|
|
|
| Optimization of Liquid Fermentation Medium for High-yield Sterol and Evaluation of Lipid-lowering Activity by Pleurotus tuber-regium |
| HU Xuan*, HU Fei-Wen*, WANG Dan, WANG Chao, YIN Liang-Hong, LIN Hai-Ping** |
| National Key Laboratory for Development and Utilization of Forest Food Resources, Zhejiang A&F University, Hangzhou 311300, China |
|
|
|
|
Abstract Hyperlipidemia is a group of blood lipid disorders caused by dyslipidemia, which seriously endangers human health. Sterols possess multiple important physiological activities, such as lipid-lowering, anticancer, and antioxidant effects. To enhance the sterol yield from the liquid fermentation of Pleurotus tuber-regium, this study optimized the culture medium formulation using single-factor experiments and response surface methodology. The lipid-lowering activity of the sterol extract was evaluated through bile salt binding capacity assays and a high-lipid human hepatoblastoma G2 (HepG2) cell model. The results showed that the optimal medium consisted of fructose 52.51 g/L, corn protein 3.14 g/L, and MgSO4 0.14 g/L. Under this formulation, the sterol yield reached 362.25 mg/L, representing a 105.70% increase compared to that before optimization. The 2.0 mg/mL sterol extract exhibited significantly higher binding rates to sodium cholate, sodium taurocholate, sodium glycocholate, and mixed bile salts compared to the 1.0 mg/mL extract (P<0.05), corresponding to 86.21%, 86.05%, 89.76%, and 87.56% of the positive control cholestyramine, respectively. Treatment with 5.0 μg/mL extract reduced total cholesterol, triglycerides, and low-density lipoprotein cholesterol levels in HepG2 high-lipid cells by 26.95%, 71.42%, and 38.40%, respectively. The expression of lipid decomposition and transport genes CPT1, PPARα, and ABCG5 was upregulated by 437.30%, 235.85%, and 71.79%, respectively. In contrast, the expression of lipid synthesis and absorption genes SREBP2, NPC1L1, and ACAT2 was downregulated by 36.32%, 33.10%, and 17.53%, respectively. The expression of the rate-limiting enzymes in cholesterol synthesis, HMGCS1 and HMGCR, was downregulated by 47.31% and 20.80%, respectively. In summary, the sterol extract from P. tuber-regium not only demonstrated strong bile salt binding capacity, effectively interfering with cholesterol absorption, but also significantly modulated the expression of lipid metabolism-related genes through multiple targets, exhibiting considerable lipid-lowering activity. The findings provide a scientific basis for the development of lipid-lowering products derived from P. tuber-regium.
|
|
Received: 31 October 2025
|
|
|
|
Corresponding Authors:
**hplin@zafu.edu.cn
|
| About author:: *These authors contributed equally to this work |
|
|
|
[1] 戴玉成, 杨祝良. 2008. 中国药用真菌名录及部分名称的修订[J]. 菌物学报, 27: 801-824.
(Dai Y C, Yang Z L.2008. A revised checklist of medicinal fungi in China[J]. Mycosystema, 27: 801-824)
[2] 杜娇. 2020. 猴头菌麦角甾醇高产发酵条件及提取工艺的研究[D]. 硕士学位论文, 山西农业大学, 导师: 孟俊龙, pp. 19-33.
(Du J.2020 Study on high-yield fermentation conditions and extraction technology of ergosterol from Hericium erinaceus[D]. Thesis for M.S., Shanxi Agricultural University, Supervisor: Meng J L, pp. 19-33.)
[3] 霍理坚, 冯祖睿, 林雅铃. 2021. 超声细胞粉碎法快速提取真菌中的麦角甾醇[J]. 南京农业大学学报, 44(6): 1090-1096.
(Huo L J, Feng Z R, Ling Y L.2021. Rapid extraction of ergosterol from fungi by ultrasonic cell disruption[J]. Journal of Nanjing Agricultural University, 44(6): 1090-1096.)
[4] 李向敏. 2015. 皱盖假芝和灵芝孢子中甾醇类化合物抗肿瘤作用机制研究[D]. 博士学位论文, 华南理工大学, 导师: 吴清平, pp. 63-74.
(Li X M.Anti-tumor effect and mechanisms of sterols from amauroderma rude and ganoderma lucidum spore[D]. Thesis for Ph. D., South China University of Technology, Suppervisor: Wu Q P, pp. 63-74.)
[5] 刘静娜, 夏文水, 张家骊. 2008. 壳聚糖物化性质对其体外结合脂类和胆酸盐能力的影响[J]. 食品科学, (1): 45-49.
