中国科学院微生物研究所、中国微生物学会主办
文章信息
- 阿拉腾珠拉, 胡永飞
- A La Teng Zhu La, HU Yongfei
- 褐藻寡糖的制备方法及生物活性研究进展
- Advances in the preparation of alginate oligosaccharides and its biological functions
- 生物工程学报, 2022, 38(1): 104-118
- Chinese Journal of Biotechnology, 2022, 38(1): 104-118
- 10.13345/j.cjb.210377
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文章历史
- Received: May 21, 2021
- Accepted: July 29, 2021
- Published: August 24, 2021
褐藻胶(alginate) 又称海藻酸盐,是一种从褐藻细胞壁中提取的天然多糖醛酸,是唯一一种在单体分子中含有羧基的多糖,其分子式为(C6H7NaO6)n。褐藻胶通过1, 4-糖苷键将α-L-古罗糖醛酸(α-L-guluronic acid, G) 和β-D-甘露糖醛酸(β-D-mannuronic acid, M) 连接成聚合长链,因此褐藻胶的组合方式主要有3种:只由M组成的多聚β-D-甘露糖醛酸片段(polymannuronate, PM); 只由G组成的多聚α-L-古罗糖醛酸片段(polyguluronate, PG); 由G和M组成的共聚物(heteropolymer, PolyMG)[1]。组成褐藻胶的M和G在结构上互为差向异构体,主要在C5位上羧基位置的不同(图 1)[2],且M和G的比率与其生物活性密切相关。
随着海藻多糖类天然产物研究的不断深入,褐藻胶被认为是一种具有重要的免疫刺激、抗凝血、抗病毒和抗癌活性的大分子化合物,然而褐藻胶分子量大、黏性大、难以跨越细胞膜和多重生物屏障,进而限制了褐藻胶的开发和应用[3]。近年来,通过糖苷键水解或生物/有机合成获得的低分子量的褐藻寡糖(alginate oligosaccharides, AOS) 受到了广泛关注,其不仅分子量较低、黏性小、易于吸收,而且表现出较强的生物活性[4]。因此,AOS受到了广泛的关注,特别在最近十年,相关研究明显增多,文章发表数量逐年上升(图 2A)。Web of Science的统计分析表明,AOS及其应用的基础研究主要集中在生物化学与分子生物学、化学、生物技术与应用微生物学、食品科学技术、微生物学、药理学与药学以及农学等相关领域(图 2B)。
AOS具有多种生物活性,包括免疫调节[5]、益生元[6]、抗炎[7]、抗细胞凋亡[8]、抗氧化[4]甚至抗肿瘤[9]等。AOS的生物活性取决于分子结构,因此制备结构新颖、聚合度明确的AOS,对于发现AOS新的生物活性与作用方式具有重要意义。本文重点综述了AOS的制备方法以及生物活性的研究进展,以期为未来AOS在药物研制、功能食品以及农业等领域的应用提供参考。
1 褐藻寡糖的制备方法AOS发挥生理功能的过程中,寡糖的聚合度、构象、取代基等与其活性密切相关。目前制备AOS的方法有化学降解法、物理降解法、生物降解法、有机合成法和生物合成法等。物理降解法有反应迅速和无环境污染等特点,化学降解制备得到的是饱和褐藻寡糖(saturated alginate oligosaccharides, SAOS),而生物降解制备得到的是不饱和褐藻寡糖(unsaturated alginate oligosaccharides, UAOS)。有机合成法是将1, 6-α-B-L-甲吡喃吡喃物作为底物,使-GGGG-和-MMMM-的寡糖链从非还原端(还原端) 组装到还原端(非还原端); 此外,MG连接成的二糖也用相同的糖基受体合成,进而制备-MGMG-片段的AOS,该方法的优点为精准合成,但产量较低[10]。生物合成法主要利用微生物产生的合成酶制备AOS,该方法可精确地控制产物的区域选择性以及形成立体化学键,但成本较高、产率低、产物品种单一[11]。以下将重点介绍化学降解法、物理降解法和生物降解法。
