山葡萄種質(zhì)資源原花青素動(dòng)態(tài)含量變化及提取、降解研究

STUDIES OF EXTRACTION AND DEGRADATION IN PROCYANIDINE，AND THE DYNAMIC CONTENTS IN GERMPLASM RESOURCES ON VITIS AMURENSIS RUPR

作者：王新偉　　導(dǎo)師：沈育杰

中國(guó)農(nóng)業(yè)科學(xué)院　　經(jīng)濟(jì)植物資源評(píng)價(jià)與利用2011屆碩士

摘　要

本文對(duì)山葡萄籽中原花青素提取、降解進(jìn)行了研究，優(yōu)化了工藝路線。以40份山葡萄種質(zhì)資源為試材，對(duì)山葡萄各個(gè)重要時(shí)期的葉片、莖皮、果實(shí)中的原花青素動(dòng)態(tài)含量變化進(jìn)行了測(cè)定，分析了原花青素在山葡萄各組織器官中的變化規(guī)律和山葡萄種質(zhì)資源間原花青素含量和分布的特點(diǎn)與差異性，主要結(jié)果如下：

1.　確定了山葡萄黃烷-3-醇的HPLC洗脫程序：流動(dòng)相配比：A：1%乙酸；B：80%乙腈，1%乙酸，0min→5min→15min→25min→30min→35min，動(dòng)相A對(duì)應(yīng)的梯度為95%→70%→50%→40%→95%→95%。

2.　原花青素的最佳提取工藝為：以乙醇為提取劑，溫度60℃，濃度70%，時(shí)間為120min，料液比為1:5，提取率為3.12%。

3.　溶劑分離法分離制備原花青素高聚體，綜合聚合度與過氧化指標(biāo)，確定優(yōu)化降解工藝為：乙酸為降解介質(zhì)，料液比6:5，降解時(shí)間20min，降解溫度70℃，聚合度從6.9下降到4.8，聚合度下降2.1。

4.　山葡萄種質(zhì)資源葉片中原花青素動(dòng)態(tài)含量變化趨勢(shì)為：萌芽期→花后一周，緩慢上升；花后一周→漿果膨大期，含量急劇上升，并達(dá)到最大值；漿果膨大期→轉(zhuǎn)色期，含量迅速下降；轉(zhuǎn)色期→成熟期，含量上升。

5.　莖皮原花青素動(dòng)態(tài)含量上升變化趨勢(shì)為：萌芽期→花后一周，緩慢上升；花后一周→漿果膨大期，含量急劇上升，并達(dá)到最大值；漿果膨大期→漿果轉(zhuǎn)色期→漿果成熟期，含量迅速下降。

6.　種子中原花青素動(dòng)態(tài)含量變化趨勢(shì)為：花后一周→漿果膨大期→漿果轉(zhuǎn)色期，含量迅速上升并在轉(zhuǎn)色期達(dá)到最大值；漿果轉(zhuǎn)色期→漿果成熟期，含量迅速下降。成熟期含量最高的種質(zhì)資源為：4N1,4N2，燕山。

7.　果皮中原花青素動(dòng)態(tài)含量變化趨勢(shì)為：花后一周→漿果膨大期，含量很低，緩慢上升；漿果膨大期→漿果轉(zhuǎn)色期，含量迅速升高；漿果轉(zhuǎn)色期→漿果成熟期，含量急劇上升，并達(dá)到頂峰。漿果成熟期含量最高的種質(zhì)資源為：086034,8558814,8558831。

8.　山葡萄成熟果實(shí)果皮中原花青素含量高于種子，且差異性極為顯著，與歐亞種釀酒葡萄相比具有明顯的種間差異性。

關(guān)鍵詞　山葡萄　原花青素　黃烷-3-醇　提取　降解　動(dòng)態(tài)含量變化

Abstract

By studying conditions of extraction of proanthocyanidins from the vitis grape seeds after fermentation，the process of separations was optimized. The changes on the concentration of proanthocyanidins in leaves,barka and fruita was studied based on forty vitis amurensis germplasm resource with different developmental stages，the variation of proanthocyanidins with various organs and various grapes species. The preliminary conclusions as followed：

1.　The extraction of flvan-3-ols process was determined：mobile phase ratio was A:1% acetic acid ;B:80% acetonitrile,1% acetic acid,0min→5min→15min→25min→30min→35min，the mobile phase gradient of the corresponding to 95%→70%→50%→40%→95%→95%.

2.　The optimum condition of the proanthocyanidins extraction were determined：temperature 60℃，concentration of alcohol 70%，extraction time 120min，rate of solution to solid 5:1.Ratio of extraction was 3.12%.

3.　Respectively used hydrochloric acid and acetic acid as acid hydrolysis medium,this experiment studied the acid hydrolysis technology for procyanidin high polymer,determined the best acid hydrolysis technology is that acetic acid concentration－55%,hydrolysis time－20min ,hydrolysis temperature－70℃. In this experimental condition,the procyanidins high polymer units decreased from 6.9 to 4.8,dropped 2.1 units by test.

4.　The trends of the procyanidin dynamic content in leaves are that: procyanidin concentration increased slowly from budding to flowering;the content of procyanidin sharply rise in berries growing stage and up to maximum;the period of berry growth and turn color，the content declined rapidly when berry maturity.

5.　The dynamic content of procyanidin in barks are that：from budding to flowering is increase slowly；in the period of berries growing the procyanidin sharply rise and up to maximum；the content declined rapidly when berry maturity.

6.　The procyanidin trends in grape seeds are：from flowering to berry growing to turn color， the content are still increase and up to maximum in turn color period； until maturity the concentration of procyanidin declined rapidly. The higher content procyanidin species in berry maturity are：4N1,4N2，Yanshan.

7.　The trends in peel are：from flowering to berry growing，the concentration is low and rise；the content rise rapidly in turn color period；from turn color to maturity the procyanidin sharply increased and reach the peak. The higher content procyanidin species in berry maturity are：086034,8558814,8558831.

The content of procyanidin in pulp during the whole fruit development is very low.

8.　The content of procyanidin in vitis grape peel is significant higher than seeds in berry maturity,and compared with the Eurasian grape species have obvious differences.

Key words　Vitis amurensis　Procyanidin　Flan-3-ols　Abstraction　Degradation Dynamic