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超高压和高温短时杀菌对NFC苹果汁贮藏期品质的影响

本站小编 Free考研考试/2021-12-26
邓红1,2,3, 雷佳蕾1, 杨天歌1, 刘旻昊1, 孟永宏,1,2,3, 郭玉蓉1,2,3, 薛佳,1,2,31 陕西师范大学食品工程与营养科学学院,西安 710119
2 国家苹果加工技术研发专业中心,西安 710119
3 西部果品资源高值利用教育部工程研究中心,西安 710119

Effect of Ultra-High Pressure and High Temperature Short-Time Sterilization on the Quality of NFC Apple Juice During Storage

DENG Hong1,2,3, LEI JiaLei1, YANG TianGe1, LIU MinHao1, MENG YongHong,1,2,3, GUO YuRong1,2,3, XUE Jia,1,2,3 1 College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi’an 710119;
2 National Research & Development Center of Apple Processing Technology, Xi’an 710119;
3 Engineering Research Center of High Value Utilization of Western China Fruit Resources, Ministry of Education, Xi’an 710119;

通讯作者: 孟永宏,Tel：029-85310517;E-mail： mengyonghong@snnu.edu.cn 薛佳,Tel：029-85310517;E-mail： cathyxue0727@snnu.edu.cn

收稿日期:2019-04-29接受日期:2019-09-3网络出版日期:2019-11-08
基金资助:国家重点研发计划.2017YFD0400700
农业部苹果产业体系项目.CARS-28


Received:2019-04-29Accepted:2019-09-3Online:2019-11-08
作者简介 About authors
邓红,Tel：029-85310517;E-mail： hongden@snnu.edu.cn。









摘要
目的 通过比较两种不同杀菌处理（高温短时High temperature short-time和超高压Ultra-high pressure杀菌）的非浓缩还原（Not from concentrate,NFC）苹果汁贮藏期品质变化,为我国NFC果汁产品标准的制定和规范市场NFC果汁产品货架期提供试验依据。方法 以‘红富士’苹果为原料制备NFC苹果汁,采用高温短时杀菌（HTST,98℃,50 s）和超高压杀菌（UHP,400 MPa,15 min）处理后,在4℃条件下冷藏,利用微生物学、化学及主成分分析（PCA）等方法以及HPLC、GC-MS等手段研究两种不同杀菌处理的NFC苹果汁贮藏期微生物、理化指标、多酚含量、酶（Polyphenol oxidase,PPO;Peroxidase,POD）活性、抗氧化活性、香气成分的变化。结果 HTST和UHP处理对细菌总数、大肠杆菌、霉菌与酵母的杀菌率均为100%,但贮藏第10、5周时果汁菌落总数分别呈显著增加（89.15%、58.65%）,大肠杆菌、霉菌与酵母增殖速度不明显;贮藏期HTST和UHP两种处理NFC苹果汁微生物种群变化小,但优势菌属不同。两种处理的NFC苹果汁贮藏期可溶性固形物、pH、总酸的变化不大,但总色差显著增大（P＜0.05）;贮藏期HTST和UHP处理NFC苹果汁中表儿茶素分别下降了33%和53%,FRAP总抗氧化能力和DPPH自由基清除率分别保持在76%、73%和77%、76%,HTST处理NFC苹果汁的多酚含量和抗氧化活性明显高于UHP处理的样品;HTST处理后贮藏期NFC苹果汁的PPO与POD完全失活,但UHP处理样品的酶活性呈现先增大后降低的趋势。HTST处理后果汁18种特征香味物质总的保留率为52%,但贮藏期香气成分保持稳定;UHP处理NFC苹果汁的香气含量（57.75 mg/100 mL）接近对照样品（57.17 mg/100 mL）,但贮藏期香气成分变化显著,降低了26.13%。结论 HTST和UHP杀菌处理的NFC苹果汁在4℃可分别贮藏9、4周,贮藏期内其品质满足商业要求,HTST处理NFC苹果汁货架期长于UHP处理。
关键词： NFC苹果汁;超高压处理;高温短时灭菌;品质;贮藏期

Abstract
【Objective】 In order to provide an experimental basis for regulating the shelf life of not from concentrate (NFC) juice products in Chinese market and formulating standard of NFC juice products in China, the quality changes of NFC apple juice treated by different sterilization methods (e.g., high temperature short-time (HTST) and Ultra-high pressure (UHP)) during storage periods were compared. 【Method】 NFC apple juice was prepared by using Fuji apple as raw material and treated by HTST at 98℃ for 50 s and UHP at 400 MPa for 15 min, respectively, before refrigerated at 4℃. Then the changes of microbial, physical and chemical indicators, polyphenols, enzymes (polyphenol oxidase and peroxidase) activity, antioxidant activity and aroma components were studied using various methods (e.g., microbial and chemical methods, principal component analysis (PCA), instrumental analysis methods of HPLC, and GC-MS). 【Result】 The sterilization rates of total bacteria, Escherichia coli, mold and yeast in NFC apple juice were 100% through HTST and UHP treatment, however, the total number of colonies increased significantly (89.15%, 58.65%) at the 10 th and 5 th week of storage, respectively. No obvious proliferation of Escherichia coli, mold and yeast was observed. The microbial communities of NFC apple juice treated by HTST and UHP during storage were of no significant changes, but the increases of dominant bacteria were different. The TSS, pH, TA, and the sensory quality (storage for 4 and 8 weeks) of NFC apple juices treated by HTST and UHP did not change significantly, but not the total color significantly (P＜0.05). During the storage period, the content of epicatechin in NFC apple juice treated by HTST and UHP decreased by 33% and 53%, respectively. The total antioxidant capacity of FRAP, scavenging rate of DPPH free radical were kept at 76%, 73%, and 77%, 76%, respectively. PPO and POD of NFC apple juice were completely inactivated after HTST treatment, but the enzymes activity (PPO and POD) in samples processed by UHP showed a trend of first increasing and then decreasing. The polyphenol content and antioxidant activity of HTST treated NFC apple juice were significantly higher than those of UHP treated samples. The total retention rate of 18 characteristic aroma substances in HTST treated NFC apple juice was 52%, but the aroma components remained stable during storage. In contrast, the aroma content of UHP treated NFC apple juice (57.75 mg/100 mL) was close to the control sample (57.17 mg/100 mL), but the aroma components significantly changed and reduced by 26.13% during storage. 【Conclusion】 NFC apple juice with HTST and UHP sterilization could maintain the commercial quality stored at 4℃ for 9 and 4 weeks, respectively, but the shelf life of HTST treated NFC apple juice was longer than that treated by UHP.
Keywords：NFC apple juice;ultra-high-pressure treatment;high-temperature short-time sterilization;quality;storage period


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本文引用格式
邓红, 雷佳蕾, 杨天歌, 刘旻昊, 孟永宏, 郭玉蓉, 薛佳. 超高压和高温短时杀菌对NFC苹果汁贮藏期品质的影响[J]. 中国农业科学, 2019, 52(21): 3903-3923 doi:10.3864/j.issn.0578-1752.2019.21.018
DENG Hong, LEI JiaLei, YANG TianGe, LIU MinHao, MENG YongHong, GUO YuRong, XUE Jia. Effect of Ultra-High Pressure and High Temperature Short-Time Sterilization on the Quality of NFC Apple Juice During Storage[J]. Scientia Agricultura Sinica, 2019, 52(21): 3903-3923 doi:10.3864/j.issn.0578-1752.2019.21.018


