【作者】 曲留柱；

【导师】 王建中；

【作者基本信息】 北京林业大学 ， 野生动植物保护与利用， 2014， 博士

【摘要】 香蕉是世界上产量最高的水果之一,也是许多发展中国家的重要食物来源之一,但它在保存和加工过程中极易褐变,不易保存。其主要原因是香蕉含有多酚氧化酶,能催化香蕉中多酚类物质褐变,破坏香蕉的营养成分、风味物质、感官色泽。为此本文针对香蕉多酚氧化酶的底物、提取方法、特性进行了研究,并据此深入分析了香蕉多酚氧化酶催化褐变机制的特点,进而探寻防控香蕉多酚氧化酶催化褐变的有效方法。主要研究成果如下：1.使用福林酚显色法检测100g鲜重香蕉中的总酚含量为36.0±1.9mg儿茶素当量。使用HPLC对多酚样品进行分离鉴定、定性、定量,结果表明每100g鲜重香蕉中含有多巴胺10.6±1.4mg,绿原酸9.5±0.1mg,儿茶素10.6±0.2mg,表儿茶素0.7±0.1mg。2.通过响应面实验法确定闪式提取香蕉中多酚氧化酶的最佳提取工艺条件为：以pH5.58的磷酸-柠檬酸为提取缓冲液,液料比为1.57,PVPP含量为1.13%,提取时间为2.5min。3.从不同底物褐变反应的酶活性及褐变现象的变化可判断香蕉多酚氧化酶的特异性底物为多巴胺。多巴胺的酶促褐变过程可划分为五个阶段。五个阶段中每一阶段都可划分为两个区间,第一个区间褐变反应表现出较高的酶活性,第二个区间褐变速率几乎趋于零；褐变产物的颜色变化依次为：橘红色、红褐色、紫黑色、灰色、黑色,达到黑色后产物颜色不再发生改变。另外香蕉中的多酚,或者不同酚类的氧化褐变产物之间存在某种协同作用,能促进褐变反应的进行。4.香蕉中的至少存在两种同工酶。他们的分子量分别为b=60000±1000Da,c=42000±2000Da。两种同工酶除最适pH相差较大外,其他特性,如：pH稳定性、最适温度、热稳定性非常相近。他们的最适pH分别是5.4和6.8；在pH5.0-9.0间有较强的稳定性,能够保持原酶液70%以上的活力。他们具有较强的耐碱性质,而耐酸性较弱。两同工酶的最适温度为30℃,在60℃以下,香蕉中多酚氧化酶的酶活力仍能保持在最大酶活力值的50%以上。当温度达到80℃以上,活性下降至最大酶活力的10%以下,能有效降低酶的活性。在90℃下处理5min后,在80℃下处理100min后,以及在100℃下处理30s后,多酚氧化酶的活性被完全抑制。5.当抗坏血酸浓度达到0.3mg/mL,柠檬酸的浓度达到2mg/mL,亚硫酸氢钠浓度达到0.3mg/mL时,都能够完全的抑制香蕉多酚氧化酶的活性。6.本文采用扫描法检测香蕉汁的褐变情况,能够准确、有效、快速检测到香蕉汁的褐变变化情况。根据此法,检测抑制香蕉汁褐变的条件有：当香蕉汁中抗坏血酸浓度达到0.4mg/mL时,在200min内,能有效的抑制香蕉汁的褐变。抗坏血酸在低浓度下,对香蕉汁的褐变抑制作用是可逆的,但是当它的浓度达到0.8mg/mL时,能对香蕉汁形成稳定的不可逆的褐变抑制作用。另外抗坏血酸具有漂白作用,能使香蕉汁的亮度超出样品的初始亮度。当柠檬酸浓度达到2.5mg/mL时,香蕉汁的pH为3.0,对香蕉汁的褐变起到有效的抑制作用。亚硫酸氢钠浓度大于0.4mg/mL时,能够有效的抑制香蕉汁的褐变,并且它在抑制褐变的过程中具有漂白作用。更多还原

【Abstract】 Bananas are one of the highest yield fruits and one of the most important food sources for many developing countries in the world. During preservation and processing, they easily get browned due to the banana polyphenol oxidase (PPO) which catalyzes the banana polyphenols to be brown. These reactions severely affect the nutrients and flavor of the bananas. We researched the substrates, extraction methods, properties and the catalytic browning mechanism of the banana polyphenol oxidase, in order to find methods to control the enzymatic browning. The main study results are shown as follows:1. The total polyphenol content of banana is36.0±1.9mg catechin equivalents/100g fresh weight banana detected with Folin-Ciocalteu reagent. The polyphenolic contents include dopamine10.6±1.4mg/100g FW, chlorogenic acid9.5±0.1mg/100g FW,(+)-catechin10.6±0.2mg/100g FW, and (-)-epicatechin0.7±0.1mg/100g FW, which were qualitatively and quantitatively analyzed by HPLC.2. The optimal extraction conditions for banana polyphenol oxidase are source of buffer solution (phosphoric acid-citric acid) without potassium chloride, pH (5.58), liquid:solid ratio (1.57), PVPP concentration (1.13%), time (2.5min), through the response surface methodology.3. The special substrate of banana PPO is dopamine, which was determined by the velocity and the color change of the browning reaction. The enzymatic browning on dopamine can be divided into5stages. The initial velocity was higher, and then turned to almost zero in each stage. The colors of the browning products of each stage are orange, reddish brown, violet black, gray, black successively. The experimental data showed that there must be a synergistic reaction which could promote the browning reaction between the polyphenols or the browning products in bananas. When the ammonium sulfate saturation was less than30%, it could remove something and lower the PPO activity or browning reaction in the extraction. 4. Two kinds of isoenzyme are found in banana PPO, whose molecular weights are b=60,000+1,000Da, c=42,000+2,000Da respectively. They have similar properties, like pH stability, optimum temperature and thermal stability, except the optimum pH.Their optimum pH is5.4and6.8respectively. The enzyme activity is strongly stable at pH5.0-9.0, during which the activity could be kept more than70%of the initial one. The banana PPO is strong in alkali-resistance, but weak in acid-resistance.The optimum temperature for banana PPO is30℃. Below60℃, the enzyme activity can still remain at more than50%of the maximum enzyme activity. When the temperature was above80℃, the enzyme activity fell to10%. If the banana PPO was processed over3s,5min,100min under100℃,90℃,80℃respectively, the PPO activity would be completely inhibited.5. When the concentration of ascorbic acid, citric acid, sodium bisulfite reached0.3mg/mL,2mg/mL,0.3mg/mL respectively, they can completely inhibit the banana PPO activity separately.6. Base on the scanning method, the banana juice browning can be detected acutely, effectively and rapidly. The methods of inhibition on banana juice browning are followed:When the concentration of ascorbic acid in banana juice reached0.4mg/mL, it can effectively control the banana juice browning within200min. Ascorbic acid’s browning inhibition property is was reversible at low concentrations, but when its concentration reache over0.8mg/mL, it can form a stable irreversible inhibition to browning. The ascorbic acid can make the brightness of the banana juice to be beyond the initial brightness of the sample, due to its bleaching effects.When the concentration of citric acid was2.5mg/mL, banana juice pH reached3.0, and the banana juice browning was inhibited effectively. Sodium bisulfite can effectively restrain the browning when its concentration is greater than0.4mg/mL. Sodium bisulfite also has the bleaching property during the process更多还原