果品及其制品展青霉素污染的發(fā)生、防控與檢測

聶繼云

（中國農(nóng)業(yè)科學院果樹研究所/農(nóng)業(yè)部果品質(zhì)量安全風險評估實驗室（興城）/農(nóng)業(yè)部果品及苗木質(zhì)量監(jiān)督檢驗測試中心（興城），遼寧興城 125100）

食品科學與工程

果品及其制品展青霉素污染的發(fā)生、防控與檢測

聶繼云

（中國農(nóng)業(yè)科學院果樹研究所/農(nóng)業(yè)部果品質(zhì)量安全風險評估實驗室（興城）/農(nóng)業(yè)部果品及苗木質(zhì)量監(jiān)督檢驗測試中心（興城），遼寧興城 125100）

展青霉素是由青霉屬、曲霉屬、絲衣霉屬等真菌產(chǎn)生的一種聚酮類次生代謝產(chǎn)物，廣泛存在于果品及其制品中，蘋果及其制品是主要污染源，也是人類膳食中展青霉素最主要的來源。展青霉素有各種急性、慢性和細胞水平的危害。國際組織和不少國家均制定了果品及其制品中展青霉素限量。擴展青霉是最主要的產(chǎn)展青霉素真菌，能污染許多種果品及其制品。擴展青霉地域分布甚廣，許多國家均分離到了其菌株。作為植物病原菌，擴展青霉往往通過果實上的傷口如受傷部位、害蟲為害部位、病菌感染部位，以及果柄、開放的萼筒、皮孔等部位入侵。過熟和長期貯藏的水果更易感染擴展青霉。擴展青霉是一種好寒性霉菌，在 0℃生長旺盛。展青霉素生物合成通路約由10步構(gòu)成，棒曲霉和擴展青霉中展青霉素生物合成基因簇包含相同的15個基因，但兩者基因序列差異很大。展青霉素的細胞毒性和遺傳毒性歸因于其與細胞中親核物質(zhì)的高反應活性，主要通過共價鍵與細胞的親核物質(zhì)，特別是與蛋白質(zhì)和谷胱甘肽的巰基結(jié)合，從而發(fā)揮毒性、破壞染色體和致突變。在其反應通路中，1個展青霉素分子最多可與3個谷胱甘肽分子反應。采前措施、采后處理和貯藏條件對控制展青霉素污染及其產(chǎn)毒真菌至關(guān)重要。使用化學殺菌劑是重要的防控策略，但過度使用殺菌劑會導致抗性菌株出現(xiàn)。使用“低風險殺菌劑”則能降低產(chǎn)生抗藥性的風險。生物防治是化學防治的替代方法或補充，可減少甚至避免使用殺菌劑。清洗、分選、整理等加工工藝有助于消減水果制品中的展青霉素污染。液液萃取和固相萃取是果品及其制品中展青霉素的經(jīng)典提取方法，但液液萃取成本高、耗時、不適于固體樣品。近年來，又研究建立了分散液液微萃取、鹽析旋渦輔助液液微萃取、基質(zhì)固相分散萃取、QuEChERS等提取方法。展青霉素通常用配紫外檢測器或二極管陣列檢測器的液相色譜儀進行定量，采用液相色譜串聯(lián)質(zhì)譜、氣相色譜串聯(lián)質(zhì)譜等儀器進行定性。另外，PCR具有快速、專一的特點，可用于對潛在的產(chǎn)展青霉素真菌進行早期檢測。

果品；果品制品；展青霉素；污染；發(fā)生；防控；檢測

Abstract:Patulin is a secondary metabolite of polyketide lactone mainly produced by species of Penicillium, Aspergillus, and Byssochylamys. It was found as a contaminant in many fruits and fruit products, the major sources of contamination are apples and apple products, which are also the most important source of patulin in human diet. Patulin has various acute and chronic effects and others at the cellular level. Today, international organizations (Codex Alimentarius Commission and European Union) and many countries across the world have set maximum levels of patulin content in fruit and fruits products. Among the different genera, themost important patulin producer is P. expansum, it can contaminate a number of fruits and fruit products, and produce mycotoxin patulin. P. expansum distributes very extensively, its strains have been isolated from many countries. As a plant pathogen, P.expansum penetrates fruits typically through wounds or injuries produced during harvest and handling, it can also penetrate through stem end, open calyx tube and lenticels of fruits, and infection sites of other primary fruit pathogens. Overmature or long-stored fruits are more susceptible to P. expansum infection. P. expansum is a psychrophile, its growth is quite strong at 0℃. The biosynthetic pathway of patulin consists of approximately 10 steps. It has been clarified that both of the patulin biosynthetic gene cluster from P.expansum and that from A. clavatus composed of the same 15 genes, but their gene sequences differed greatly. The genotoxic and cytotoxic properties of patulin are due to its high reactivity to cellular nucleophiles. Patulin is believed to exert its toxic,chromosome-damaging, and mutagenic activity mainly by covalent binding to cellular nucleophiles, in particular to the thiol groups of proteins and glutathione (GSH). In the major reaction pathways, up to three molecules of glutathione can bind to one molecule patulin. To control the contamination of patulin and the growth of moulds producing it, pre-harvest measures, post-harvest treatments,and storage conditions deserve special attention. The use of chemical fungicides is an important strategy, but the overuse of fungicides will lead to the emergence of fungicide-resistant strains. Because their way of action reduces the risk of resistant population emergence, “l(fā)ow risk fungicides” are more suitable and efficient. Using biocontrol agents are alternative or complementary treatments that permit to decrease fungicide doses or even avoid the use of chemicals. Some stages of manufacturing process (such as washing, sorting and trimming) are highly efficient in reducing the levels of patulin in fruit products. Liquid-liquid extraction (LLE) and solid-phase extraction (SPE) are classical methods to extract patulin in fruits and fruit products. However, LLE is expensive, time consuming, and unsuitable for the treatment of solid sample. In the last years, some other extraction measures have been studied and developed, including DLLME, IL-DLLME, BS-DLLME, salting out-VALLME, MSPD, and QuEChERS. LC-UV or LC-DAD procedure is routinely used for quantitative determination of patulin, and methods to confirm the presence of patulin usually include more specific detection techniques such as LC-MS/MS and GC-MS/MS. PCR method is a fast and specific method of early detecting the potential patulin producing fungi.

