锦橙(Citrus sinensis L.Osbeck cv.Jincheng)属甜橙类,其中‘北碚447’锦橙是甜橙中的优良品种,果大皮薄、水分充足、风味浓郁,富含抗氧化活性物质,具有预防糖尿病、心血管疾病和癌症的作用[1-2]。柑橘果实风味品质主要由挥发性香气物质、糖、酸、异味等决定[3-4]。随着贮藏期延长,由于自身呼吸消耗和生理衰老等[5],果实风味和感官接受度不断下降,异味物质的积累缩短了果实风味寿命[6]。目前柑橘集中上市带来巨大的销售压力,且消费市场对果实品质要求更高,因此开发保持柑橘采后风味品质的保鲜剂、延长贮藏期具有重要意义;随着消费者健康、环保意识提升,寻找天然无毒、无污染的保鲜剂成为发展趋势。

草酸(oxalic acid,OA)作为一种存在于生物体的有机酸,具有一定的抗氧化活性[7]。研究表明,草酸处理能够提高血橙花色苷含量和糖酸比[8],有效抑制芒果[9]、荔枝[10]、猕猴桃[11]、香蕉[7]等果实采后乙烯的生成,提高果实抗氧化能力,延缓果实衰老进程。目前草酸调控柑橘品质的研究较少[12],而草酸处理对柑橘采后挥发性香气物质积累及乙醇代谢的影响尚不清楚。

本研究拟通过不同质量浓度草酸对锦橙进行处理,运用GC-MS和实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)技术探究草酸处理对锦橙采后挥发性香气物质含量及乙醇代谢的影响,筛选利于锦橙采后风味品质保持的方法,以期为锦橙采后天然保鲜剂开发提供理论依据。

1 材料与方法

1.1 材料与处理

‘北碚447’锦橙于2018年11月28日采自重庆市北碚区歇马镇,选择成熟度、大小一致且无机械损伤、无病虫害的果实为试材。果实采收后当天运回实验室,将样品分为4组,分别浸泡10 min,OA1组:0.5 g/L草酸;OA2组:1 g/L草酸;OA3组:2 g/L草酸;CK组:清水。果实晾干后用厚0.02 mm聚乙烯薄膜袋单果包装,随后置于冷库(温度6～8 ℃、湿度85%～90%)中贮藏120 d。每30 d取样分析,每组设3个重复,每个重复取10个果,取部分果肉用液氮速冻后于-80 ℃保存,部分果肉榨汁后测定相关指标。

1.2 仪器与设备

Pocket Brix-Acidity Meter Master kit手持式折光仪,日本爱拓公司;Eppendorf Top Buret M数字滴定仪,德国艾本德公司;Agilent 7890B-5977A气相色谱-质谱联用仪(GC-MS),美国安捷伦公司。

1.3 实验方法

1.3.1 感官评价

感官评定采用描述测试法[13],12名评定人员于相同评鉴环境不同品评间对贮藏120 d的锦橙果实进行评价。评分标准如表1所示。

1.3.2 挥发性香气成分和含量测定

称取3 g经液氮研磨成粉末的果肉样品,加入2 mL饱和氯化钠与20 μL环己酮溶液(稀释1 000倍),在40 ℃、200 r/min下温育5 min后顶空萃取25 min,上机解析5 min。GC条件:色谱柱DB-5(60 m×0.25 mm×0.5 μm),进样口温度240 ℃,氦气流速1 mL/min,柱温50 ℃,保持1 min,以3 ℃/min升至155 ℃,后以20 ℃/min升至255 ℃,保持2 min。MS条件:EI离子源温度230 ℃,四级杆温度150 ℃,质谱29～500 amu。利用安捷伦系统图谱库(W10 N14.L)匹配及保留指数信息[14]来进行定性分析,内标法进行定量分析。挥发性成分的香气特征参考TERRY等[14]。

1.3.3 乙醇含量测定

称取3 g经液氮研磨成粉末的果肉样品,加入2 mL饱和氯化钠溶液,于40 ℃、200 r/min下温育10 min后顶空萃取20 min,上机解析5 min[13,15]。GC条件:色谱柱DB-WAX(60 m×0.25 mm×0.5 μm),进样口温度240 ℃,氦气流速1 mL/min,柱温30 ℃,保持7 min,以20 ℃/min升至200 ℃,保持5 min。MS条件:EI离子源温度230 ℃,四级杆温度150 ℃,质谱29～500 amu。绘制标曲进行定性和定量分析。