0 引言

【研究意义】中国的苹果优势明显,产量占世界各地苹果总产量的47.8%,其中陕西2017年苹果产量达到1.15×107 t,居全国第一[1,2],除鲜食外,苹果汁是其重要的加工产品。按照加工工艺的不同,苹果汁可分为非浓缩还原汁（Not from concentrate,简称NFC）和浓缩还原汁（From concentrate,简称FC）两大类。NFC果汁具有新鲜水果的营养和口感,总体品质均明显高于FC果汁,符合人们健康保健的消费需求,具有极大的开发前景[3,4]。但是目前由于关键的杀菌工艺和设备不完善使得NFC果汁普遍存在保质期相对较短、成本高等问题,限制了NFC果汁产业的发展[5]。因此,研究不同杀菌方式对NFC苹果汁贮藏品质的影响,对延长NFC果汁货架期、提高其市场价值有重要意义。【前人研究进展】很多对NFC果汁的研究主要集中在非热处理[6,7,8]（超高压（Ultra-high pressure,UHP）、脉冲电场（Pulsed electric field,PEF）、高压均质（High pressure homogenization,HPH）等）技术与工艺参数、NFC果汁品质分析[9]、NFC果汁鉴伪[10,11]等方面;如关云静[12]研究了高压均质对NFC芒果汁微生物和品质的影响,发现HPH可有效杀灭NFC芒果汁中自然菌群和接种的大肠杆菌,对理化性质（pH、TSS和TA）和维生素C含量没有显著影响,但果汁悬浮稳定性提高;苟小菊等[13]分析了16个苹果品种非浓缩还原汁（NFC）的理化特征,结果发现NFC苹果汁可分为3大类,同一大类中各品种之间不同理化指标差异较小,可作为NFC果汁生产和智能复配的可替代品种。目前在苹果NFC果汁贮藏期品质变化方面的研究不多见,CAO等[14]研究了高静水压加工混浊与澄清草莓汁贮藏过程中质量的变化,KRYSTIAN等[15]研究了超临界二氧化碳（SCCD,10—60 MPa/45℃/30 min）对苹果浊汁的影响以及浑浊苹果汁在4℃储存10周其酶活（多酚氧化酶（PPO）、过氧化物酶（POD））、酚类、维生素C、糖类、物理化学性质的变化,发现糖和总多酚没有显著变化,但施加的压力对多酚的降解有重要影响,且两种酶与绿原酸、儿茶酚存在协同作用。【本研究切入点】目前,我国NFC果汁产品缺少国家标准,市场已有产品由于生产工艺不同导致货架期差异很大,消费者难以判断品质优劣;同时也缺少关于不同杀菌方法对NFC苹果汁贮藏品质的影响以及货架期预测方面的研究。【拟解决的关键问题】利用化学、微生物学及主成分分析（PCA）等方法以及HPLC、GC-MS等手段研究两种不同杀菌处理的NFC苹果汁贮藏期微生物、理化指标、多酚、抗氧化活性、香气成分的变化,探明NFC果汁生产中两种杀菌方法对品质的影响,为我国NFC果汁产品标准的制定提供依据。

1 材料与方法

试验于2017年10月至2018年12月在陕西师范大学食品学院进行。

1.1 试验材料

原材料：红富士苹果于2017年9月中旬购于华润万家超市,并于4℃冷藏保存。试剂：没食子酸、绿原酸、咖啡酸、4-羟基苯甲酸、儿茶素、表儿茶素、芦丁、槲皮素、根皮苷,均为色谱纯,Sigma-Aldrich（中国）有限公司;食品级抗坏血酸、氯化钙、Trolox、TPTZ、DPPH、PVPP、乙酸、氯化钠、乙酸钠、邻苯二酚、过氧化氢、愈创木酚、聚乙二醇6000、硫酸铁、乙醇等其他化学品（分析纯）购自天津福晨有限公司;2-辛醇、甲醇均为色谱级,上海阿拉丁生化科技股份有限公司;平板计数琼脂、结晶紫中性红胆盐琼脂和孟加拉红培养基均为生化试剂,北京奥博星生物技术有限公司。

1.2 仪器与设备

HPP.L1-600MPa-10L超高压处理装置（天津华泰森宇生物工程技术有限公司）,HU-780WN榨汁机（韩国首尔Hurom电器公司）,Testo 106电子探针温度计（德国Testo有限公司）,PHS-3C pH计（上海仪电科学仪器股份有限公司）,MASTER-53α手持折光仪（日本ATAGO（爱拓）公司）,3k30超高速低温离心机（美国Sigma公司）,NS800色差仪（中国深圳3nh有限公司）,1260 Infinity安捷伦高效液相色谱仪（上海安捷伦科技有限公司）,C18液相色谱柱（150 mm×4.6 mm,5 μm,上海安捷伦科技有限公司）,DYCP-32B琼脂糖凝胶电泳仪（北京六一生物科技有限公司）,GC/MS QP2010气相色谱质谱联用仪（东京岛津电子有限公司）,AOC 6000自动进样器（东京岛津电子有限公司）,SPME萃取头（50/30 μm,DVB/CAR/PDMS,1 cm,美国Supelco公司）等。

1.3 试验方法

1.3.1 NFC苹果汁的制备与杀菌处理

（1）NFC苹果汁制备 选用新鲜的没有机械损伤且成熟度好的‘红富士’苹果,用流动水洗净果皮上的泥沙等杂质,削皮去核后切块,在护色液（0.5% VC+0.25% CaCl2）中浸泡30 s进行护色处理;后将苹果块投入HU-780WN榨汁机榨汁,用100目筛进行粗滤除杂,杀菌后灌装封口,于4℃冷藏备用[9,13]。

（2）HTST杀菌处理 参照文献[9,13]和预试验结果确定条件进行杀菌,NFC苹果汁用实验室规模的巴氏灭菌器在98℃下巴氏灭菌50 s,使用电子探针温度计在线监控果汁的中心温度,待果汁中心温度达到要求后计时。将杀菌的果汁在汁温不低于80℃条件下热灌装到灭菌过的玻璃瓶中并迅速封口,冷却至低于40℃后在4℃冰箱冷藏保存。

（3）UHP杀菌处理 参照文献[16]和预试验结果确定条件进行杀菌,10 L UHP设备用于塑料瓶装NFC苹果汁UHP杀菌处理,蒸馏水用作压力传递介质。加压速率约为120 MPa/min,减压时间小于3 s。最终压力400 MPa,处理时间15 min是压力的持续时间,不包括压力增加和释放时间。样品的初始温度为10℃,整个处理过程中样品温度不超过25℃。

1.3.2 NFC苹果汁品质分析

（1）NFC苹果汁理化指标的检测 NFC苹果汁pH的测定、可溶性固形物（total soluble solid,TSS）的测定、可滴定酸度TA的检测均与文献[17]方法相同。

（2）NFC苹果汁色泽和酶活性的检测 使用NS800比色计测量NFC苹果汁的色泽,其颜色表示为L*（明度指数）,a*（红值,当其为正值时,数值越大,颜色越红）和b*（黄值,正值时数值越大,颜色越黄,b*为负值时绝对值越大,颜色越蓝）[17]。酶活性（多酚氧化酶（PPO）和过氧化物酶（POD））的测定方法与文献[18]方法相同。