Key words:fruit; fruit product; patulin; contamination; occurrence; control; determination

0 引言

展青霉素（patulin，PAT）也叫棒曲霉素，為真菌次生代謝產(chǎn)物[1]。展青霉素最早于20世紀40年代作為抗生素分離自灰黃青霉（Penicillium griseofulvum）和擴展青霉（P. expansum）[2]。許多早期研究都針對其抗菌活性的利用，例如用于治療普通感冒和皮膚真菌感染[3-4]。20世紀50年代和60年代，情況變得明了，除抗菌、抗病毒和抗原生動物外，展青霉素對植物和動物也有毒性，排除了其作為抗生素的臨床用途，1960年代被重新分類為毒素[5]。展青霉素廣泛存在于果品和果品制品中，對人體健康和經(jīng)濟造成嚴重影響[6]，其含量可達數(shù)百ppb[7-8]，甚至高達1 650 ppb[9]。其中，水果包括蘋果[1-2,7-18]、梨[2,7,12-15,17,19]、葡萄[7,10,12-15,17,19]、桃[7,13,15,17,19]、櫻桃[13,15]、李子[13,15]、杏[7,12-15,17,19]、草莓[12-13,15,19]、桑葚[15]、獼猴桃[15]、油桃[15]、橄欖[15]、藍莓[19]、黑穗醋栗[13]、榅桲[11,14]、橙子[13]、芒果[12]、菠蘿[13]、香蕉[12-13]、榛子[20]等；水果制品以蘋果汁為主[1-2,11-14,16-19,21-29]，此外，還有梨汁[8,12-13,16,21,28-29]、葡萄汁[24-25,28]、桃汁[13,16,29]、櫻桃汁[28]、杏汁[25,29]、橙汁[24-25,28]、菠蘿汁[25,28]、荔枝汁[25]、西番蓮汁[28]、黑穗醋栗汁[28]、混合果汁[8,25,30]、水果飲料[29]、蘋果酒[1-2,12,28]、葡萄酒[31]、蘋果醬[8]、梨醬[8]、草莓醬[28]、藍莓醬[28]、黑穗醋栗醬[28]、榅桲醬[11]、蘋果泥[1,12-13,16,18,23,28]、梨泥[13]、桃泥[13]、蘋果沙司[1]、無花果干[21]等。蘋果及其制品是主要污染源，也是人類膳食中展青霉素最主要的來源[28]。除單獨污染外，展青霉素還能與桔青霉素和黃曲霉毒素共生，例如，蘋果中與桔青霉素共生[10]，榛子中與黃曲霉毒素（B1、B2、G1、G2）共生[20]。

1 展青霉素的主要特性

展青霉素為無色針狀結(jié)晶，熔點110.5—112℃，易溶于水、乙醇、丙酮、乙酸乙酯和氯仿，微溶于乙醚和苯，不溶于石油醚，最大紫外吸收波長276 nm；酸性條件下較穩(wěn)定，堿性條件下不穩(wěn)定[32-33]。pH 3.3—5.5水溶液中的展青霉素在 105℃—125℃下穩(wěn)定，因此，熱處理（巴氏殺菌）不能使蘋果制品（如蘋果汁、蘋果酒）中的展青霉素完全失活[26]。與黃曲霉毒素、伏馬毒素、赭曲霉素等主要真菌毒素一樣[2]，展青霉素也是聚酮類代謝產(chǎn)物[2,34-35]，為一種高反應活性[29]α,β-不飽和 γ-內(nèi)酯[36]，分子式為 C7H6O4，分子量為154，化學名稱為4-羥基-4氫-呋喃[3,2-碳]吡喃-2[6 氫]-酮[26]，化學結(jié)構(gòu)[1]見圖 1。

圖1 展青霉素的化學結(jié)構(gòu)[1]Fig. 1 Chemical structure of patulin[1]

