本文主要研究内容
作者孔婕(2019)在《百里酚和水杨酸对腐皮镰孢和匍枝根霉的协同抑制作用及在番茄采后保鲜中的应用》一文中研究指出:植物源保鲜剂,因其没有化学抑菌剂所带来的环境污染、农药残留及抗药性等问题,已成为国内外天然防腐保鲜剂开发研究的方向和热点。但由于其天然产物价格居高不下,造成使用成本较高,尤其是附加值较低的农产品很难推广使用。番茄果实营养丰富,但属于果皮薄且多汁的品种,采后贮藏过程当中容易受机械损伤、微生物感染等发生腐烂,采后损失率极高。因此,如何减少植物源保鲜剂在番茄等生鲜果蔬中的使用量,降低其使用成本是一个亟待解决的问题。本研究选取11种已报道具有抑菌活性的植物源保鲜剂,针对果蔬采后两种常见的腐败真菌,同时也是番茄采后主要的腐败菌,腐皮镰孢菌(Fusarium solani CICC 2603=ATCC 36031,以下简写F.solani)和匍枝根霉(Rhizopus stolonifer ATCC 12939,以下简写R.stolonifer),进行抗菌活性研究。根据最小抑菌浓度(Minimum Inhibitory Concentration,MIC),按照棋盘法对11种植物源抑菌剂进行复配,发现对F.solani和R.stolonifer均有协同抑菌作用的组合仅有1组,即百里酚和水杨酸(记为STSA),其在协同抑菌作用中的MIC值比单独使用两种抑菌剂时的MIC值降低了74.62%87.69%,有效降低了单个活性成分的作用浓度。通过对F.solani和R.stolonifer的菌丝和细胞形态的观察,发现STSA作用后细胞结构和形态发生严重变化,菌丝体表面出现一些隆起,显示出不规则的收缩,出现皱褶;细胞壁变薄,细胞质基质减少,大量空泡在细胞质中融合,形成较大的空泡。同时,STSA、百里酚和水杨酸均对F.solani和R.stolonifer的细胞膜的结构和功能都造成了不同程度的损伤。对两种真菌进行碘化丙啶(propidium iodide,PI)染色后观察发现,随着STSA、百里酚和水杨酸作用浓度的增加,两种真菌中被染色的细胞比例均有所增加,并且呈浓度依赖性(r值均在0.91以上),表明F.solani和R.stolonifer的细胞膜完整性被破坏,使得PI可以穿过细胞膜与核酸结合显示红色荧光。水杨酸在STSA对F.solani细胞膜完整性损伤的协同作用中占主要地位;百里酚和水杨酸在STSA对R.stolonifer细胞膜完整性损伤的协同作用中贡献度相当。随着抑菌进程,两种真菌细胞在260 nm和280 nm处的泄漏量均有所增加,各组在1 h?24 h的作用时间之间具有显著差异(P(27)0.05),增加比例最高分别达79.52%和82.65%,表明抑菌剂造成两种真菌细胞内的核酸和蛋白质的泄漏量增加。且百里酚和水杨酸在浓度为0.5和1 MIC的STSA对F.solani和R.stolonifer细胞内核酸泄露的协同作用中贡献度相当,但百里酚在浓度为2MIC的STSA对F.solani细胞内核酸泄露的协同作用中起主要作用,水杨酸在STSA对于R.stolonifer细胞内蛋白质泄露的协同作用中起主要作用。同时,F.solani和R.stolonifer细胞膜中麦角固醇的含量随着抑菌剂浓度的增加而降低,并呈现浓度依赖性(r值在-0.8211和-0.8975之间),且百里酚和水杨酸在STSA分别对两种菌麦角固醇含量的影响的贡献度相当。STSA通过阻碍细胞膜中麦角固醇的合成,破坏了两种真菌的细胞膜的正常功能,并最终抑制F.solani和R.stolonifer的生长。此外,STSA、百里酚和水杨酸分别对F.solani和R.stolonifer的线粒体的结构和功能产生严重影响。三种抑菌剂分别作用F.solani和R.stolonifer后,两种真菌的线粒体膜电位均显著升高,表现为膜电势超级化,且呈现浓度依赖性(r值在0.9244到0.9972之间),表明两种真菌的线粒体膜结构被改变。且相同浓度抑菌剂处理后,F.solani线粒体膜电位均高于R.stolonifer,说明F.solani的线粒体膜电位所受影响更大。研究抑菌剂对三羧酸(TCA)循环的关键酶活性的影响发现,柠檬酸合成酶(CS)、异柠檬酸脱氢酶(IDH)、α-酮戊二酸脱氢酶(α-KGDH)、琥珀酸脱氢酶(SDH)和苹果酸脱氢酶(MDH)的活性均受到抑菌剂不同程度的抑制,酶活性下降率在71.45%到98.38%之间。同时,抑菌剂导致F.solani和R.stolonifer细胞内TCA循环中的柠檬酸(CA)含量分别下降65.64%(F.solani)和90.80%(R.stolonifer),表明抑菌剂严重影响了TCA循环的正常进行,进而损伤了两种真菌的线粒体功能。此外,两种真菌经抑菌剂处理后发现,其细胞内活性氧(ROS)的积累有明显增加,增长率最大值分别为86.71%(F.solani)和97.78%(R.stolonifer);并且,丙二醛(MDA)大量生成,增长率最大值分别为73.54%(F.solani)和80.00%(R.stolonifer)。综上所述,STSA导致F.solani和R.stolonifer细胞膜损伤,致使其细胞内容物(核酸和蛋白质)的大量泄漏,麦角固醇合成严重受阻,线粒体膜发生了电势超级化改变了线粒体的膜结构,TCA循环中关键酶活性降低,进而影响了ATP合成和NADH产生,破坏TCA循环,ROS和MDA的大量累积进一步造成线粒体功能障碍和细胞氧化损伤,细胞的过氧化继续影响细胞内线粒体内关键酶的活性,加剧膜的损伤,最终将导致F.solani和R.stolonifer细胞死亡。