（3）NFC苹果汁抗氧化活性与多酚组分的检测 参照文献[19]的方法检测NFC苹果汁DPPH自由基清除率和FRAP总抗氧化活性。以绿原酸、表儿茶素、儿茶素、没食子酸、4-羟基苯甲酸、咖啡酸、芦丁、槲皮素和根皮苷为标准品,参照邓红等[20]的方法对多酚组分进行测定。

（4）NFC苹果汁的香气成分的检测 采用顶空固相微萃取法（HS-SPME）富集提取苹果汁中香气成分,参考郭静等[21]的方法,使用气相色谱-质谱联用仪（GC/MS）检测贮藏期NFC苹果汁的香气成分。GC/MS QP2010气相色谱-质谱联用仪与AOC 6000自动进样器组合应用于苹果汁香气成分的快速检测和分析。

香气成分的定性分析主要是根据气相色谱-质谱中NIST 14质谱数据库、匹配度和保留时间对各个物质进行检索,选择匹配度大于85%的物质作为有效的香气成分。香气成分的定量分析方法是每次检测均加入相同质量浓度的2-辛醇作为内标物进行定量,按式（1）计算苹果汁中香气物质的含量,取3次检测结果的平均值为最终分析结果。

香气物质含量=（各物质的峰面积/内标物的峰面积）×内标物质量浓度 （1）

（5）NFC苹果汁的微生物指标与细菌多样性的检测 菌落总数的测定、霉菌酵母的测定、大肠菌群的测定等参照国标及文献[22]进行,重复3次,取平均值。细菌多样性的检测方法[23,24]如下：

①基因组DNA的提取和PCR扩增 样本的基因组DNA进行提取采用CTAB（Hexadecyl trimethyl ammonium bromide,十六烷基三甲基溴化铵）方法,之后检测DNA的纯度和浓度利用琼脂糖凝胶电泳。不同区域的16S rRNA 均为扩增特异性引物。聚合酶链式反应（polymerase chain reaction,PCR）反应体系：15 μL Phusion? High-Fidelity PCR Master Mix;0.2 μmol?L-1正向和反向引物,以及约10 ng模板DNA。PCR的扩增程序为：98℃ 1 min;98℃ 10 s,50℃退火30 s,72℃延伸30 s,30个循环。最后72℃保持5 min。

②PCR产物的混样、纯化以及文库构建和测序 使用2%浓度的琼脂糖凝胶对PCR产物进行电泳检测;根据PCR产物浓度进行等量混样,充分混匀后使用1×TAE浓度2%的琼脂糖胶电泳纯化PCR产物,剪切回收目标条带。使用Thermofisher 公司的Ion Plus Fragment Library Kit 48 rxns建库试剂盒进行文库的构建,构建好的文库经过Qubit定量和文库检测合格后,基于Thermofisher的Ion S5TMXL上机测序,进行单端测序。

对有效数据进行操作分类单元（operational taxonomic units,OTU）聚类和物种分类分析。一方面,为了得到样本内物种丰富度和均匀度信息,不同样本或分组间的共有和特有OTUs信息,对OTUs进行丰度、Alpha多样性计算、Venn图和花瓣图等分析。另一方面,可以对OTUs进行多序列比对并构建系统发生树,通过主坐标分析（principal co-ordinates analysis,PCoA）、无度量多维标定法（non-metric multi-dimensional scaling,NMDS）等降维分析和样本聚类树展示,可以探究不同样本或组别间群落结构的差异。

1.4 数据统计分析

所有试验均重复三次取平行样进行分析。使用Origin 8.0和SPSS 12.0软件进行数据分析,结果表示为平均值±标准差（SD）,P＜0.05被认为是影响显著。主成分分析（principal component analysis,PCA）被用作评估每个数据集以检测潜在异常值的探索性技术。

2 结果

2.1 HTST和UHP两种处理的NFC苹果汁贮藏期pH、可溶性固形物和可滴定酸度的变化

由表1可知,两种方法杀菌后NFC苹果汁的pH、TA、TSS与对照基本无差异,贮藏期HTST和UHP处理后的NFC苹果汁中的可溶性固形物没有显著变化,pH下降幅度稍大（P＜0.05）,可滴定酸度增大不明显。

Table 1
表1
表1超高压与高温短时杀菌处理的NFC苹果汁4℃贮藏期pH、可滴定酸度、可溶性固形物的变化
Table 1Changes on pH, TA, TSS of NFC apple juice after HTST and UHP treatment during storage period at 4℃
不同处理
Different treatment		贮藏时间（周） Storage time (week)
12345710
pHControl4.01±0.3a
UHP4.01±0.2a3.98±0.2abcd3.99±0.1abc4.00±0.1ab3.95±0.2d——
HTST4.01±0.2a3.99±0.1abc3.98±0.1abcd3.99±0.1abc3.97±0.1bcd3.95±0.1d3.96±0.2cd
可滴定酸度
TA		Control0.342±0.01abc
UHP0.339±0.01bc0.338±0.01c0.340±0.01abc0.339±0.02bc0.342±0.02abc——
HTST0.341±0.02abc0.340±0.01abc0.341±0.01abc0.342±0.01abc0.343±0.01ab0.342±0.01abc0.344±0.01a
可溶性固形物TSSControl11.9±0.2a
UHP12.0±0.1a11.9±0.1a12.0±0.1a11.8±0.1a11.9±0.1a——
HTST11.9±0.1a11.9±0.1a11.8±0.2a12.0±0.1a11.9±0.1a11.9±0.1a12.0±0.1a
UHP,超高压杀菌处理;HTST,高温短时杀菌处理。“—”表示未检测。Control为第1周第1天制备的NFC苹果汁,未杀菌样品的分析结果;其余均为NFC苹果汁杀菌后贮藏期每周第1天的分析结果。同列不同小写字母表示差异显著（P＜0.05）。下同
UHP indicate ultra-high pressure, and HTST indicate high temperature short-time; “—”indicate no detection. Control is the analysis results of NFC apple juice prepared on the first day of the first week. The other data are analysis results on the first day of every week after the sterilization of NFC apple juice during the storage period. Different letters of the same column data indicate significant differences (P＜0.05). The same as below

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2.2 两种处理的NFC苹果汁贮藏期色泽和酶活性的变化

2.2.1 两种处理的NFC苹果汁贮藏期色泽的变化 NFC苹果汁在4℃贮藏期间的颜色参数（L*、a*、b*值）如表2所示。与对照相比,HTST处理的NFC苹果汁的L*值（亮度）略有下降,UHP处理后苹果汁的L*值接近未杀菌处理的果汁,但贮藏期HTST处理的NFC苹果汁L*值（亮度）下降幅度小于UHP处理;两种处理的NFC苹果汁的a*值（红值）和b*值（黄值）没有显着差异,但与对照相比,两种处理之后a*值和b*值有明显降低（P＜0.05）;然而贮藏期a*值和b*值的变化不大,两种处理的NFC苹果汁颜色发生小幅变化,但仍在可接受范围内。

Table 2
表2
表2超高压与高温短时杀菌处理的NFC苹果汁4℃贮藏期色值的变化
Table 2Changes on color of NFC apple juice after HTST or UHP treatment during storage period at 4℃
不同处理
Different treatment		贮藏时间（周）Storage time (week)
12345710
L*Control33.26±0.05ab
UHP33.48±0.04a32.66±0.03ab30.3±0.09c30.77±0.06c29.89±0.03c——
HTST31.69±0.04bc31.55±0.05bc31.70±0.15bc30.35±0.14c30.22±0.12c30.19±0.06c30.01±0.11c
a*Control-1.77±0.05a
UHP-2.49±0.02ef-2.53±0.01f-2.44±0.04ef-2.31±0.04cde-2.17±0.01bc——
HTST-2.37±0.04cdef-2.35±0.06cdef-2.40±0.08def-2.32±0.08bcde-2.28±0.01bcde-2.19±0.03bcd-2.08±0.12b
b*Control3.24±0.07a
UHP2.59±0.02b2.65±0.02b2.60±0.12b2.55±0.02b2.50±0.04b——
HTST2.59±0.11b2.50±0.06b2.61±0.14b2.54±0.09b2.51±0.04b2.48±0.08b2.44±0.11b

