黄芪多糖在草菇冷藏保鲜中的应用.pdf

52 食用菌学报 2023.30（5）：2023.05.收稿日期：2023-04-28原稿；2023-05-08修改稿基金项目：国家自然科学基金（32072642）作者简介：郑婷婷（1997），女，在读硕士，主要从事食用菌遗传育种研究。*本文通信作者 E-mail:;5258006黄芪多糖在草菇冷藏保鲜中的应用郑婷婷1,2，黄天宇1，龚 明1*，王瑞娟1，吴莹莹1,万佳宁1，汪 滢1*（1上海市农业科学院食用菌研究所，农业农村部南方食用菌资源利用重点实验室，国家食用菌工程技术研究中心，国家食用菌加工技术研发分中心，上海市农业遗传育种重点实验室，上海 201403；2上海海洋大学食品学院，上海 201306）摘 要：分析4 处理24、48 h的草菇（Volvariella volvacea）渗出液代谢组；用200 molL-1黄芪多糖溶液浸泡草菇后在4 放置24、48 h，测定草菇-葡萄糖苷酶活性和转录组，检测糖基转移酶基因VVO_02307表达量。结果表明：4 处理24、48 h草菇渗出液代谢组分明显不同；与4 处理24 h草菇比较，处理48 h的草菇中蔗糖含量较低，亚油酸含量较高。与对照比较，黄芪多糖溶液浸泡的草菇在4 处理24 h时自溶不明显，-葡萄糖苷酶活性显著降低。转录组分析结果显示，黄芪多糖增强草菇对低温胁迫的耐受性，可能是通过提高N-聚糖生物合成通路活力实现的。研究结果可为草菇低温保鲜技术研发提供参考。关键词：草菇；黄芪多糖；代谢组；转录组；-葡萄糖苷酶Use of Astragalus Polysaccharide in Preservation of Volvariella volvacea at 4 ZHENG Tingting1,2,HUANG Tianyu1,GONG Ming1*,WANG Ruijuan1,WU Yingying1,WAN Jianing1，WANG Ying1*1Institute of Edible Fungi,Shanghai Academy of Agricultural Sciences;Key Laboratory of Edible Fungi Resources and Utilization(South),Ministry of Agriculture and Rural Affairs,P.R.China;National Engineering Research Center of Edible Fungi;National R&D Center for Edible Fungi Processing;Key Laboratory of Agricultural Genetics and Breeding of Shanghai,Shanghai 201403,China;2 College of Food Science,Shanghai Ocean University,Shanghai 201306,ChinaAbstract:The metabolites of Volvariella volvacea exudates stored at 4 for 24 and 48 h were analyzed,respectively.Using distilled water as the blank control,V.volvacea fruiting bodies were immersed in 200 molL-1 Astragalus polysaccharide(AST)solution prior to storage at 4 for 24 and 48 h,and then determined for-glucosidase activity,analyzed for transcriptome,and measured for the expression level of glycosyltransferase gene VVO_02307.The results showed that metabolic components in the exudate of V.volvacea stored at 4 for 24 h were significantly different with those in the exudate of V.volvacea stored at 4 for 48 h.Compared with V.volvacea stored at 4 for 24 h,V.volvacea stored at 4 for 48 h showed a lower sucrose content and a higher linoleic acid content.Compared with the control,the autolysis of the AST solution treated V.volvacea was not obvious at 4 for 24 h,and the activity of-glucosidase decreased significantly.Transcriptomic analysis showed that AST was very likely to promote the tolerance of V.volvacea to cold stress by improving N-glycan biosynthesis activity.These results provided a reference for developing low-temperature 53 郑婷婷，等：黄芪多糖在草菇冷藏保鲜中的应用第 5 期preservation technology of V.volvacea.Key words:Volvariella volvacea;Astragalus polysaccharide;metabolome;transcriptome;-glucosidase草菇（Volvariella volvacea）是高温型草腐食用真菌，适宜生长温度为（321），其栽培始于我国1-2。草菇味道鲜美，口感细腻，富含蛋白质、氨基酸和维生素C等营养物质，具有促进人体新陈代谢、增强免疫力等功效3-5。