展青霉素屬中等毒性物質(zhì)，雄鼠經(jīng)口 LD50為 35 mg·kg-1[37]；暫定最大耐受日攝入量（PMTDI）成人為0.4 μg·(kg bw)-1[8,25,30,38-41]、兒童為 0.2μg·(kg bw)-1[8,25,30]，該攝入量依據(jù)展青霉素的最大無作用量（NOEL）43 μg·(kg bw)-1和安全系數(shù)100制定[39,42]。國際組織（國際食品法典委員會[43]、歐盟[44]）和不少國家均制定了食品中展青霉素限量[1,11,45]（表1）。展青霉素是可能致癌物[1]，國際癌癥研究中心將其歸入第3類物質(zhì)[46]，即對人體致癌性尚未歸類的物質(zhì)[47]。展青霉素對動物有各種急性、慢性和細胞水平的危害[27-28,48]。急性癥狀包括緊張、抽搐、肺充血、水腫、充血、胃腸道擴張、腸出血和上皮細胞變性。慢性癥狀包括遺傳毒性、神經(jīng)毒性、免疫毒性、免疫抑制、致畸和致突變。細胞水平的危害有原生質(zhì)膜破裂以及蛋白質(zhì)、DNA和RNA合成受抑制等。展青霉素對高等植物也有毒[28]，例如，能抑制種子萌發(fā)[3,49]和引起植株萎蔫[3]，對植物（伊樂藻和番茄）原生質(zhì)體流動有強抑制作用[3]，可導致春小麥節(jié)間長度、小花數(shù)、種子重量和種子數(shù)減少[50]。展青霉素對微生物的毒性也有報道。它能抑制革蘭氏陰性細菌和革蘭氏陽性細菌的生長，人們測試了超過75種細菌，無一能完全抵抗其毒性[3]；展青霉素還能抑制酵母的生長[51]。

表1 國際組織和一些國家制定的食品中展青霉素限量（μg·kg-1或μg·L-1）Table 1 Maximum levels (μg·kg-1or μg·L-1) established by countries and international organizations

2 展青霉素的發(fā)生流行

展青霉素由曲霉屬（Aspergillus）、絲衣霉屬（Byssochlamys）、擬青霉屬（Paecilomyces）、青霉屬（Penicillium）等真菌產(chǎn)生[2,52]，絲衣霉是無性型擬青霉[36]。曲霉屬中的棒曲霉（A. clavatus）、巨曲霉（A.giganteus）、A. longivesica，青霉屬中的P. carneum、棒束青霉（P. clavigerum）、嗜糞青霉（P.concentricum）、P. coprobium、P. dipodomyicola、擴展青霉、櫟實青霉（P. glandicola）、唐菖蒲青霉（P.gladioli）、灰黃青霉、P. marinum、P. paneum、P.sclerotigenum、狐糞青霉（P. vulpinum），絲衣霉中的雪白絲衣霉（B. nivea），以及擬青霉中Paecilomyces saturatus的某些菌株，均能產(chǎn)生展青霉素[2,52]。據(jù)報道[53]，中國青霉屬真菌中橘灰青霉（P. aurantiogriseum）、擴展青霉、櫟實青霉、灰黃青霉、多毛青霉（P.hirsutum）、楊奇青霉（P. janczewskii）、梅林青霉（P.melinii）、婁地青霉（P. roqueforti）、小刺青霉（P.spinulosum）、狐糞青霉等均能產(chǎn)生展青霉素。

圖2 被擴展青霉污染的蘋果[2,36]Fig. 2 Apples contaminated by P. expansum[2,36]

擴展青霉是食品中展青霉素的主要來源[28,54]。通常，水果（特別是蘋果）受該病菌影響最大，被認為是到目前為止，展青霉素進入食物鏈的主要途徑[55-56]。擴展青霉寄主極廣，能侵染蘋果[7,15-16,22-24,26,28,57-59]、梨[7,15,22-24,26,28,57-59]、杏[7,15,24,26,28,57-59]、桃[7,15,26,28,57,59]、葡萄[7,15,23-24,26,28,59]、櫻桃[15,22,28,58]、草莓[15,28,57]、香蕉[24,28]、油桃[15,28]、李子[15,28]、桑葚[15,28]、獼猴桃[15,57]、黑穗醋栗[28,58]、薄殼山核桃[28,58]、榛子[28,58]、菠蘿[24]、橙子[24]、樹莓[28]、柿子[28]、藍莓[28]、青梅[59]、杏仁[28]、榅桲[58]、花楸漿果[58]、橡實[58]、核桃[58]、橄欖[15,59]等果品，以及蘋果汁、梨汁、葡萄汁、櫻桃汁、醋栗汁、草莓醬、梨泥等果品制品[15,58-59]，并產(chǎn)生展青霉素。擴展青霉地域分布甚廣，歐洲、美洲、亞洲、大洋洲和非洲的許多國家均分離到了其菌株[53,58,60]。中國青霉屬真菌中，狐糞青霉罕見，梅林青霉、多毛青霉和櫟實青霉較罕見，余者均分布廣或較廣[53]。另外，果品及其制品展青霉素污染存在地區(qū)差異。以報道最多的蘋果汁為例，中國陜西生產(chǎn)的蘋果汁其展青霉素含量平均僅為8.44 μg·kg-1[17]，西班牙加泰羅尼亞地區(qū)銷售的蘋果汁展青霉素含量略高于該水平（平均含量11.7 μg·kg-1）[18]，而南非[9]和突尼斯[8]的蘋果汁展青霉素污染情況則要嚴重得多，展青霉素平均含量分別高達 210 μg·kg-1和 45.7 μg·kg-1。