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2.2.2 贮藏期两种处理的NFC苹果汁酶活性的变化 HTST处理可以完全杀灭NFC苹果汁中PPO和POD酶的活性,且整个贮藏期均未检测出来;但UHP处理后果汁中多酚氧化酶和过氧化氢酶的活性均升高（高于对照Control）;随着贮藏时间的增加,UHP处理后NFC苹果汁的酶活性略有下降,在贮藏期的第5周,PPO和POD活性与对照相比分别下降了17.4%和14.3%（表3）。

Table 3
表3
表3超高压与高温短时杀菌处理的NFC苹果汁4℃贮藏期酶活（多酚氧化酶和过氧化氢酶）的变化
Table 3Changes on PPO and POD of NFC apple juice after HTST or UHP treatment during storage period at 4℃
酶活
Enzymes activity		不同处理Different treatment贮藏时间（周） Storage time (week)
12345710
PPO
(△OD420?min-1·g-1)		Control0.023±0.0021bc
UHP0.047±0.0017a0.029±0.001b0.022±0.0015bc0.026±0.0021bc0.019±0.0012c——
HTSTNDNDNDNDNDNDND
POD
(△OD470?min-1·g-1)		Control0.14±0.0136cd
UHP0.19±0.0175a0.15±0.0092bc0.14±0.011cd0.17±0.0121ab0.12±0.0075d——
HTSTNDNDNDNDNDNDND
ND表示未检测出PPO与POD酶活 ND indicated that enzyme activity of PPO and POD was not detected

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2.3 两种处理的NFC苹果汁贮藏期多酚组分和抗氧化活性的变化

2.3.1 两种处理的NFC苹果汁贮藏期多酚组分的变化 在本研究中,从NFC苹果汁中成功分离出了9种酚类物质,含量最高的两个单酚被确定为绿原酸和表儿茶素,两种处理的NFC苹果汁在贮藏期间的HPLC色谱图如图1所示。通过HPLC检测,对照样品、HTST和UHP处理的苹果汁的总多酚含量分别为224.80、173.80和127.68 μg?mL-1;在HTST和UHP处理后,与对照样品相比,NFC苹果汁中绿原酸和4-羟基苯甲酸的含量略有增加,儿茶素、表儿茶素、芦丁和根皮苷的含量显著下降;UHP处理后总酚类物质显著减少,一些单酚类物质如表儿茶素、儿茶素、芦丁和根皮苷在超高压处理后显著降低,可能是因为多酚氧化酶和过氧化物酶在超高压处理后的NFC苹果汁中具有较高的残留活性,多酚氧化酶和过氧化氢酶等内源酶暴露于其基质中,这两者都可以氧化并诱导单酚物质的降解并形成聚合物。HTST处理后,NFC苹果汁中绿原酸和表儿茶素的含量高于超高压处理,可能是酚类物质的提取性能因热处理过程中植物细胞壁的破坏而发生变化,多酚物质被释放出来,导致其含量较高[25]。HTST和UHP处理的果汁中绿原酸分别从113.96、98.99 μg?mL-1降解至97.93和97.93 μg?mL-1;表儿茶素分别下降了33%和53%;4-羟基苯甲酸的降解率分别为0.05%和0.34%;槲皮素下降0.05%和0.50%;没食子酸减少0.29%和0.34%。在贮藏期间,NFC苹果汁中大多数多酚物质的降解主要由非氧化反应引起,槲皮素的降解可能是由于槲皮素糖苷的不稳定性引起。

图1

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图1在4℃下贮藏期超高压与高温短时灭菌处理的苹果汁中9种单体酚含量的HPLC色谱图

A：超高压处理的NFC苹果汁第一周的HPLC色谱图;B：高温短时杀菌处理的NFC苹果汁第一周HPLC色谱图;C：贮藏期高温短时杀菌处理的NFC苹果汁的HPLC色谱图;D：贮藏期超高压处理的NFC苹果汁的HPLC色谱图。图A、B中色谱峰上数字1-9分别表示没食子酸、4-羟基苯甲酸、绿原酸、咖啡酸、儿茶素、表儿茶素、芦丁、槲皮素、根皮苷
Fig. 1HPLC chromatogram of 9 monomeric phenolic contents in NFC apple juice treated with ultra-high pressure and high temperature short-time sterilization during storage at 4℃

A: HPLC chromatogram of NFC apple juice by high temperature short-time sterilized at the first week; B: HPLC chromatogram of NFC apple juice by ultra-high pressure treated at the first week; C: HPLC chromatogram of NFC apple juice by high temperature short-time sterilization during storage; D: HPLC chromatogram of NFC apple juice by ultra-high pressure treated during storage. Number 1-9 of spectrum peak on the figure A and B indicated gallic acid, 4-hydroxybenzoic acid, chlorogenic acid, caffeic acid, catechin, epicatechin, rutin, quercetin, phloridin


2.3.2 两种处理的NFC苹果汁贮藏期抗氧化活性的变化 由图2可知,UHP与HTST处理的NFC苹果汁的DPPH自由基清除率和FRAP总抗氧化能力随着贮存期的延长而降低;贮藏期结束时,FRAP总抗氧化能力分别保持在73%和76%,DPPH自由基清除率分别保持在76%和77%。

图2

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图2超高压与高温短时杀菌处理的NFC苹果汁4℃贮藏期抗氧化活性（DPPH自由基清除率和FRAP总抗氧化能力）变化

Fig. 2Changes of DPPH radical scavenging rate and FRAP total antioxidant capacity in NFC apple juices during storage time at 4℃ after UHP and HTST treatments



2.4 两种处理的NFC苹果汁贮藏期香气成分的变化

通过SPME/GC-MS对UHP和HTST处理的NFC苹果汁贮藏期香气成分的变化进行分析,从贮藏期NFC苹果汁的挥发性成分的总离子流色谱图（省略）中定量检测到HTST、UHP处理的NFC苹果汁中分别有54和44种芳香族化合物（表4）。其中,酯类物质分别为36、24种,醇类物质分别为6、7种,醛类物质分别为7、6种,酮类物质分别为2、2种,萜烯类物质分别为1、4种,醚类物质均为1种,其中酯类物质占苹果汁中总挥发性化合物的很大比例。通过UHP处理的苹果汁中的芳烃化合物含量为酯（31.61 mg/100mL）>醇（14.81 mg/100 mL）>醛（14.48 mg/100 mL）>萜烯类（1.73 mg/100 mL）>酮（0.11 mg/100 mL）>乙醚（0.066 mg/100 mL）;HTST处理的苹果汁中的芳香化合物依次为酯（10.38 mg/100 mL）>醛（9.36 mg/100 mL）>醇（2.85 mg/100 mL）>萜烯（1.50 mg/100 mL）>酮（0.10 mg/100 mL）>乙醚（0.04 mg/100 mL）。