草菇子实体在4 储藏会自溶软化6-8，其低温保鲜技术研究一直是学界关注热点。抑制-葡萄糖苷酶和中性蛋白酶的活性可有效延长草菇子实体低温保藏时间9-10。低温条件下纤维素酶活性较高可能是导致草菇低温自溶的重要原因10。一种草菇纤维二糖水解酶基因VvCBHI-I在里氏木霉（Trichoderma reesei）异源表达，可显著提高低温条件下里氏木霉纤维素酶复合物糖化水解效率11。这些研究结果表明，低温时草菇纤维素酶系统活性较高，降解细胞壁多糖，导致草菇低温自溶。因此，添加外源多糖可能通过保护细胞壁而提高草菇对低温的耐受性。黄芪多糖有助于山羊精液冷冻保存，可提高小叶杨耐低温胁迫能力12-13，其在食品尤其是草菇保鲜中的应用研究鲜有报道。笔者首先在4 保藏草菇，分析渗出液代谢组；然后用黄芪多糖溶液浸泡草菇后在4 保藏，测定草菇-葡萄糖苷酶活性和转录组，研究黄芪多糖对草菇的保鲜机制，以期为草菇低温保鲜技术研发提供参考。1 材料与方法1.1 供试菌株草菇（V.volvacea）菌株V23由国家食用菌种质资源库（上海）保藏并提供，在上海市农业科学院庄行综合试验站草菇菌种生产基地栽培。1.2 4 处理草菇渗出液代谢组分析选取栽培9 d且无损伤、大小一致的蛋形期子实体，分别在4 放置24、48 h，收集子实体渗出液，在80 mg渗出液中加入预冷体积比为221的甲醇乙腈水溶液，14 000 g离心20 min，将上清液真空干燥，送上海派森诺生物科技股份有限公司，采用LC-MS/MS质谱检测非靶标代谢组10，每个处理设6个重复。以正交偏最小二乘法判别分析（orthogonal partial least squares discriminant analysis,OPLS-DA）得到的变量权重值（variable importance in projection,VIP）1 和P1 和 P1 和 P1 and P0.05),red connected lines indicate positive correlation and blue connected lines indicate negative correlation;D.heatmap analysis of differential metabolites in metabolic pathway(ko01100).图1 4 处理草菇渗出液的代谢组分析结果Fig.1 Metabolomic analysis of exudate of V.volvacea stored at 4 55 郑婷婷，等：黄芪多糖在草菇冷藏保鲜中的应用第 5 期2.2 黄芪多糖对草菇低温耐受性的影响渗出液代谢组分析结果显示，多糖含量减少可能不利于草菇对低温胁迫的耐受性（图1D），因此笔者用200 molL-1黄芪多糖溶液浸泡草菇，然后在4 保藏。已有研究表明，-GC抑制剂可增强草菇对低温胁迫的耐受性10，用-GC抑制剂浸泡的草菇在4 保藏24 h时，-GC活性显著降低。与对照比较，黄芪多糖溶液浸泡的草菇4 保藏24 h时自溶不明显（图 2A），-GC活性显著降低（图2B），说明黄芪多糖能够增强草菇对冷胁迫的耐受性；48 h时草菇表面凹陷并产生渗出液，-GC活性显著上升。注：A.草菇形态；B.草菇-葡萄糖苷酶（-GC）活性；A中CK为蒸馏水处理，AST为黄芪多糖处理；B中0 h为蒸馏水处理，24、48 h为黄芪多糖处理；*表示与对照比较差异显著（P 0.05）。Notes:A.fruiting bodies of V.volvacea under different treatments,where AST indicates Astragalus polysaccharide and CK indicates the control of distilled water;B.-glucosidase(-GC)activity of V.volvacea soaked with 200 mol L-1 Astragalus polysaccharide at 4 for 24 h and 48 h,where distilled water treatment was used as the blank control at 0 h,and*indicates a significant difference compared with the control at P0.05.图2 低温处理对草菇形态和-葡萄糖苷酶活性的影响Fig.2 Effect of cold stress on-GC activity of V.volvacea2.3 草菇转录组分析结果热图分析结果显示，低温处理改变草菇的基因表达谱(图3A)。主成分分析结果显示，黄芪多糖处理草菇与对照(48 h)差异明显（图3B）。Venn分析结果显示，黄芪多糖处理草菇具有520个显著差异表达基因，而对照(48 h)则有770个显著差异表达基因（图3C），表明黄芪多糖影响草菇低温胁迫应答代谢过程。注：A.不同处理草菇基因表达热图；B.基因表达主成分分析结果；C.差异表达基因Venn分析结果；AST为黄芪多糖处理；CK为蒸馏水处理。Notes:A.heatmap analysis of V.volvacea gene expression under different treatments at 4,where AST indicates Astragalus polysaccharide and CK is the control of distilled water;B.principal component analysis of gene expression;C.Venn diagram of differentially expressed genes.图3 黄芪多糖处理48 h草菇转录组分析结果Fig.3 Transcriptome analysis of V.volvacea treated with Astragalus polysaccharide at 4 for 48h 56 第 30 卷食 用 菌 学 报如图4A所示，黄芪多糖处理草菇基因表达上调的是簇4（cluster 4），而对照蒸馏水处理草菇基因表达上调的是簇6（cluster 6）。簇4富集分析结果显示，N-聚糖生物合成（N-glycan biosynthesis）、赖氨酸合成（lysine biosynthesis）、精氨酸合成（arginine biosynthesis）等通路活力上调。