图1 Levansucrase水解蔗糖合成levan的机制
Fig.1 Mechanism for the synthesis of levan from sucrose by levansucrase
摘 要：果聚糖levan是一类典型的果糖聚合物，主要由微生物产生的levan蔗糖酶以蔗糖为底物来合成的，其主链由果糖以β-（2，6）糖苷键键合而成，并伴有少量β-（2，1）糖苷键的支链。本文从来源、功能与应用的角度总结了levan的研究进展，并对其发展趋势进行了展望。
关键词：果聚糖；来源；功能；应用
*通信作者
Levan是一类典型的果糖聚合物，其主链由果糖以β-（2，6）糖苷键键合而成，并伴有少量β-（2，1）糖苷键的支链。Levan的来源相当广泛，它不仅存在于植物[1]中，也可以由微生物[2]代谢产生。微生物来源的levan的合成与levan蔗糖酶（levansucrase，β-2，6-fructan，EC 2.4.1.10）有关，这是一类属于糖苷酶家族GH68的果糖基转移酶（fructosyltransferase），具有水解蔗糖和转移果糖基的双重活性[3]（如图1所示）。
由于微生物发酵制备levan的周期短、成本低且工艺简便，因此成为工业化生产levan的主要途径。前期陆娟等总结了levan生产与应用方面的研究进展[4]，而本文将从来源、功能与应用角度对levan的国内外研究成果进一步综合论述。
图1 Levansucrase水解蔗糖合成levan的机制
Fig.1 Mechanism for the synthesis of levan from sucrose by levansucrase
1.1 微生物来源
微生物是自然界中levan的主要来源，1990年Han YW总结了28个种/属的levan产生菌[2]，随着对levan研究的不断深入，乳杆菌（Lactobacillus）、明串珠菌（Leuconostoc）、链球菌属（Streptococcus）等一些被公认可安全食用的益生菌也被发现可以产生levan。到目前为止，产levan菌株的覆盖面已达到了近30个属、40余种，相关报道如表1所示。
表1 产levan菌株的生物分类学地位
Table 1 Biological taxonomies of strains producing levan
注：“-”未进一步鉴定具体种不可知。
中文菌名 参考文献Acetobacter Acetobacter pasteurianus 巴氏醋酸杆菌 [5] Acetomonas sp. - 醋单胞菌属 [6] Achromobacter sp. - 无色杆菌属 [2] Actinomyces Actinomyces viscosus 粘性放线菌 [7] Aerobacter Aerobacter aerogenes 产气气杆菌 [8] Aerobacter levanicum 产左聚糖气杆菌 [9] Arthrobacter Arthrobacter ureafaciens 产脲节杆菌 [10] Arthrobacter nicotinovorans 食烟碱节杆菌 [11] Aspergillus Aspergillus sydowii 聚多曲霉 [5] Aspergillus versicolor 花斑曲霉 [5] Azotobacter Azotobacter chroococum 褐球固氮菌 [12] Bacillus Bacillus amyloliquefaciens 解淀粉芽胞杆菌 [13] Bacillus circulans 环状芽胞杆菌 [14] Bacillus licheniformis 地衣芽孢杆菌 [15] Bacillus megaterium 巨大芽孢杆菌 [16] Bacillus mesentericus 炭疽芽孢杆菌 [17] Bacillus subtilis 枯草芽孢杆菌 [18] Bacillus polymyxa 多黏芽孢杆菌 [19] Corynebacterium Corynebacterium laevaniformans 产左聚糖棒状杆菌 [20] Corynebacterium beticola 栖甜菜棒状杆菌 [21] Erwinia Erwinia amylovora 解淀粉欧文氏菌 [22] Erwinia herbicola 草生欧文氏菌 [23] Geobacillus