为了评估这些化合物在4周储存期间对HTST与UHP处理的苹果汁质量的影响,对18种关键化合物进行了主成分分析（PCA）。PC1和PC2的贡献率分别为64.4%和21.1%,累积贡献率大于70%,如分数图（图3-A）和变量负荷图（图3-B）所示。由PC1和PC2定义的样本越集中（图3-C）,相似性越高。未灭菌处理的果汁、UHP与HTST处理的果汁之间存在轻微分离。在4周的储存期间,通过HTST处理的NFC苹果汁,相对于UHP处理的NFC苹果汁,在储存期间香气保持更稳定。图3-B显示对PC1最重要的贡献是己醛、（E）-2-己烯醛、乙酸丁酯、乙酸丙酯和丁酸丁酯,PC2的主要贡献者是乙酸己酯。结合图3-A和3-B,可以看出HTST处理的NFC苹果汁与PC1呈负相关,因此,己醛、（E）-2-己烯醛、乙酸丁酯,乙酸丙酯和丁酸丁酯含量均较低。未杀菌处理和UHP处理的NFC苹果汁与PC1呈正相关,表明己醛、（E）-2-己烯醛、乙酸丁酯、乙酸丙酯和丁酸丁酯含量均较高。未杀菌处理的NFC苹果汁在PC1上得分最高,而UHP加工苹果汁在第4周得分最低。结果表明,在4周贮藏期间,HTST处理的果汁中乙酸己酯含量没有显著变化,而UHP处理的果汁中乙酸己酯含量显著下降。同时,热图可以更直观地显示18种关键香味物质浓度水平,图3-C显示了在4周储存期间未杀菌处理、HTST和UHP处理的NFC苹果汁中18种主要香气成分的含量变化。根据含量/颜色规则,可知HTST果汁的香气成分的变化相对较小,并且在果汁中未检测到乙醛。由于乙醛含量低,香气值低,对香气几乎没有影响。在UHP处理的果汁中,1-己醇在第2周显著降低,之后变化稳定,可能是由于酶促褐变过程产生的化合物与气味活性化合物之间的相互作用,从而影响了挥发性释放,导致气味减弱。总之,贮存期间,乙醛和乙酸己酯含量降低,丁酸2-甲基己酯含量增加,NFC苹果汁的绿色气味减少,甜味物质增加。

图3

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图3超高压与高温瞬时灭菌处理的NFC苹果汁4℃贮藏期香气成分的变化

Fig. 3Changes in aroma components of NFC apple juice treated by ultra-high pressure and high temperature at 4℃ during storage

A：主成分分析PCA评分图;B：PCA负荷变量图;C：两个处理的苹果汁在贮藏期间18种主要香气成分变化的热图。Control：未处理的NFC苹果汁;HHP1、2、3、4W：其超高压处理的NFC苹果汁贮藏1、2、3、4周;HTST1、2、3、4W：超高温瞬时杀菌处理的NFC苹果汁贮藏1、2、3、4周A: Principal component analysis PCA score map; B: PCA load variable map; C: Heat map of 18 major aroma components during storage of two treated apple juices. CK: Untreated NFC apple juice; HHP1, 2, 3, 4W: NFC apple juice treated with ultra-high pressure during storage 1, 2, 3 and 4 weeks; HTST1, 2, 3, 4W: NFC apple juice treated with ultra-high temperature instantaneous sterilization 1, 2, 3 and 4 weeks. (E)-2-己烯醛,Hexenal：己烯醛,Acetaldehyde：乙醛,1-hexanol：1-己醇,2-methyl-1-butanol：2-甲基-1-丁醇,1-butanol：1-丁醇,2-methyl butyrate：2-甲基-丁酯,Hexyl butyrate：丁酸己酯,Butyl 2-methyl butyrate：2-甲基丁酸丁酯,Hexyl acetate：乙酸己酯,Ethyl hexanoate：己酸乙酯,Butyl butyrate：丁酸丁酯,Butyl acetate：乙酸丁酯,Amyl acetate：乙酸戊酯,Isoamyl formate：甲酸异戊酯,Ethyl 2-methyl butyrate：2-甲基丁酸乙酯,Ethyl butyrate：丁酸乙酯,Propyl acetate：乙酸丙酯