STSA对F.solani和R.stolonifer的体外抑菌作用表明,STSA对两种腐败真菌的菌丝生长抑制率均达到100%,对其孢子萌发抑制率达到96%以上;番茄接菌试验结果表明,STSA对F.solani的抑菌效果优于R.stolonifer;对番茄伤口接种F.solani的保护作用优于治疗作用,对伤口接种R.stolonifer的番茄的保护作用和治疗作用的差异不显著;结合感官评价,浓度为1 MIC的STSA对好果的保鲜效果更好,2 MIC的STSA更适用于有腐烂情况发生的番茄保鲜;进一步将不同浓度的STSA加入到可食性虫胶中,制成涂膜液,覆盖于番茄表面。研究发现,STSA虫胶涂膜和对照组相比,其透湿性和透氧性均显著降低(P(27)0.05),在一定程度上阻隔了番茄果实内部的水分散失和气体交换;浓度为5 MIC的STSA虫胶涂膜番茄在贮藏期间,腐烂率与失重率最低(P(27)0.05),番茄的可溶性固形物含量下降最慢,采后感官品质维持最好,不同浓度的STSA虫胶涂膜对番茄采后的颜色没有影响;对番茄硬度损失具有较好抑制作用的浓度为1MIC和5MIC STSA,能将可滴定酸含量维持在较高的水平的浓度为10 MIC,延迟总酚峰值出现的有效浓度是1 MIC,有利于番茄风味的保存;对降低贮藏期间番茄体内MDA累积的有效浓度是5 MIC,有效防止了果实衰老和膜损伤,诱导番茄体内两种抗病性相关酶(多酚氧化酶和苯丙氨酸解氨酶)活性在短时间内升高,提高果实的免疫力,提高果实采后抵抗病原真菌侵染的能力,从而减少采后贮运过程中的损失。本课题的研究结果将拓展植物活性成分的应用前景,为番茄采后保鲜提供基础数据和科学的理论依据,并为果蔬采后贮藏及运输提供借鉴作用。
Abstract
zhi wu yuan bao xian ji ,yin ji mei you hua xue yi jun ji suo dai lai de huan jing wu ran 、nong yao can liu ji kang yao xing deng wen ti ,yi cheng wei guo nei wai tian ran fang fu bao xian ji kai fa yan jiu de fang xiang he re dian 。dan you yu ji tian ran chan wu jia ge ju gao bu xia ,zao cheng shi yong cheng ben jiao gao ,you ji shi fu jia zhi jiao di de nong chan pin hen nan tui an shi yong 。fan jia guo shi ying yang feng fu ,dan shu yu guo pi bao ju duo zhi de pin chong ,cai hou zhu cang guo cheng dang zhong rong yi shou ji xie sun shang 、wei sheng wu gan ran deng fa sheng fu lan ,cai hou sun shi lv ji gao 。yin ci ,ru he jian shao zhi wu yuan bao xian ji zai fan jia deng sheng xian guo shu zhong de shi yong liang ,jiang di ji shi yong cheng ben shi yi ge ji dai jie jue de wen ti 。ben yan jiu shua qu 11chong yi bao dao ju you yi jun huo xing de zhi wu yuan bao xian ji ,zhen dui guo shu cai hou liang chong chang jian de fu bai zhen jun ,tong shi ye shi fan jia cai hou zhu yao de fu bai jun ,fu pi lian bao jun (Fusarium solani CICC 2603=ATCC 36031,yi xia jian xie F.solani)he pu zhi gen mei (Rhizopus stolonifer ATCC 12939,yi xia jian xie R.stolonifer),jin hang kang jun huo xing yan jiu 。gen ju zui xiao yi jun nong du (Minimum Inhibitory Concentration,MIC),an zhao qi pan fa dui 11chong zhi wu yuan yi jun ji jin hang fu pei ,fa xian dui F.solanihe R.stoloniferjun you xie tong yi jun zuo yong de zu ge jin you 1zu ,ji bai li fen he shui yang suan (ji wei STSA),ji zai xie tong yi jun zuo yong zhong de MICzhi bi chan du shi yong liang chong yi jun ji shi de MICzhi jiang di le 74.62%87.69%,you xiao jiang di le chan ge huo xing cheng fen de zuo yong nong du 。