Geobacillus Stearothermophilus 嗜热脂肪土芽孢杆菌 [24] Gluconoacetobacter Gluconoacetobacter xylinus 葡萄糖木质醋酸菌 [25] Gluconobacter Gluconobacter oxydans 氧化葡萄糖酸杆菌 [26] Gluconobacter frateurii 弗托氏葡萄糖酸杆菌 [27] Gluconobacter cerinus 蜡状葡萄糖酸杆菌 [27] Halomonas sp. - 盐单胞菌属 [28] Lactobacillus Lactobacillus gasseri 加氏乳杆菌 [29] Lactobacillus reuteri 罗伊氏乳杆菌 [30] Lactobacillus sanfranciscensis 旧金山乳杆菌 [31] Leuconostoc Leuconostoc Mesenteroides 肠膜明串珠菌 [32] Microbacterium Microbacterium laevaniformans 产左聚糖微杆菌 [33] Odontomyces Odontomyces viscosus 粘性牙霉菌 [34] Paenibacillus Paenibacillus polymyxa 多粘类芽孢杆菌 [35] Phytomonas Phytomonas pruni 桃李植物单胞菌 [16] Pseudomonas Pseudomonas syringae 丁香假单胞菌 [36] Pseudomonas aureofaciens 金色假单胞菌 [37] Pseudomonas mors-prunorum 死李假单胞菌 [38] Pseudonomas chlororaphis 绿针假单胞菌 [39] Pseudonomas fluorescens 荧光假单胞菌 [39] Rahnella Rahnella aquatilis 水生拉恩氏菌 [40] Rothis Rothis dentocariosa 频齿罗氏菌 [2] Saccharomyces Saccharomyces cerevisiae 酿酒酵母 [41] Serratia sp. - 沙雷氏菌属 [42] Streptococcus Streptococcus salivarius 唾液链球菌 [43] Xanthomonas sp. - 黄单胞菌属 [2] Zymomonas Zymomonas mobilis 运动发酵单胞菌 [44]属种
发酵单胞菌（Zymomonas）是最早被发现可以产levan的细菌，其中的Z. mobilis至今依然是大规模生产levan的重要工业微生物。值得关注的是，乳杆菌产生的levan具有益生元的作用[31]、而明串珠菌合成的levan具有良好的安全性[32]，预示此类果聚糖在食品、医药领域具有潜在的应用价值。
1.2 植物来源
植物来源的levan主要存在于草本植物。Challinor SW等从粗茎早熟禾（Poa trivialis）中分离得到了一种与菊粉（inulin）结构类似的多糖类物质，通过对其乙酰基和甲基衍生物的分析表明是果聚糖levan[1]。Ende VD等发现levan在常绿植物富贵草（Pachysandra terminalis）中也有积累[45]。此外，通过分子生物学的手段，Banguela等将G. diazotrophicus中编码levansucrase的基因导入烟草中并成功获得表达，烟叶中levan含量最高可占转基因烟叶的70 %[46]。
2.1 抗癌、抗肿瘤作用
Leibovici等认为，Levan的抗肿瘤效果来自于果聚糖对肿瘤细胞的直接作用[47]，每天以静脉注射的方式将levan与传统抗肿瘤药物（环磷酰胺、甲氨蝶呤、长春新碱、5-氟尿嘧啶）分别或共同作用于患有路易士肺癌的老鼠，发现levan与环磷酰胺联合使用比单独使用所发挥的抗肿瘤效果更佳，2 mg levan配以10 mg环磷酰胺作用于受试对象时，其癌细胞的生长可完全被抑制，这说明除了自身具有的肿瘤细胞生长抑制作用外，levan还能消除细胞毒素剂对免疫系统的毒害作用[48]。Liu等发现，levan的乙酰化、磷酸化和苯基化衍生物普遍拥有比天然levan更高的抑制肿瘤细胞增殖的能力，可能是在levan中引入特殊基团后，其供电子能力得到的提高，从而增强了多糖分子与免疫细胞上受体的亲和力[35]，而另一种理论认为，levan衍生物可以通过线粒体途径破坏DNA，进而诱导肿瘤细胞发生凋亡的[49]。