果品主要真菌毒素污染檢測、風(fēng)險評估與控制研究進(jìn)展

李志霞，聶繼云，閆震，張曉男，關(guān)棣鍇，沈友明，程楊

（中國農(nóng)業(yè)科學(xué)院果樹研究所/農(nóng)業(yè)部果品質(zhì)量安全風(fēng)險評估實驗室（興城），遼寧興城 125100）

果品主要真菌毒素污染檢測、風(fēng)險評估與控制研究進(jìn)展

李志霞，聶繼云，閆震，張曉男，關(guān)棣鍇，沈友明，程楊

（中國農(nóng)業(yè)科學(xué)院果樹研究所/農(nóng)業(yè)部果品質(zhì)量安全風(fēng)險評估實驗室（興城），遼寧興城 125100）

果品在生產(chǎn)、貯運過程中易發(fā)生真菌性病害，不但可引起腐爛或腐敗，帶來嚴(yán)重的經(jīng)濟(jì)損失，部分霉菌還可能產(chǎn)生真菌毒素對人體健康造成潛在危害。真菌毒素是一類由絲狀真菌在適宜條件下產(chǎn)生的有毒次級代謝產(chǎn)物，是繼農(nóng)藥殘留、重金屬污染后，影響果品質(zhì)量安全的又一類關(guān)鍵風(fēng)險因子，具有強毒性。大量研究表明，真菌毒素可致DNA損傷，低濃度下即可對人和動物健康造成危害，使肝臟、腎臟和胃腸道發(fā)生病變，并且可致癌、致畸、致突變等，研究真菌毒素的污染狀況，進(jìn)行精準(zhǔn)檢測、風(fēng)險評估和控制，對于果品質(zhì)量安全研究具有重要意義。展青霉素（Patulin，PAT）、黃曲霉毒素（Aflatoxins，AF）、鏈格孢毒素（Alternaria toxins）和赭曲霉毒素A（Ochratoxin A，OTA）是存在于果品中的主要真菌毒素種類，國際癌癥研究機構(gòu)（IARC）分別將PAT、AFB1、AFM1和OTA列為第3類、1類、2B類和2B類致癌物質(zhì)。通常果品中檢出的真菌毒素含量極低，因此對檢測方法的要求較高，目前主要的分析方法有薄層色譜法、高效液相色譜（含質(zhì)譜聯(lián)用）技術(shù)、氣相色譜（含質(zhì)譜聯(lián)用）技術(shù)、毛細(xì)管電泳技術(shù)等，但往往由于化學(xué)結(jié)構(gòu)和性質(zhì)各異，無法采用一種標(biāo)準(zhǔn)方法完成對所有真菌毒素的定量測定。因此，篩選準(zhǔn)確、高效、快速的檢測方法也是該領(lǐng)域當(dāng)今的研究熱點。迄今為止，已有80個國家和地區(qū)制定了果品中真菌毒素的限量標(biāo)準(zhǔn)來保護(hù)消費者健康，但均未涉及鏈格孢霉毒素。許多國家均不同程度地開展了真菌毒素風(fēng)險評估研究，基于毒理學(xué)數(shù)據(jù)的評估結(jié)果表明，大多情形下通過果品攝入的真菌毒素水平極低，不會對居民健康產(chǎn)生危害。果品中的真菌毒素可采用化學(xué)、物理、生物等方法進(jìn)行防治、降解和控制，但無法將被真菌毒素污染后產(chǎn)品中的毒素完全脫除，果品中真菌毒素污染重在“防”而非“除”。 本文從果品中主要真菌毒素的種類、污染狀況、毒性、檢測方法、限量標(biāo)準(zhǔn)、風(fēng)險評估及控制技術(shù)等方面進(jìn)行概述，同時，對果品中真菌毒素的重點研究方向進(jìn)行了展望，以期為該領(lǐng)域研究者提供參考。

果品；真菌毒素；展青霉素；黃曲霉毒素；鏈格孢毒素；赭曲霉毒素A；風(fēng)險評估

0 引言

中國是果品生產(chǎn)大國，2014年水果（含瓜類）和堅果產(chǎn)量分別達(dá)2.5×108t和3.6×106t，果品總產(chǎn)量居世界首位[1]。果品是國民日均膳食中除蔬菜和糧食外攝入量（推薦300 g）最多的農(nóng)產(chǎn)品，其質(zhì)量安全關(guān)乎國計民生和社會安定，成為政府高度重視、社會廣泛關(guān)注的熱點和焦點[2]。越來越多的研究表明，真菌毒素污染是繼農(nóng)藥殘留、重金屬污染后，影響果品質(zhì)量安全的又一類關(guān)鍵風(fēng)險因子，不僅為害人體健康，也直接造成嚴(yán)重的經(jīng)濟(jì)損失和頻繁的國際貿(mào)易糾紛，引起了國際社會的廣泛關(guān)注[3-6]。

