展青霉素(patulin),又称棒曲霉素,主要是由青霉属(Penicillium)、曲霉属(Aspergillus)、丝衣霉属(Byssochlamys)和拟青霉属(Paecilomyces)中的某些真菌产生的一种聚酮类次级代谢产物[1]。展青霉素的分子式为C7H6O4(相比分子质量154.12),是一类结构简单、性质稳定、水溶性较强的化合物[2-3]。1943年,Raistrick等[4]首次从灰黄青霉(Penicillium griseofulvum)中分离出该化合物,将其命名为patulin。实际上,关于展青霉素的相关研究最早可追溯到19世纪末[5],早于青霉素(penicillin)的发现。它具有广谱的抗生活性,最初被尝试用于感冒等疾病的治疗[6];但随后的研究发现,其使用过程中对人和动物产生较强的毒副作用;因此,在1963年被归类为真菌毒素[7]。展青霉素可侵害消化系统、神经系统、生殖系统的多个器官,引起急性和慢性症状,危害健康[8]。

展青霉素主要污染水果及其加工制品。扩展青霉(Penicillium expansum)是造成食品中展青霉素污染的主要真菌。P. expansum主要通过伤口等途径侵染成熟后的水果,寄主范围包括苹果、桃、梨、葡萄、猕猴桃、山楂等常见大宗和加工水果,是最重要的采后病原菌之一[2]。扩展青霉在侵染过程中产生大量的展青霉素,在果实的腐烂部位积累,并向未发病部位扩散,伴随鲜食流通和加工过程进入食物链,危害消费者的健康。果汁、果酱、果泥等加工制品是展青霉素污染的主要食品类别。世界卫生组织(World Health Organization,WHO)建议日常饮食中展青霉素的每天摄入量不超过0.4 μg/kg(以体质量计)[3]。目前,全球主要国家和地区均对水果制品中展青霉素的含量制订了严格的限量标准[9]。GB 2761—2017《食品安全国家标准 食品中真菌毒素限量》中对以苹果和山楂为原料制备的水果制品、饮料和酒类中展青霉素的最高限量标准均为50 μg/kg。然而,展青霉素污染仍是全球范围内普遍存在的问题,超标情况时有发生[10]。我国是水果生产和消费的第一大国,其中苹果汁的生产和出口量均居世界首位[11]。因此,展青霉素污染给我国水果及加工产业造成了严重的影响。本研究对展青霉素的生物合成和调控机制,以及其污染防控策略的相关研究进展进行了阐述,以期减少和降低展青霉素对生鲜果品及其加工制品的污染。

1 展青霉素的生物合成途径

20世纪70～80年代,人们通过突变株代谢产物分析等方法推测展青霉素的生物合成途径包括 10 步催化反应,但有些步骤是否存在分支途径还存在争议[1,9]。随后,部分可能参与展青霉素合成的催化酶及编码基因被陆续鉴定[11]。全基因组测序技术的快速发展推动了展青霉素生物合成机制的研究,展青霉素生物合成途径得到了全面解析。

1.1 展青霉素生物合成基因簇

真菌中与次生代谢产物合成途径相关基因通常在染色体上以基因簇的形式存在[12]。这些基因在特定的染色体区段内彼此相邻,有利于代谢途径的调控。基于全基因组测序技术,目前展青霉素生物合成基因簇已经在扩展青霉(P. expansum)、棒曲霉(Aspergillus clavatus)、灰黄青霉(P. griseofulvum)等霉菌的基因组中被鉴定[13-16]。在已测序的物种中,完整的展青霉素生物合成基因簇均由15个基因(PatA～PatO)组成,整个基因簇的长度约40 k碱基对。然而,不同种类的产展青霉素真菌中簇内基因的排列顺序并不相同[15,17-18]。