擴展青霉能引起蘋果、梨、櫻桃、杏、獼猴桃、桃等許多水果發(fā)生青霉病[57,61-63]，造成貯藏期和貨架期損失[64]。果實發(fā)病后，出現(xiàn)褐色軟腐，產(chǎn)生綠色或藍色分生孢子梗和分生孢子，形成膿皰[2,36]（圖2）。擴展青霉往往通過傷口或受傷部位侵入，也可通過果柄、開放的萼筒和皮孔侵入，還可從果實上害蟲為害部位和其他病原菌感染部位侵入[27,65]。采前產(chǎn)生的傷口和采收、分級、包裝、貯藏、運輸過程中造成的機械傷是擴展青霉的重要入侵點[66]，任何機械損傷都會增加果實感染擴展青霉的敏感性[27]。過熟或長期貯藏的水果更容易感染擴展青霉[67]。對于樹上的果實，即使上面有擴展青霉分生孢子，果實采收前這些分生孢子通常也不會生長，但這些果實如果受到病蟲為害或掉落地上，果實采收后就可能發(fā)病，并產(chǎn)生展青霉素[27]。擴展青霉的生長主要受溫度以及 O2和 CO2濃度等環(huán)境條件的影響[37]：作為一種好寒性霉菌，擴展青霉生長溫度最低可達-6℃，在 0℃下生長強勁，最適生長溫度為25℃，最高生長溫度為35℃；擴展青霉對O2要求低，即使O2濃度低至2.1%，其生長也幾乎不受影響；空氣中的CO2能刺激擴展青霉生長，CO2濃度高至15%仍有刺激作用，但較高的CO2濃度會使擴展青霉生長速率降低。

圖3 展青霉素生物合成通路[2]Fig. 3 Scheme of patulin biosynthetic pathways[2]

3 展青霉素的生物合成

展青霉素的生物合成通路（圖3）約由10步構(gòu)成[2]?，F(xiàn)已明確棒曲霉和擴展青霉中的展青霉素生物合成基因簇，均包含相同的15個基因[35,68]，但兩者基因序列差異很大（圖 4）。擴展青霉中的展青霉素生物合成基因簇聚集在一個41 kb的DNA區(qū)域。其中，PatL編碼一個轉(zhuǎn)錄因子；PatM、PatC和PatA編碼轉(zhuǎn)運蛋白；PatB編碼羧酸酯酶；PatD編碼依賴Zn的乙醇脫氫酶；PatE編碼葡萄糖-甲醇-膽堿（GMC）氧化還原酶（該酶催化展青霉素生物合成通路的最后一步，即由ascladiol生成展青霉素）；PatG編碼一個脫羧酶（即6-甲基水楊酸脫羧酶[69]，該酶呈現(xiàn)一個氨基羥化酶保守域，極有可能參與了6-甲基水楊酸脫羧生成間甲酚的過程）；PatH和PatI編碼細胞色素P450（P450負責間甲酚羥化為間羥基苯甲醇和間羥基苯甲醇羥化為龍膽醇）；PatJ編碼一個加雙氧酶；PatK是擴展青霉的展青霉素生物合成基因簇中的骨干基因，編碼6-甲基水楊酸聚酮合成酶（6-MSAS）；PatN編碼isoepoxydon脫氫酶；PatO編碼異戊醇氧化酶；PatF功能未知，有一個類似SnoaL的結(jié)構(gòu)域。雖然意大利青霉（P. italicum）和指狀青霉（P. digitatum）中也發(fā)現(xiàn)了展青霉素基因的直系同源物，但均無骨干基因PatK，且意大利青霉中僅鑒定出了 PatC、PatD和PatL 3個基因，這兩種青霉菌都不產(chǎn)生展青霉素[68]。

圖4 擴展青霉和棒曲霉中展青霉素基因簇示意圖[35]Fig. 4 Schematic representation of the patulin gene clusters in P. expansum and A. clavatus[35]

4 展青霉素的作用機制

展青霉素的遺傳毒性和細胞毒性是由于其與細胞親核物質(zhì)的高反應活性[70]。展青霉素與蛋白質(zhì)和谷胱甘肽[71-72]的巰基反應快，而與氨基[73]的反應要慢得多。在高濃度展青霉素處理的中國倉鼠V79肺成纖維細胞中觀察到了 DNA鏈斷裂、DNA氧化性修飾和DNA-DNA鉸鏈，證明在細胞系統(tǒng)中展青霉素直接與DNA反應[72]。展青霉素誘導細胞遺傳損傷的機制[70]是交鏈的姐妹染色單體在有絲分裂期沒有很好地分離，被拉到相反的兩極，形成后期橋，后期橋在胞質(zhì)分裂期轉(zhuǎn)變?yōu)楹速|(zhì)橋；DNA損傷引起的細胞周期紊亂導致中心體擴增，產(chǎn)生多極紡錘體；陰性著絲粒微核通過核質(zhì)橋斷裂產(chǎn)生或在鉸鏈 DNA修復和復制過程中產(chǎn)生，而陽性著絲粒微核則可能是有絲分裂紊亂的結(jié)果。展青霉素是致突變真菌毒素，特別是在谷胱甘肽濃度低的細胞中[74]。例如，展青霉素處理的人體肝癌HepG2細胞[75]和中國倉鼠V79肺成纖維細胞[46]染色體畸變率增加；展青霉素能誘導中國倉鼠V79肺成纖維細胞產(chǎn)生陰性著絲粒微核和陽性著絲粒微核[71]。