1.3.4 可溶性固形物和可滴定酸含量测定

锦橙榨汁后用手持式折光仪测定可溶性固形物(total soluble solids,TSS)和可滴定酸(titratable acids,TA)含量,重复测定3次。

1.3.5 抗坏血酸含量测定

采用2,6-二氯靛酚滴定法[16]测定果汁中抗坏血酸(ascorbic acid,ASA)含量,重复测定3次。

1.3.6 呼吸强度测定

采用静置碱液吸收法[17]测定果实呼吸强度,重复测定3次。

1.3.7 qRT-PCR测定基因表达水平

锦橙果肉总RNA提取及cDNA合成分别参考天根植物多糖多酚总RNA提取试剂盒(DP441型)和Tiangen FastKing RT Kit (with gDNase)(KR116型)试剂盒的说明操作。乙醇代谢相关基因引物序列参考王晶等[13]。参照宝生物SYBR®Premix Ex Taq® Ⅱ的说明操作,在qRT-PCR仪上反应。反应体系:10 μL SYBR Green I mix,0.8 μL正向引物,0.8 μL反向引物,1.5 μL cDNA模板,6.9 μL ddH2O。反应程序:95 ℃、30 s;95 ℃、5 s,58 ℃、30 s,经40个循环反应。融解曲线绘制:95 ℃、10 s,降温到65 ℃后开始以0.5 ℃每步升温,并维持5 s采集荧光信号,反应至95 ℃结束。每个样品3次重复。采用2-ΔΔCt 法进行相对定量分析。

1.3.8 数据分析

所得数据采用Microsoft Excel 2016软件进行统计处理,采用SPSS 25.0软件的Duncan法进行显著性分析(P<0.05),采用Origin 8.0绘图。

2 结果与分析

2.1 草酸处理对锦橙感官品质的影响

锦橙贮藏120 d时果实感官品质如表2所示。与CK组相比,OA2、OA3组果实新鲜度无显著变化,OA1组显著下降;OA1和OA3组果实气味显著提高,OA2组无显著变化;OA1、OA2组果实水分含量明显增加;OA2组果实风味和口感显著提高,OA1、OA3组无显著变化。综合分析认为,OA2组果实整体感官品质最佳,能够保持果实较高水分含量、风味和口感。KAUR等[18]也发现草酸处理能够保持梨果实感官品质。

2.2 草酸处理对锦橙挥发性香气成分和含量的影响

如表3所示,贮藏期间锦橙果肉中挥发性香气物质总量减少,由初始值512.67 μg/g降低至贮藏120 d时的155.95 μg/g,降低约2.3倍。贮藏120 d的锦橙果肉中共鉴定出51种挥发性香气成分,含量最高的是萜烯类物质,占挥发性香气物质总量的75.77%～88.78%,其中D-柠檬烯含量最高,与前人研究结果一致[19]。贮藏120 d时OA1、OA2、OA3组果实挥发性香气物质总量分别为205.56、226.48、187.23 μg/g,为CK组的131.81%、145.23%、120.06%,说明草酸处理延缓了锦橙采后挥发性香气物质总量的下调。

贮藏120 d的4组样品中酮类物质含量较高的是己酮、香叶基丙酮,主要表现为果香、青草香。醛类物质含量较高的是(E)-2-十一烯醛、(E)-2-己烯醛,主要表现为叶香、甜香。酯类物质含量较高的是乙酸异丁酯、己酸乙酯、辛酸乙酯,主要表现为果香、甜香、草本味。醇类物质含量较高的是芳樟醇,具有花香。萜烯类物质含量较高的是D-柠檬烯、α-姜烯、月桂烯,主要表现为柚香、香料味、清新味。与CK组相比,OA1组D-柠檬烯含量无显著变化,其他含量较高的10种挥发性香气物质显著增加;OA2组含量较高的11种挥发性香气物质显著增加;OA3组D-柠檬烯、月桂烯含量显著降低,芳樟醇含量无显著变化,其他含量较高的8种挥发性香气物质显著增加。结果表明,OA2组草酸处理能更好地维持锦橙果肉中挥发性香气物质含量。此外,OA2组DL-香芹酮、4-松油醇、甲基二氢呋喃硫醇、可巴烯、α-水芹烯含量明显低于CK组,表现为薄荷、茴香、霉味、肉味、香料味、木质味、蔬菜味,这些气味特征大多不利于果实风味的提升。综合感官评定结果认为OA2组草酸处理效果最佳。