Table 4
表4
表4UHP与HTST杀菌的NFC苹果汁贮藏期香气成分的变化
Table 4Changes of aroma content in NFC apple juice after HTST or UHP treatment during storage period at 4℃
不同处理
Different treatment		不同贮藏时间（周）的质量浓度（mg/100 mL）
Mass concentration of aroma at different storage time (week)
1234
酯类Ester
乙酸乙酯
Ethyl acetate		Control0.36
UHP0.080.080.200.09
HTST00.050.040.04
乙酸丙酯
Propyl acetate		Control0.28
UHP0.450.430.310.37
HTST0.130.140.160.14
丁酸乙酯
Ethyl butyrate		Control0.28
UHP0.620.650.630.69
HTST0.100.160.20.21
丙酸丙酯
Propyl propionate		Control0.13
UHP0.170.170.180.20
HTST0000.08
乙酸丁酯
Butyl acetate		Control3.68
HHP4.224.113.564.00
TP2.593.382.502.66
2-甲基丁酸乙酯
Ethyl 2-methylbutyrate		Control0.15
UHP0.280.300.300.33
HTST0.030.050.030.04
甲酸异戊酯
Isoamyl formate		Control7.42
UHP8.437.716.496.37
HTST4.736.684.624.67
丁酸丙酯
Propyl butyrate		Control0.83
UHP1.441.511.431.74
HTST0.470.740.500.50
丙酸丁酯
Butyl propionate		Control0.43
UHP0.350.350.330.35
HTST0.160.240.160.17
乙酸戊酯
Amyl acetate		Control0.56
UHP0.890.720.600.48
HTST0.390.610.390.39
己酸甲酯
Methyl caproate		Control0.04
UHP0.040.030.020
HTST00.010.010.01
2-甲基丁酸丙酯
Propyl 2-methylbutyrate		Control0.62
UHP0.790.874.590.92
HTST0.120.190.130.13
不同处理
Different treatment		不同贮藏时间（周）的质量浓度（mg/100 mL）
Mass concentration of aroma at different storage time (week)
1234
2-戊醇丙酸酯
Pentylester; sec-amyl propionate		Control0.16
UHP0.150.1400
HTST0000
丁酸丁酯
Butyl butyrate		Control1.14
UHP1.471.541.421.53
HTST0.510.730.490.49
己酸乙酯
Ethyl hexanoate		Control0.49
UHP0.510.390.450.27
HTST00.140.170.15
2-甲基丁酸丁酯
Butyl 2-methylbutyrate		Control0.87
UHP1.040.991.011.07
HTST0.100.180.110.10
苯甲酸甲酯
Methyl benzoate		Control0.26
UHP0.04000
HTST0000
己酸丙酯
Caproic acid propyl
ester grade I		Control0.19
UHP0.510.410.360.33
HTST0.080.110.070.06
丙酸己酯
Hexyl propionate		Control0.12
UHP0.160.080.060
HTST0.040.060.030
2-甲基丁酸-2-甲基丁酯
2-Methylbutyl -2-methylbutyrate		Control0.01
UHP0.140.130.130.16
HTST0.010.010.20
2-甲酸丁酸戊酯
2-Methanoic acid
amyl butyrate		Control0.04
UHP0.060.050.050.04
HTST0000
2-甲基丙酸己酯
Hexyl 2-methylpropionate		Control0.06
UHP0.050.260.050
HTST00.3400
丁酸己酯
Hexyl butyrate		Control0.94
UHP1.040.530.230
HTST0.310.370.210.18
2-甲基丁酸己酯
Hexyl 2-methyl butyrate		Control1.61
UHP1.691.331.330
HTST0.280.3400.19
葵酸甲酯
Methyl decanoate		Control0.11
UHP0.04000
HTST0000
不同处理
Different treatment		不同贮藏时间（周）的质量浓度（mg/100 mL）
Mass concentration of aroma at different storage time (week)
1234
己酸己酯
Hexyl hexanoate		Control0.15
UHP0.140.060.070.04
HTST0.090.090.070.07
十二烷酸甲酯
Methyl laurate		Control0.28
UHP00.0400
HTST0000
癸酸癸酯
n-Capricacidn-decylester		Control1.31
UHP0000
HTST0000
肉豆蔻酸甲酯
Methyl myristate		Control0.68
UHP0.050.020.030
HTST00.0800
二十烷酸甲酯
Methyl eicosanoate		Control0.28
UHP0000
HTST0000
（Z）-9-十六碳烯酸甲酯
Methyl-(z)-9-hexadecenoate		Control0.79
UHP0000
HTST0.130.080.020
（Z）-9-十八碳烯酸甲酯
Methyl-(z):9-Octadecenoic acid,		Control0
UHP0.250.120.030
HTST0000
（Z）-十六烷-11-烯酸乙酯
(Z)-Hexadecane-11-ethyl oleate		Control1.26
UHP0.650.280.050.04
HTST0.320.140.030.03
十六烷酸甲酯
Methyl-hexadecanoate		Control0.24
UHP0.080.0400
HTST0.040.0300
（Z,Z）-9-十六碳烯酸-9-十六烯己酯
(Z,Z)-9-Hexadecanoic acid-9-hexadecanohexyl este		Control0
UHP0.06000
HTST0000
戊酸乙酯
Ethyl valerate		Control0
UHP0..030.0200
HTST0000
2-甲基丙酸丁酯
2-Methyl butyl propionate		Control0
UHP0.020.0200
HTST0000
乙酸庚酯
Hepty lacetat		Control0
UHP0.02000
HTST0000
不同处理
Different treatment		不同贮藏时间（周）的质量浓度（mg/100 mL）
Mass concentration of aroma at different storage time (week)
1234
二十二酸壬酯
Nonyl Do decanoate		Control0
UHP0000
HTST0.02000
3-甲基-1-丁醇丙酸酯
3-Methyl-1-butanol propionate		Control0
UHP0000.16
HTST0.0500.060
2,6,10,14-四甲基-十五烷酸甲酯
2,6,10,14-Tetramethyl-pentadecanoic acid methyl ester		Control0
UHP0000
HTST0.12000
丁酸甲酯
Methyl butyrate		Control0
UHP00.0300.03
HTST00.010.010.01
2-甲基丁酸甲酯
Methyl 2-methyl butyrate		Control0
UHP00.030.030.03
HTST0000
正丁酯
n-Butyl ester		Control0
UHP00.0500.05
HTST0000
醇类Alcohols
乙醇
Ethanol		Control0.06
UHP0.020.020.040.02
HTST0.02000
1-丁醇
1-Butanol		Control0.44
UHP0.690.710.710.89
HTST0.570.560.640.63
2-甲基-1-丁醇
2-Methyl-1-butanol		Control0.66
UHP0.910.990.961.23
HTST0.710.750.740.74
（E）-2-己烯-1-醇
(E) -2-hexene-1-ol		Control0.28
UHP000.221.18
HTST000.080
1-己醇
1-Hexanol		Control3.94
UHP13.083.994.884.75
HTST1.471.681.761.44
2-乙基-1-己醇
2-Ethyl-1-hexanol		Control0.28
UHP000.100
HTST0000
（S）-2-戊醇
(S) -2-Pentanol		Control0
UHP0.030.040.040.05
HTST00.020.020.02
不同处理
Different treatment		不同贮藏时间（周）的质量浓度（mg/100 mL）
Mass concentration of aroma at different storage time (week)
1234
（S）-2-庚醇
(S) -2-Heptanol		Control0
UHP0.070.070.060.10
HTST0.040.060.040.04
2-甲基-1,3-戊二醇
2-Methyl-1,3-pentanediol		Control0
UHP000.040
HTST0.020.0200.02
1-戊醇
1-Pentanol		Control0
UHP00.030.120.05
HTST0.030.030.050.04
1-丙醇
1-Propanol		Control0
UHP00.0400
HTST0000
1-辛醇
1-Octanol		Control0
UHP000.020
HTST0000
2-甲基-1-丙醇
2-Methyl-1-propanol		Control0
UHP0000.03
HTST0000
二环丙基甲醇
Dicyclopropyl methanol		Control0
UHP0000.05
HTST0000
醛类Aldehyde
乙醛
Acetaldehyde		Control0.05
UHP00.020.020.02
HTST0000
戊醛
Pentanal		Control0.08
UHP000.040
HTST0.020.020.020.02
己醛
Hexanal		Control6.71
UHP9.328.005.577.04
HTST3.363.981.532.97
（E）-2-己烯醛
(E) -2-Hexenal		Control3.52
UHP4.794.622.383.59
HTST1.051.500.210.69
（E）-2-辛烯醛
(E) -2-Octene aldehyde		Control0.17
UHP0.040.050.070.05
HTST0000
壬醛
Nonyl aldehyde		Control0.40
UHP0.060.030.150.03
HTST0.0900.060.06
不同处理
Different treatment		不同贮藏时间（周）的质量浓度（mg/100 mL）
Mass concentration of aroma at different storage time (week)
1234
癸醛
Decyl aldehyde		Control1.31
UHP0.220.080.110
HTST0.090.060.020.02
丁醛
Butyraldehyde		Control0
UHP0.020.020.020.02
HTST0.010.0100.01
庚醛
Heptanal		Control0
UHP0.0200.050
HTST00.020.030.04
酮类Ketones
丙酮
Acetone		Control0.06
UHP00.020.060
HTST0000
2-辛酮
2-Octanone		Control0.07
UHP0.060.060.090.09
HTST0.060.060.060.06
6-甲基-5-庚烯-2-酮
6-Methyl-5-heptene-2-one		Control0.11
UHP0.050.060.080.13
HTST0.040.050.030.04
顺式-5-甲基-2-（1-甲基乙基）-
环己酮
Cis-5-methyl-2-(1-methylethyl)-
cyclohexanone		Control0.07
UHP0000
HTST0000
（Z）-6,10-二甲基-5,
9-十一碳二烯-2-酮
（Z）-6,10-Dimethyl-5,9-
undecadien-2-one		Control0.05
UHP000.020
HTST0000
2-庚酮
2-Heptanone		Control0.05
UHP000.030
HTST0000
2-（1,5-二甲基-己基）-环丁酮
2-hexyl-1,5-dimethyl-
Cyclobutanone		Control0.05
UHP000.020
HTST0000
萜烯类Terpene
D-柠檬烯
d-Limonene		Control0.58
UHP000.110.07
HTST000.060
长叶烯
d-Longifolene		Control0.08
UHP0000
HTST0000
不同处理
Different treatment		不同贮藏时间（周）的质量浓度（mg/100 mL）
Mass concentration of aroma at different storage time (week)
1234
反式-α-香柠檬烯
Trans-alpha-citrine		Control0.05
UHP00.040.040
HTST0.040.040.030
α-法尼烯
αFarnesene		Control1.70
UHP1.731.421.341.10
HTST1.411.651.271.46
2,5,5-三甲基-1,6-庚二烯2,5,5-Trimethyl-1,6-heptadienControl0
UHP0000
HTST0.040.050.040.04
酸类Acid
正癸酸
n-Decanoic acid		Control0.07
UHP0000
HTST0000
醚类Ethers
草蒿脑
Estragole		Control0.11
UHP0.070.060.050.05
HTST0.020.030.010.01
茴香脑
Anethole		Control0.24
UHP000.030
HTST0000
反式芳樟醇氧化物（呋喃型）
Trans linalool oxide (furan type)		Control0
UHP0000.03
HTST0.02000