展青霉素的細胞毒性模式是通過C-6和C-2不飽和雜環(huán)內(nèi)酯與蛋白質(zhì)、酶和其他細胞成分的巰基相互作用，破壞細胞功能[72,76]。展青霉素主要通過共價鍵結(jié)合到細胞的親核物質(zhì)上，特別是結(jié)合到蛋白質(zhì)和谷胱甘肽（GSH）的巰基上[71,77-78]，從而發(fā)揮毒性[79-80]、破壞染色體[46,71,81]和致突變[74]。展青霉素與谷胱甘肽反應的主要通路和加合物結(jié)構(gòu)見圖5，圖中*表示手性碳原子[76]，加合物的命名按文獻[82]進行。由圖5可知，一個展青霉素分子可與多達3個谷胱甘肽分子結(jié)合。展青霉素最初與一個巰基反應形成的加合物比展青霉素本身具有更高的與巰基和氨基的反應活性，但有兩個或三個巰基的加合物沒有或僅有較低的進一步反應活性[82]。谷胱甘肽發(fā)揮著展青霉素防護劑的作用[72,83]，其與展青霉素反應的加合物可用高效液相色譜（HPLC）結(jié)合生化檢測器（BCD）和電噴霧離子化串聯(lián)質(zhì)譜（ESI-MS/MS）的方法加以檢測[38]。外源性抗氧化劑維生素E對展青霉素引起的遺傳毒性和細胞毒性有防御作用[75]。而谷胱甘肽合成抑制劑丁硫氨酸亞砜亞胺（BSO）則會引起細胞谷胱甘肽含量減少，從而增加展青霉素的細胞毒性和遺傳毒性[74,83]。

5 展青霉素的污染防控

使用化學殺菌劑是控制農(nóng)產(chǎn)品采后真菌病害的重要策略[29,84-86]，但因?qū)娊】档年P(guān)注，采后化學藥劑使用要求越來越嚴，英國禁止采后使用殺菌劑[40]，在歐洲和美國，一些最有效的殺菌劑已被取消[29]。過度使用殺菌劑會導致抗殺菌劑菌株的出現(xiàn)[87]?？┚嬉种茢U展青霉分生孢子萌發(fā)和菌絲體生長[88]，能降低擴展青霉產(chǎn)生抗藥性的風險，被稱為低風險殺菌劑[89]。病菌的不同種群對同一種殺菌劑的敏感性有差異，為保證防控效果，可多種殺菌劑活性成分混合使用[87]。由于抗性菌株的發(fā)展，一些殺菌劑已失去效力[29,89-91]，宜用敏感殺菌劑代替。例如，不少擴展青霉菌株已對噻菌靈產(chǎn)生抗性[92-93]，而抗噻菌靈的擴展青霉分離株對抑霉唑敏感[92]；咯菌腈能控制抗噻菌靈擴展青霉引起的青霉病[94]。因此，可用抑霉唑或咯菌腈替代噻菌靈。有的殺菌劑甚至還會促進病菌的發(fā)展和展青霉素的產(chǎn)生，應避免使用。例如，噻菌靈能刺激擴展青霉孢子的形成，進而促進擴展青霉的繁殖[95]；多菌靈、克菌丹和乙嘧酚磺酸酯能刺激某些擴展青霉菌株產(chǎn)生展青霉素[96]。一些殺菌劑替代品也有很好的防治效果。3%次氯酸鈉溶液浸果5 min能完全抑制受感染蘋果中互隔交鏈孢霉（Alternaria alternata）、黃曲霉（A. flavus）、黑曲霉（A. niger）、枝孢樣枝孢霉（Cladosporium cladosporioides）、鐮刀霉（Fusarium）、擴展青霉和桃軟腐病菌（Rhizopus stolonifer）在 25℃下的生長和為害[97]。2%—5%的醋酸溶液能抑制蘋果上擴展青霉的生長和展青霉素的產(chǎn)生[55]。

圖5 展青霉素與谷胱甘肽反應的主要通路[76]Fig. 5 Major pathways of the reaction of patulin with GSH[76]

生物防治是化學防治的替代方法或補充，可減少甚至避免使用殺菌劑[90]。使用微生物拮抗劑控制水果采后病害是最有前途的殺菌劑替代方案[98-99]。水果表面存在細菌、酵母等微生物群落，對擴展青霉有顯著的拮抗活性[84,100]。有的生防劑其效果可與殺菌劑[29,100]、氣調(diào)貯藏結(jié)合殺菌劑相媲美[87]。研究表明，清酒假絲酵母（Candida sake）[90,100]、西弗假絲酵母（Candida ciferrii）[62]、淺白隱球酵母（Cryptococcus albidus）[101]、羅倫隱球酵母（Candida laurentii）[62,102]、植物乳桿菌（Lactobacillus plantarum）[40]、乳酸菌（Lactic acid bacteria）[103]、核果梅奇酵母（Metschnikowia fructicola）[29]、成團泛菌（Pantoea agglomerans）[90]、卡利比克畢赤酵母（Pichia caribbica）[48]、熒光假單胞菌（Pseudomonas fluorescens）[99]、水拉恩氏菌（Rahnella aquatilis）[104]、膠紅酵母（Rhodotorula mucilaginosa）[105]等拮抗菌對蘋果上的擴展青霉都有顯著防效。生防劑與低風險殺菌劑結(jié)合使用效果更好[89]，如生防酵母結(jié)合啶酰菌胺和嘧菌環(huán)胺[106]、丁香假單胞菌（Pseudomonas syringae）結(jié)合嘧菌環(huán)胺[89]等。許多拮抗菌能降解展青霉素，將其轉(zhuǎn)化為ascladiol、desoxypatulinic acid等毒性更低的物質(zhì)[107-109]，可用于展青霉素污染處理[110]，如動物雙歧桿菌（Bifidobacterium animalis）[111]、植物乳桿菌[40]、美極梅奇酵母（Metschnikowia pulcherrima）和核果梅奇酵母[29]、卡利比克畢赤酵母[48]、奧默畢赤酵母（Pichia ohmeri）[112]、粘紅酵母（Rhodotorula glutinis）[113]、釀酒酵母（Saccharomyces cerevisiae）[107,114]、氧化葡糖桿菌（Gluconobacter oxydans）[108]、紅冬孢酵母（Rhodosporidium kratochvilovae）[109]、沼澤生紅冬孢酵母（Rhodosporidium paludigenum）[115]等。屎腸球菌（Enterococcus faecium）能與展青霉素結(jié)合而將其從水溶液中清除[116]。一些植物源性成分也有很好的防效。柚皮苷、橙皮苷、新橙皮苷、櫻桃苷、橙皮素葡萄糖苷等黃烷酮及其葡萄糖苷酯能抑制擴展青霉、土曲霉（A. terreus）和黃褐絲衣菌（B. fulva）產(chǎn)生展青霉素，使其積累量減少95%以上[26]。槲皮素和傘形花內(nèi)酯能控制擴展青霉生長和展青霉素積累，可代替?zhèn)鹘y(tǒng)化學殺菌劑，用于蘋果采后青霉病的防控[41,117]。0.2%檸檬精油和 2%橙子精油均對抑制蘋果中擴展青霉產(chǎn)生展青霉素有非常好的效果[97]。