2.3 草酸处理对锦橙乙醇含量的影响

乙醇代谢产生乙醛、乙醇是柑橘采后产生异味的重要原因[6],低温贮藏能有效降低果实乙醇的积累[20]。如表4所示,贮藏期间锦橙果肉中乙醇含量增加,由初始值37.99 μg/g增长至贮藏120 d时的740.15 μg/g,增长约19倍,与OBENLAND等[15]研究结果相似。贮藏120 d时,OA1、OA2、OA3组果实乙醇含量分别为56.09、52.85、66.33 μg/g,为CK组的7.58%、7.14%与8.96%。结果表明,草酸处理显著降低了锦橙采后乙醇含量,减少了异味的产生,与感官评定中风味评价结果相互印证。

2.4 草酸处理对乙醇代谢相关基因表达水平的影响

乙醇代谢途径已阐明[6,21-22]:丙酮酸经丙酮酸脱羧酶(pyruvate decarboxylase,PDC)催化生成乙醛,乙醛经乙醇脱氢酶(alcohol dehydrogenase,ADH)催化生成乙醇。随后乙醇酯化产生酯类物质[6,22],该类物质主要表现为果香味、甜味,对果实风味形成有一定正向作用,但过量积累则会引起风味劣变[20]。贮藏期间PDC、ADH表达水平明显上调,表明其正向调节乙醇的合成。如图1所示,OA2组草酸处理后,PDC表达量在贮藏30、60 d时显著降低,分别为CK组的0.81、0.54,贮藏90 d时显著增加,为CK组的1.14,其他时期无显著变化;ADH表达量在贮藏30、60、90、120 d时显著降低,分别为CK组的0.34、0.17、0.81、0.79。前人研究发现褪黑素[13]、草酸[23]分别处理柑橘、猕猴桃后通过抑制PDC、ADH表达量、降低酶活性,导致乙醇含量减少。因此,草酸处理可能通过降低PDC、ADH表达量来抑制乙醇合成。此外,OA2处理后果实挥发性香气物质中酯类含量增加,而感官评定中果实风味显著提升,推测草酸处理还诱导了乙醇酯化反应,进一步降低乙醇积累,草酸处理后引起的酯类含量增加对锦橙风味起正向作用。

2.5 草酸处理对锦橙TSS和TA含量的影响

贮藏期间锦橙果肉TSS含量呈先上升后下调趋势,可能是贮藏过程中酸类物质向糖类转换[24]、果实衰老进程中细胞壁成分分解或水分蒸发[8]等引起糖分升高,而后由于呼吸消耗使糖分下调[25]。如图2所示,整个贮藏过程中OA2组TSS含量始终高于CK组,且在60、120 d时有显著差异。贮藏期间果实TA含量整体呈下调趋势,可能与参与三羧酸循环为细胞供能、氨基酸代谢[25]及挥发性香气物质的合成[26]有关。贮藏30 d时OA2组TA含量高于CK组,而贮藏30 d后OA2组TA含量低于CK组,且在60 d时有显著差异。总的来说,草酸处理后锦橙果肉TSS含量增加、TA含量降低,较高的固酸比使果实风味变甜、口感更佳,与前人在草酸处理血橙中的研究结果相似[8]。

2.6 草酸处理对锦橙ASA含量的影响

ASA是植物中重要的抗氧化活性物质[27],能够清除多种活性氧自由基[28]、延缓衰老。如图3所示,贮藏过程中,锦橙果肉ASA含量呈先升高后降低的趋势。贮藏60 d时,OA2组果实ASA含量达到峰值,为26.68 mg/100 mL,随后下降至17.89 mg/100 mL。与CK组相比,贮藏60、90 d时OA2组果实ASA含量显著增加,其他时期无显著变化。结果表明,草酸处理后锦橙ASA含量增加,可能是草酸利用其抗氧化活性参与清除果实衰老进程中的部分活性氧自由基,从而使参与清除活性氧的ASA损耗减少,引起ASA含量增加。

2.7 草酸处理对锦橙呼吸强度的影响

呼吸作用是植物生理活动所需能量来源,呼吸强度是衡量呼吸强弱的重要指标[29]。如图4所示,贮藏30 d时果实存在一个呼吸峰,随后开始下降,可能是果实采摘后贮藏前期生理活动旺盛,而后由于持续低温和衰老等引起生理活性降低所致。贮藏期间OA2组果实的呼吸强度始终低于CK组,且在贮藏30、90、120 d时存在显著差异。结果表明,草酸处理能够降低果实呼吸强度,减少呼吸消耗,与较高的TSS含量结果相互印证。