1.2 展青霉素生物合成的关键酶

基因功能注释表明展青霉素生物合成基因簇共包括11个催化酶编码基因(PatB、PatD、PatE、PatF、PatG、PatH、PatI、PatJ、PatK、PatN、PatO),3个转运蛋白编码基因(PatA、PatC 和PatM)和1个转录因子编码基因(PatL)。转录组测序、基因表达和基因敲除分析表明簇内所有基因都参与了展青霉素的生物合成途径,其中绝大部分成员是展青霉素合成的必需基因[14-15,19]。笔者团队以分离于苹果果实的P. expansum T01菌株为研究对象,在完成全基因组测序的基础上,通过基因敲除、底物饲喂和体外催化等方法对展青霉素生物合成途径中的关键催化酶及其编码基因进行了较为系统的研究[15,19]。目前,已经明确PePatK、PePatG、PePatH和PePatI编码的6-甲基水杨酸合酶、6-甲基水杨酸脱羧酶、间甲酚甲基羟化酶和间羟基苯甲醇羟化酶催化展青霉素合成的第1～4步[19]。其中,分子遗传学和生化证据表明第4步反应产物为龙胆醇,而不是间羟基苯甲醛,解决了长期以来在该分支步骤上的争议。此外,PePatN、PePatF、PePatD和PePatE编码的异环氧菌素脱氢酶、新展青霉素合成酶、乙醇脱氢酶和葡萄糖-甲醇-胆碱氧化还原酶负责催化第7～10步合成反应[19-20]。合成途径中第5和6步的催化酶还不确定,推测功能未知的PePatJ和 PePatO(异戊醇氧化酶)可能参与第 6步合成反应[19]。

1.3 展青霉素生物合成的亚细胞位点

真菌次生代谢合成途径中的酶通常定位在不同的亚细胞位点,这样有利于特定生化反应的高效发生,也能避免代谢产物对细胞自身产生毒性[21-22]。研究发现,展青霉素合成基因簇编码的15个蛋白也定位在不同的亚细胞位点[19]。其中,大部分的催化酶(PePatB、PePatD、PePatF、PePatG、PePatK和PePatN)定位在细胞质,催化第3和 4步反应的2个羟化酶PePatH和PePatI定位在内质网中,可能参与第6步反应的PePatO和PePatJ定位在液泡中[19]。值得关注的是,催化展青霉素合成最后一步反应的PePatE定位在细胞壁,将无细胞毒性的中间产物 E-ascladiol 转化为终产物展青霉素。终产物的胞外合成可能是真菌进化产生的一种自我保护机制[19]。这些催化酶的亚细胞定位对其催化相应步骤的生化反应至关重要。改变PePatH和PePatE的亚细胞定位后,P. expansum中展青霉素的合成途径被中断,不能产生终产物[19]。除参与合成途径的催化酶外,与中间产物转运相关的PePatC和PePatM定位于细胞质膜和液泡,PePatA定位于内质网,转录因子PePatL定位于细胞核[19]。

2 展青霉素生物合成的调控机制

展青霉素的生物合成受控于一套多层次的调控网络,合成途径特异性调控因子、环境信号-全局性调控因子以及一些表观遗传因子都参与其中[2,23](图1)。

2.1 合成途径特异性调控因子

真菌次级代谢基因簇通常会编码一个或多个途径特异性调控因子,直接参与基因簇基因的转录调控。已有研究表明,途径特异性调控因子存在于60%的真菌次级代谢基因簇中,其中大多数属于次级代谢物生物合成的正调控因子[24]。在丝状真菌中,最常见的途径特异性调控因子是Zn(II)2Cys6型锌指转录因子,如黄曲霉(Aspergillus flavus)中的黄曲霉毒素转录因子AflR。AflR能够特异性识别并结合黄曲霉毒素合成基因簇基因启动子区的保守序列5’-TCGN5CGR-3’,并正调控基因簇基因的表达和黄曲霉毒素的生物合成[12]。在P. expansum中,展青霉素合成基因簇也编码一个该类型的转录因子PePatL[15]。PePatL定位于细胞核,其编码基因的敲除会导致展青霉素合成基因簇内其他基因的表达量显著下调,造成P. expansum完全丧失产毒能力[15]。目前,PePatL在基因启动子区的结合位点尚不清楚。

