Studies On The Mechanism Of Quality Deterioration And Preservation Technology Of Fresh-cut Zizania Latifolia

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.