【作者】 汪开拓；

【导师】 郑永华；

【作者基本信息】 南京农业大学 ， 食品科学， 2010， 博士

【摘要】 杨梅为中国亚热带特色水果,果实色泽鲜艳、风味浓郁、且富含人体必需的多种维生素和矿物质以及其他营养物质,深受广大消费者喜爱。但杨梅果实采收于初夏高温多雨季节,采后呼吸作用旺盛,且果实组织娇嫩,含水量高,无外果皮保护,极易受机械损伤和病原菌侵染,常温货架期极短。由桔青霉(Penicillium citrinum Thom)和杨梅轮帚霉(Verticicladiella abietina (Peak) Hughes)引起的绿霉病是杨梅果实采后主要的真菌性病害。化学杀菌剂可有效降低杨梅果实采后绿霉病的发病率,但由于公众对农药残留和病菌耐药性的担忧,化学杀菌剂的应用日益受到限制,这迫使人们寻求安全高效的杨梅防腐保鲜新方法。本论文以“乌种”果实(Myrica rubra sieb.et Zucc. Cv. Wumei)为试材,研究了热空气(hot air treatment, HAT)和茉莉酸甲酯(methyl jasmonate, MeJA)处理对杨梅果实采后绿霉病的抑制效果及其内在机理,并采用杨梅果实肉柱活体培养的方法比较两处理间的优劣,在此基础上探索了两者复合处理对减轻杨梅果实采后病害的效果。此外,还研究了乙醇单独熏蒸处理和与热空气复合处理对杨梅果实采后绿霉病以及果实自然腐烂的影响,以期为杨梅果实保鲜新技术的开发提供依据。研究结果分述如下：(1)利用响应曲面法(RSM)建立了不同条件下热空气处理(45-55℃,105-195min)对杨梅果实品质影响的二次多项式的数学模型,经检验该模型极显著,拟合度高。同时,通过模型的对曲面图和等高线分析,明确了处理时间与处理温度对果实品质指标的影响,并在此基础上优化了热空气处理的条件：温度为48℃,处理时间为180 min。采用48℃、3h热空气处理杨梅果实能够降低贮藏期间由P.citrinum导致的绿霉病发病率。其原因一方面是由于热空气处理可直接诱导果实中几丁质酶、p-1,3葡聚糖酶和POD活性等抗病相关酶活性的上升和木质素的合成,抑制SOD、CAT和APX等活性氧代谢酶活性的下降,从而提高果实的抗病性。但热处理诱导杨梅果实的抗病性是逐渐发展的,在处理后6h才完全呈现出来。此外,热处理能够明显诱导杨梅果实HSP70的合成,此蛋白可能起到了分子伴侣的作用从而提高了果实抗病性。另一方面,热空气处理能够显著抑制P. citrinum的孢子萌发、芽管生长和菌丝扩展。这说明热处理减少杨梅果实腐烂主要通过间接诱导果实抗病性和直接抑制病原菌生长两方面来起作用。(2)10μmol/L的MeJA处理可以有效控制杨梅果实采后腐烂的发生,并维持了果实TSS、TA含量和pH值。而高浓度的MeJA处理(100和1000μmol/L)则反而促进果实的腐烂。10μmol/L MeJA处理能够有效的抑制采后杨梅果实在1℃贮藏期间总酚、总黄酮、总花色苷和总胡萝卜素含量的下降,同时10μmol/L MeJA也能够促进了果实主要酚类单体物质如没食子酸、原儿茶酸、杨梅黄酮、槲皮素-3-O-芸香苷和花色苷类主要单体矢车菊-3-葡萄糖苷的积累,提高了果实清除自由基能力和总还原力,从而维持了果实的抗氧化能力。杨梅果实的多酚类物质的合成与苯丙烷类代谢酶密切相关,这其中与PAL和DFR相关度最高。10μmol/L MeJA处理可以诱导杨梅果实苯丙烷类代谢酶活性的提高,从而促进了果实多酚类物质的合成。(3) 10μmol/L MeJA处理能够显著抑制由P citrinum导致的杨梅果实采后绿霉病的发病率。该处理可启动杨梅果实的Priming反应,使得果实在受到病菌侵染时能够迅速启动防御反应,诱导抗病相关酶几丁质酶、p-1,3-葡聚糖酶、PAL和POD蛋白的合成并提高它们的活性,刺激PPP途径促进总酚、木质素和植保素等抗病相关物质的合成,从而提高果实的抗病性。体外研究表明,10μmol/L MeJA对P. citrinum的孢子萌发和芽管生长无显著影响。这些结果可以推断MeJA抑制杨梅腐烂的机理主要是通过间接的诱导果实Priming反应从而使果实“储存”了抗病性,而不是直接的抑制病原菌生长。(4)经48℃、3h热空气处理的杨梅肉柱细胞中的总蛋白以及HSP70和几丁质酶蛋白合成明显增加。但放线菌素D(限制mRNA转录,actidione D)和环己亚胺(限制蛋白翻译合成,cycloheximide)明显抑制了热处理时杨梅肉柱细胞中蛋白合成,这说明热空气处理能直接在转录和翻译水平上诱导杨梅果实细胞HSP70和抗病相关蛋白的合成。但伴随热激蛋白和抗病相关蛋白合成,杨梅肉柱中葡萄糖和蔗糖含量以及氨基酸含量明显降低,这暗示杨梅果实抗病性的诱导是一个不可逆的能量和物质消耗的过程。而采用10μmol/L MeJA处理不能直接诱导杨梅肉柱细胞中总蛋白以及HSP70和几丁质酶蛋白的合成,同时MeJA处理对杨梅肉柱中草酸、酒石酸、苹果酸、可溶性糖和氨基酸含量无显著影响,但提高了柠檬酸含量。联系第五章关于MeJA诱导杨梅果实Priming反应的结果,我们认为MeJA处理在诱导杨梅果实抗病性的同时能够较好的保持果实品质。(5) 10μmol/L MeJA和48℃热空气复合处理比两者单独处理更为显著地抑制了杨梅果实在1℃贮藏期间腐烂的发生。热空气单独处理及其与MeJA的复合处理促进了杨梅果实在贮藏前期的呼吸作用,但却抑制了果实乙烯产生和膜脂过氧化,减少了MDA的积累,从而延缓了果实的衰老进程。同时,两者复合处理较单一处理在整个贮藏过程中更为有效的诱导了杨梅果实中几丁质酶、p-1,3-葡聚糖酶和PAL活性以及总酚含量的上升,从而提高了果实抗病性。此外,复合处理最有效的抑制了杨梅果实在贮藏期间硬度的下降和维生素C的损失,并维持了果实TSS、TA含量、pH值和DPPH自由基清除力。因此,与单一处理相比,复合处理更为显著的抑制了杨梅果实采后腐烂的发生并维持了果实品质。(6) 500μl/L乙醇单独处理有效抑制了采后杨梅果实腐烂的发生,并维持了果实TSS和TA含量,同时抑制了果实内源乙醇含量的上升。500μl/L乙醇与48℃热空气复合处理比单一处理更为显著地抑制了杨梅果实在1℃贮藏时由V. abietina, P. citrinum或T. viride引起的果实腐烂。同时,该复合处理最有效的控制了杨梅果实在货架期结束后自然腐烂的发生和微生物污染,并且最好的维持了果实品质。所以乙醇与热空气复合处理对控制杨梅果实采后腐烂和维持果实品质的效果要优于单一处理。更多还原