Yoo等发现，来自M. laevaniformans （Mw=710，000）、R. aquatilis（Mw=380，000）和Z. mobilis （Mw=570，000）的levan对肉瘤180细胞有强烈的生长抑制作用，其平均抑制活性可达到66 %，而G. xylinus产生的levan（Mw=40，000）抗肿瘤效果显著低于上述3种levan，仅为42 %[25]，并且随着levan分支化程度的降低，其抗肿瘤活性也会不同程度地下降[50]，由此可见，Levan的抗肿瘤活性与多糖的分子量和分支化程度有关。
2.2 抗菌、抗病毒作用
研究表明，levan对食源性致病菌普遍具有抗菌作用，尤其是低分子量levan可明显降低面包中腐败菌和致病菌的存活率，levan诱导产生渗透压的变化、或者通过减少水分活度从而干扰细菌对营养成分的吸收是可能的主因[51]。Esawy从不同蜂蜜中分离得到多株产levan的B. subtilis，其中来自菌株K、M、E的levan可抑制致病性禽流感病毒HPAI和H5N1的活性，而菌株A、M的levan对40型腺病毒有抑制活性[52]。
2.3 降血糖作用
2011年，Dahech等将levan每天以小鼠体重5 %的剂量通过口服途径作用于四氧嘧啶诱导的糖尿病小鼠，60天后伴随着受试对象的肝糖元水平提高了52 %，血浆中的葡萄糖水平相应地降低了52 %，同时，小鼠肝、肾、胰腺和心脏中硫代巴比妥酸反应物质（TBARS）的含量下降了31 %、41 %、39 %和25 %，而超氧化物歧化酶（SOD）和过氧化氢酶（CAT）水平均有一定程度的提高，更重要的是，levan显著降低了受试小鼠肝脏、肾中碱性磷酸酶（ALP）、天冬氨酸转氨酶（AST）和乳酸转氨酶（ALT）的活性、胆红素、肌氨酸酐和尿素水平等指标。表明levan可将异常的氧化指标恢复到正常水平，这是首次发现levan对高血糖和糖尿病引发的氧化应激具有一定的控制作用[53]。
2.4 降血脂、降胆固醇作用
为了考察levan对高胆固醇饮食小鼠的降胆固醇作用，研究人员每天以体重5 %剂量将levan通过口服途径作用于受试对象，60天后样品组小鼠（同时摄入levan和高胆固醇食物）血液中的高密度脂蛋白胆固醇（HDL-cholesterol）水平与对照组（仅摄入高胆固醇食物）相比有所上升，而低密度脂蛋白胆固醇（LDL-cholesterol）水平大幅下降，其作用机理是levan扰乱体内类固醇肝肠循环的结果，此项研究为levan对与动脉粥样硬化有关的氧化应激的保护作用提供了依据[54]。
2.5 免疫增强作用
Levan的免疫增强作用较多被证实于鱼类的免疫系统。被嗜水气单胞菌（Aeromonas hydrophila）感染的鲤鱼（Cyprinus carpio）和露斯塔野鲮（Labeo rohita）经长期饲喂一定浓度（0.5 %和1.25 %）的levan后，受试对象存活率大幅提高，数据显示，前者血液中红血球数量和血红蛋白含量明显增加[55]，而后者肝脏中浸润了白细胞但肾小管只发生了中度退化[56]，不仅如此，Levan对暴露于亚致死剂量氟虫腈（Fipronil，一种苯基吡唑类杀虫剂）环境下的鲤鱼同样有免疫刺激和保护作用[57]。
2.6 同种移植排斥延迟作用
同种移植排斥反应是同种器官或组织移植时，受体免疫系统对移植物产生的排异反应，而levan可延缓这种排斥反应，延长移植物的存活时间。当高分子量Levan每日以15 mg～30 mg剂量作用于Balb/c和C57BL受体小鼠时，可分别延长两者的排斥反应3.6 d和5.6 d[58]，levan的这种抑制功效既不是果聚糖对移植床的生理破坏所产生的，也不是机械性干预的结果[59]，血液中被激活的单核细胞对移植物的浸润作用是关键因素[60]。
2.7 减肥作用
高分子量levan的减肥作用是近年来被发现的另一种益生功能，相关实验表明，当levan以体重10 %剂量作用于高脂肪饮食诱导引发的肥胖症老鼠4周后，受试对象的体重明显降低，该效果与levan摄入量呈一定剂量效应[61]。另一项研究显示，饲喂levan与发酵红参组合物可有效降低肥胖小鼠的体重、总胆固醇和空腹血糖水平，并减少体内脂肪含量。上述结果表明，以单独或复合形式向食物中添加levan均有助于抑制由饮食引发的肥胖症[62]。
2.8 益生元作用
