【作者】 罗海波；

【导师】 郁志芳；

【作者基本信息】 南京农业大学 ， 食品科学， 2011， 博士

【摘要】 茭白作为一种健康、天然的蔬菜,由于其色泽洁白、质地脆嫩、气味清香、味道鲜美和营养丰富等特点,适合加工成鲜切产品,但切分造成的机械损伤极易诱导切割面褐变和木质化等品质劣变现象的发生,商品价值急剧下降。本研究旨在通过对鲜切茭白品质和生理生化特性的探索,揭示其品质劣变机理,科学选择控制其品质劣变的适宜技术。主要研究结果如下：1、新鲜茭白含水量为94.46%,游离氨基酸、还原糖含量分别为5.34、16.37 mg.g-1FW,粗脂肪、粗纤维、木质素和可溶性固形物含量分别为0.29%、0.69%、0.37%和4.6%,抗坏血酸含量为27.6μg.g-1FW,其它成分与文献报道相近。综上所述,茭白营养价值较高,作为特色水生蔬菜有进一步发展的潜力。鲜切茭白酚提取物定性鉴定结果表明其主要酚类物质是咖啡酸、没食子酸和愈创木酚,分别占茭白酚提取物总量的25.57%、29.54%和22.100%,三种酚均可作为鲜切茭白POD作用的底物,咖啡酸同时也是木质素合成的前体物质,表明鲜切茭白不仅具有很强的褐变潜力,而且具备了快速木质化的物质基础。鲜切茭白冷藏期间品质和生理生化指标测定结果显示,与完整去壳茭白相比,切分显著加速了茭白白度(WI)、硬度、pH、总醣、还原糖、抗坏血酸及游离酚含量的下降和失重率、纤维素、木质素含量的增加；呼吸速率显著提高,切分后立即测定即从70.96增加到168 mgCO2.kg-1.h-1,且整个贮藏过程均显著高于完整茭白；O2.-生成速率加快,H2O2和MDA积累显著增多,但相对电导率在整个贮藏过程中与完整茭白差异不明显。此外,切分还显著增加了SOD、POD、APX、PAL、4CL及CAD活性,减小了CEL活性,但对PPO及CAT活性影响不大。相关性分析发现,鲜切茭白贮藏过程中WI值和木质素含量与POD活性均显著相关(r=-0.9130,r=0.9985)。以上结果表明,鲜切茭白品质的快速劣变是一个极其复杂的过程,是多种生理和生化代谢反应共同作用的结果。2、研究了鲜切茭白POD粗酶液特性。结果表明,鲜切茭白POD粗酶活力可达34.05 U.mg-1 protein,最适pH值为6.0,pH5.0～7.0范围内较稳定；最适温度为40℃60℃以上温度下稳定性较差;H2O2浓度在12.25 mmol.L-1以下时,POD活力随H2O2浓度增加直线上升(r=0.9786),浓度进一步增加POD活力上升不明显,浓度超过14.71mmol.L-1时对POD活力有抑制作用。鲜切茭白POD对H202的Kin为4.91 mmol.L-1,与不同酚类底物亲和力顺序为焦性没食子酸>咖啡酸>愈创木酚>表儿茶酚>儿茶酚>没食子酸＞酪氨酸、肉桂酸,且与愈创木酚结合时表现出最大酶活力。经硫酸铵沉淀、Sephadex G-25脱盐和DEAE Sepharose Fast Flow离子交换色谱分离提纯了完整和鲜切茭白POD,得到了一个阳离子组分(PODc)和两个阴离子组分(PODaⅠ和Ⅱ),阳离子组分占总酶活的76%。阳离子组分经Sephadex G-100柱层析纯化后,完整和鲜切茭白POD分别纯化了99.48和61.51倍,回收率分别为9.68%和9.32%。SDS-PAGE图谱显示完整茭白POD为单一条带,根据蛋白质分子量标准计算其分子量为22.1 kDa,而鲜切茭白POD为两条带,分子量分别为20.6、22.1 kDa。Native-PAGE图谱显示完整茭白POD含有2条同工酶谱带(PODc1-2),鲜切茭白POD含有3条同工酶谱带(PODc1-3),表明鲜切诱导了茭白POD同工酶的合成。POD的酶学特性研究表明,完整和鲜切茭白POD最适pH值均为6.0,且分别在pH4.0～8.0和pH4.0～9.0范围内较稳定；完整茭白最适温度为40～60℃,鲜切茭白最适温度为40℃；完整和鲜切茭白热稳定性均较差,60℃保温1min残余酶活仅分别为3.87%和2.21%。完整和鲜切茭白POD对H202的Km分别为0.98和1.43 mmol.L-1,对咖啡酸和愈创木酚的Km分别为2.5、3.33 mmol.L-1和6.67、10 mmol.L-1。AA、L-cys和N-AC对鲜切茭白POD抑制效果最好,0.02%浓度可抑制鲜切茭白POD活力99.9%以上NaHSO3对鲜切茭白POD也有较强抑制作用；4-HR对抑制鲜切茭白POD活力有一定效果,但有效浓度较高,0.12%对鲜切茭白POD的抑制为69.3%；而CA、CC和EDTA-2Na在试验浓度下对鲜切茭白POD几乎没有抑制作用。3、根据NCBI中已报道的水稻、高粱、玉米和燕麦4种植物POD基因的保守区域设计简并引物,利用PCR和RACE技术对茭白POD基因进行克隆和分析。结果表明,茭白POD基因cDNA全长1318bp,含有621bp的ORF,编码207个氨基酸,推测该蛋白的相对分子质量为22.87 kDa,理论等电点为6.74。经生物学软件分析该基因与禾本科中高粱、水稻和玉米POD的亲缘关系较近,相似度达到80%以上。该基因的克隆与cDNA序列分析为采后茭白中POD的基因工程操作奠定了基础。4、研究了化学物质对鲜切茭白品质及生理生化的影响。结果显示,4-HR、CC、AA、N-AC和复合处理均显著抑制了鲜切茭白的褐变；失重率贮藏15天后分别为对照的95.6%、75.3%、74.5%、76.5%和57.9%；硬度分别为入贮时的66.1%、91.0%、80.4%、74.6%和86.7%,对照仅为63.6%；纤维素、木质素含量比入贮时分别增加了25.5%、36.8%、41.3%、17.1%、24.5%和138%、140%、120%、93.5%、75.4%,而对照分别增加了43.3%和149%；AA含量保留率分别为70.5%、75.1%、89.2%、83.3%和82.6%,对照仅保留57.5%；pH值、还原糖及FP含量维持在相对高的水平,呼吸速率、相对电导率及02-和MDA含量维持在相对低的水平；POD活力分别为入贮时的3.07、1.95、1.73、2.01和1.35倍,对照为3.88倍；PAL和PPO活力显著低于对照而CEL、SOD、CAT和APX活力显著高于对照。以上结果表明,试验所选化学物质均能有效延缓鲜切茭白褐变和生理代谢,提高抗氧化酶类活力和活性氧自由基清除能力,最终维持鲜切茭白较好的品质。由于复合处理表现出最好的效果,提示复合处理可用于商业上控制鲜切茭白褐变和维持其品质。5、研究了外源植物生长调节剂对鲜切茭白木质化及活性氧代谢的影响。结果表明,GA3、SA及6-BA处理显著抑制了鲜切茭白木质素含量的增加,对纤维素含量也有一定抑制作用,维持相对高的游离酚含量和纤维素酶活力。外源植物生长调节剂处理还抑制了PAL、4CL、CAD及POD活性。GA3处理在贮藏前9天抑制了SOD,APX及CAT活性,导致鲜切茭白O2.-和H2O2的积累增多；SA处理在贮藏显著提高了SOD活性,而对APX及CAT活性影响不明显,从而抑制O2-产生但促进H202的积累；6-BA处理显著提高了SOD,APX及CAT活性,O2-和H2O2的积累减少。以上结果表明GA3、SA及6-BA处理能有效抑制鲜切茭白的木质化,其机制可能主要是由于抑制木质化相关酶PAL、4CL、CAD及POD的活性而不是降低活性氧伤害。更多还原

