蝙蝠蛾拟青霉菌丝体多糖-纳米硒的制备、结构表征及其对MC3T3-E1细胞增殖分化的影响  

Preparation and Structural Characterization of Selenium Nanoparticles Stabilized by Paecilomyces hepiali Mycelium Polysaccharide and Its Effects on the Proliferation and Differentiation of MC3T3-E1 Cells

作　　者：陈旭洁 梁慧嘉 焦春伟 何春艳 陈少丹 陈秋颜 谢意珍 高雄 CHEN Xujie;LIANG Huijia;JIAO Chunwei;HE Chunyan;CHEN Shaodan;CHEN Qiuyan;XIE Yizhen;GAO Xiong(Guangdong Yuewei Edible Fungi Technology Co.,Ltd.,Guangzhou 510663,China;Guangdong Yuewei Biotechnology Co.,Ltd.,Zhaoqing 526000,China;National Health Commission Science and Technology Innovation Platform for Nutrition and Safety of Microbial Food,Guangdong Provincial Key Laboratory of Microbial Safety and Health,State Key Laboratory of Applied Microbiology Southern China,Institute of Microbiology,Guangdong Academy of Sciences,Guangzhou 510070,China)

机构地区：[1]广东粤微食用菌技术有限公司,广东广州510663 [2]广东粤微生物科技有限公司,广东肇庆526000 [3]广东省科学院微生物研究所,华南应用微生物国家重点实验室,广东省微生物安全与健康重点实验室,国家卫健委微生物食品营养与安全科技创新平台,广东广州510070

出　　处：《食品工业科技》2025年第7期123-132,共10页Science and Technology of Food Industry

基　　金：广州开发区国际科技合作项目(2020GH07);肇庆市新型研发机构(ZQ01);国家自然科学基金面上项目(82373770);广东省基础与应用基础研究基金项目(2023A1515012155,2023A1515030151)。

摘　　要：本研究制备了蝙蝠蛾拟青霉菌丝体多糖-纳米硒(selenium nanoparticles,SeNPs),并探索其在促进骨形成方面的潜力。通过透析、醇沉、除蛋白以及阴离子交换柱层析,从蝙蝠蛾拟青霉菌丝体水提取物中分离纯化得到多糖组分PHPW,采用苯酚硫酸法、高效凝胶渗透色谱法和高效阴离子交换色谱法分析PHPW的基本理化特性;以PHPW为模板,通过氧化还原法制备PHPW-SeNPs,采用纳米粒度仪、透射电子显微镜、X射线衍射仪、X射线光电子能谱仪、傅里叶变换红外光谱仪表征PHPW-SeNPs结构,并评价其稳定性;采用CCK-8法、碱性磷酸酶(alkaline phosphatase,ALP)检测试剂盒及实时定量PCR技术评价PHPW-SeNPs对MC3T3-E1细胞增殖分化的影响。结果表明,PHPW是一种以葡萄糖为主的中性多糖,其多糖含量和重均分子量分别为99.1%和29.4 kDa。利用200μg/mL PHPW制备获得的PHPW-SeNPs粒径最小且分散性好,呈现零价态、无定形、球状的结构,分析测定的元素中含有碳(63.1%)、氧(3.5%)和硒(33.4%),PHPW中的羟基可通过类似氢键作用稳定SeNPs。PHPW-SeNPs溶液在pH6~8、4℃避光条件下具有较好的稳定性。PHPW-SeNPs在1.25~20μmol/L浓度范围内,MC3T3-E1细胞增殖率呈剂量、时间依赖性提高,当浓度为20μmol/L时,MC3T3-E1细胞ALP活力增强至对照组的1.8倍,骨形态发生蛋白2(bone morphogenetic protein 2,BMP2)、RUNT相关转录因子2(Runt-related transcription factor 2,RUNX2)、osteorix(OSX)及ALP mRNA表达量分别上调至对照组的2.9、4.7、3.2和3.8倍。综上所述,以多糖组分PHPW为模板可成功制备稳定性较好的PHPW-SeNPs,其能够显著促进MC3T3-E1细胞增殖、分化,在预防骨质疏松方面具有良好的应用潜力,有望开发成促进骨健康的保健品。The study prepared Paecilomyces hepiali mycelium polysaccharide-stabilized selenium nanoparticles(SeNPs)and explored its potential to promote bone formation.A polysaccharide fraction(PHPW)was isolated and purified from P.hepiali mycelium water extract using dialysis,alcohol precipitation,deproteinization,and anion exchange column chromatograph.The physicochemical properties of PHPW were analyzed using the phenol-sulfuric acid method,highperformance gel permeation chromatograph,and high-performance anion exchange chromatograph.Subsequently,PHPW was used as a template to prepare PHPW-SeNPs using an oxidation-reduction method.The structural properties of the PHPW-SeNPs were characterized by nanoparticle size analyzer,transmission electron microscopy,X-ray diffraction,X-ray photoelectron spectroscopy,and Fourier transform infrared spectroscopy,and its storage stability was further investigated.Subsequently,the effects of PHPW-SeNPs on MC3T3-E1 cells proliferation and differentiation were evaluated using the Cell Counting Kit-8(CCK-8)assay,alkaline phosphatase(ALP)assay,and real-time quantitative PCR.The results showed that PHPW was a neutral polysaccharide primarily composed of glucose,and its polysaccharide content and weight-average molecular weight were 99.1%and 29.4 kDa,respectively.PHPW-SeNPs prepared using 200μg/mL of PHPW exhibited the smallest particle size and good dispersion,presenting zero-valent,amorphous,and spherical structures.Among the analyzed elements,carbon,oxygen,and selenium accounted for 63.1%,3.5%,and 33.4%of the total,respectively.The SeNPs were stabilized by the hydroxyl groups in PHPW through hydrogen bond-like interactions.The stability of the PHPW-SeNPs solution was observed to exhibit good stability at a pH value range of 6~8 and in the dark at 4℃.The PHPW-SeNPs increased the proliferation rate of MC3T3-E1 cells in a dose-and time-dependent manner at a concentration range of 1.25~20μmol/L.Compared with the control group,PHPW-SeNPs at 20μmol/L significantly enhanced the ALP activity o

关 键 词：蝙蝠蛾拟青霉 菌丝体多糖 纳米硒 结构特性 MC3T3-E1细胞 成骨分化 

分 类 号：TS201.4[轻工技术与工程—食品科学]