作为一种特殊的外源性碳源，高分子量Levan （2 000 ku）可大幅提高双歧杆菌在牛奶发酵过程中的细胞增殖效果，进一步研究发现，当以levan为碳源时，发酵液上清中存在β-果聚糖水解酶活性，双歧杆菌因为具有上述果聚糖水解酶活性，可以利用低聚果糖或寡聚果糖[63]进行增殖。有趣的是，Levan对微生物的这种促生长作用是有选择性的，数据显示，长期食用levan的罗斯肉鸡盲肠内的乳杆菌、双歧杆菌等有益菌数量均有所增加，而大肠杆菌、产气荚膜梭菌等有害菌浓度却在降低[64]。
2.9 成膜特性
纯粹的levan因易碎而难于单独成膜，但Barone等指出，向levan中添加20 %（质量分数）甘油不但可有效地提高其塑化效果，而且增加了levan结构中氢键的相互作用，从而有更有助于levan的成膜[65]。以Levan和少量蒙脱黏土（montmorillonite clay）为原料制备成的纳米复合材料可形成一种网状结构，这种特殊结构极大地改善了levan的热学和力学特性[66]。
2.10 用于蛋白的分离、纯化和固定
凝集素（Lectin）是一类可特异性识别碳水化合物，并与其可逆结合的蛋白，Angeli等利用levan和磁铁的混合物对Cramoll 1（一种凝集素）有特异性吸附的特性，将制备步骤由原来的三步简化为两步，操作难度降低的同时还大大减少了制备成本[67]，而levan与聚乙二醇（PEG）混合组成的双水相系统则表现出对生物蛋白的分离特性，但分离效果受pH影响较大[68]。此外，带有磁性的levan颗粒还可作为一种基质在胰蛋白酶固定化中发挥积极的作用，被固定的酶衍生物反复使用10次后仅损失16 %的活性[69]。
2.11 其他功能与应用
除了上述功能与应用外，根据来源与特性的不同，Levan还可在代血浆[70]、纳米载体[71]、静电纺丝[72]以及化妆品[73]等诸多方面发挥作用。
Levan是一种天然的果糖聚合物，存在于多种植物和微生物发酵产物中，虽然在甘蔗汁加工业中是不受欢迎的微生物代谢产物，但在医药临床和食品加工方面具有一定的应用价值，近年来受到研究者的关注。有关levan的报道最早可追溯到20世纪30年代，至今相关研究已遍布产量优化、结构解析、作用机理、功能特性等多个领域，然而，依然存在一些问题有待解决：①levan在现有培养基中的产量普遍较低，有必要从培养基组成、培养条件、变异诱导以及异源表达等方面入手进一步提高产量；②酶法产糖是目前levan的主要制备途径，因此有必要对温度、pH、离子强度等环境因素研究以保持或提高酶的稳定性与活性；③大多数levan的产生菌属于致病菌，有必要筛选类似Lactobacillus的高安全性levan产生菌，从而更好地应用于食品与医疗行业。
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Progress in the Study of Functionality and Application of Microbial Levan
Abstract：Levan，a typical fructose polymer with β-2，6-glycosidic linkage in the backbone and a few β-2，1-glycosidic linkage in the branches，is mainly synthesized by microbial levansucrase with sucrose as the substrate. In this article，the progress in the research and development of levan resource，functionality and application was reviewed and the future perspective is also predicted.
Key words：levan；resource；functionality；application
DOI：10.3969/j.issn.1005-6521.2016.04.046
基金项目：“十二五”国家科技支撑计划课题：发酵乳制品乳酸菌菌种与发酵剂的研究与开发（2013BAD18B01）；“十二五”国家863项目：优良益生菌高效筛选与应用关键技术（2011AA100901）
收稿日期：2014-11-18