真菌毒素（Mycotoxin）是一類由絲狀真菌在適宜條件下產(chǎn)生的有毒次級代謝產(chǎn)物，可自然發(fā)生于果品生產(chǎn)、采收、貯藏和運輸過程的各個環(huán)節(jié)[6]，其共同毒性主要是致DNA損傷和細(xì)胞毒性，低濃度下即可對人和動物健康造成危害，使肝臟、腎臟和胃腸道發(fā)生病變，甚至致癌、致畸、致突變等[7-9]。關(guān)于果品中的真菌毒素，不少發(fā)達(dá)國家均不同程度地開展了真菌毒素產(chǎn)生、鑒定、監(jiān)測、防控等相關(guān)研究[10-15]。目前，中國對真菌毒素檢測識別的研究大多集中在谷物、糧食、糧油等農(nóng)產(chǎn)品上[16-18]，果品中對蘋果及制品展青霉素污染研究相對較多[19-20]，近年來開始有對蘋果汁中鏈格孢霉毒素和果酒中赭曲霉毒素 A的少量報道[21-22]，中國果品真菌毒素研究遠(yuǎn)不能滿足政府監(jiān)管、產(chǎn)業(yè)發(fā)展和公眾消費的需求。因此，有步驟、有重點地開展果品真菌毒素研究具有重要的現(xiàn)實和科學(xué)意義。

1 果品中主要真菌毒素的種類、污染及其毒性

影響果品質(zhì)量安全且普遍存在于果品中的真菌毒素主要包括展青霉素、黃曲霉毒素、鏈格孢毒素和赭曲霉毒素A，其毒性特點各不相同。

1.1 展青霉素（Patulin，PAT）

PAT又稱棒曲霉素、珊瑚青霉毒素，是由曲霉和青霉等真菌產(chǎn)生的一種次級代謝產(chǎn)物，主要由擴(kuò)展青霉產(chǎn)生[23]。PAT首先在霉?fàn)€蘋果和蘋果汁中被發(fā)現(xiàn)，由于果皮受傷被病原菌侵染即可誘導(dǎo)其產(chǎn)生，同時極易感染周圍的健康果肉組織[24]，且在冷藏條件下、加工過程中和制品中均能夠穩(wěn)定存在[25-26]，因此PAT被認(rèn)為是全球果品中最重要的毒素種類，不但廣泛存在于蘋果及其制品中，在梨、草莓、藍(lán)莓、櫻桃等果品中也均有檢出[5,12,27]。據(jù)報道，新鮮水果中很少有PAT產(chǎn)生，霉菌侵染的果品中腐爛部位PAT含量最高，可高達(dá)1 000 μg·kg-1以上；整果平均含量可達(dá)21—746 μg·kg-1；用腐爛原料果制成的水果制品中 PAT可達(dá)0.79—140 μg·kg-1[5,27]。

PAT在20世紀(jì)60年代被重新分類界定為真菌毒素。因?qū)θ梭w具有潛在致癌性，國際癌癥研究機構(gòu)（IARC）將其列為第3類[28]。毒理學(xué)試驗表明，PAT具有影響生育、免疫、遺傳、神經(jīng)系統(tǒng)、致癌、致畸等毒理作用[29-30]。按物種和暴露量不同，PAT的半致死劑量LD50范圍約為15—25 mg·kg-1[5]，對人體的危害很大，急性癥狀包括肺和腦水腫，肝、脾和腎功能損害，以及胃腸功能紊亂、免疫系統(tǒng)受損等；慢性癥狀包括神經(jīng)麻痹、致畸、胚胎毒性、致原生質(zhì)膜破裂、使DNA、RNA和蛋白質(zhì)合成受阻等[3,31]。

1.2 黃曲霉毒素（Aflatoxins，AF）

AF是一類化學(xué)結(jié)構(gòu)相似的二氫呋喃香豆素的衍生化合物，主要由黃曲霉和寄生曲霉產(chǎn)生。1960年，英國有10萬只火雞死于“火雞X病”，進(jìn)一步研究發(fā)現(xiàn)這些火雞的死因是吃了被黃曲霉污染的花生粕，由此AF被發(fā)現(xiàn)并得到廣泛研究[32]。AF主要有B1、B2、G1和G24種，以AFB1毒性最大和研究最多。最易受AF污染的是糧食作物，近年來，堅果、新鮮水果及其干制品中也陸續(xù)有AF檢出，特別是干果和堅果中較常見。JUAN等[33]研究表明，摩洛哥拉巴特-薩累地區(qū)的核桃、開心果、葡萄干、無花果干中AFB1平均含量為0.16—367.6 μg·kg-1。IQBAL等[34]對巴基斯坦2個省96個棗樣品及57個棗制品進(jìn)行了AF含量檢測，39.6%的棗樣品和31.6%的棗制品均有AF檢出，含量在2.76—4.96 μg·kg-1，分別有13.7%和17.0%的樣品污染水平超過了AFB1和AF總量的限量值。BAMBA等[35]發(fā)現(xiàn)檸檬被病原菌Aspergillus flavus侵染后，AF含量可達(dá)141.3—811.7 μg·kg-1。

AF毒性極強，1993年AFB1被IARC劃定為1類致癌物，是目前已知最強致癌物之一；2002年AFM1被列為2B類致癌物[36-37]。按物種和暴露量不同，AFB1的LD50在0.3—18 mg·kg-1左右[5]，對哺乳動物、鳥類、魚類具有致癌、致畸、致突變和肝毒性，這和 AFB1可與細(xì)胞DNA結(jié)合的特性有關(guān)[38]。AFB1毒性極強，是氰化鉀的10倍、砒霜的68倍，在亞洲和非洲進(jìn)行的許多流行性病學(xué)研究表明，食物中AF含量與肝細(xì)胞癌癥發(fā)生呈正相關(guān)[39]。