【Abstract】 Chinese bayberry is a subtropical fruit native to China and grown in eastern and southern China. The fruit is very popular with consumers for its attractive red to purple color, appealing flavor and rich in vitamins and minerals and other nutritive properties. Chinese bayberry matures and is commercially harvested in the hot and rainy early summer season. After harvest, the fruit is susceptible to mechanical injury and pathogenic attack, with extreme short shelf-life under ambient temperature mainly due to its soft texture, high water content and lack of epicarp protection, as well as high respiration rate. Green mold decay caused by Penicillium citrinum Thom and Verticicladiella abietina (Peak) Hughes is the major postharvest fungal disease on Chinese bayberries and can be effectively controlled by synthetic chemical fungicides. However, with increasing consumer concern over chemical residues on foods and pathogen resistance to fungicides, there is an urgent need to search for effective and safe alternatives to control postharvest diseases of Chinese bayberries.The present study was designed to investigate the effect of hot air treatment (HAT) and methyl jasmonate (MeJA) on inhibiting major fungal disease of green mold decay on Chinese bayberry fruit (Myrica rubra sieb.et Zucc. Cv. Wumei) and the possible mechanisms involved, and to compare the costs and benefits between MeJA and HAT using the flesh column in vivo incubation method. Furthermore, the effectiveness of HAT in combination with MeJA in controlling postharvest disease on Chinese bayberry fruit was also assessed. In addition, the effect of ethanol vapor treatment (EVT) alone or in combination with HAT on postharvest mold and natural decay, as well as microbial loads on Chinese bayberries was investigated. The results were as follows:(1) The effect of hot air treatment (HAT) on postharvest decay and quality of Chinese bayberry fruit (Myrica rubra Seib & Zucc.) was investigated and optimized by response surface methodology (RSM) under different time-temperature combinations. A central composite rotatable design (CCRD) was applied with treatment temperature (ranging from 45 to 55℃) and exposure time (from 105 to 195 min) as independent variables while postharvest decay, firmness, Vc content and TSS/TA of Chinese bayberry after storage at 1℃for 7 days followed by 20℃for 1 day as investigated responses. The results indicated that the response and variables were fitted well to each other by multiple regressions, and overall optimization showed the lowest incidence of decay and highest values of firmness, Vc content and TSS/TA were achieved by an air treatment at treatment temperature of 48℃and exposure time of 180 min. HAT at 48℃for 3 h enhanced the resistance of Chinese bayberry fruit against green mold decay caused by P. citrinum and reduced the severity of the disease. The HAT-treated fruit demonstrated a gradual development of disease resistance against fungus P. citrinum infection and reached a maximum at 6 h after the treatment, being associated with a rapid and strong activation of defense enzymes including chitinase,β-1,3-glucanase, POD and PPO, and an increase in lignin content, and an inhibition of SOD, CAT and APX decrease. Moreover, the results have shown that the HAT treatment could significantly promote accumulation of HSP70 during the whole incubation at 1℃, which acts as a molecular chaperone that played a key role in induced resistance in Chinese bayberry fruit. In addition, the in vitro experiment showed that HAT significantly inhibited spore germination, germ tube elongation and mycelial growth of P. citrinum. These results indicate that HAT can effectively reduce fruit decay possibly by directly inhibiting pathogen growth and indirectly inducing disease resistance.