is at an extraction temperature of 82℃, extraction time of 5.6 h, and a liquid-solid ratio of 56 mL/g, reaching 1.90%. 【Conclusion】 The cultivation conditions and polysaccharide extraction process of G. tsugae were clarified, and it was confirmed that it has antioxidant activity in vitro. Its protective effect on H₂O₂-induced HepG2 is related to oxidative stress.

Key words: Ganoderma tsugae, agronomic traits, polysaccharides, extraction process, antioxidant activity

张鑫, 邬柯奕, 祝嗣臣, 李玉, 李兰洲. 栽培条件对松杉灵芝多糖含量的影响及其抗氧化活性分析[J]. 生物技术通报, 2024, 40(11): 47-57.

ZHANG Xin, WU Ke-yi, ZHU Si-chen, LI Yu, LI Lan-zhou. Effect of Cultivation Conditions on the Content of Polysaccharide in Ganoderma tsugae and its Antioxidant Activity[J]. Biotechnology Bulletin, 2024, 40(11): 47-57.

图/表 11

表1 Box-Behnken设计实验因素与水平

Table 1 Factors and levels in Box-Behnken design

因素Factor	水平Level
因素Factor	-1	0	1
A 提取温度Extraction temperature/℃	70	80	90
B 提取时间Extraction time/h	4	5	6
C 液料比Liquid-solid ratio/（mL·g^-1）	40	50	60

图1 不同栽培条件松杉灵芝子实体形态

Fig. 1 Morphologies of G. tsugae fruiting bodies under different cultivation conditions

表2 不同栽培条件松杉灵芝子实体农艺性状

Table 2 Agronomic traits of G. tsugae fruiting bodies under different cultivation conditions

组别 Group	栽培条件 Cultivation conditions	子实体质量 Quality/g	菌盖长度 Length/cm	菌盖宽度 Width/cm	生物学效率 Biological efficiency/%
1	遮阴率0%	89.85±14.64 a	14.10±1.98 a	7.95±1.55 a	8.99±1.46 a
2	遮阴率90%	94.26±11.16 ab	15.44±2.36 a	8.82±1.84 a	9.43±1.11 ab
3	温度25℃	84.70±10.27 a	12.80±2.73 b	8.02±1.75 a	8.47±1.03 a
4	温度35℃	110.83±14.42 c	15.27±1.91 a	8.74±2.02 a	10.10±1.44 b
5	湿度70%	97.70±13.78 ab	12.78±2.28 b	7.73±1.46 a	9.77±1.38 ab
6	湿度90%	104.83±13.21 b	12.27±2.73 b	9.08±2.38 a	10.48±1.32 b
7	CO₂浓度0.02%	112.41±14.32 c	15.42±2.02 a	9.04±1.62 b	11.24±1.43 c
8	CO₂浓度0.04%	80.57±11.86 a	11.78±2.23 c	7.07±1.45 c	8.06±1.19 a
9	遮阴率60%，温度30℃，湿度80%，CO₂浓度0.03%	112.10±15.52 c	16.20±2.29 d	8.29±1.57 b	11.21±1.55 c

图2 不同栽培条件松杉灵芝子实体多糖含量 A：遮阴率；B：温度；C：湿度；D：CO2浓度

Fig. 2 Polysaccharide contents of G. tsugae fruiting bodies under different cultivation conditions A: Shading rate; B: temperature; C: humidity; D: CO2

图3 不同栽培条件松杉灵芝水提物体外抗氧化活性 A：总抗氧化能力；B：DPPH自由基清除率；C：OH·自由基清除率；D：抗超氧阴离子能力

Fig. 3 Antioxidant activityies in vitro of water extracts of G. tsugae under different cultivation conditions A: Total antioxidant capacity; B: DPPH radical scavenging rate; C: OH· radical scavenging rate; D: ability to resist superoxide anions

图4 不同提取条件对多糖得率的影响 A：提取温度；B：提取时间；C：液料比

Fig. 4 Effects of different extraction conditions on extraction of polysaccharide A: Extraction temperature. B: Extraction time. C: Liquid-solid ratio

表3 RSM实验设计和结果

Table 3 RSM design and results

实验号 No.	因素Factors			多糖提取率 Extraction rate of polysaccaride/%
实验号 No.	A：提取温度Temperature/℃	B：提取时间Time/h	C：液料比Liquid-solid ratio/（mL·g^-1）	多糖提取率 Extraction rate of polysaccaride/%
1	70	4	50	1.27
2	90	4	50	1.45
3	70	6	50	1.41
4	90	6	50	1.52
5	70	5	40	1.49
6	90	5	40	1.61
7	70	5	60	1.46
8	90	5	60	1.62
9	80	4	40	1.55
10	80	6	40	1.71
11	80	4	60	1.46
12	80	6	60	1.58
13	80	5	50	1.8
14	80	5	50	1.81
15	80	5	50	1.81
16	80	5	50	1.82
17	80	5	50	1.82

表4 回归模型方差分析

Table 4 Analysis of variance for quadratic regression model

来源Source	平方和Sum of squares	自由度Df.	均方Mean square	F	P	显著性Significance
A	0.0091	1	0.0091	8.53	0.0223	*
B	0.0153	1	0.0153	14.34	0.0068	**
C	0.0085	1	0.0085	7.91	0.0260	*
AB	0.0042	1	0.0042	3.96	0.0870
AC	0.0081	1	0.0081	7.59	0.0283	*
BC	0.0009	1	0.0009	0.84	0.3891
A²	0.2605	1	0.2605	243.98	< 0.0001	* *
B²	0.0995	1	0.0995	93.21	< 0.0001	* *
C²	0.0041	1	0.0041	3.85	0.0905
模型Model	0.4363	9	0.0485	45.40	< 0.0001	Significant
残差Residual	0.0075	7	0.0011
失拟项Lack of Fit	0.0041	3	0.0014	1.60	0.3229	Not significant
纯误差Pure Error	0.0034	4	0.0009
总离差Cor Total	0.4438	16
R² = 0.9832	Adj.R² = 0.9615

图5 各因素交互作用对GTP提取率影响的响应面图和等高线图 A、B、C为多糖提取过程中提取温度、提取时间和液料比三个因素的三维响应面图；D、E、F为多糖提取过程中提取温度、提取时间和液料比三个因素的等高线图

Fig. 5 Response surface diagram and contour map of the effects of interactions of various factors on GTP extraction rates A, B, and C are the three-dimensional response surface diagrams of extraction temperature, extraction time and solid-liquid ratio in the extraction process of polysaccharide. D, E, and F are contour plots of extraction temperature, extraction time, and liquid-material ratio in the extraction process of polysaccharide

图6 GTP对H2O2处理HepG2细胞活力的影响

Fig. 6 Effects of GTP on viability of HepG2 cells treated with H2O2 Compared with Control group，### P < 0.001；** P < 0.01, and *** P < 0.001, compared with H2O2 group

图7 Nrf2、HO-1、SOD1蛋白表达

Fig. 7 The protein expressions of Nrf2, HO-1, and SOD1 # P < 0.05 and ### P < 0.001, compared with control group, * P < 0.05, *** P < 0.001, compared with H2O2 group

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栽培条件对松杉灵芝多糖含量的影响及其抗氧化活性分析

Effect of Cultivation Conditions on the Content of Polysaccharide in Ganoderma tsugae and its Antioxidant Activity

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