2.2 环境信号-全局性调控因子

真菌的生长环境对其次级代谢物的生物合成具有重要的影响,常见的环境信号包括光、温度、pH值、氮源、碳源、硫源以及氧化胁迫等[24]。环境因子对次级代谢相关途径的调控一般是通过各种环境信号通路中的全局性调控因子来实现的,比如Velvet复合体(LaeA-VeA-VelB)能够响应外界的光信号来调控真菌的生长发育和次级代谢物的合成[25],PacC响应环境pH值信号并调控真菌次级代谢基因簇的表达[26],CreA和MetR能够分别应答环境碳源和硫源信号并参与真菌毒素生物合成的调控[27-28]。

2.2.1 光照-Velvet复合体

光照作为最重要的环境因子之一,参与了真菌众多性状的调控,包括生长、形态建成和次级代谢物合成等过程。Velvet复合体是真菌光信号调控系统中最核心的调控元件,能够与多个光受体蛋白互作,感知不同光信号,并进入细胞核调控真菌发育和次级代谢基因簇内基因的表达[25,29]。在P. expansum中,蓝光能够显著抑制展青霉素的生物合成[30]。Velvet复合体中的3个成员LaeA、VeA和VelB在P. expansum中均被鉴定到,其中PeLaeA可以通过调控展青霉素合成基因簇中的15个基因的表达来影响展青霉素的合成[31]。同时,PeVeA和PeVelB对展青霉素的合成均具有正向调节作用[19,32]。此外,P. expansum中Velvet蛋白家族的中的其他2个成员PeVelC和PeVosA也被鉴定,其中PeVelC参与了展青霉素的合成[19]。

2.2.2 pH值-PacC

环境pH值是影响真菌生长发育和次级代谢的另一个重要因素。P. expansum中展青霉素合成的最适pH值在4.0～5.0[33]。pH值-PacC信号途径是真菌感知和传导环境pH值的重要途径,包含6个Pal蛋白(PalA、PalB、PalC、PalH、PalI和PalF)和1个Cys2His2型转录因子PacC[34-35]。在酸性条件下,PacC主要以闭合非活性的形式存在,当pH值升高至中性或碱性后,PacC转变成开放式结构,经过两步蛋白酶切形成具有活性的转录因子,进入细胞核,调控下游基因的表达[36]。在P. expansum中,PePacC的缺失导致展青霉素的合成受阻,当环境pH值大于5.0时,展青霉素的合成完全被阻断,且15个展青霉素合成基因的表达都显著下调[37]。其中,9个基因的启动子区存在PacC的保守结合序列5’-GCCARG-3’,可能受到PePacC的直接调控作用。

2.2.3 碳源-CreA

碳源是真菌生命活动的重要能量来源,同时也作为信号分子影响真菌的代谢过程。Zong等[33]发现不同碳源条件下,P. expansum的展青霉素合成及基因簇基因表达水平存在明显差异,单糖和二糖相对多糖更有利于展青霉素合成。CreA是真菌碳同化和代谢通路的核心调控因子,负责启动下游多个水解酶的转录,调控真菌的碳源利用过程[38-39]。在P. expansum中,蔗糖作为碳分解代谢物阻遏(carbon catabolite repression, CCR)系统中的代谢物,与展青霉素的生物合成密切相关[31]。进一步研究发现蔗糖能诱导creA的表达,而creA的敲除会导致P. expansum产毒能力严重下降[27]。与上述全局性调控因子不同,creA敲除突变株中大部分展青霉素合成基因簇基因能够正常表达,这种不一致的调控模式可能与转录后调控或代谢产物的反馈调节有关。

2.2.4 硫源-MetR

硫是生物体的重要元素之一,参与了诸多重要的细胞过程。无机硫离子和有机硫分子作为硫源可以被丝状真菌利用以维持自身正常的生命活动[40]。硫代谢阻遏途径(sulfur metabolite repression,SMR)是调控真菌适应不同硫源,选择利用最适硫源的关键系统,MetR是SMR途径中重要的正调控因子,负责调控硫酸盐同化和甲硫氨酸合成相关的催化酶[41-42]。在P. expansum中,PeMetR的缺失导致展青霉素产量减少到野生型菌株的2%,而外源添加甲硫氨酸能够部分恢复突变株的产毒能力[28]。此外,PeMetR介导的硫同化途径中关键酶PesB、PesC、PesA、PesF、PeArS和PeTaD都参与了P.expansum中展青霉素的生物合成,证实PeMetR介导的硫元素的同化代谢对展青霉素的生物合成至关重要[28]。