3 结论

草酸处理能够有效维持锦橙果实感官品质,增加锦橙果肉中挥发性香气物质含量,保持果实较高风味和口感,其中以质量浓度为1 g/L的草酸处理效果最佳。贮藏过程中,草酸处理维持了果实较高TSS含量和较低TA含量,使果实风味更甜。此外,草酸处理显著增加了锦橙ASA含量、抑制了果实呼吸强度,通过维持较高ASA含量、降低呼吸消耗来延缓果实衰老进程。另外,草酸处理显著抑制PDC、ADH的转录水平,诱导乙醇合成显著降低。GOLDENBERG等[22]发现,异味感知不仅取决于乙醇的积累,还与萜烯类物质含量的下降密切相关。锦橙果肉中含量最高的挥发性香气物质是萜烯类。因此,草酸处理可能通过维持较高的挥发性香气物质含量和较低的乙醇含量,降低了异味产生和感知,保持锦橙采后较高的风味品质。总之,锦橙采后草酸处理能够有效改善果实风味品质、延缓风味劣变,提升果品市场价值和消费体验。

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Effects of postharvest application of oxalic acid on flavor quality and ethanol metabolism in Citrus sinensis L. Osbeck cv. Jincheng

HONG Min1,2, WANG Jing1, WANG Rikui1,2, FENG Yu1,2, ZHOU Lian1,2, DENG Tujing1, YAO Shixiang3, XIAO Yunhui4, HE Mingyang1,2,4,5*

1(Citrus Research Institute, Southwest University, Chongqing 400712, China)2(Citrus Research Institute, Chinese Academy of Agricultural Sciences, Chongqing 400712, China)3(College of Food Science, Southwest University, Chongqing 400715, China) 4(Ganzhou Agricultural and Rural Bureau, Ganzhou 341001, China)5(Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China)

ABSTRACT At present, the consumption market of citrus has higher requirements on fruit quality, but the flavor and sensory acceptability of postharvest fruits decline continuously. To explore the approaches to maintain fruit postharvest flavor quality, Citrus sinensis L. Osbeck cv. Jincheng was treated with different oxalic acid concentrations. GC-MS was used to test the contents of aroma volatiles and ethanol in postharvest fruits. The expression levels of genes involved in ethanol metabolism were tested by real-time quantitative PCR (qRT-PCR). The results showed that oxalic acid treatment could maintain higher sensory quality, aroma volatiles content and lower ethanol content in Jincheng fruits, and the 1 g/L (OA2) oxalic acid treatment showed the best effects. After storage for 120 days, the aroma volatiles content of OA2 treated group was 226.48 μg/g, which was 145.23% of CK group. And the ethanol content was 52.85 μg/g, which was 7.14% of CK group. After OA2 treatment, the expression levels of PDC and ADH genes involved in ethanol metabolism were significantly decreased. In addition, the soluble solids and ascorbic acid contents of Jincheng fruits were increased, and the fruit respiratory intensity was inhibited after OA2 treatment. These results suggested that postharvest application of oxalic acid could effectively improve Jingcheng fruit quality and reduce the off-flavors generation and perception. This study provides a theoretical basis for the research of citrus postharvest flavor quality maintenance technology and natural preservative.

Key words Jincheng; oxalic acid; flavor quality; aroma volatiles; ethanol metabolism

DOI：10.13995/j.cnki.11-1802/ts.031836

引用格式：洪敏,王晶,王日葵,等.草酸处理对锦橙采后风味品质及乙醇代谢的影响[J].食品与发酵工业,2023,49(10):71-77.HONG Min,WANG Jing,WANG Rikui, et al.Effects of postharvest application of oxalic acid on flavor quality and ethanol metabolism in Citrus sinensis L. Osbeck cv. Jincheng[J].Food and Fermentation Industries,2023,49(10):71-77.

第一作者：硕士,实验师(贺明阳副研究员为通信作者,E-mail:hemingyang@cric.cn)

基金项目：国家重点研发计划(2021YFD1600804);四川省科技计划(2021YFQ0072);中央高校基本科研业务费专项资金(XDJK2020C027);西南大学实验技术研究项目(SYJ2022018);中国博士后科学基金(2018M633178)

收稿日期：2022-04-06,改回日期:2022-05-06