【作者】 郭松年；

【导师】 刘兴华；

【作者基本信息】 西北农林科技大学 ， 食品科学， 2008， 博士

【摘要】 石榴汁的诱人色泽主要由其花色苷决定,是石榴汁的重要商品性状之一,花色苷也是石榴汁的重要功能成分。但是,花色苷是一类不稳定的物质,在生产和贮藏中容易发生化学变化而降解或聚合。因此研究石榴汁花色苷的稳定性和降解规律对于石榴汁的生产和贮藏具有重要的理论价值和现实意义。本文以陕西临潼地区产的石榴为试材,对石榴汁花色苷的稳定性和热降解规律、辅色素对花色苷稳定性和色泽的影响、石榴汁PPO的性质及其对花色苷降解的作用、石榴汁的酶解工艺及加工过程对花色苷的影响、石榴汁及花色苷提取物的抗氧化性等进行了研究;采用大孔树脂和葡聚糖凝胶LH-20对石榴汁花色苷进行了纯化,并用液质联用技术对其组分进行了鉴定。主要研究结果如下:（1）石榴汁花色苷对热不稳定,其Hunter a*值随加热时间延长、温度升高呈下降趋势,而Hunter b*值呈上升趋势;石榴汁花色苷的稳定性受pH值和光照的影响较大,pH>5和光照均促进其降解。参阅相关文献,推测石榴汁花色苷热稳定性较低的原因是其分子中缺乏酰化结构的缘故。石榴汁花色苷降解符合动力学一级反应,其反应活化能E₀为52.67 kJ/mol,反应常数k₀为6.37×10⁶,建立了石榴汁花色苷热降解的预测模型。经验证,模型与实测值拟合较好,表明该模型是合理的。（2） EDTA、SDS和植酸对石榴汁花色苷有一定的辅色作用,能增加石榴汁色泽的热稳定性,其中浓度为20mg/L的植酸效果最好;吐温-80、氨基酸、芦丁均无辅色作用,不影响石榴汁花色苷的热稳定性;单宁有辅色作用,但不影响其热稳定性;当Vc浓度大于1.06mg/L时,能显著降低石榴汁花色苷的稳定性;葡萄糖、果糖使石榴汁的Huntera*值增大,蔗糖使其Hunter a*值有所减小;蔗糖和低浓度的葡萄糖对石榴汁花色苷的热稳定性无影响,当葡萄糖浓度为150mg/L时,对花色苷有保护作用,同时色泽破坏程度也减轻;果糖对石榴汁花色苷热稳定性有不利影响。（3）石榴汁PPO对一元酚、二元酚和多元酚均有催化氧化作用,在所选试验底物中,以邻苯二酚为底物时活性最大。以邻苯二酚为底物,在pH7、30℃条件下活性最强,温度在80℃以上时PPO活力基本丧失。PPO可直接催化石榴汁花色苷降解,酚类物质对其有一定的辅助催化作用。（4）籽粒的清洗时间对石榴汁花色苷的含量影响不大,冷压榨有利于花色苷的保存,热压榨对花色苷的保存有不利影响。EDTA、V_C、L-半胱氨酸都有一定的护色作用,其中L-半胱氨酸护色的效果最好,最佳浓度0.025%。运用三元二次中心组合响应面法确定的最优酶解澄清工艺条件为:加酶量0.98ml/L、酶解温度40.5℃、酶解时间127min,该条件下酶解的石榴汁透光率为95.31%,花色苷保存率为82.5%。真空旋转蒸发浓缩时石榴汁花色苷保存率几乎为100%, Hunter L*、Hunter a*值几乎不变化;而常压加热浓缩、微波加热浓缩的石榴汁花色苷保存率显著降低,Hunter L*、Hunter a*值均减小;微波加热浓缩对石榴汁花色苷具有显著的破坏作用。（5） HPD-100大孔树脂是分离纯化石榴汁花色苷较为适宜的树脂类型,其最佳吸附时间为3h,酸性乙醇最佳洗脱浓度为80%,可重复利用次数为4次,经纯化后花色苷的色价为16.96。采用HPLC-DAD-MS技术可以有效地对石榴汁花色苷进行分离鉴定;净皮甜品种石榴汁花色苷的主要组分为:飞燕草-3-葡萄糖苷、飞燕草-3,5-二葡萄糖苷、矢车菊-3-葡萄糖苷、矢车菊-3,5-二葡萄糖苷、天竺葵-3-葡萄糖苷和天竺葵-3,5-二葡萄糖苷6种花色苷,其中矢车菊-3,5-二葡萄糖苷和矢车菊-3-葡萄糖苷是含量最多的组分。（6）各品种石榴汁花色苷和总酚含量范围分别为2.1 mg/L～146.09mg/L和1.10g/L～1.70g/L;各品种石榴汁对DPPH·都有良好的清除效果;总酚含量与总还原力具有显著正相关性,与DPPH·清除率、O₂^-·清除率也具有很好的相关性;花色苷含量与·OH清除率相关性较好,而与其他3个抗氧化相关指标的相关性较小。在所选4个抗氧化指标中,除DPPH·清除率与总还原能力、O₂^-·清除率与·OH清除率具有显著相关性外,其余各指标之间的相关性都很小。石榴汁花色苷提取物具有良好的抗氧化能力,其总还原力及对·OH、O₂^-·、DPPH·的清除能力随着浓度增大而增大,具有明显的量效关系,对·OH、O₂^-·、DPPH·的IC₅₀分别为135mg/L、72.3 mg/L和8.56mg/L。更多还原