1.3 鏈格孢霉毒素（Alternaria toxins）

鏈格孢菌可在水果運輸和儲存過程中或低溫潮濕環(huán)境下產(chǎn)生并繁殖，導(dǎo)致水果腐敗。鏈格孢菌產(chǎn)生的真菌毒素代謝物按結(jié)構(gòu)可分為3類：四氨基酸衍生物—細(xì)交鏈格孢菌酮酸（Tenuazonic acid，TeA）；二苯并吡喃酮衍生物—交鏈孢酚（Alternariol，AOH）、交鏈孢酚單甲醚（Alternariol monomethyl ether，AME）和鏈格孢霉素（Altenuene，ALT）；二萘嵌苯類衍生物—細(xì)格菌毒素Ⅰ、Ⅱ、Ⅲ（AltertoxinⅠ、Ⅱ、Ⅲ，ATX-Ⅰ、ATX-Ⅱ、ATX-Ⅲ）等[40]。果品中最為常見的類型為AOH、AME和TeA，迄今為止，已在草莓、黑莓、紅醋栗、藍(lán)莓、蘋果、柑桔、橄欖等果品[41-46]及制品中[47-48]檢測到鏈格孢霉毒素，漿果類水果的檢出率較高，AOH、AME、TeA檢出率達(dá)25%—75%，含量最高可達(dá)2778 μg·kg-1，腐爛部位含量更高[41-42]。

鏈格孢霉毒素對細(xì)菌和哺乳動物具有致癌、致畸、致突變和細(xì)胞毒性，AOH和AME急性毒性較弱但可顯示協(xié)同效應(yīng)，TeA毒性最強[5]。PANIGRAHI等[49]試驗顯示，TeA、AOH、ATX I 和ALT的 LD50分別為75、100、200和375 μg·mL-1。LIU等[50]發(fā)現(xiàn)，河南林縣食管癌人群高發(fā)與當(dāng)?shù)丶Z食被鏈格孢霉毒素污染有關(guān)。ZHOU和QIANG[51]研究表明，TeA在濃度12.5—400 g·mL-1時，可抑制白鼠成纖維細(xì)胞、倉鼠肺細(xì)胞、人體肝細(xì)胞的再生和總蛋白含量。有研究者認(rèn)為，TeA可能與發(fā)生在非洲的人類血液紊亂疾病“奧尼賴?。╫nyalai）”有關(guān)[52]。

1.4 赭曲霉毒素A（Ochratoxin A，OTA）

OTA于1965年首次從赭曲霉中分離出來，是由曲霉屬和青霉屬產(chǎn)生的有毒代謝產(chǎn)物，由一個氯化的二氫異香豆素衍生物通過一個肽鍵與L-β-苯丙氨酸的7-羧基相連而形成，因此對溫度和水解作用表現(xiàn)極為穩(wěn)定[5]。研究[53-54]表明，焙烤只能使OTA毒性減少20%，蒸煮對OTA毒性不具有破壞作用，溫度達(dá)到100—200℃都不能完全分解。OTA廣泛分布于自然界，果品中以干果、葡萄及其制品污染率最高，研究最廣。果酒和果醋、干果中OTA污染發(fā)生率可達(dá)50%—100%，含量分別為0.2—6.4 μg·L-1和0.1—6 900 μg·L-1[53,55-56]。另外，在被病原菌侵染的桃、櫻桃、草莓、蘋果和柑橘等水果中也發(fā)現(xiàn)有少量的OTA，且去除腐爛部位后仍可進(jìn)一步侵染，含量為0.15—29.2 μg·kg-1[57-58]。

OTA具有強烈的腎毒性、肝毒性和免疫毒性，并有致癌、致畸和致突變作用，被IARC劃定為2B類致癌物[56,59]。在已發(fā)現(xiàn)的真菌毒素中，OTA被認(rèn)為僅次于黃曲霉毒素而列第二位。由于其毒性較高，JECFA建議其每日耐受攝入量為 0.2—14 ng·kg-1bw·d-1[60]，EFSA建議其每日耐受攝入量為17 ng·kg-1bw·d-1[61]。前人研究[62]發(fā)現(xiàn)，巴爾干地方性腎炎（BEN）和尿路癌（UT）的發(fā)生可能與OTA攝入有密切關(guān)系，患病人群血液中 OTA含量明顯高于未感染人群，腎臟是OTA毒性作用的主要靶器官。也有研究者[63]認(rèn)為，這2種疾病的發(fā)生與馬兜鈴酸（Aristolochic acids）的攝入有相關(guān)性，而非OTA。