(Liu J N, Xia W S, Zhang J L. 2008 Effects of chitosans physico-chemical properties on binding capacities of lipid and bile salts in vitro[J]. Food Science, (1): 45-49.)
[6] 刘淑敏. 2016. 不同茶类浸提液及茶多酚的生物活性和机理研究[D]. 博士学位论文, 华南理工大学, 导师: 黄惠华, pp. 27-42.
(Liu S M.2016. Study on the biological activities and functional mechanisms of different tea extracts and tea polyphenols[D]. Thesis for Ph. D., South China University of Technology, Supervisor: Huang H H, pp. 27-42.)
[7] 刘晓峰, 李玉, 孙晓波, 等. 1998. 侧耳属真菌化学成分及药用活性研究进展[J]. 吉林农业大学学报, (3): 99-105.
(Liu X F, Li Y, Sun X B, et al. 1998. Research progress on chemical constituents and medicinal activities of Pleurotus fungi[J]. Journal of Jilin Agricultural University, (3): 99-105.)
[8] 刘艳如, 林勇, 郑怡. 2010. 深层发酵培养的虎奶菇多糖对3种小球藻的凝集作用[J]. 福建师范大学学报(自然科学版), 26(2): 106-110.
(Liu Y R, Lin Y, Zheng Y.2010. Agglutination of three kinds of chlorella cells by polysaccharides from Pleurotus tuber-regium by submerged fermentation[J]. Journal of Fujian Normal University (Natural Science Edition), 26(2): 106-110.)
[9] 刘媛. 2024. 虎奶菇甾醇提取分离及其生物活性研究[D]. 硕士学位论文, 浙江农林大学, 导师: 王超, pp. 14-32.
(Liu Y.2024. Extraction and separation of sterols from Pleurotus tuber-regium(Fr.) Sing and biological activities[D]. Thesis for M.S., Zhejiang A&F University, Supervisor: Wang C, pp. 14-32.)
[10] 马海霞, 司静, 戴玉成, 等. 2022. 中国海南省木生大型真菌多样性研究[J]. 菌物学报 41: 695-712.
(Ma H X, Si J, Dai Y C, et al.2022. Diversity of wood-inhabiting macrofungi in Hainan Province, South China[J]. Mycosystema, 41: 695-712.)
[11] 陶咏真, 张俐娜, 韩露. 2009. 水溶性高支化多糖及硫酸酯衍生物抗癌活性研究[J]. 高分子学报, 1(9): 852-859.
(Tao Y Z, Zhang L N, Han L.2009. Studies on antitumor activity of a water-soluble hyperbranched polysaccharide and its sulfated derivatives[J]. Acta Polymerica Sinica, 1(9): 852-859.)
[12] 王娟, 刘军权, 陈复兴. 2014. β-谷甾醇对人共刺激细胞杀伤胃癌SGC-7901细胞的影响及其机制的探讨[J]. 免疫学杂志, 30(7): 578-584.
(Wang J, Liu J Q, Chen F X.2014. The effect of β-sitosterol on cytotoxicity of human costimulatory cells against gastric cancer SGC-7901 cells and its mechanism[J]. Journal of Immunology, 30(7): 578-584.)
[13] 王军芳, 宋国月, 高悦, 等. 2023. 野生菌核侧耳生物学特性及驯化栽培[J]. 菌物学报, 42(1): 395-407.
(Wang J F, Song G Y, Gao Y, et al.2023. Biological characteristics and domestication cultivation of wild Pleurotus tuber-regium[J]. Mycosystema, 42(1): 395-407.)
[14] 王莉莉, 赵庆佳, 张娜, 等. 2022. 不同提取方法对红松籽油甾醇浸提及功能性质影响的研究[J]. 中国粮油学报, 37(6): 125-134.
(Wang L L, Zhao Q J, Zhang N, et al.2022. Effects of different extraction methods on extraction effectand functions of korean pine oil sterol[J]. Journal of the Chinese Cereals and Oils Association, 37(6): 125-134.)
[15] 王倩, 张佳婵, 王昌涛. 2019. 碳氮比对真菌发酵桑枝-燕麦麸皮的活性成分和抗氧化能力的影响[J]. 食品工业科技, 40(2): 93-99+106.(Wang Q, Zhang J C, Wang C T. 2019. Effects of C/N ratio on the changes of active substances and antioxidant capacity of fungus fermented mulberry-oat bran[J]. Science and Technology of Food Industry, 40(2): 93-99+106.)