【Abstract】 Zizania latifolia (Griseb.) Turcz.ex Stabf, a healthy and natural vegetable, with the characteristics of white color, tender texture, attractive aroma, delicious flavor and rich in nutrients, is very suitable to be processed into fresh-cut product, but mechanical tissue damage induces enzyme activity which results in tissue damage that affects the commercial value of Z. latifolia. This thesis touched the quality, physiological and biochemical property of Z. latifolia after cutting and during refrigerated storage in order to obtain that how the quality, physiological and biochemical of fresh-cut Z. latifolia are changed, what browning and lignification mechanisms are and how we could control them. The main research results are as following:1. The fresh Z. latifolia contain 94.49% water, reducing sugar and free amino acid content were 39.6,5.34 mg.g-1FW. Crude fat, crude fiber, lignin and soluble solids contents were 0.29%,0.69%, 0.37% and 4.6%, respectively. Ascorbic acid content of 27.6μg.g-1FW. Other components were similar with the reported. In a word, Z. latifolia had higher nutritional values as a aquicolous vegetable.The phenols of fresh Z. latifolia were identified by combination of effect of acid and base, scanning spectrum and high performance liquid chromatography (HPLC). The results showed that the main phenols of Z. latifolia were gallic acid, caffeic acid and guaiacol, which accounted for 25.57%,29.54% and 22.10%, respectively, of the total content of Z. latifolia phenol extracts. All these phenols can be used as substrate for the fresh-cut Z. latifolia POD, while caffeic acid also was the precursor substance of lignin synthesis. These results indicated that fresh-cut Z. latifolia not only have very strong browning potential, but also have the material base of rapid lignification.The quality, physiological and biochemical changes of fresh-cut Z. latifolia were investigated. The results showed that fresh-cut significantly promoted the decrease of whiteness index (WI), firmness, pH, total sugar, reducing sugar, ascorbic acid and free phenol contents and the increase of weight loss, cellulose and lignin contents compared with the whole Z. latifolia. The respiratory rate significantly increased from 70.96 to 168 mgCO2.kg-1.h-1 when immediately determined after fresh-cut, and it was constantly higher than the whole Z. latifolia during whole storage time; Fresh-cut accelerated the accumulation of O2-, H2O2 and MDA significantly, while the relative conductivity was no significant difference compared with the whole Z. latifolia. In addition, fresh-cut were also significantly increased the SOD, POD, APX, PAL,4CL and CAD activities and decreased the CEL activity, but no significant difference on PPO and CAT activity between the fresh-cut and the whole Z. latifolia were found. These results indicated that the rapid quality deterioration of fresh-cut Z. latifolia is an extremely complex process, which result from variety of physiological and biochemical metabolic reactions.2. The characteristics of the POD in fresh-cut Z. latifolia was investigated. The results showed that the POD activity in fresh-cut Z. latifolia can reach 34.05 U.mg-1 protein. POD had its greatest activity at pH 6.0 and temperature 40.0℃, and lost its activity quickly when pH below 5.0 or temperature above 60℃. The POD activity increased fast when H2O2 was below 12.25 mmol.L-1, and increased a