1.1 化学降解法化学降解法已被广泛用于AOS的制备,主要包括酸水解、碱水解和氧化降解法,其中酸水解法是制备AOS的传统方法。酸水解法通常用到盐酸、草酸、甲酸和硫酸等[12]。在酸水解反应过程中,反应条件以及褐藻胶的来源和组成对AOS的聚合度有较大的影响。在100 ℃下,使用1 mol/L草酸制备AOS,大约30%的褐藻胶发生水解反应(degrees of polymerizations, DP 10–30)[13]。然而褐藻胶具有耐酸性,不能被无机酸完全降解[14]。Chandı́a等[15]用90%的甲酸水解6 h (100 ℃),再用1.5 mol/L甲酸水解2 h (100 ℃),褐藻胶能完全水解。酸水解褐藻胶的具体反应步骤如下:糖苷氧给予共轭酸质子; 共轭酸形成的非还原端基末端和碳-正氧鎓离子断裂; 后向碳-氧离子快速加水,形成还原端基[3]。碱水解也是制备AOS常用的方法。然而,在碱性条件下,利用β-消除反应,使褐藻胶糖苷键断裂,会导致褐藻胶的结构发生变化,所以一般不用此方法。酸和碱水解法具有成本低、操作方便,可获得褐藻胶大分子固有结构特征的AOS,且在一次降解中可获得不同聚合度的寡糖(DP 2–6)[16],但易产生有毒的降解产物,腐蚀仪器,造成环境污染。氧化降解(H2O2) 法是一种绿色制备AOS的方法,可直接醇沉淀制备AOS,且制备的AOS中没有双键,副产物是H2O,具有较高的回收率和纯度,同时避免了其他杂质对环境的污染[17]。目前,H2O2降解褐藻胶的确切机制尚不清楚,可能是H2O2的羟基自由基首先从M或G残基中提取C-1质子,诱导褐藻胶糖苷键的结构重排和降解[18]。
1.2 物理降解法制备褐藻寡糖的物理方法有很多,常用的包括超声波、紫外照射和辐照法(γ射线和微波),此外还有离子体处理、水热处理和亚临界水解法等。在能源效率方面,γ射线辐照被认为是目前最有效的物理降解方法之一[19],且不受温度、环境或添加剂的影响; 微波辐射是一种方便、快速和环保的降解褐藻胶的方法,可避免脱盐[20]; 二氧化钛存在条件下,紫外照射褐藻酸钠水溶液3 h以上,其降解产生的AOS的相对分子质量较低,G/M比率与酸降解法产物相似[21]。亚临界水通常是在沸点(100 ℃) 和临界温度(374 ℃) 之间的压力下的液态水。在高温(150–374 ℃) 时,水发生自电离,产生酸性水合氢离子和碱性氢氧根离子[22]。亚临界水解法(180–250 ℃) 可选择性地降解褐藻胶中M-M、M-G、G-M和G-G之间的糖苷键,进而制备AOS[23]。物理法具有反应迅速和无环境污染等优点,但该方法成本较高、降解效率较低、反应机理不明确,仍需进一步研究。
1.3 生物降解法生物降解法利用微生物或者具有特异性的褐藻胶裂解酶对底物进行酶解[24]。褐藻胶裂解酶是多糖裂解酶家族(PL家族,包括PL5、6、7、14、15、17和18) 的成员,可通过β-消除作用于褐藻胶的β-1, 4-糖苷键,并在糖苷键所在的C4和C5之间形成双键,进而制备出一系列不同分子量的AOS混合物[24]。褐藻胶裂解酶通过β-消除降解褐藻胶的具体反应步骤如下(图 3):(1) 中和褐藻胶的羧基负电荷并降低H-5质子的pKa; (2) 碱催化C-5上的质子,并形成羧酸根二价阴离子的中间体; (3) 在C-4和C-5之间双键的形成中提供质子,导致4-O-糖苷键的消去反应[21]。在自然界中,褐藻胶裂解酶分布广泛,主要来源于海洋微生物、海洋动物、真菌和病毒等[25]。根据底物的特异性,可将其分为三类:G区域特异性裂解酶(PG裂解酶,EC4.2.2.11)、M区域特异性裂解酶(PM裂解酶,EC4.2.2.3) 和双功能裂解酶[21]。按褐藻胶裂解酶作用方式可分为内切酶和外切酶,其中内切酶是从褐藻胶的内部降解,制备出以二糖、三糖和四糖为主要产物的UAOS,而外切酶是从褐藻胶的末端切除单体或二聚体[26]。表 1总结了不同来源的褐藻胶和褐藻胶裂解酶以及降解产物的相关信息。褐藻胶裂解酶具有较高的底物特异性,是制备具有特殊结构低聚糖的理想选择。然而,目前关于褐藻胶裂解酶具有高度底物特异性的研究报道较少。