(2) The effects of MeJA on postharvest decay and antioxidant capacity in harvested Chinese bayberry fruit were investigated. MeJA at 10μmol/L was most effective in reducing fruit decay; quality parameters including pH value, total soluble solids, and titratable acidity were not significantly affected by MeJA treatment. Fruit treated with 10μmol/L MeJA exhibited significantly higher levels of total phenolics, flavonoids, anthocyanins and carotenoids as well as individual phenolic compounds including gallic acid, protocatechuic acid, myricetin, quercetin-3-O-rutinoside, and cyanidin-3-glucoside than the controls. These fruits also maintained significantly higher antioxidant activity as measured by scavenging capacity against DPPH, superoxide, and hydroxyl radicals and by the reducing power test compared to the controls. These results indicate that MeJA can effectively reduce fruit decay and improve antioxidant capacity of Chinese bayberry fruit.Polyphenolic biosynthesis is the result of coordinated action of phenylpropanoid related enzymes including PAL, C4H,4-CL、DFR and CHS. The results indicated that both PAL and DFR were key enzymes that determined the polyphenolic biosynthesis in Chinese bayberry fruit. MeJA treatment could induce the activities of a series of phenylpropanoid related enzymes, and thus promote the polyphenolic biosynthesis.(3) The effect of MeJA on postharvest decay of Chinese bayberry fruit caused by P. citrinum, and its ability to induce biochemical defense responses in fruits, were assessed. The results demonstrated that treatment with 10μmol/L MeJA could significantly inhibit the green mold decay caused by P. citrinum on Chinese bayberry fruit. MeJA treatment in reducing the decay on Chinese bayberries could be related to the priming for defense responses, which indirectly triggered the new biosynthesis of pathogenesis-related (PR) proteins in Chinese bayberries and stimulated biosynthesis of tatol phenolics, lignin and phytoalexin when the fruit was challenged or infected by pathogen, rather than a direct activation of defense responses. In addition, the in vitro experiment showed that MeJA was not directly capable of inhibiting spore germination and germ tube elongation of P. citrinum. Thus, these results MeJA can effectively reduce fruit decay possibly by priming action for "preserve the disease resistance", rather than a direct inhibitory effect on pathogen growth.(4) After 3h treatment of the HAT at 48℃, the biosynthesis of total protein as well as chitinase and PAL protein in flesh column of Chinese bayberry were markedly promoted. However, after heating for 3 h, the intensity of the immune signal of chitinase and PAL protein in fruit column incubated in CHX (inhibiting protein synthesis) or actidione D (inhibiting mRNA transcription) began to get weaker, indicating the heat treatment might confer disease resistance through the accumulation of PR protein and HSP70 at the transcriptional and translational level. Furthermore, the development of disease resistance was accompanied by significantly lower