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2.5 两种处理的NFC苹果汁贮藏期主要微生物指标的变化与多样性分析

2.5.1 主要微生物指标的变化 在本研究中,HTST与UHP处理可使NFC苹果汁中的菌落总数、霉菌酵母和大肠菌群完全失活。在4℃的储存期间,大肠菌群的水平一直是低于检测限,第5周超高压处理的NFC苹果汁的菌落总数数值为2 log CFU/mL（表5）。这表明在4周储存期间由超高压处理得到的NFC苹果汁是微生物安全的,而在9周储存期间通过高温短时灭菌处理的NFC苹果汁也是微生物安全的。目前已经证明超高压与高温短时灭菌处理在减少食物中微生物数量方面的有效性,但是物理处理（热、辐射等）和化学处理后的孢子很可能具有非常高的抗性休眠形式,并且它们的耐压性随着营养物质丰富而增加,因此一些微生物将通过自我修复方式来恢复生长[26],所以第5周和第10周NFC果汁菌落总数增殖明显,分别达89.15%、58.65%。

2.5.2 细菌多样性的分析 由图4可知,检测到未杀菌处理NFC苹果汁的主要的分类单位（operational taxonomic units,OTU）是变形杆菌门（Proteobacteria,75.71%）、生氧光细菌门（Oxyphotobacteria,19.21%）、厚壁菌门（Firmicutes,0.49%）和其他类（4.24%）;检测到超高压NFC苹果汁的主要OTU是变形杆菌门（Proteobacteria,75.03%）、生氧光细菌门（Oxyphotobacteria,19.90%）、厚壁菌门（Firmicutes,0.21%）和其他类（4.60%）;UHP处理NFC苹果汁在贮藏期微生物种群的主要变化是第4周变形杆菌相对丰度变化剧烈,显著增加,在贮藏期微生物种群基本没有大的变化,其OTU保持相对稳定。这一结果与PULIDO等[27]研究结果类似,他们对细菌群落的焦磷酸测序分析显示,在贮藏期间,UHP处理的芒果果浆的放线菌门（45.63%）、厚壁菌门（42.55%）、变形菌门（10.68%）和拟杆菌门（1.0%）的起始对照样品中的相对丰度发生了变化,UHP处理还诱导变形菌的强烈增加,然后是厚壁菌的晚期恢复和较小程度的放线菌的变化;他们还发现乳酸杆菌是对照样品在早期储存期间和在晚期储存期间在UHP处理的样品中检测到的主要OTU。试验分析结果显示HTST处理NFC苹果汁的主要OTU是变形杆菌门（Proteobacteria,64.63%）、厚壁菌门（Firmicutes,20.91%）、生氧光细菌门（Oxyphotobacteria,10.46%）、拟杆菌门（Bacteroidetes,1.60%）。

图4

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图4基于16S rRNA基因焦磷酸测序分析的DNA排名前10的物种OTU相对丰度

Control：未处理的NFC苹果汁;HHP1、2、3、4、5：贮藏期超高压处理的NFC苹果汁1、2、3、4、5周;HTST1、3、5、7、10：超高温瞬时杀菌处理的NFC苹果汁1、3、5、7、10周。A-E依次是OTU按门、纲、目、科、属分类水平分类 CK: Untreated NFC apple juice; HHP1, 2, 3, 4, 5: NFC apple juice treated with ultra-high pressure during storage 1, 2, 3, 4, and 5 weeks; HTST1, 3, 5, 7, 10: NFC apple juice treated with ultra-high temperature instantaneous sterilization 1, 3, 5, 7, and 10 weeks. A-E: OTU is classified according to the door, outline, purpose, and department, respectively
Fig. 4The relative abundance of the top 10 species of OTU based on the 16S rRNA gene pyrosequencing analysis of DNA



Table 5
表5
表5超高压与高温短时灭菌处理的NFC苹果汁4℃贮藏期微生物指标的变化
Table 5Microbial counts in NFC apple juice after HTST or UHP treatment during storage period at 4℃ (log CFU/mL)
不同处理
Different treatment		贮藏时间 (周) Storage time (week)
12345710
菌落总数
Total number
of colonies		Control3.41±0.16a
UHPNDNDNDND2.00±0.18b——
HTSTNDNDNDNDNDND3.04±0.31b
霉菌酵母
Mold and
yeast		Control4.78±0.11a
UHPNDNDNDNDND——
HTSTNDNDNDNDNDNDND
大肠杆菌
Escherichia coli		Control3.08±0.07a
UHPNDNDNDNDND——
HTSTNDNDNDNDNDNDND

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未杀菌处理的果汁和贮藏期UHP处理的果汁中检测到的优势菌属为未确认的立克次氏体目属（unidentified Rickettsiales）,未确认的生氧光细菌属（unidentified Oxyphotobacteria）,假单胞菌属（Pseudomonas）;HTST处理的果汁贮藏期检测到的优势菌属为假单胞菌属（Pseudomonas）,未确认的生氧光细菌属（unidentified Oxyphotobacteria）,微小杆菌属（Exiguobacterium）,未确认的立克次氏体目属（unidentified Rickettsiales）,鞘氨醇单胞菌属（Sphingomonas）。

3 讨论

3.1 不同杀菌工艺对NFC苹果汁理化指标与色泽影响

前人研究已经表明苹果品种和成熟期对苹果汁理化指标有很大影响[9,13],对同一个品种NFC苹果汁,本试验说明HTST与UHP杀菌处理对理化指标则没有明显影响,此结果与关云静[12]研究相似,CHEN等[28]研究了高静水压处理和超高温处理对生姜浊汁和木瓜饮料质量和货架期的影响,发现了同样的结果。其次,对果蔬汁的品质进行评价时,pH作为一项重要指标,对判断果蔬汁的变质有指示性作用;分析认为贮藏期UHP和HTST处理的NFC苹果汁pH的降低可能是由于乳酸菌、酵母菌等微生物随着贮藏时间延长其代谢产生了酸性物质,同时伴随一系列生化反应所导致。胡盼盼等[29]研究也发现HTST处理、巴氏杀菌和热杀菌技术处理的鲜榨苹果汁在贮藏期间pH均有不同程度的降低,而且HTST处理的苹果汁在贮藏期间内pH值变化幅度最小,与本试验结果一致。