Alginate sources | Enzyme sources | AOS products | References |
Laminaria japonica (M/G=2.28 MW=300 kDa) | Vibrio sp. 510 | ΔGG, ΔMM, ΔMG, ΔGGG, ΔMMG, ΔMGM, ΔMGG, ΔGMG, and ΔMMM | [34] |
Laminaria japonica (M/G=1.86) | Pseudomonas sp. HZJ216 | Average MW=1.2 kDa, DP 2-6 M/G=1/2.6 | [35] |
Alginate | Streptomyces violaceoruber | DP 2–6 | [36] |
Alginate | Gracilibacillus A7 | Average DP 3.3 | [37] |
Alginate | Vibrio sp. 510 | 2 202 and 2 324 Da | [38] |
Sodium alginate | Microbulbifer sp. ALW1 | DP 2 and 3 | [39] |
Sodium alginate (Macrocystis pyrifera, M/G=77/23) | Cellulophaga sp. NJ-1 | DP 2–6 | [40] |
Sodium alginate | Flavobacterium sp. LXA | DP 6 | [8] |
Sodium alginate (M/G=2.28) | Pseudomonas sp. HZJ 216 | DP 2–3 | [41] |
Sodium alginate | Pseudoalteromonas sp. strain No. 272 | DP 2–4 | [42] |
Sodium alginate | Alteromonas sp. No. 1786 | DP 2–9 | [43] |
Sodium alginate (M/G=60/4) | Sphingobacterium | DP 2–4 | [4] |
Sodium alginate | Pseudoalteromonas elyakovii IAM 14594 | DP 4 and 5 (12.6%), DP 6 (35.2%), DP 7 (51.0%), DP > 8 (1.2%) | [44] |
Sodium alginate | Vibrio sp. QY102 | DP 3 (77.8%), DP 4 (16.5%), DP 5 (3.6%), and DP 6 (2.1%) | [6] |
Sodium alginate | Isoptericola halotolerans CGMCC 5336 | DP 2–4 | [45] |
Sodium alginate, polyM/polyG | Vibrio weizhoudaoensis M0101 | DP 2–9 | [46] |
Sodium alginate, polyM (99%) | Serratia marcescens NJ-07 | DP 1–5 | [47] |
Sodium alginate (Macrosystis pyrifera, M/G=77/23), polyM/polyG (99%) | Vibrio sp. NJU-03 | DP 2–4 | [48] |