2.3 表观遗传因子

真菌次级代谢基因簇的表达依赖于染色质状态[43]。因此,组蛋白修饰在丝状真菌次级代谢物合成的过程中发挥着重要的作用。

2.3.1 组蛋白甲基化

组蛋白甲基化是发生在组蛋白球状结构域以及暴露在核小体表面N末端的甲基化修饰,是一种重要的表观遗传调控机制[44]。H3K4甲基化是最重要的组蛋白甲基化修饰类型之一。H3K4甲基化由Set1/MLL关联复合体COMPASS催化完成[45]。Xu等[46]通过基因敲除和H3K4甲基化水平检测,发现PeSet1敲除导致P. expansum中展青霉素的合成量减少近96%,并伴随着H3K4me1/me2/me3修饰的完全丧失。PeSet1敲除突变株中展青霉素基因簇中15个基因和全局性调控基因(PeLaeA、PeVeA和PeVelB)都显著下调,且关键基因PePatK、PePatG和PePatL不同区段的H3K4甲基化水平均降低,表明PeSet1正向调控展青霉素的生物合成以及基因簇中基因区段的H3K4甲基化水平[46]。

2.3.2 组蛋白乙酰化

蛋白质乙酰化在转录、信号通路和代谢等过程中均具有重要调控作用[47-48]。同其他表观修饰过程一样,组蛋白的乙酰化也是可逆的,在乙酰化酶和去乙酰化酶的作用下维持蛋白乙酰化修饰的动态平衡[49]。研究发现,表观修饰识别蛋白SntB能够调控全局性调控因子PePacC、PeCreA和PeLaeA的表达,进而调控展青霉素的生物合成[50]。SntB是酵母Snt2的同源蛋白,Snt2与组蛋白去乙酰化酶Rpd3形成复合体,可以结合在氧化胁迫和饥饿胁迫应答基因的启动子区并调控基因表达[51]。但SntB是否通过蛋白质乙酰化修饰调控展青霉素的生物合成还有待进一步解析。

3 展青霉素污染的防控策略

展青霉素污染防控研究可划分为2个方面,即生鲜果品或原料果的源头防控和加工制品中毒素的直接脱除。控制病原菌的侵染和展青霉素的产生是实现源头控制展青霉素污染的有效手段,而针对已经污染的果蔬产品,则可以利用物理、化学或生物方法对展青霉素进行直接脱除(图2)。

3.1 源头防控技术

3.1.1 物理和化学防控

产毒菌株的传统控制方法主要包括物理方法(加热、辐照、巴氏杀菌、控制贮藏条件等)和化学方法(ClO2、二苯胺、多菌灵、苯并咪唑类化学杀菌剂等)[52]。加热、辐照和巴氏杀菌能够灭活真菌孢子,但同时也会影响水果风味。低温和气调贮藏配合作用能有效抑制P. expansum的生长,然而冷藏后的水果一旦重新放置于室温,病菌的生长会被重新激活[53]。此外,化学杀菌剂的滥用易导致病原菌耐药性的产生,随之而来的农药残留问题也会带来新的安全隐患。因此,传统的控制技术应用过程中的弊端逐渐显现,亟须研发替代性的新控制技术。

3.1.2 生物防控

生物防治主要是通过微生物之间的相互作用,利用拮抗菌抑制病原菌,从而控制采后病害和毒素的产生。在不同种类的拮抗微生物中,酵母菌由于抑菌效果好、安全性高、营养要求低、耐受能力强等优点成为控制水果采后病害的常用拮抗菌[54-55]。Rhodotorula spp., Sporobolomyces spp., Candida spp.和Pichia spp.等多种酵母拮抗菌在水果青霉病防控中已有报道,主要生防机制包括营养和空间竞争、诱导果实抗病性、分泌抗菌物质等[56-57]。除活体的拮抗菌外,许多生物源活性物质因在抗病原真菌方面具有高效、绿色、环保的优势,受到人们的广泛关注[58]。比如黄酮类物质、壳聚糖、水杨酸、纳他霉素、部分植物精油(茶树精油、茴香精油)均能抑制P. expansum的孢子萌发和菌丝生长,减少病害的发生,从源头降低展青霉素污染风险[2,59]。