试验发现HTST处理苹果汁的亮度（L*值）下降,其L*值与未杀菌处理的NFC苹果汁有显著差异,可能是由于热处理后NFC苹果汁中的蛋白质变性,生成了不溶性的变性蛋白质[30],所以导致其L*值低于对照样品。与HTST处理的果汁相比,UHP处理的NFC苹果汁L*值的降低程度较小,ZHOU[31]和WANG等[32]也有类似的发现,他们发现采用高静水压处理的紫甘薯花蜜和高静水压处理的桑椹汁的颜色与未经处理的果汁没有显著差别。但贮藏期HTST处理的NFC苹果汁L*值的降幅低于UHP处理,可能是由于HTST杀灭了果汁中的过氧化物酶和多酚氧化酶等的活性,阻止了酶促褐变的发生,减缓了果汁的变色。LIU等[33]也发现UHP处理的黄瓜汁的L*值在储存20 d期间趋于降低,表明果汁色泽有所减弱。本试验两种处理后NFC苹果汁的a*和b*值降低,其原因可能是由于HTST和UHP处理使细胞被裂解,色素分子逐步向外扩散渗漏到细胞间隙,使a*和b*值下降;CAO等[14]报道了混浊草莓汁的L*、a*和b*值在储存4个月期间趋于减少,这可能是由于非酶促反应,如氨基和还原糖之间的美拉德反应和抗坏血酸的降解所造成的。当然,多酚氧化酶和过氧化氢酶残留活性引起的酚类化合物的酶促褐变也会使L*值降低。HUANG等[34]也获得了类似的结果,HTST处理的杏花蜜中的多酚氧化酶和过氧化氢酶完全失活,酶促褐变受到抑制。

3.2 不同杀菌工艺对NFC苹果汁抗氧化活性、酶活性、多酚的影响

在本试验过程中发现,HTST处理的NFC苹果汁在贮藏过程中抗氧化活性的损失低于UHP处理的苹果汁,这可能是HTST处理的高温降低了NFC苹果汁中氧气的溶解量;同时UHP与HTST处理的苹果汁中多酚和抗坏血酸的降解[35]也是抗氧化活性降低的重要因素。PATRAS等[36]通过研究得到了相同的结果,他们发现热杀菌处理（70℃/2 min）胡萝卜泥的抗氧化活性高于未杀菌处理和超高压（400 MPa/15 min/ 20℃）处理的样品。本试验中由于HTST处理的NFC苹果汁的酶被完全钝化,因此其抗氧化活性的降解主要是贮藏过程中自身的有氧降解和无氧降解造成的,在苹果汁贮藏期最开始,包装瓶中可能残留的氧气导致了有氧降解,当果汁中的氧气完全消耗完或低至某一浓度时,在酸性物质的催化下就会发生无氧降解,且有氧降解的速率远高于无氧降解速率。杀菌工艺对苹果汁酶活性的影响与他人[34]研究结论一致,HTST处理可使PPO和POD完全失活,UHP处理则有残留活性,从而引起一些酶促褐变反应发生。UHP处理后NFC苹果汁酶活性的变化,可能是由于多酚氧化酶和过氧化氢酶不仅具有相当高的耐高压性,而且在压力400 MPa的处理条件下,试验中使用的VC和氯化盐（CaCl2）组合护色剂对果汁中的PPO和POD有激活作用,在一定程度上提高了酶的活性,从而促进多酚氧化酶与酚类化合物的反应,产生了酚类化合物的降解;其次是组合护色剂对酶的活力中心的结构有较大影响[37],这些酶的二级和三级结构的变化以及UHP处理后NFC苹果汁中酶的提取率增加也会引起酶活性提高,因为酶的构象变化可通过改变其底物特异性和功能来增加或降低酶活性[38]。不同杀菌工艺对NFC苹果汁多酚的影响试验结果与KRYSTIAN等[15]的报道基本一致,超高压力是多酚降解的主要原因;GLISZYNSKA -SWIGLO等[39]发现通过脉冲电场处理番茄汁后,在4℃下贮存56 d过程中,绿原酸、芦丁和槲皮素含量均下降,咖啡酸浓度略有增加;在室温下储存11个月的商品苹果汁中对香豆酸、阿魏酸、槲皮素和山奈酚的含量没有显著变化,而其他单酚物质的含量下降,所有这些变化最终导致商业苹果汁中总多酚含量的减少。

3.3 不同杀菌工艺对NFC苹果汁香气成分的影响

香气是果汁产品可接受度的重要指标,而杀菌工艺对NFC苹果汁香气成分的影响比较大。酯类和醛类是果汁中重要的风味成分,本试验保留了苹果挥发物中的18种关键香气活性物质,包括乙酸丙酯、丁酸乙酯、2-甲基丁酸乙酯、乙酸丁酯、己酸乙酯、乙酸己酯、丁酸己酯,甲酸异戊酯、己醛、乙醛、（E）-2-己烯醛、1-丁醇、1-己醇、2-甲基-1-丁醇、乙酸戊酯、丁酸丁酯、2-甲基丁酸丁酯和2-甲基丁酸己酯;这与YI等[40]研究结果相似。香气阈值最高的3种物质是乙酸己酯、己醛和2-甲基乙基丁酸酯,香气阈值分别为0.002、0.005和0.00013 mg?L-1,是苹果汁中最重要的气味来源[41]。HTST处理后,NFC苹果汁中18种特征香味物质总的保留率为52%,而UHP处理18种关键香气物质含量均有所增加,所以UHP处理能更好地保留NFC苹果汁的果香。KAKIUCHI等[42]也发现热杀菌后的苹果汁中鉴定出39种挥发性化合物,特征芳香物质含量显著降低。但ZHAO等[43]的研究不同,他们发现在超滤和超高压协同处理后的新鲜苹果汁中7种主要的挥发性芳香族化合物无明显变化。

3.4 不同杀菌工艺对NFC苹果汁微生物的影响

本试验HTST、UHP处理的NFC苹果汁9/4周贮藏期均能保证其微生物安全性。LANDL等[44]报道通过超高压（400 MPa/5 min/20℃）处理和巴氏灭菌 （75℃/10 min）的Granny Smith苹果酱的菌落总数和霉菌酵母的微生物计数均低于检测限,可保质21 d;PATTERSON等[45]也发现500 MPa和600 MPa（1 min/ 20℃）高压处理下,胡萝卜汁在4℃下储存22 d,微生物的生长非常低。已有研究显示UHP杀菌有局限性,其压力、时间等条件对微生物生长影响较大,果汁不能长期贮藏的原因是UHP对某些孢子致死效果相对较差,需要进一步探讨UHP与HTST等协同杀菌技术条件,发挥非热杀菌的优势。

4 结论

本研究发现高温短时杀菌（HTST、98℃、50 s）和超高压杀菌（UHP、400 MPa、15 min）两种处理的非浓缩还原苹果汁贮藏期有相似的理化稳定性、感官品质和抗氧化能力,但与对照相比,HTST处理的NFC苹果汁在储存期间具有更好的颜色稳定性;UHP处理不能完全使NFC苹果汁中的酶灭活,多酚氧化酶和过氧化物酶残留活性高于HTST处理,从而诱导更多的酶促褐变过程与酚类化合物降解,HTST处理则可最大限度地减少颜色损失和酶促褐变。冷藏期间,HTST处理的NFC苹果汁中的香气成分含量比UPP处理变化显著,而UHP处理显着降低了NFC苹果汁中总酚类物质（127.68 μg?mL-1）的含量,且HTST和HPP处理后表儿茶素发生显著降解。HTST和UHP处理完全灭活细菌、大肠杆菌、酵母和霉菌,但在储存4、9周后,菌落总数显著升高（分别升高89.15%、58.65%）,而大肠杆菌、霉菌/酵母没有显著的生长。因此,在相同的储存时间下,HTST处理的NFC苹果汁的整体质量特性优于UHP处理;相同的品质下,HTST处理后NFC苹果汁的保质期长于UHP。

（责任编辑 赵伶俐）

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