glucose, sucrose and aimino acid contents, possibly indicating the induced resistance was an irreversible process of consuming substrates and energy.On the other hand, the in vivo incubation experiment showed 10μmol/L MeJA treatment could not directly induced biosynthesis of total protein as well as chitinase and PAL protein in flesh column of Chinese bayberry. Meanwhile, MeJA did not affect the oxalic acid, tartaric acid, and malic acid as well as soluble sugars and aimino acid contents, but promoted the content of citric acid. To take the MeJA-induced Priming mechanism mentioned in Chapter 5 into account, between benefits of MeJA-mediated resistance and the costs of substrates and energy in Chinese bayberry fruit brought into a state of equilibrium. (5) The effect of HAT (48℃for 3h) alone or in combination with MeJA (10μmol/L) on postharvest decay and physiological response, and their effects on fruit quality in harvested Chinese bayberries were investigated. The results showed that the combined treatment of HAT and MeJA resulted in a remarkably improved control of fruit decay compared with treatment of HAT or MeJA alone during the storage at 1℃. HAT alone or its in combination with MeJA could promote the respiration rate over the earlier period of the storage, inhibit the ethelyne production and membrane lipid peroxidation, thus retard the senescence process as confirmed by lower MDA content. Meanwhile, the combined treatment induced higher activities of the defense-related enzymes including chitinase,β-1,3-glucanase and PAL in Chinese bayberry fruit than applying either alone, which were closely related to the enhanced disease resistance. In addition, HAT in combination with MeJA showed an improved effect on inhibiting the decrease of firmness and Vc content during the storage. Moreove, the combined treatment maintained TSS and TA contents as well as pH value, further higher scavenging capacity against DPPH radicals. Thus, these results suggest that the combination of HAT treatment and MeJA might be a more useful technique to reduce fruit decay and maintain quality in Chinese bayberries during postharvest storage compared with the HAT or MeJA alone.(6) Results demonstrated that EVT at 500μL/L for 3 h was found most effective in reducing natural decay without impairing quality. Furthermore, the effect of EVT (500μL/L for 3 h) alone or in combination with hot air treatment HAT (48℃for 3 h) on postharvest decay and microbial loads in Chinese bayberries was investigated. Treatment with ethanol vapor or hot air alone both resulted in significantly lower decay incidence caused by V. abietina, P. citrinum or Trichoderma viride compared with the control. The combined treatment showed the lowest incidence of fruit decay caused by these pathogens. This treatment also significantly inhibited spore germination and germ tube elongation of the pathogens in vitro than EVT or HAT alone. Meanwhile, the combined treatment exhibited the lowest natural decay incidence and microbial loads on Chinese bayberries without impairing fruit quality. These results suggest the usefulness of the combined treatment for reducing decay and maintaining quliaty in harvested Chinese bayberries.更多还原