3.2 展青霉素的脱除技术

3.2.1 物理脱除技术

物理脱除方法主要包括辐照、吸附、超声波处理等。Funes等[60]采用35.8 J/cm2的脉冲光辐照处理苹果汁,30 s后展青霉素含量减少了78%。Diao等[61]使用波长254 nm、强度3.8 mW/cm2的紫外线降解苹果汁中的展青霉素,5 min后毒素质量浓度从99.42 μg/L降低至16.98 μg/L,但经过紫外线辐照后,苹果汁的颜色和抗坏血酸含量等指标发生显著变化,果汁品质受到影响。通过吸附剂脱除真菌毒素具有经济有效、操作简单等优点。近年来,海藻酸盐凝胶[62]、壳聚糖基材料[63]、磁性碳纳米管[64]和半胱氨酸功能化的UiO-66(NH2)吸附剂[65]等展现出较好的展青霉素吸附能力。其中,半胱氨酸功能化UiO-66(NH2)吸附剂对苹果汁中的展青霉素吸附量可达4.18 μg/mg[65]。Zhang等[64]合成的新型磁性多壁碳纳米管吸附剂,在1 h内对展青霉素的结合能力达到0.64 μg/mg。新型吸附剂作用时间短、吸附效率高,能够多次循环使用。然而,它们在吸附毒素的过程中也会吸附果汁等产品中的部分营养物质,影响产品品质,吸附的特异性有待加强。

3.2.2 化学脱除技术

化学脱除方法是通过氧化剂、食品添加剂、巯基类物质等与展青霉素发生反应,破坏其毒性官能团,进而达到脱除毒素的目的。研究发现,氨、高锰酸钾、抗坏血酸、苯甲酸钠、山梨酸钾、臭氧等物质均能降低反应体系中的展青霉素含量[52]。氨和高锰酸钾通过氨化和强氧化作用对展青霉素的脱除效果显著,脱除效率接近100%[66]。丙基巯基化SBA-15可以与展青霉素的共轭双键发生反应从而脱除展青霉素,脱除效率可达94%[67]。臭氧能够灭活产毒真菌,对处理果蔬及产品影响较小,因此,广泛应用于果蔬保鲜[68]。同时,臭氧也可以用来脱除真菌毒素。Diao等[69]发现用臭氧处理被展青霉素污染过的苹果汁,15 min后75%左右的展青霉素被脱除。化学脱除展青霉素效果明显,但在使用化学物质前需要充分考虑其安全性,并对化学物质脱毒的反应机制进行深入研究。同时,明确反应产物的毒性也十分必要。

3.2.3 生物脱除技术

生物法脱除包括微生物降解法和生物酶降解法。许多酵母、细菌甚至部分霉菌已经被证实对展青霉素具有较强的直接降解作用[2,52]。然而,在水果加工产业中,除某些发酵型食品外,大部分情况下活体微生物不便直接用于毒素的脱除。与直接使用微生物脱除毒素相比,生物酶法具有效率更高、专一性更强和适用范围更广等特点,更具应用前景。Tang等[70]从Rhodotorula mucilaginosa酵母中分离到一个乳清酸磷酸核糖转移酶并用于苹果汁中展青霉素的脱除。在25℃条件下,该酶对苹果汁中展青霉素的脱除率达80%以上。Wang等[71]在研究Pichia caribbica酵母降解展青霉素的机制过程中发现S-腺苷蛋氨酸依赖的甲基转移酶可以直接降解展青霉素。笔者团队从具有展青霉素降解作用的酵母菌Candida guilliermondii中克隆到一个短链脱氢酶基因CgSDR,并通过基因工程技术获得了CgSDR蛋白[72-73]。CgSDR能够高效降解苹果汁中的展青霉素,降解产物为无细胞毒性的E-alscladiol,而且降解过程对果汁品质没有影响[74]。此外,将酶固定化技术与生物酶脱除技术相结合,可提高生物酶活性的稳定性,降低生产成本。使用磁性Fe3O4颗粒固定化的短链脱氢酶不仅能够保持较高的展青霉素降解活性,其稳定性和重复使用性较游离酶也得到了显著提升[75]。