little afterwards, reached the maximum at the H2O3 level of 14.71 mmol.L-1, then decreased. The Km for H2O2 was 4.91 mmol.L-1. The combining capability of POD to various phenolic substrates were in the order of Pyrogallic acid> Caffeic acid> Guaiacol> Epicatechin> Catechol> Gallic acid> Tyrosine, Cinnamic acid. The POD shows the highest activity when combined with Guaiacol.POD from whole and fresh-cut Z. latifolia were purified using a combination of (NH4)2SO4 fractionation and anion exchange chromatography, resulting in one cationic (PODc) and two anionic fractions (PODaⅠandⅡ). The cationic fraction, which accounted for 76% of recovered activity, was further purified by gel filtration. The whole and fresh-cut Z. latifolia POD activity were purified 99.48 and 61.51-fold, respectively, and with 9.68% and 9.32% recovery, respectively. The purified whole Z. latifolia POD showed a single protein band while the fresh-cut Z. latifolia POD showed two protein bands on SDS-PAGE. The molecular weight of whole Z. latifolia POD was 22.1 kDa and that of fresh-cut Z. latifolia POD were 20.6 kDa and 22.1 kDa, respectivrly. Native-PAGE showed the purified whole Z. latifolia POD contains two POD isoenzymes (PODc1-2), while the fresh-cut Z. latifolia POD contains three POD isoenzymes (PODc1-3), which indicated that fresh-cut caused the de novo induction of PODc3 in Z. latifolia.Both the purified PODs showed optimum pH of 6.0, and they were stable in the pH range 4.0～8.0 and 4.0～9.0, respectively. The optimum temperature for the whole Z. latifolia POD was 40～60℃while for the fresh-cut Z. latifolia POD was 40℃. Both the purified PODs were very thermolabile and retained only 3.87% and 2.21%, respectively, of the activity for 1 min at 60℃. The purified whole and fresh-cut Z. latifolia showed the apparent Km values of 0.98 and 1.43 mmol.L-1, respectively, for H2O2, and of 2.5,3.33 mmol.L-1 and 6.67,10 mmol.L-1, for caffeic acid and guaiacol, respectively. AA, L-cys and N-AC have the best effect on inhibit the activity of fresh-cut Z. latifolia POD, which can inhibit more than 99.9% of the POD activity only at 0.02% concentration; NaHSO3 also has a strong inhibitory effect on fresh-cut Z. latifolia POD; 4-HR has some effect on inhibit the fresh-cut Z. latifolia POD, but a higher effective concentration was needed,0.12% concentration can inhibition 69.3% of the POD activity; However, CA, CC, and EDTA-2Na were no inhibiting effect at the test concentration on fresh-cut Z. latifolia POD.3. With degenerated primers designed according to conservative region of peroxidase gene sequence of Oryza sativa, Sorghum bicolor, Zea mays and Avena saliva L. on NCBI, the fragment of POD gene was cloned from Z. latifolia by polymerase chain reaction (PCR) method and the 3’ and 5’ end by rapid amplification of cDNA end (RACE) strategy. The results showed that the POD cDNA of Z. latifolia was 1 318 bp in length and it has an opening-reading frame (ORF) of 621 bp, which encoded a protein of 207 amino acid residues with molecular weight of 22.87 kDa and theoretical isoelectric point of 6.74. The similarly between the POD of Z. latifolia with that of S. bicolor, O. sativa and Z. mays was over 80%. The cloning and sequence analysis of the POD cDNA gene established base