tong guo dui F.solanihe R.stoloniferde jun si he xi bao xing tai de guan cha ,fa xian STSAzuo yong hou xi bao jie gou he xing tai fa sheng yan chong bian hua ,jun si ti biao mian chu xian yi xie long qi ,xian shi chu bu gui ze de shou su ,chu xian zhou zhe ;xi bao bi bian bao ,xi bao zhi ji zhi jian shao ,da liang kong pao zai xi bao zhi zhong rong ge ,xing cheng jiao da de kong pao 。tong shi ,STSA、bai li fen he shui yang suan jun dui F.solanihe R.stoloniferde xi bao mo de jie gou he gong neng dou zao cheng le bu tong cheng du de sun shang 。dui liang chong zhen jun jin hang dian hua bing ding (propidium iodide,PI)ran se hou guan cha fa xian ,sui zhao STSA、bai li fen he shui yang suan zuo yong nong du de zeng jia ,liang chong zhen jun zhong bei ran se de xi bao bi li jun you suo zeng jia ,bing ju cheng nong du yi lai xing (rzhi jun zai 0.91yi shang ),biao ming F.solanihe R.stoloniferde xi bao mo wan zheng xing bei po huai ,shi de PIke yi chuan guo xi bao mo yu he suan jie ge xian shi gong se ying guang 。shui yang suan zai STSAdui F.solanixi bao mo wan zheng xing sun shang de xie tong zuo yong zhong zhan zhu yao de wei ;bai li fen he shui yang suan zai STSAdui R.stoloniferxi bao mo wan zheng xing sun shang de xie tong zuo yong zhong gong suo du xiang dang 。sui zhao yi jun jin cheng ,liang chong zhen jun xi bao zai 260 nmhe 280 nmchu de xie lou liang jun you suo zeng jia ,ge zu zai 1 h?24 hde zuo yong shi jian zhi jian ju you xian zhe cha yi (P(27)0.05),zeng jia bi li zui gao fen bie da 79.52%he 82.65%,biao ming yi jun ji zao cheng liang chong zhen jun xi bao nei de he suan he dan bai zhi de xie lou liang zeng jia 。ju bai li fen he shui yang suan zai nong du wei 0.5he 1 MICde STSAdui F.solanihe R.stoloniferxi bao nei he suan xie lou de xie tong zuo yong zhong gong suo du xiang dang ,dan bai li fen zai nong du wei 2MICde STSAdui F.solanixi bao nei he suan xie lou de xie tong zuo yong zhong qi zhu yao zuo yong ,shui yang suan zai STSAdui yu R.stoloniferxi bao nei dan bai zhi xie lou de xie tong zuo yong zhong qi zhu yao zuo yong 。tong shi ,F.solanihe R.stoloniferxi bao mo zhong mai jiao gu chun de han liang sui zhao yi jun ji nong du de zeng jia er jiang di ,bing cheng xian nong du yi lai xing (rzhi zai -0.8211he -0.8975zhi jian ),ju bai li fen he shui yang suan zai STSAfen bie dui liang chong jun mai jiao gu chun han liang de ying xiang de gong suo du xiang dang 。