簇6富集分析结果显示，类固醇生物合成（steroid biosynthesis）、抗生素生物合成（biosynthesis of antibiotics）等通路活力上调（图4B、C）。黄芪多糖处理的代谢通路类型和数量均明显多于对照（图4B、C）。以上结果证实，N-聚糖生物合成、碱性氨基酸合成通路活力上调可能与黄芪多糖提高草菇对低温耐受性相关。注：A.差异表达基因趋势分析结果；B.簇4差异表达基因热图分析结果；C.簇6差异表达基因热图分析结果；B和C中不同颜色代表不同归类富集分析簇；AST为黄芪多糖处理；CK为蒸馏水处理。Notes:A.trend analysis of differentially expressed genes,where AST indicates Astragalus polysaccharide and CK is the control of distilled water;B.heatmap analysis of differentially expressed genes in cluster 4;C.heatmap analysis of differentially expressed genes in cluster 6;in B and C,different colors represent different classification enrichment clusters.图4 差异表达基因趋势分析和富集分析结果Fig.4 Trend and enrichment analysis of differentially expressed genes 57 郑婷婷，等：黄芪多糖在草菇冷藏保鲜中的应用第 5 期N-聚糖生物合成（map00510）的 KEGG 通路注释结果显示，两个富集基因 VVO_02307 和VVO_05240编码N-聚糖生物合成限速酶糖基转移酶（glycosyltransferase）的两个亚基。采用转录组数据的FPKM（fragments per kilobase of exon model per million mapped fragments,FPKM）进行基因表达分析，结果显示黄芪多糖处理显著提高VVO_02307和VVO_05240表达量(图5)。如图6所示，与对照（0 h）比较，黄芪多糖处理显著提高VVO_02307表达量。以上结果进一步提示N-聚糖生物合成通路活力上调有助于促进草菇耐受低温胁迫。注：CK为蒸馏水处理；AST为黄芪多糖处理；*表示差异显著(P 0.05)。Notes:AST indicates Astragalus polysaccharide and CK is the control of distilled water；*indicates a significant difference at P0.05.图5 糖基转移酶基因FPKM分析结果Fig.5 FPKM expression of glycosyltransferase genes VVO_02307 and VVO_05240注：CK为蒸馏水处理；AST为黄芪多糖处理；不同小写字母表示差异显著(P 0.05)。Notes:AST indicates Astragalus polysaccharide and CK is the control of distilled water;different lowercase letters indicate a significant difference at P0.05.图 6 糖基转移酶基因VVO_02307表达情况Fig.6 Expression analysis of glycosyltransferase gene VVO_023073 讨论低温胁迫导致草菇细胞壁多糖降解10，4 处理草菇的蔗糖、亚油酸等代谢物质含量增加分别反映低温对草菇细胞壁和细胞膜的损坏程度。细胞壁作为细胞最外层的保护结构，其多糖的适度消耗是一种应激反应，可能有助于草菇耐受低温胁迫。低温处理48h的渗出液明显增多，推测主要原因是细胞膜结构破坏。黄芪多糖处理的草菇对低温耐受性增强与-葡萄糖苷酶活性降低相关，这与抑制-葡萄糖苷酶活性可阻止草菇低温自溶的结论一致10。草菇可能通过添加的黄芪多糖上调多糖代谢通路活力，抵抗低温胁迫。前期研究结果表明，草菇泛素化结合酶E2（UBEV2）抑制剂L345-0044处理后，从表型上可以看出草菇对低温耐受性增强，这与N-聚糖生物合成通路活力上调密切关联16-17。FPKM分析结果显示，L345-0044处理显著提高VVO_02307和VVO_05240表达量10，与黄芪多糖处理的结果一致。结合定量PCR验证结果说明黄芪多糖处理的草菇N-聚糖生物合成通路活力上调，其可能有助于增强细胞结构或者功能18，进而改善草菇对低温耐受性。草菇对低温耐受性增强可能减弱细胞壁多糖降解应激反应，抑制-葡萄糖苷酶活性10，具体机制还有待进一步研究。草菇适宜在偏碱性环境中生长，最适pH为7.07.8，而渗出液pH呈酸性，这可能与细胞壁和细胞膜裂解释放酸性代谢物质相关。黄芪多糖处理的草菇碱性氨基酸合成通路活力上调，可能释放碱性物质，中和冷胁迫过程中释放的酸性物质。冷胁迫时渗出液pH与正常状态下的细胞液pH是否一致，还有待进一步研究。据报道黄芪多糖通过调控MAPK信号通路减轻脂多糖引起的炎症19。黄芪多糖处理的草菇MAPK信号通路活力出现上调，其改变草菇对低温耐受性的应答模式机制有待进一步研究。58 第 30 卷食 用 菌 学 报参考文献1 张萍,牛磊,王爱云,等.草菇菌丝体深层培养条件研究J.河南农业科学,2005,34(11):79-81.2 张树庭.草菇M.香港:香港中文大学出版社,1975:143-147.3 GALAZ P,VALDENEGRO M,RAMREZ C,et al.Effect of drum drying temperature on drying kinetic and polyphenol contents in pomegranate peelJ.Journal of Food Engineering,2017,208:19-27.4 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