采前措施、采后處理和貯藏條件對控制產(chǎn)展青霉素真菌生長和展青霉素污染十分重要。以蘋果為例，為保證果品質(zhì)量，一些采前措施值得特別注意[27]。包括選擇抗病蟲和果皮堅實的品種；清理和銷毀果園內(nèi)的爛果、爛枝；保持樹冠通風、透光；對引起果實腐爛的病蟲害和產(chǎn)展青霉素霉菌入侵點進行防控；使用殺菌劑防止采收期間和采收后霉菌的發(fā)生和生長；施用鈣肥和磷肥，改善果實細胞結(jié)構(gòu)，降低對果實腐爛的敏感性；不將礦質(zhì)元素含量少的果實用于長期貯藏（超過3—4個月）；果實充分成熟后采摘；采收、運輸和裝卸過程中避免對果實造成機械損傷；淘汰落地果、有病害的果和有機械損傷的果。蘋果貯藏庫應清潔衛(wèi)生，可采取清潔劑和高壓熱水清洗后，噴灑0.025%次氯酸鈉溶液消毒[27]。蘋果貯藏期間，盡可能降低庫溫和 O2水平[118]。蘋果裝入聚乙烯包裝袋后貯藏，可避免使用化學藥劑，易于控制擴展青霉生長和展青霉素產(chǎn)生，與不裝袋相比，展青霉素產(chǎn)生量至少可降低99.5%[119]。氣調(diào)貯藏能很好地控制蘋果上擴展青霉生長和展青霉素產(chǎn)生，在低或超低 O2和 CO2條件下，1℃氣調(diào)貯藏 2—2.5個月的金冠蘋果，均未檢測到展青霉素[120]。冷藏后再在室溫下貯藏，蘋果上擴展青霉的生長會被重新激活[87]，因此，蘋果在冷藏結(jié)束后應盡快消費或加工。加工前貯藏是控制加工用蘋果展青霉素積累的一個關(guān)鍵點。蘋果加工前，最好在10℃以下冷藏[121]；盡可能縮短室溫貯藏時間，或不進行室溫貯藏，如室溫貯藏，時間不能超過48 h[122]。在蘋果加工過程中，選果、清洗和整理是除去蘋果中展青霉素最關(guān)鍵的步驟[27,123-124]。選果就是剔除腐爛嚴重的果實，蘋果加工前應進行選果[56]。清洗，特別是高壓水沖洗，是控制展青霉素污染的好辦法，能清除果實的腐爛部分和高達54%的展青霉素[123]。蘋果中展青霉素污染主要集中在肉眼看得見的腐爛部位，去除腐爛部位能顯著減少展青霉素污染[125]。鑒于展青霉素能滲透到腐爛部位附近1—2 cm左右的健康組織中[126-127]，將腐爛部位周圍2 cm內(nèi)的健康組織一并去除，通?？梢员苊庹骨嗝顾匚廴綶127-128]。清除腐爛和受損的果實或清理掉霉爛的部分，能顯著降低蘋果制品中的展青霉素水平[42]。

水果加工過程中一些加工工藝對展青霉素有消減作用。輻照能使蘋果汁中的展青霉素發(fā)生降解，用2.5 kGy的劑量輻照展青霉素起始濃度為2 mg·kg-1的蘋果濃縮汁，可使展青霉素完全消失[129]。多波長紫外光照射也能使蘋果汁中的展青霉素發(fā)生降解，其降解過程遵循一級時間動力學方程[124]。巴氏殺菌、酶處理、微孔過濾、蒸發(fā)等加工步驟能在一定程度上降低果品制品中的展青霉素水平[130]。巴氏殺菌能破壞擴展青霉的孢子[121]，因而能降低隨后擴展青霉產(chǎn)生展青霉素的風險。與超濾相比，采用回轉(zhuǎn)式真空過濾的傳統(tǒng)澄清方法去除展青霉素效果更好[123]。但超濾后用吸附樹脂處理能顯著降低蘋果汁的展青霉素水平，并能改善蘋果汁的色澤和澄清度[131]。展青霉素溶于水[32-33]，用硫脲改性殼聚糖樹脂（TMCR）能有效去除水溶液中的展青霉素，在pH 4.0和25℃條件下，24 h能吸附1.0 mg·g-1[132]。經(jīng)丙硫醇功能化的介孔二氧化硅 SBA-15（SBA-15-PSH），其硫醇官能團能與展青霉素的共軛雙鍵系統(tǒng)進行Michael加成反應，從而降低受污染蘋果汁和水溶液中的展青霉素水平[133]。交聯(lián)黃原酸化殼聚糖樹脂（CXCR）是清除蘋果汁中展青霉素的適宜吸附劑，其最適條件為pH 4和30℃下吸附18 h[134]。雪白絲衣霉（B. nivea）和黃褐絲衣菌（B. fulva）均為耐熱菌，在層壓紙板和聚對苯二甲酸乙二醇酯瓶包裝的蘋果清汁和濁汁中均能產(chǎn)生展青霉素，果汁生產(chǎn)企業(yè)應采取措施控制這兩種真菌的子囊孢子[135]。高靜水壓（HHP）也可用于蘋果飲料展青霉素污染控制[136]。