4 结论与展望

由真菌毒素污染引起的食品安全问题越来越得到人们的重视。近年来,展青霉素的相关研究逐渐成为热点,文章发表数量呈逐渐上升趋势。生物合成基因簇的鉴定和P. expansum遗传转化技术的建立为展青霉素产生机制的研究奠定了基础。目前,展青霉素生物合成途径中8步反应的催化酶已经明确,另外2步反应的催化酶及3个转运蛋白的功能需要进一步的解析。此外,多个重要的展青霉素生物合成调控因子及其调控机制已被揭示,这些调控因子之间互作的调控网络还需进一步深入研究。通过解析展青霉素的产生机制,一方面可以鉴定阻断毒素合成的关键内部因子,挖掘毒素合成抑制剂的靶点;另一方面可以发现抑制毒素合成的关键外源条件,通过对环境因子的调控来控制毒素的产生,为展青霉素污染防控技术的研发提供理论支撑。在展青霉素污染防控策略方面,无论是源头防控还是直接脱除,基于生物的方法都是未来的发展方向。生物防控技术具有绿色、安全的优势,但在防控效果的稳定性、成本控制及使用便捷性等方面还需进一步加强,以满足实际生产的需求,实现商业化的应用。

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Production Mechanisms and Control Strategies of Patulin in Food

LI Boqiang1,2, CHEN Yong1,2, XING Mengyang1,2, TIAN Shiping1,2,3

(1.Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China;2.China National Botanical Garden, Beijing 100093, China;3.College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing 100049, China)

Abstract：Patulin, a polyketide secondary metabolite produced by fungi, is a major mycotoxin in fruits and their processed products. Penicillium expansum is the primary agent for patulin contamination in food. P. expansum infects most bulk and special fruits, causes serious postharvest decay, and simultaneously produce patulin. The circulation and processing of fresh fruits can introduce patulin into the food chain, which pose a potential threat to the health of consumers. Patulin may damage vital organs in both humans and animals and causes acute and chronic symptoms. The food safety issues caused by patulin contamination are attracting increasing attention, and patulin-related research has become a hot topic. The research advances in patulin biosynthesis and regulation mechanisms were comprehensively summarized. The gene cluster of patulin biosynthesis, catalytic enzymes in different steps of the biosynthetic pathway, and the molecular pathway of patulin biosynthesis and intermediate product transport were introduced. The complex regulatory network of patulin biosynthesis from the aspects of specific regulatory factor of biosynthetic pathway, global regulatory factors responding to environmental signals such as light, pH value, carbon source, and sulfur source, as well as epigenetic factors involved in histone methylation and acetylation modifications were described. The source control and removal strategies for patulin contamination in food were elaborated. The main control technologies based on physical, chemical, and biological principles were summarized, respectively, and the advantages and disadvantages of different control strategies were raised. Finally, the relationship between the research of patulin biosynthetic mechanisms and the development of contamination control technologies was discussed. The green and safe biological control methods as the promising technology was emphasized.

Keywords：Penicillium expansum; secondary metabolism; biosynthetic pathway; regulatory factor; blue mold; biological detoxification

中图分类号：TS201.3

文献标志码：A

doi：10.12301/spxb202300267

文章编号：2095-6002(2023)04-0016-10

引用格式：李博强,陈勇,邢孟阳,等.食品中展青霉素的产生机制及污染防控策略[J]. 食品科学技术学报,2023,41(4):16-25.

LI Boqiang, CHEN Yong, XING Mengyang, et al. Production mechanisms and control strategies of patulin in food[J]. Journal of Food Science and Technology, 2023,41(4):16-25.

收稿日期：2023-05-05

基金项目：北京市科技计划项目(Z201100008920007);中国博士后基金项目(2022M723369)。

Foundation: Beijing Science and Technology Program (Z201100008920007); China Postdoctoral Science Foundation (2022M723369).

第一作者：李博强,男,研究员,博士,主要从事果蔬采后病害和真菌毒素污染的发生机制及控制技术方面的研究。

(责任编辑：李 宁)