STSAtong guo zu ai xi bao mo zhong mai jiao gu chun de ge cheng ,po huai le liang chong zhen jun de xi bao mo de zheng chang gong neng ,bing zui zhong yi zhi F.solanihe R.stoloniferde sheng chang 。ci wai ,STSA、bai li fen he shui yang suan fen bie dui F.solanihe R.stoloniferde xian li ti de jie gou he gong neng chan sheng yan chong ying xiang 。san chong yi jun ji fen bie zuo yong F.solanihe R.stoloniferhou ,liang chong zhen jun de xian li ti mo dian wei jun xian zhe sheng gao ,biao xian wei mo dian shi chao ji hua ,ju cheng xian nong du yi lai xing (rzhi zai 0.9244dao 0.9972zhi jian ),biao ming liang chong zhen jun de xian li ti mo jie gou bei gai bian 。ju xiang tong nong du yi jun ji chu li hou ,F.solanixian li ti mo dian wei jun gao yu R.stolonifer,shui ming F.solanide xian li ti mo dian wei suo shou ying xiang geng da 。yan jiu yi jun ji dui san suo suan (TCA)xun huan de guan jian mei huo xing de ying xiang fa xian ,ning meng suan ge cheng mei (CS)、yi ning meng suan tuo qing mei (IDH)、α-tong wu er suan tuo qing mei (α-KGDH)、hu po suan tuo qing mei (SDH)he ping guo suan tuo qing mei (MDH)de huo xing jun shou dao yi jun ji bu tong cheng du de yi zhi ,mei huo xing xia jiang lv zai 71.45%dao 98.38%zhi jian 。tong shi ,yi jun ji dao zhi F.solanihe R.stoloniferxi bao nei TCAxun huan zhong de ning meng suan (CA)han liang fen bie xia jiang 65.64%(F.solani)he 90.80%(R.stolonifer),biao ming yi jun ji yan chong ying xiang le TCAxun huan de zheng chang jin hang ,jin er sun shang le liang chong zhen jun de xian li ti gong neng 。ci wai ,liang chong zhen jun jing yi jun ji chu li hou fa xian ,ji xi bao nei huo xing yang (ROS)de ji lei you ming xian zeng jia ,zeng chang lv zui da zhi fen bie wei 86.71%(F.solani)he 97.78%(R.stolonifer);bing ju ,bing er quan (MDA)da liang sheng cheng ,zeng chang lv zui da zhi fen bie wei 73.54%(F.solani)he 80.00%(R.stolonifer)。zeng shang suo shu ,STSAdao zhi F.solanihe R.stoloniferxi bao mo sun shang ,zhi shi ji xi bao nei rong wu (he suan he dan bai zhi )de da liang xie lou ,mai jiao gu chun ge cheng yan chong shou zu ,xian li ti mo fa sheng le dian shi chao ji hua gai bian le xian li ti de mo jie gou ,TCAxun huan zhong guan jian mei huo xing jiang di ,jin er ying xiang le ATPge cheng he NADHchan sheng ,po huai TCAxun huan ,ROShe MDAde da liang lei ji jin yi bu zao cheng xian li ti gong neng zhang ai he xi bao yang hua sun shang ,xi bao de guo yang hua ji xu ying xiang xi bao nei xian li ti nei guan jian mei de huo xing ,jia ju mo de sun shang ,zui zhong jiang dao zhi F.solanihe R.stoloniferxi bao si wang 。