【Abstract】 The attractive red color of pomegranate juice is the important commercial character anddetermined by its anthocyanins.Anthocyanins which are unsteady and easy to degrade inproducing and storage are important functional components of pomegranate juice.It hasimportant realistic meaning and theory value to study the stability and degradation ofpomegranate anthocyanins.In this paper pomegranate planted in Lintong of shanxi province was took asexperimental material.Stability and degradation kinetics of pomegranate juice anthocyanins,the effect of many copigments on stability of anthocyanins and color of pomegranate juice,characteristics of polyphenoloxidase（PPO）and the role in anthocyanins degradation,enzymatic clarification of pomegranate juice and the effect of several production procedureson the stability of its anthocyanins,antioxidant activity of pomegranate juice andanthocyanins extract were studied.Anthocyanins of pomegranate juice were purified bymacroporous resin and Sephadex LH-20 and identified by HPLC-DAD-MS.The main resultswere as follows:（1）Anthocyanins in pomegranate juice is instable to heat,its Hunter a* value has adowntrend with increases of heating time and temperature,while the Hunter b* has anincreased trend.The stability of pomegranate juice anthocyanins is influenced deeply by pHvalue and light.It can induce degradation of anthocyanins when pH＞5 or presence of light.The degradation of anthocyanin in pomegranate is followed first-order reaction kinetics,andthe thermal degradation activation energy of anthocyanins in pomegranate juice is46.1602kJ/mol,and the rate constant （k₀）is 6.37×10⁶.The theoretical value and experimentalvalue are propinquity,which indicate that the model is appropriate.（2）EDTA,SDS and phytic acid have certain copigment effects and could increaseheating stability of anthocyanins of pomegranate juice,and phytic acid whice concentration is20mg/mL has best effct.Tween-80,amino acids and rutin have no copigment effect and donot affect the heating stability of pomegranate juice anthocyanins.Tannic acid has copigmenteffect but has no effect on stability of anthocyanins of pomegranate juice.Vc decrease thestability of pomegranate juice anthocyanins significantly when the concentration is over 1.06mg/L.Glucose and fructose increase the Hunter a* value of the pomegranate juice whilesucrose decrease the value.Sucrose and low concentration glucose have no effect on heatingstability of pomegranate juice anthocyanins.Glucose has protecting effect on anthocyaninswhen the concentration is 150mg/L.Fructose shows a disadvantage impact and the higher theconcentration is the more disadvantage effect is.（3）The PPO of pomegranate juice could katalyze monohydric phenol,dihydric phenoland polyhydric phenol.PPO has maximum activity when substrate is catechol.At thecondition of pH7&30℃PPO activity is biggest.PPO loses activity when temperature is over80℃.PPO of the pomegranate juice can directly catalyze degradation of pomegranate juiceanthocyanins and Phenolic compounds may also have certain catalysis in the process of PPOcatalytic degradation of anthocyanins.（4）Cleaning time of arils （seed sacs）has little effect on the pomegranate juiceanthocyanins content.Cold squeezing has advantage effect on content of anthocyanins whileheat squeezing has disadvantage effect.EDTA,Vc,L-cysteamine has certain effect of colorpreservation,L-cysteamine was the best.The optimum concentration was 0.025%.Theoptimum condition for clarification by a two-factor central composite design was as following:pectinase quantity was 0.98 ml/L,hydrolyze temperature is 40.5℃,Hydrolyze time is 127min,under the conditions,the light transmittance of the pomegranate juice was 95.31%,theretention rate of anthocyanins was 82.5%.It is observed that the retention rate ofanthocyanins was almost 100% and Hunter L*,Hunter a* has no change in rotary vacuumconcentrating process.The retention rate of anthocyanins,Hunter L* and Hunter a* reducedsignificantly in microwave and atmospheric heating concentrating process.There isremarkable degradation effect in microwave concentrating process.（5）HPD-100 is the suitable macroporous resin in purifying pomegranate juiceanthocyanins.The optimal adsorbing time is 3h and optimal concentration of acid ethanol is80%.The color value is 19.96 after purifying by HPD-100.HPLC-DAD-MS was used topurify and identify the pomegranate juice anthocyanins.The composition of pomegranatejuice anthocyanins are delphinidin-3-glycoside,delphinidin-3,5-di-glycoside,cyanidin-3-glycoside,cyanidin-3,5-di-glycoside,pelargonidin-3-glycoside,and pelargonidin-3,5-di-glycoside.Cyanidin-3-glycoside,cyanidin-3,5-di-glycoside are the main anthocyanins in thejuice.（6）The contents of anthocyanins and polyphenol in pomegranate juice ranged from2.1mg/L to 146.09mg/L and 1.10g/L to 1.70g/L respectively.All varieties of pomegranatejuice show good effect on scavenging capacity to DPPH·.There was significant positivecorrelation between total phenol content and reducing power.There was also good correlation between the total phenol and scavenging rate of DPPH·and O₂^-·.Anthoeyanins andscavenging rate of·OH shows good correlation but shows little correlation with other indices.Among the 4 antioxidation indices,besides scavenging rate of DPPH·and reducing power,scavenging rate of O₂^-·and scavenging rate of·OH show significant correlation both,there isno significant correlation each other.The extract of pomegranate juice anthocyanins showsgood antioxidant activity.Reducing power and scavenging rate of·OH,O₂^-·,DPPH·ofanthocyanins extract increase with the increase of the concentration,and there are significantdose-effect relationship between them.The IC₅₀of·OH,O₂^-·,DPPH·are 135mg/L,72.3mg/L,8.56mg/L.更多还原