Sodium alginate, polyM/polyG | Wenyingzhuangia fucanilytica | DP 2–6 (sodium alginate) DP 2–4 (polyM/polyG) | [49] |
Sodium alginate (Macrosystis pyrifera, M/G=77/23), polyM and polyG (95%; M/G=97/3, 3/97; DP 39; MW=7 200 Da) | Flammeovirga sp. NJ-04 | DP 2–4 | [50] |
Sodium alginate, polyM | Bacillus sp. Alg07 | DP 2–4 (sodium alginate) DP 2–3 (polyM) | [51] |
PolyG containing 79% L-guluronic acid prepared from sodium alginate (M/G=0.2) | Flavobacterium multivolum | DP 2–7 | [14] |
PM, PG | Marinimicrobium sp. H1 | DP 2–4 | [52] |
PM, PG | Cellulophaga algicola | ΔGG, ΔGGG, and ΔGGGG | [53] |
此外,还可以利用微生物直接降解褐藻胶,比酶促降解更加简单,不需要进行酶的分离纯化等,且微生物法生产条件可控、可规模化生产、产品品质可控、生产周期较短。研究发现,可利用微生物混合菌降解北方海带Laminaria hyperborea叶柄中的褐藻胶[27]。从人类肠道微生物中分离得到拟杆菌门(Bacteroidetes) 成员中的单形拟杆菌(Bacteroides uniformis) L8和卵形拟杆菌B. ovatus G19可通过分泌酶产生AOS[21, 28]。此外,降解褐藻胶的细菌还包括糖球菌属(Gracilibacillus A7)[29]、枯草芽孢杆菌(B. subtilis) KCTC11782BP[30]、海滨芽孢杆菌(B. litoralis) M3[31]、黄杆菌(Flavobacterium sp.) LXA[32]、食琼脂假交替单胞菌(Pseudoalteromonas agarovorans) CHO-12[33]等。
2 褐藻寡糖的主要生物活性 2.1 抗肿瘤活性AOS可通过抑制肿瘤细胞增殖和迁移,以及调节免疫防御反应等发挥抗肿瘤活性。已有研究证明,AOS可通过抑制Hippo/YAP/c-Jun信号通路来减弱人前列腺癌细胞的增殖、迁移和侵袭[9]。由交替假单胞菌Pseudoalteromonas sp.褐藻胶裂解酶制备的古罗糖醛酸寡糖(α-L-guluronic acid AOS, GAOS) 和甘露糖醛酸寡糖(β-D-mannuronicacid AOS, MAOS) 通过上调人单核细胞合成细胞毒性细胞因子,增强人白血病细胞U937的防御机制,这些作用被肿瘤坏死因子(tumor necrosis factor-α, TNF-α) 的抗体所抑制[54]。50 mg/kg GAOS能有效抑制肿瘤细胞对细胞外基质的粘附,抑制肿瘤细胞在植入组织中的浸润,减少荷瘤小鼠体内免疫抑制细胞和炎症细胞的积累[55]。因此,AOS的抗肿瘤作用和免疫调节作用是密不可分的。噬琼胶菌(Agarivorans sp.) L11褐藻胶裂解酶制备的AOS (DP 2–5) 可抑制人骨肉瘤MG-63细胞的生长[56]; 多元醇化的MAOS在肿瘤组织中选择性地诱导TNF-细胞毒因子,进而对小鼠MH134肝癌有局部治疗作用[57]。此外,连续2年每日口服10 mg AOS可提高骨肉瘤患者抗氧和抗炎功能,并减少肿瘤平均体积,降低局部复发率[56]。因此,AOS提高机体的抗氧化和抗炎能力可能是AOS抗肿瘤的另一种重要作用。