STSAdui F.solanihe R.stoloniferde ti wai yi jun zuo yong biao ming ,STSAdui liang chong fu bai zhen jun de jun si sheng chang yi zhi lv jun da dao 100%,dui ji bao zi meng fa yi zhi lv da dao 96%yi shang ;fan jia jie jun shi yan jie guo biao ming ,STSAdui F.solanide yi jun xiao guo you yu R.stolonifer;dui fan jia shang kou jie chong F.solanide bao hu zuo yong you yu zhi liao zuo yong ,dui shang kou jie chong R.stoloniferde fan jia de bao hu zuo yong he zhi liao zuo yong de cha yi bu xian zhe ;jie ge gan guan ping jia ,nong du wei 1 MICde STSAdui hao guo de bao xian xiao guo geng hao ,2 MICde STSAgeng kuo yong yu you fu lan qing kuang fa sheng de fan jia bao xian ;jin yi bu jiang bu tong nong du de STSAjia ru dao ke shi xing chong jiao zhong ,zhi cheng tu mo ye ,fu gai yu fan jia biao mian 。yan jiu fa xian ,STSAchong jiao tu mo he dui zhao zu xiang bi ,ji tou shi xing he tou yang xing jun xian zhe jiang di (P(27)0.05),zai yi ding cheng du shang zu ge le fan jia guo shi nei bu de shui fen san shi he qi ti jiao huan ;nong du wei 5 MICde STSAchong jiao tu mo fan jia zai zhu cang ji jian ,fu lan lv yu shi chong lv zui di (P(27)0.05),fan jia de ke rong xing gu xing wu han liang xia jiang zui man ,cai hou gan guan pin zhi wei chi zui hao ,bu tong nong du de STSAchong jiao tu mo dui fan jia cai hou de yan se mei you ying xiang ;dui fan jia ying du sun shi ju you jiao hao yi zhi zuo yong de nong du wei 1MIChe 5MIC STSA,neng jiang ke di ding suan han liang wei chi zai jiao gao de shui ping de nong du wei 10 MIC,yan chi zong fen feng zhi chu xian de you xiao nong du shi 1 MIC,you li yu fan jia feng wei de bao cun ;dui jiang di zhu cang ji jian fan jia ti nei MDAlei ji de you xiao nong du shi 5 MIC,you xiao fang zhi le guo shi cui lao he mo sun shang ,you dao fan jia ti nei liang chong kang bing xing xiang guan mei (duo fen yang hua mei he ben bing an suan jie an mei )huo xing zai duan shi jian nei sheng gao ,di gao guo shi de mian yi li ,di gao guo shi cai hou di kang bing yuan zhen jun qin ran de neng li ,cong er jian shao cai hou zhu yun guo cheng zhong de sun shi 。ben ke ti de yan jiu jie guo jiang ta zhan zhi wu huo xing cheng fen de ying yong qian jing ,wei fan jia cai hou bao xian di gong ji chu shu ju he ke xue de li lun yi ju ,bing wei guo shu cai hou zhu cang ji yun shu di gong jie jian zuo yong 。
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