2 果品真菌毒素分析方法

通常，果品中真菌毒素含量極低，需篩選準(zhǔn)確、靈敏的方法對其進(jìn)行檢測，且由于不同真菌毒素的化學(xué)結(jié)構(gòu)和性質(zhì)各異，無法采用一種標(biāo)準(zhǔn)方法完成對所有真菌毒素的定量測定[64]。目前，真菌毒素檢測常用的樣品前處理方法有液液萃取技術(shù)（liquid-liquid extraction）、固相萃取技術(shù)（solid phase extraction，SPE）、超臨界流體萃取技術(shù)（supercritical fluid extraction，SFE）、凝膠色譜凈化技術(shù)（gel permeation chromatography，GPC）和免疫親和層析凈化技術(shù)（immunoaffinity cleanup，IAC）等；主要檢測分析方法有薄層色譜法、高效液相色譜法（含液質(zhì)聯(lián)用技術(shù)）、氣相色譜法（含氣質(zhì)聯(lián)用技術(shù)）、毛細(xì)管電泳技術(shù)等，其中以LC-MS-MS的應(yīng)用前景最廣闊。

2.1 薄層色譜法（thin-layer chromatography，TLC）

TLC是檢測真菌毒素的一種傳統(tǒng)方法，其優(yōu)點是可同時做大量樣品、成本較低，缺點是樣品前處理相對繁瑣復(fù)雜、所用溶劑和展開劑毒性較大、靈敏度較低，因此實際應(yīng)用受到一定限制。但隨著與SPE、IAC等前處理手段聯(lián)用、前處理自動化研究等，TLC法在真菌毒素多殘留檢測方面仍有一定的應(yīng)用前景。WELKE等[65]采用TLC法結(jié)合電荷耦合裝置（CCD）對蘋果汁樣品中的PAT含量進(jìn)行了測定，檢出限低至0.005 μg·L-1。ELHARIRY等[66]采用TLC-PCR-RAPD方法對腐爛蘋果不同部位及所得果汁中的 PAT污染情況進(jìn)行了測定，并對分離的8個條帶進(jìn)行了基因測序。SANTOS等[67]采用IAC-TLC法對生咖啡中的OTA進(jìn)行測定，方法回收率為98.4%—103.8%，檢出限達(dá)到 0.5 μg·kg-1。

2.2 高效液相色譜法（high-performance liquid chromatography，HPLC）

HPLC法測定真菌毒素是20世紀(jì)70年代中期發(fā)展起來的，具有分離和檢測效能高、分析快速等特點，是目前真菌毒素檢測最重要的方法，應(yīng)用極為廣泛。某些自身產(chǎn)生熒光的真菌毒素如AFs、OTA等，可直接用配有熒光檢測器（fluorescence detector，F(xiàn)LD）的HPLC進(jìn)行分析，分子中不含發(fā)色基團(tuán)的毒素（如伏馬菌素）或本身可產(chǎn)生熒光但強度較弱的毒素（如AFBl和AFGl）進(jìn)行HPLC分析時，需經(jīng)柱前或柱后衍生、優(yōu)化流動相條件（如使用離子對試劑、改變流動相 pH 等）以增強熒光信號方能定量檢測[3]。MYRESIOTIS等[68]采用QuEChERS 結(jié)合HPLC-DAD方法對石榴及石榴汁中AOH、AME和TEN等3種鏈格孢霉毒素進(jìn)行檢測，結(jié)果顯示方法線性關(guān)系在0.9937以上，RSDs在0.53%—2.52%，回收率在82.0%—109.4%，AOH和AME檢出限為0.015 μg·g-1，TEN檢出限為0.02 μg·g-1。HPLC與質(zhì)譜技術(shù)聯(lián)用可在提高分析靈敏度和可靠性的同時，又能檢測并鑒定多種不同種屬的真菌毒素，因此，LC-MS或LC-MS/MS技術(shù)已越來越多地用于真菌毒素多殘留測定。SULYOK等[69]采用半定量LC-MS-MS方法，對160個水果和堅果樣品中 23種真菌毒素進(jìn)行了多殘留檢測；PERRE等[41]也建立了半定量LC-TOF-MS方法，對50個漿果樣品中AOH、AME、OTA、FB1、FB2、FB3等6種真菌毒素進(jìn)行了分析；ZWICKEL等[70]建立的HPLC-MS/ MS方法可對果蔬汁和酒中12種鏈格孢霉毒素進(jìn)行同時檢測，檢出限和定量限分別為0.10—0.59 μg·L-1和0.4—3.1 μg·L-1。國內(nèi)，研究者先后對水果及果汁中多種真菌毒素的HPLC（MS/MS）檢測技術(shù)進(jìn)行了有益探索和分析[15,21-22,71]，這些研究為今后果品中真菌毒素的篩查與分析提供了參考和依據(jù)。