2.2 免疫调节功能天然多糖和寡糖具有作为免疫调节剂的潜力。AOS在免疫调节中关键功能之一是诱导细胞因子的活性[58]。在动物试验中发现,AOS可提高断奶仔猪血清中白介素(interleukin, IL)-6、IL-10、免疫球蛋白(immunoglobulin, Ig) 和IgA的浓度,并提高了小肠中分泌型免疫球蛋白A的含量[59],进而表明AOS可提高免疫球蛋白和细胞因子,增强断奶仔猪的免疫功能。在RAW264.7巨噬细胞中,具有较高分子量和M/G比值的褐藻胶可诱导TNF-α的分泌,而由Pseudoalteromonas sp. No. 272褐藻胶裂解酶制备的AOS比未降解的褐藻胶更能增强TNF-α的诱导活性[60]。不同方法制备的AOS与诱导细胞因子分泌的结构与功能之间有密切的相关性。目前,越来越多的研究关注于AOS不饱和末端结构与其活性之间的关系。Pseudoalteromonas sp. 褐藻胶裂解酶制备的AOS不饱和结构在RAW264.7巨噬细胞中诱导TNF-α的分泌中起了关键作用,且GAOS(G8) 和MAOS(M7) 对TNF-α、IL-1α、IL-1β和IL-6的诱导作用最强。这可能是由于MAOS呈“带”状相对较直的聚合物,而GAOS呈“脊柱”状。因此,G8和M7在整个构象上的这种差异可能导致最有效的GAOS比MAOS多一个残基,然而酸降解法制备的SAOS显示出较低的活性。因而,酶降解法制备的AOS中存在不饱和末端结构是激活巨噬细胞的必要条件[61]。此外,Xu等[5]也证明了GOS可提高RAW264.7巨噬细胞中诱导型一氧化氮合酶(inducible nitric oxide synthase, iNOS) 的表达,促进一氧化氮(nitric oxide, NO) 的生成,并刺激活性氧(reactive oxygen species, ROS) 和TNF-α的产生。但Yamamoto等[62-63]研究发现MAOS(M3-M6) 刺激RAW264.7巨噬细胞产生细胞因子的活性均高于GAOS(G3-G6)。其中,M3与G3之间的差异最明显。然而,目前尚不清楚MOS和GOS对RAW264.7巨噬细胞上的识别位点和具体机制上的区别。上述研究结果可证明,AOS的分子构象和分子量的大小可能是影响其诱导细胞因子活性的重要因素,并证明了AOS可通过激活巨噬细胞并诱导多种具有免疫活性的细胞因子,进而增强宿主免疫系统的能力。
巨噬细胞通过识别入侵机体的各类病原微生物及其产物,并以吞噬的方式将其消化和清除,进而保护机体免受病原微生物的感染[58]。激活巨噬细胞可产生ROS和各种细胞因子,还可通过分泌iNOS催化L-精氨酸产生NO。酶降解法制备的AOS(DP 2–5) 通过促进RAW264.7巨噬细胞中iNOS的表达来诱导NO的生成,因此NO可作为炎症介质和特异性免疫调节剂[64]。方伟珊等[58]在体外和体内试验中证明:Pseudoalteromonas sp. strain 272褐藻胶裂解酶制备的GAOS(DP 2–8) 介导RAW264.7巨噬细胞的免疫调节功能可分为以下几个步骤(图 4):(1) 表面Toll样受体4 (toll like receptor 4,TLR4) 识别CAOS; (2) TLR4激活磷脂酰肌醇-3-激酶(phosphatidylinositol-3-kinase, PI3K) 并诱导蛋白激酶B (protein kinase B, Akt or PKB) 磷酸化; (3) 磷酸化的Akt促进核转录因子κB抑制蛋白(inhibitory protein, IκB) 磷酸化; (4) 进而释放核转录因子κB (nuclear factor-κB, NF-κB) 并转位进入细胞核内; (5) 同时被激活的Akt还能通过磷酸化下游的雷帕霉素靶体蛋白(mechanistic target of rapamycin, mTOR) 及核糖体S6蛋白激酶(70 S6K); (6) 激活下游的转录因子,促进iNOS和TNF-α mRNA的转录,从而增加NO及TNF-α的产生。促分裂原活化蛋白激酶(mitogen-activated protein kinases, MAPKs) 位于TLR4下游信号通路,在机体固有和适应性免疫系统中起着重要的调节作用[58]。