[16] 巫光宏, 伍志权, 何典路, 等. 2007. 虎奶菇菌核多糖对四氧嘧啶诱导糖尿病小鼠的降血糖作用[J]. 华南农业大学学报, (4): 100-104.
(Wu G H, Wu Z Q, He D L, et al. 2007. Hypoglycemic effect of Pleurotus tuber-regium sclerotial polysaccharides on alloxan-induced diabetic mice[J]. Journal of South China Agricultural University, (4): 100-104.)
[17] 吴锦忠, 黄丽敏, 张志强. 2002. 虎奶菇挥发油成分分析[J]. 康复学报, 12(1): 34-36.
(Wu J Z, Huang L M, Zhang Z Q.2002. Component analysis of essential oil from Pleurotus tuber-regium (Fr)Sing[J]. Journal of Rehabilitation Medicine, 12(1): 34-36.)
[18] 肖志彬, 贾韩学, 刘小雷. 2015. β-谷甾醇药理活性的研究现状[J]. 世界最新医学信息文摘, 15(8): 66-68.
(Xiao Z B, Jia H X, Liu X L.2015. The current research status of pharmacological activity of β-sitosterol[J]. World's Latest Medical Information Digest, 15(8): 66-68.)
[19] 杨皓瑜, 程艳芬, 王胜男, 等. 2023. 香菇中麦角甾醇提取、表征及抗氧化、降胆固醇特性[J]. 食用菌学报, 30(2): 75-84.
(Yang H Y, Cheng Y F, Wang S N, et al.2023. Extraction, characterization of ergosterol from Lentinula edodes and its antioxidant and cholesterol-lowering properties[J]. Journal of Edible Fungi, 30(2): 75-84.)
[20] 杨兰. 2009. 虎奶菇的生药鉴定及化痰平喘的药理作用和化学成分研究[D]. 硕士学位论文, 广东药学院, 导师: 房志坚, pp. 7-17.
(Yang L.2009. Pharmacognosy-identification and studies of pharmacological action and chemical components on dissipating phlegm to relieve asthma on the sclerotium of Pleurotus tuber-regium (Fr.) Sing[D]. Thesis for M.S., Guangdong Pharmaceutical University, Supervisor: Fang Z J, pp. 7-17.)
[21] 袁兰兰. 2020. 基于细胞模型和代谢组学技术的黑灵芝甾醇类化合物降胆固醇作用机制研究[D]. 硕士学位论文, 南昌大学, 导师: 谢建华, pp. 63-72.
(Yuan L L.2020. Study on cholesterol-lowering mechanism of sterol compounds in Ganoderma lucidum based on cell models and metabolomic analysis[D]. Thesis for M.S., Nanchang University, Supervisor: Xie J H, pp. 63-72.)
[22] 赵亚东. 2011. 不同培养料对秀珍菇生长发育、产量及胞外酶的影响[D]. 硕士学位论文, 南京农业大学, 导师: 赵明文, pp. 20-22.
(Zhao Y D.2011. The effect of different culture medium on production and extracellular enzyme activity of Pleurotus geeseteranus[D]. Thesis for M.S., Nanjing Agricultural University, Supervisor: Zhao M W, pp. 20-22.)
[23] 张俊峰, 张忠, 汪雯翰. 2020. 桑黄菌丝体和子实体中次级代谢产物及其活性的比较[J]. 菌物学报, 39(2): 398-408.
(Zhang J F, Zhang Z, Wang W H.2020. Comparison of active secondary metabolites between mycelia and fruiting bodies of Sanghuangporous sanghuang[J]. Mycosystema, 39(2): 398-408.)
[24] 张忠, 吴迪, 王雨阳. 2021. 猴头菌高产麦角甾醇液体发酵工艺优化[J]. 菌物学报, 40(8): 2159-2170.
(Zhang Z, Wu D, Wang Y Y.2021. Optimization of liquid fermentation process of ergosterol produced by Hericium erinaceus[J]. Mycosystema, 40(8): 2159-2170.)
[25] 郑林. 2011. 中国被毛孢甾醇提取最优条件的正交法研究[D]. 硕士学位论文, 上海师范大学, 导师: 杨晓彤, pp. 33-37.
(Zheng L.2011. Study of the best method to extract sterols from the mycelium of Hirsutella sinensis by orthogonal design[D]. Thesis for M.S., Shanghai Normal University, Supervisor: Yang X T, pp. 33-37.)
[26] 邹思, 易骏, 吴岩斌. 2020. 虎奶菇菌核总三萜提取工艺优化及抗氧化活性研究[J]. 海峡药学, 32(1): 25-29.