2.3 氣相色譜法（gas chromatography，GC）

某些食品中真菌毒素的GC測定方法已標(biāo)準(zhǔn)化，一般用于食品中真菌毒素的定期鑒別與定量，通常與質(zhì)譜技術(shù)聯(lián)用，使用火焰離子檢測器（flame ionization detector，F(xiàn)ID）或傅里葉變換紅外光譜技術(shù)（fourier transform infrared spectroscopy，F(xiàn)TIR）對目標(biāo)物質(zhì)進(jìn)行分析[64]。大多數(shù)真菌毒素不具有揮發(fā)性，需衍生后再使用 GC法進(jìn)行測定。KHARANDI等[72]利用QuEChERS前處理、N,O-雙（三甲基硅烷基）三氟乙酰胺衍生和GC-MS方法對蘋果汁中PAT進(jìn)行測定，回收率為79.9%—87.9%，檢出限和定量限分別為0.4 μg·L-1和 1.3 μg·L-1，RSDs低于 9.5%。JIMéNEZ和MATEO[73]采用HPLC和GC方法同時對香蕉中由鐮刀菌產(chǎn)生的單端孢霉烯族毒素等 11種真菌毒素進(jìn)行檢測，發(fā)現(xiàn)大多數(shù)真菌毒素采用HPLC法測定效果更好，僅2種毒素可用GC法測定?？傮w而言，鑒于HPLC法低成本、靈敏、高效等優(yōu)勢，其在真菌毒素檢測中大范圍推廣應(yīng)用的可能性更大。

2.4 毛細(xì)管電泳技術(shù)（capillary electrophoresis，CE）

CE是20世紀(jì)80年代發(fā)展起來的一種新型液相分離技術(shù)，具有分離模式多、分離效率高、分析速度快、試劑和樣品用量少、易于調(diào)控、對環(huán)境污染小等優(yōu)點，目前在很多領(lǐng)域均有應(yīng)用，但對果品中真菌毒素的檢測僅見蘋果汁中 PAT和果酒中 OTA的報道。MURILLO-ARBIZU等[74-75]分別采用毛細(xì)管微乳電動色譜法和毛細(xì)管膠束電動色譜法測定了 20份市售蘋果汁中的PAT含量，檢出限分別為3.2 μg·L-1和0.7 μg·L-1，定量限分別為8.0 μg·L-1和2.5 μg·L-1，回收率分別為75.3%和80.2%。GONZáLEZ-PE?AS等[76]和LUQUE等[77]分別采用CE-DAD法和毛細(xì)管膠束電動色譜法測定了 27份加度葡萄酒以及從果品等食品分離出的真菌條帶中的OTA含量，同時與HPLC方法進(jìn)行比較驗證，證明CE可有效檢測真菌毒素，但與LC-MS-MS方法相比檢出限較高，用于實際樣品檢測時靈敏性可能達(dá)不到要求。

3 果品真菌毒素限量標(biāo)準(zhǔn)

為保護(hù)消費者健康，許多國家和國際組織均制定了水果及相關(guān)產(chǎn)品中真菌毒素的限量標(biāo)準(zhǔn)。黃曲霉毒素是最受關(guān)注的真菌毒素種類，近年來其他真菌毒素限量標(biāo)準(zhǔn)的制定也得到了較快發(fā)展[6]，但迄今尚無國家和地區(qū)制定果品中鏈格孢霉毒素限量。國際食品法典委員會（Codex Alimentarius Commission，CAC）分別于 1981年、1987年、1995年和 2003年舉行了4次全球范圍的真菌毒素限量標(biāo)準(zhǔn)調(diào)查，并隨后發(fā)布了相關(guān)限量標(biāo)準(zhǔn)[78-81]。根據(jù)聯(lián)合國糧食及農(nóng)業(yè)組織（Food and Agriculture Organization，F(xiàn)AO）的最新調(diào)查[81]，制定果品中真菌毒素限量標(biāo)準(zhǔn)的國家和地區(qū)共涉及80個，中國在2011年對食品中真菌毒素限量標(biāo)準(zhǔn)（GB 2761—2011）[82]進(jìn)行了修訂。國際組織和各國關(guān)于果品中真菌毒素的限量標(biāo)準(zhǔn)制定情況見表1。

目前已制定的果品真菌毒素限量標(biāo)準(zhǔn)，大多對所有果品、干果與堅果中的AF和OTA以及果汁、果醬、果酒或蘋果制品中的PAT關(guān)注度較高。主要真菌毒素中，制定 AF（包括AFB1、AFB1+B2+G1+G2、AFM1等）限量標(biāo)準(zhǔn)的國家最多，涉及77個國家和地區(qū)，僅CAC、白俄羅斯和南斯拉夫未制定；其次為PAT，涉及55個國家和地區(qū)；再次為OTA和ZEA，分別涉及36個和3個國家和地區(qū)；DON僅烏克蘭有制定；T-2僅亞美尼亞有制定。歐盟（European Union，EU）、烏克蘭和伊朗制定的果品真菌毒素限量標(biāo)準(zhǔn)涉及的產(chǎn)品種類和毒素種類最為全面。相對而言，EU制定的果品真菌毒素限量標(biāo)準(zhǔn)較為嚴(yán)格，也有個別國家對指定產(chǎn)品的毒素限量比EU更嚴(yán)格，如摩洛哥規(guī)定開心果和杏仁中AFB1的限量為1 μg·kg-1；克羅地亞規(guī)定杏仁、榛子、核桃中AFB1+B2+G1+G2的限量為3 μg·kg-1。大多數(shù)國家制定的AFB1限量為5 μg·kg-1，AFB1+B2+ G1+G2為10或20 μg·kg-1，PAT為50 μg·kg-1；中國制定的標(biāo)準(zhǔn)包括熟制堅果及籽類中 AFB1限量為 5 μg·kg-1，除果丹皮外的水果及制品、果汁、果酒中PAT的限量為50 μg·kg-1（表1）。