for gene engineering operation of POD from the postharvested Z. latifolia.4. The effect of antibrowning agents alone or in combination treatment on physiological, biochemical and quality of fresh-cut Z. latifolia were investigated. The results showed that the 4-HR, CC, AA, N-AC and combination treatments retarded browning of fresh-cut Z. latifolia significantly. After 15 days storage at 1±0.5℃, the weight loss were 95.6%,75.3%,74.5%,76.5% and 57.9%, respectively, of the control. Firmness were 66.1%,91.0%,80.4%,74.6% and 86.7%, respectively, compared to that at the beginning, while it was only 63.6% in the control. Cellulose and lignin contents were increased by 25.5%,36.8%,41.3%,17.1%,24.5% and 138%,140%,120%,93.5%,75.4%, respectively, and them were increased by 43.3% and 149% in the control, respectively. Ascorbic acid retention rate were 70.5%, 75.1%,89.2%,83.3% and 82.6%, respectively, and it was 57.5% in the control. All treatments maintained relatively high pH value, reducing sugar and free phenolic contents and relatively low respiratory rate, relative leakage rate, superoxide anion radical (O2-) and malondialdehyde (MDA) contents. POD activities were 3.07,1.95,1.73,2.01 and 1.35 folds, respectively, compared to that at the beginning, and it was 3.88 fold in the control. These treatments also inhibited activities of PAL and PPO while promoted CEL, SOD, CAT and APX activities compared with the control. The present findings indicate that the tested antibrowning agents could retard physiological metabolism and browning on cut surface, improve antioxidant enzymes activities and free radicals scavenging activities, and therefore maintain a better quality of fresh-cut Z. latifolia during storage at 1℃. Since the combination of antibrowning agents gives even better effect, it is suggested that the combination treatment could be commercially used to control browning and to maintain quality in fresh-cut Z. latifolia.5. The effect of exogenous plant growth regulator on reducing lignification in fresh-cut Z. latifolia and the possible mechanisms involved were investigated. The results showed that treatment of Z. latifolia slices with GA3, SA and 6-BA significantly slowed down the increase in lignin and cellulose contents, maintained relatively high free phenol content and cellulase activity. These treatments also inhibited phenylalanine ammonia-lyase (PAL),4-coumarate CoA ligase (4CL), cinnamyl alcohol dehydrogenase (CAD) and peroxidase (POD) activities. GA3 treatment promoted the accumulation of O2 and H2O2 over the first 9 days of storage; SA treatment inhibited the accumulation of O2-throughout storage time but promoted the accumulation of H2O2 over the first 9 days of storage; 6-BA treatment significantly inhibited O2- and H2O2 accumulation during the whole storage time, indicating that diminished oxidative damage is not the major mechanism for reducing lignification in fresh-cut Z. latifolia. These results suggested that GA3, SA and 6-BA treatments can effectively inhibits lignification in fresh-cut Z. latifolia. It is postulated that the control of the lignification by GA3, SA and 6-BA in fresh-cut Z. latifoli is mainly due to inhibited the activities of PAL,4CL, CAD and POD but not diminished the oxidative damage.更多还原