(Zou S, Yi J, Wu Y B.2020. Optimization of extraction process of total triterpenoids and antioxidant activity of the sclerotia from Pleurotus tuber-regium[J]. Strait Pharmacy, 32(1): 25-29.)
[27] 邹胜龙, 黄志毅, 龚红. 2020. 植物甾醇对快大黄鸡生长性能、屠宰性能、血清生化指标及抗氧化指标的影响[J]. 广东饲料, 29(6): 21-25.
(Zou S L, Huang Z Y, Gong H.2020. Effects of phytosterol on growth performance, slaughter performance, serum biochemical indexes and antioxidant indexes of Fast-Growing Yellow chicken[J]. Guangdong Feed, 29(6): 21-25.)
[28] 朱戎, 陈向东, 兰进. 2003. 药用真菌液体发酵研究进展[J]. 中药材, 26(1): 55-57.
(Zhu R, Chen X D, Lan J.2003. Research progress in liquid fermentation of medicinal fungi[J]. Chinese Medicinal Materials, 26(1): 55-57.)
[29] Ayobami E M, Nisaudah R, Uz R Z, et al.2020. Fatty acids and sterol rich stem back extract of Shorea roxburghii attenuates hyperglycemia, hyperlipidemia, and oxidative stress in diabetic rats[J]. European Journal of lipid Science and Technology, 122: 11-12.
[30] Bakratsas G, Polydera A, Katapodis P.2021. Recent trends in submerged cultivation of mushrooms and their applications a source of nutraceuticals and food additives[J]. Future Foods, 4: 100086.
[31] Bamigboye C O, Oloke J K, Dames J F.2019. Development of high yielding strain of Pleurotus tuber-regium: Fructification, nutritional and phylogenetic studies[J]. Journal of Food Science and Technology, 56(8): 3597-3608.
[32] Brunham L B, Steven B, Andrew M, et al.2018. Role of genetics in the prediction of statin-associated muscle symptoms and optimization of statin use and adherence[J]. Cardiovascular Research, 114(8): 1073-1081.
[33] Cofán M, Ros E.2019. Use of plant sterol and stanol fortified foods in clinical practice[J]. Current Medicinal Chemistry, 26(37): 6691-6703.
[34] Cusack L K, Fernandez M L, Volek J S.2013. The food matrix and sterol characteristics affect the plasma cholesterol lowering of phytosterol[J]. Advances in Nutrition, 4(6): 633-643.
[35] Handelsman Y.2011. Role of bile acid sequestrants in the treatment of type 2 diabetes[J]. Diabetes Care, 2(5): 244-250.
[36] Jenkins D J A, Kendall C W C, Nguyen T H.2008. Effect of plant sterols in combination with other cholesterol-lowering foods[J]. Metabolism, 57(1): 130-139.
[37] Liang T Y, Wong T W, Guan L, et al.2011. Effect of phytosterols and their oxidation products on lipoprotein profiles and vascular function in hamster fed a high cholesterol diet[J]. Atherosclerosis, 219(1): 124-133.
[38] Liu Y W, Mei H C, Su Y W.2014. Inhibitory effects of Pleurotus tuber-regium mycelia and bioactive constituents on LPS-treated RAW 264.7 cells[J]. Journal of Functional Foods, 7: 662-670.
[39] Milovanovic I, Zengin G, Maksimovic S.2021. Supercritical and ultrasound-assisted extracts from Pleurotus pulmonarius mushroom: Chemical profiles, antioxidative, and enzyme inhibitory properties[J]. Journal of the Science of Food and Agriculture, 101(6): 2284.
[40] Sungmin L, Bu H, Yun Y.2020. Hypolipidemic roles of casein-derived peptides by regulation of trans-intestinal cholesterol excretion and bile acid synthesis[J]. Nutrients, 12(4): 3058-3058.
[41] Trautwein A E, Duchateau E, Lin Y.2003. Proposed mechanisms of cholesterol-lowering action of plant sterols[J]. European Journal of Lipid Science and Technology, 105(3-4): 171-185.
[42] Wu F, Zhou L W, Yang Z L, et al.2019. Resource diversity of Chinese macrofungi: Edible, medicinal and poisonous species[J]. Fungal Diversity, 98: 1-76.
[43] Yaxmehen O B, Anuar K, Monserratte R.2018. Familial combined hyperlipidemia: Current knowledge, perspectives, and controversies[J]. Revista de Investigacion Clinica; Organo del Hospital de Enfermedades de la Nutricion, 70(5): 224-236. |
|
|
|