4 果品真菌毒素污染風(fēng)險暴露評估

真菌毒素作為最主要的世界性農(nóng)產(chǎn)品質(zhì)量安全風(fēng)險因子之一，其風(fēng)險評估研究在全球范圍內(nèi)受到關(guān)注，也引起了科學(xué)家們的極大興趣。盡管各類食品中真菌毒素的污染情況已有廣泛研究，但目前國內(nèi)外關(guān)于真菌毒素風(fēng)險評估的報道主要集中在兩個方面：一是全膳食暴露風(fēng)險評估的研究[84-86]，二是不同人群攝入糧食產(chǎn)品中某（幾）種真菌毒素的膳食風(fēng)險研究[87-88]，關(guān)于果品中真菌毒素的膳食暴露風(fēng)險評估研究尚在探索階段。與其他危害因子風(fēng)險評估程序一致，果品中真菌毒素風(fēng)險評估包括危害識別、危害描述、暴露評估和風(fēng)險描述4個步驟，暴露評估是研究的核心內(nèi)容?，F(xiàn)有研究多為結(jié)合食品消費量和真菌毒素污染水平計算估計膳食攝入量的點評估方法，以及基于@RISK分析軟件的概率評估技術(shù)[60,89-90]，得出的膳食攝入量數(shù)據(jù)再與基于毒理學(xué)數(shù)據(jù)推導(dǎo)或制定出的人類健康指導(dǎo)值（表2）進(jìn)行比較，來評估人體膳食暴露風(fēng)險。

近幾年來，研究者針對不同人群，主要圍繞蘋果及制品中的PAT[30,84,89-90]、干果及葡萄制品中的OTA[56,85,91]、堅果和干果中的AF[84,91]等產(chǎn)品種類和真菌毒素種類進(jìn)行了膳食暴露風(fēng)險評估，結(jié)果顯示，除極少量高污染樣品、嬰幼兒和兒童等高風(fēng)險人群、P97.5或P99.9高百分位點值等情形下的膳食風(fēng)險可能超出推薦限值外，其他大多數(shù)情形下果品中真菌毒素污染水平極低，不會對居民健康產(chǎn)生危害，一般嬰幼兒和兒童的風(fēng)險高于成人。健康指導(dǎo)值常用每日耐受攝入量（The tolerable daily intake，TDI）、臨時每日耐受攝入量（provisional tolerable daily intake，PTDI）、臨時最大每日耐受攝入量（provisional maximum tolerable daily intake，PMTDI）、臨時每周耐受攝入量（provisional tolerable weekly intake，PTWI）等表示。目前報道的果品中真菌毒素膳食風(fēng)險評估結(jié)果如表 3所示。鑒于目前真菌毒素毒理學(xué)資料、膳食數(shù)據(jù)和污染數(shù)據(jù)的缺乏，科學(xué)家認(rèn)為真菌毒素膳食暴露風(fēng)險評估研究應(yīng)進(jìn)一步深入開展，同時亟需根據(jù)評估結(jié)果對真菌毒素限量標(biāo)準(zhǔn)進(jìn)行完善[6,60,83]。

表1 國際組織和各國制定的果品中真菌毒素限量標(biāo)準(zhǔn)[78,82-83]Table 1 Maximum tolerated levels for mycotoxins in fruits and the products worldwide

續(xù)表1 Continued table 1

續(xù)表1 Continued table 1

續(xù)表1 Continued table 1

表2 食品添加劑聯(lián)合專家委員會建議的主要真菌毒素健康指導(dǎo)值[60]Table 2 The Health-based guidance value for main mycotoxins made by JECFA

5 果品真菌毒素污染控制技術(shù)

果品中碳水化合物、水分等較高，極易被真菌病害侵染，侵染后因霉變、腐爛和產(chǎn)生真菌毒素等可造成巨大的經(jīng)濟(jì)損失。如何有效地削減和控制果品中真菌毒素的污染，保障其質(zhì)量安全是農(nóng)業(yè)和食品加工領(lǐng)域亟待解決的重要課題。李培武等[2]認(rèn)為，真菌毒素的控制應(yīng)包括控制霉菌生長、控制菌株產(chǎn)毒和消減毒素3個層面，目前研究最多的是毒素消減或脫毒技術(shù)，控制毒素產(chǎn)生和消減技術(shù)通常采用化學(xué)、物理或生物

學(xué)的方法，無論何種方法，對果品中的真菌毒素應(yīng)重在“防”而非“除”。

表3 果品中真菌毒素膳食暴露風(fēng)險評估報道Table 3 Reported dietary intake assessment and risks of mycotoxins in fruits and the products