6 展青霉素的分析檢測

薄層色譜法（TLC）是最早用于展青霉素檢測的方法。由于樣品前處理費時、雜質(zhì)干擾嚴重、靈敏度低、只能半定量、與5-羥甲基糠醛等存在共萃取現(xiàn)象，該方法已甚少采用。目前，展青霉素檢測主要采用LC-UV、LC-DAD、LC-MS/MS、GC、GC-MS、GC-MS/MS等氣相/液相色譜法或氣相/液相色譜質(zhì)譜聯(lián)用法，檢出限一般在 ppb級[137-142]，最低可至 0.09 ppb[143]。展青霉素是小分子量的極性化合物，有強紫外吸收，適于用配紫外檢測器（UV）或二極管陣列檢測器（DAD）的液相色譜儀（LC）檢測，但存在 5-羥甲基糠醛和酚類物質(zhì)干擾的問題[138-139]。展青霉素的LC-MS/MS檢測通常采用大氣壓離子源（API源）的負離子源模式（如ESI源、APCI源和APPI源），采用 ESI源時會出現(xiàn)很強的基質(zhì)效應，而采用 APCI源時基質(zhì)效應可忽略[139]。采用 GC、GC-MS和GC-MS/MS檢測展青霉素時，需進行分析前衍生化，費時、繁瑣，而采用進樣口衍生則可節(jié)約衍生試劑和樣品制備時間[141]。除前述方法外，展青霉素檢測還可采用膠束動電毛細管色譜法（MEKC），如MEKC-DAD（UV）、DLLME-MEKC-DAD等，檢測限可低至不足1 ppb[144-146]。值得注意的是，在檢測蘋果濁汁展青霉素含量時，相比于濁汁的液相部分，濁汁的固體部分富含蛋白質(zhì)，展青霉素極有可能與蛋白質(zhì)相互作用而結(jié)合在一起，使高達20%的展青霉素未被檢測到，從而導致毒性水平的低估[147]。

展青霉素檢測中普遍采用液液萃?。↙LE）或固相萃?。⊿PE）[140,144,148-150]。由于需使用更多的有機溶劑，LLE萃取成本高而且耗時，用碳酸鈉凈化還會使展青霉素發(fā)生降解（因為展青霉素在酸性介質(zhì)中更穩(wěn)定）[142]。與LLE萃取相比，SPE更簡便，回收率高，污染小。采用SPE時，提取相中加入NaH2PO4有利于 pH保持微酸性，以免展青霉素降解[151]?；诜肿佑≯E聚合物（MIPs）的 SPE，模板分子識別選擇性和親和力高[152]，有良好的選擇性和穩(wěn)定性，更高效[153]，已成功用于蘋果汁中真菌毒素的檢測[153-155]。分散液液微萃?。―LLME）提取時間短、操作簡單、富集因子和回收率高，離子液體（IL）具有對空氣和水分穩(wěn)定，不揮發(fā)，熱穩(wěn)定性好，粘度可調(diào)，與水和有機溶劑混溶等優(yōu)點，可作為 DLLME的提取溶劑[140]。在DLLME基礎(chǔ)上，還發(fā)展出了二元溶劑分散液液微萃?。˙S-DLLME）[150]。通常，傳統(tǒng)固體、半固體和粘性生物樣品的分析中，樣品制備、提取和分離往往需要好幾步，而基質(zhì)固相分散（MSPD）可將所有這些步驟合并為一步完成[142]。除上述方法外，人們也對鹽析旋渦輔助液液微萃?。╯alting out-VALLME）[156]、QuEChERS[138]、基于環(huán)糊精的聚合物[157]等進行了研究，效果良好。

與普遍采用的色譜方法和色譜質(zhì)譜聯(lián)用方法相比，免疫學方法具有方便、靈敏、可小型化、允許實時檢測的特點[158]。自MCELROY和WEISS[159]制備出多克隆抗體，建立展青霉素檢測的酶聯(lián)免疫法（ELISA）以來，已開發(fā)出了表面等離子體共振免疫分析法、化學發(fā)光免疫分析法、近紅外熒光免疫分析法、石英晶體微天平（QCM）免疫分析法、熒光免疫分析法等多種測定展青霉素的免疫學方法[158,160-161]。然而，免疫學方法使用的免疫抗體由動物產(chǎn)生，昂貴且不可再生，為提高檢測性能，展青霉素免疫學檢測可改用合成的生物受體，如寡核苷酸適配體[158]。與抗體相比，寡核苷酸適配體親和力高、穩(wěn)定、可與多種目標物結(jié)合、合成簡單[162-163]。用 DNA適配體PAT-11建立的基于酶-發(fā)色底物系統(tǒng)的展青霉素檢測方法，線性范圍在0.05—2.5 μg·L-1，檢出限低至0.048 μg·L-1[158]。展青霉素的近紅外熒光免疫分析法也很靈敏，檢出限可低至0.06 μg·L-1[161]。此外，還有基于分子印跡溶膠-凝膠聚合物的石英晶體微天平（MIP-QCM）傳感器展青霉素檢測技術(shù)[164]。

展青霉素是一種相當穩(wěn)定的化合物，無可靠的方法加以清除，而利用快速、專一的方法對潛在的展青霉素產(chǎn)毒真菌進行早期檢測，可以在展青霉素達到不可接受的水平前，甚至是展青霉素合成前，阻止其進入食物鏈[165]。PCR是檢測食品中產(chǎn)展青霉素真菌的有效方法[166]，針對真菌毒素生物合成或調(diào)控通路中的目標基因開發(fā)探針[167-170]，擴展青霉DNA檢測限可低至ng·mg-1級[165,171]，建立的（多重）實時PCR方法可同時檢測多種，甚至數(shù)十種產(chǎn)展青霉素真菌[172-173]。ELISA也可用于真菌鑒別[37]，用常見的食源性真菌橘灰青霉（P. aurantiogriseum）制備的抗原可與測試的45種青霉屬真菌中的43種反應[174]。

7 展望

果樹種類和品種多，地域分布廣，對展青霉素產(chǎn)毒真菌的敏感性存在差異。開展果樹抗展青霉素產(chǎn)毒真菌鑒定，篩選抗性品種和抗性種質(zhì)資源，可為抗性品種的培育和推廣創(chuàng)造有利條件。許多真菌都能產(chǎn)生展青霉素，但不同真菌之間以及同一種真菌不同菌株之間產(chǎn)毒能力有異。有必要針對主要果品及其主產(chǎn)區(qū)，開展展青霉素產(chǎn)毒真菌收集、分離和鑒定評價研究，建立展青霉素產(chǎn)毒真菌資源庫，明確展青霉素優(yōu)勢產(chǎn)毒菌株及其區(qū)域分布，并以其為對象，有針對性地進行展青霉素污染防控研究。現(xiàn)已明確展青霉素生物合成通路約由10步構(gòu)成，棒曲霉和擴展青霉的展青霉素生物合成基因簇均包含相同的15個基因。如能針對展青霉素生物合成通路中的關(guān)鍵步驟、關(guān)鍵酶和關(guān)鍵基因展開研究，提出阻遏展青霉素生物合成的技術(shù)和產(chǎn)品，必將為展青霉素污染防控開辟更廣闊的前景。果品及其制品一旦被展青霉素污染就很難徹底清除，因此，預防和控制產(chǎn)毒真菌侵染是展青霉素污染防控的關(guān)鍵，除栽培抗病品種和加強田間管理外，化學防控和生物防控是重要技術(shù)手段。為應對展青霉素產(chǎn)毒真菌抗藥性發(fā)展，應持續(xù)從新研發(fā)的藥劑中篩選高效、低毒的低風險殺菌劑。使用微生物拮抗劑是水果采后病害防控最有前途的殺菌劑替代方案。有鑒于此，應高度重視生物拮抗劑，特別是拮抗菌的發(fā)掘與利用，包括單獨使用和與低風險殺菌劑、貯藏技術(shù)等的結(jié)合使用。開發(fā)和利用對展青霉素產(chǎn)毒真菌有很好抑制作用的植物源性成分也屬生物防控范疇，已多有報道。對于果品制品的展青霉素污染防控，就加工環(huán)節(jié)而言，主要是展青霉素污染脫除與消減技術(shù)和產(chǎn)品的研究。關(guān)于展青霉素檢測技術(shù)，今后的發(fā)展方向是快速、高效、經(jīng)濟、環(huán)保，特別是提取、凈化技術(shù)和產(chǎn)品、現(xiàn)場檢測技術(shù)以及與其他毒素和污染物的同時檢測技術(shù)的研發(fā)。此外，展青霉素產(chǎn)毒真菌的早期檢測技術(shù)也是一個值得關(guān)注的領(lǐng)域。展青霉素污染主要發(fā)生在腐爛、變質(zhì)的果實和果品制品中，目前雖有不少關(guān)于果品及其制品展青霉素污染的報道，但關(guān)于展青霉素污染風險評估的報道尚不多見，今后這方面的研究應予以加強，特別是中國這樣一個果品生產(chǎn)、消費和加工大國。

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（責任編輯 趙伶俐）

Occurrence, Control and Determination of Patulin Contamination in Fruits and Fruit Products

NIE JiYun
(Institute of Pomology, Chinese Academy of Agricultural Sciences/Laboratory of Quality & Safety Risk Assessment for Fruit(Xingcheng), Ministry of Agriculture/Quality Inspection and Test Center for Fruit and Nursery Stocks, Ministry of Agriculture(Xingcheng), Xingcheng 125100, Liaoning)

2017-04-26；接受日期：2017-07-24

國家現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系（CARS-27）、國家農(nóng)產(chǎn)品質(zhì)量安全風險評估重大專項（GJFP2017003）、中國農(nóng)業(yè)科學院科技創(chuàng)新工程（CAAS-ASTIP）

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