5.1 化學(xué)方法

化學(xué)方法即使用殺菌劑、防腐劑、添加劑、抗氧化劑等化學(xué)物質(zhì)來抑制真菌和毒素生長或消減的方法。從安全角度來講，雖然并不提倡使用這些化學(xué)物質(zhì)，但考慮到實際生產(chǎn)中真菌病害侵染往往可造成較大的經(jīng)濟(jì)損失，同時相比之下真菌毒素的毒性較這些化學(xué)物質(zhì)要高得多，因此，目前化學(xué)處理仍為果品中主要使用的真菌病害及其毒素控制方法[6]。MALMAURET等[97]研究表明，許多不使用化學(xué)殺菌劑的有機蘋果中真菌毒素污染比法國傳統(tǒng)生產(chǎn)的蘋果中高。類似的結(jié)果分別在西班牙、意大利、比利時等國家也有報道[30,90,98-99]。其他化學(xué)物質(zhì)如芳香物質(zhì)、精油等也被用于霉菌和真菌毒素的防控中，NERI等[100]用反式-2-己烯醛處理接種青霉菌（P. expansum）的梨果實，發(fā)現(xiàn)霉菌生長受到顯著抑制。GEMEDA等[101]用精油處理曲霉的試驗發(fā)現(xiàn)，3種精油均可不同程度地抑制霉菌生長和AF的產(chǎn)生，效果優(yōu)于合成的防腐劑。在發(fā)現(xiàn)更為有效的控制真菌毒素方法前，化學(xué)方法可能還將在今后較長時間內(nèi)普遍應(yīng)用。事實上，只要按照國家規(guī)定的濃度、用量和安全間隔期使用，殺菌劑、防腐劑、添加劑、抗氧化劑等物質(zhì)對果品的安全及人體健康產(chǎn)生的危害是很有限的[102]。

5.2 物理方法

物理方法主要有剔除霉粒法、物理吸附法、淘洗法、輻照法、高溫高壓法等，傳統(tǒng)而言，熱處理為真菌毒素消減最常用的方法，包括熱水浸洗、干熱處理、蒸汽加熱、熱水噴淋處理或日光晾曬等，研究者分別在蘋果、荔枝、芒果、西瓜、桃、梨、草莓等果品上進(jìn)行了相關(guān)試驗，表明這些熱處理方法可將真菌毒素污染水平降低 20%—70%，但不能完全脫除[6]。近幾十年來，輻照法因節(jié)約能源、無二次和交叉污染、無殘留、工藝簡單、快速高效等優(yōu)點，被廣泛用于食品和農(nóng)產(chǎn)品真菌毒素降解領(lǐng)域，其中最為常用的是γ射線輻照。AZIZ等[103]研究表明，采用1.5—3.5 kGy的γ射線對草莓、杏、李、桃、葡萄、棗、無花果、蘋果、梨、桑葚進(jìn)行輻照處理后，樣品中PAT、OTA、AF等多種真菌毒素污染均有顯著降低，未經(jīng)輻照的樣品污染概率為輻照樣品的5.4倍，但同樣不能徹底脫除。有人曾對輻照技術(shù)處理后產(chǎn)品的營養(yǎng)水平和安全性提出質(zhì)疑，F(xiàn)AO/IAEA/WHO聯(lián)合專家委員會研究小組得出結(jié)論：食品總體平均吸收劑量不超過10 kGy時沒有毒理學(xué)危險，同時營養(yǎng)學(xué)上也是安全的。目前許多國家和地區(qū)已制定了相關(guān)的食品輻照標(biāo)準(zhǔn)[104]。

5.3 生物防治

目前采用的主要生物防治方法為生物競爭抑毒技術(shù)和生防微生物及其活性物質(zhì)抑毒技術(shù)，后者在果品上較為常見。細(xì)菌和酵母是目前研究最多的生防微生物資源，其作用機理可能是誘導(dǎo)寄主防御系統(tǒng)、分泌分解酶、抵御活性氧傷害、緩解果實被氧化等[105]。目前，不少國家均有生防菌產(chǎn)品用于果品生產(chǎn)或貯藏環(huán)節(jié)的真菌病害防治，如德國的 Shemer?和Boni-Protect?，西班牙的 Candifruit?，美國的Aspire?和BIOSAVE?，中國的枯草芽孢桿菌等，應(yīng)用廣泛。SPADARO等[106]采用3種拮抗酵母和殺菌劑對4個品種蘋果中的擴(kuò)展青霉和PAT的控制進(jìn)行研究，發(fā)現(xiàn)拮抗酵母和殺菌劑均可不同程度地降低PAT污染濃度，其中AL27的抑菌效果與殺菌劑接近，可使侵染青霉病的金冠蘋果在22℃下保存7 d和1℃下保存56 d而不產(chǎn)生PAT。AHMED等[107]證實，Stifenia?和Scala?兩種生防菌可顯著抑制葡萄真菌病害以及葡萄和酒中OTA的發(fā)生，處理后可將OTA含量降低70%—84%。有研究者認(rèn)為，生物防治可能是未來果品中真菌毒素控制的最具競爭力的技術(shù)之一[6,105]。

6 展望

真菌毒素分布廣泛，對人和動物健康的潛在危害不容忽視，已引起世界各國的高度關(guān)注。果品作為人類最為重要的膳食來源之一，保證其質(zhì)量安全的重要性日益凸顯，精準(zhǔn)快速檢測技術(shù)、風(fēng)險評估、脫除技術(shù)成為今后關(guān)注的熱點問題。隨著國家對果品質(zhì)量安全科學(xué)研究的逐步加強和風(fēng)險評估的日益強化，中國對果品真菌毒素的研究也在不斷深入，但與發(fā)達(dá)國家相比尚存在較大差距，今后還需從以下幾個方面加強相關(guān)工作：第一，從果品和毒素種類看，目前研究主要集中在蘋果及其制品中的PAT、干果和葡萄及制品中的 OTA上，其他果品和毒素涉及極少，應(yīng)拓展對其他果品中如鏈格孢霉毒素、單端孢霉烯族毒素等的相關(guān)研究。第二，從毒素產(chǎn)生情況看，目前多為對病原菌的識別和發(fā)生規(guī)律研究，各毒素產(chǎn)毒與作用機理尚不完全明確，應(yīng)進(jìn)一步深入研究。第三，從檢測技術(shù)看，多集中在對傳統(tǒng)的實驗室分析技術(shù)優(yōu)化方面，快速檢測技術(shù)與配套產(chǎn)品的研發(fā)不能滿足實際需求，可能成為今后的研究重點。第四，從安全性角度看，果品真菌毒素限量標(biāo)準(zhǔn)的制修訂研究，建立既符合國際通行規(guī)則又具有本國國情的風(fēng)險評估技術(shù)，多種真菌毒素的混合污染[35,84]和聯(lián)合毒性對產(chǎn)品安全性的影響等，也是今后的重要研究內(nèi)容。第五，從毒素控制技術(shù)看，應(yīng)基于果品真菌毒素的產(chǎn)生規(guī)律、風(fēng)險評估等基礎(chǔ)研究，建立預(yù)警防控體系，實現(xiàn)與科學(xué)監(jiān)管、生產(chǎn)指導(dǎo)的對接。

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（責(zé)任編輯 趙伶俐）

Progress in Research of Detection, Risk Assessment and Control of the Mycotoxins in Fruits and Fruit Products

LI ZhiXia, NIE JiYun, YAN Zhen, ZHANG XiaoNan, GUAN DiKai, SHEN YouMing, CHENG Yang
(Institute of Pomology, Chinese Academy of Agricultural Sciences/Laboratory of Quality & Safety Risk Assessment for Fruit (Xingcheng), Ministry of Agriculture, Xingcheng 125100, Liaoning)

Fungi are major pathogens to fruit spoilage in the production, storage and transportation, and also are responsible for significant financial losses. In addition to their ability to cause fruit spoilage, some fungi may produce mycotoxins with potential harm to human health. Mycotoxins are a diverse group of toxic secondary metabolites produced by filamentous fungi under appropriate conditions. Followed by pesticide and heavy metal, the mycotoxins are considered as another important risk factor which can directly affect the quality and safety of fruits and fruit products. Numerous studies show that the mycotoxins can cause DNA damage and are harmful to human and animal health even at low concentrations. They caused liver, kidneys and gastrointestinal tractlesions or may be carcinogenic, teratogenic and mutagenic. Therefore, it is important to investigate the occurrence, accurate detection, risk assessment and control technology of mycotoxins in fruit and fruit products. The most common mycotoxins associated with fruits are patulin (PAT), aflatoxins (AF), alternaria toxins and ochratoxin A (OTA) which are respectively classified into 3, 1, 2B and 2B carcinogen by the International Agency for Research on Cancer (IARC). Usually, equipments with high standard configurations are needed for mycotoxin detection due to the extremely low concentrations in fruits and their products. Currently the main detection methods for mycotoxins include thin-layer chromatography, high performance liquid chromatography and mass spectrometry, gas chromatography and mass spectrometry, capillary electrophoresis technology, and so on. However, because of the different chemical structure and properties of special mycotoxin, it is incapable to use a standard method for the simultaneous quantitative determination of all mycotoxins. Therefore, it is a research hotspot to screen accurate, efficient and rapid detection methods for mycotoxins. To date, a total of 80 countries and regions have set the mycotoxin limits in fruits and fruit products to protect the health of consumers. It is to be regretted that there were no regulations for Alternaria toxins yet. Risk assessment results based on toxicological data in many countries were shown that dietary intakes of the mycotoxins from fruits and their products were very low in most cases and may not threaten the human health. Although the mycotoxins in fruits and their products could be prevented and degraded by chemical, physical or biological methods, there has not been an effective technology to complete detoxification in infected products. Hence, it is crucial to prevent mycotoxin production in fruits rather than remove. This review summarized the main mycotoxin types, occurrence, toxicities, detection methods, limit standards, risk assessments and control technologies in fruit and fruit products. And finally, the future research directions of fruit mycotoxins were prospected in order to provide a reference for researchers in this field.

fruit and fruit products; mycotoxin; patulin; aflatoxin; alternaria toxin; ochratoxin A; risk assessment

2016-05-13；接受日期：2016-08-01

國家農(nóng)產(chǎn)品質(zhì)量安全風(fēng)險評估重大專項（GJFP2016003）、中國農(nóng)業(yè)科學(xué)院科技創(chuàng)新工程項目（CAAS-ASTIP）

聯(lián)系方式：李志霞，Tel：0429-3598191；Fax：0429-3598185；E-mail：lizhixia@caas.cn。通信作者聶繼云，Tel：0429-3598178；Fax：0429-3598185；E-mail：jiyunnie@163.com