GAOS可激活RAW264.7巨噬细胞中MAPK信号通路中p38、c-Jun N端激酶(c-Jun N-terminal kinase, JNK) 和细胞外信号调节的蛋白激酶(extracellular signal-regulated kinase, ERK),GAOS通过识别TLR4介导MAPK/JNK磷酸化水平,进而激活下游的转录因子(AP-1) 调节相关基因的转录,此外GAOS所激活的p38和ERK的磷酸化也参与了免疫介质的产生。然而这种激活方式不一定要通过TLR4所介导的信号通路[58],因此,TLR4并不是GAOS激活巨噬细胞的唯一受体。且已有研究发现,1 mg/mL GAOS还可诱导巨噬细胞补体受体的表达,增加对经IgG调理的金黄色葡萄球菌和大肠杆菌的胞内杀菌作用和吞噬能力[65]。此外,GAOS也能诱导巨噬细胞产生ROS。ROS是先天免疫系统的自由基效应因子,可以直接抑制病原体的繁殖[65]。GAOS还能识别RAW264.7细胞表面TLR2受体[61]。除了信号通路的激活外,GAOS还可调节与抗氧化活性、糖酵解、细胞骨架过程和翻译延伸等相关的通路[66]。
2.3 抗氧化活性AOS具有清除自由基的作用,可作为抗氧化剂。利用由产微球茎菌(Microbulbifer sp.) ALW1褐藻胶裂解酶制备的AOS由多数二糖和少数三糖组成,其具有清除自由基(DPPH、ABTS+和羟基) 和还原能力[39]。与壳聚糖和褐藻多糖相比,AOS具有较强的羟自由基清除能力,但对Fe2+的清除能力和脂质过氧化的抑制能力较差[67]。由鞘氨醇杆菌(Sphingobacterium)褐藻胶裂解酶制备的AOS (DP 2–4)能完全抑制铁诱导的乳化亚油酸氧化过程中TBARS的形成,且优于抗坏血酸。这可能是由于AOS的共轭烯酸结构有助于清除自由基的活性[4]。
AOS的抗氧化活性与AOS中G和M的相对含量无关[68]。C5羧基的方向是G和M之间唯一的结构差异,它们的空间排列不会影响AOS的抗氧化活性,但其制备方法、浓度和分子量可影响AOS的抗氧化活性。研究发现,低分子量(小于10 kDa) 的GAOS和MAOS能够发挥更高的抗氧化活性[69]。通过酸水解法制备的SAOS的抗氧化活性和作用机理与未降解的褐藻胶相似,然而通过酶降解法制备的AOS因具有共轭烯酸结构,进而具有较好的抗氧化活性。因此,通过酶降解法制备的AOS抗氧化活性高于褐藻胶和酸水解法制备的单糖或寡糖[4]。
AOS的抗氧化机理可能是从氢键中提取氢,氢与自由基加成结合到共轭烯酸结构中,产生共振稳定的加成产物,进而清除自由基[70]。在动物机体内,AOS具有抵抗氧化应激和防止氧化损伤的作用。酶降解法制备的AOS可阻断由H2O2诱导的氧化应激中内质网和线粒体产生的半胱天冬酶级联反应[8]。AOS可通过抑制GP91(phox) 和4-羟基壬烯醛的表达,进而减轻心脏氧化应激,从而增加阿霉素损伤小鼠的存活率[35]。与高脂组相比,UAOS可显著降低小鼠肝脏过氧化氢和丙二醛(malondialdehyde, MDA) 的水平,减轻高脂诱导的ROS的形成[71],然而SAOS对抗氧化能力没有影响。AOS可提高断奶仔猪血清中超氧化物歧化酶、过氧化氢酶活性和总抗氧化(total antioxidation capability, T-AOC) 能力以及肠道组织中T-AOC能力,并降低血清和肠道组织中MDA的含量[59, 72]。此外,在肉鸡日粮中添加MAOS也可提高血清和胸肌中的谷胱甘肽过氧化物酶的浓度,并降低肝脏和胸肌中MDA的含量,进而提高肉鸡抗氧化能力[73]。因此,AOS可通过酶促和非酶促抗氧化系统来提高动物的机体抗氧化防御能力,并防止动物机体组织的脂质过氧化。
2.4 抗炎症活性炎症是机体抵御病原体入侵的重要防御机制,可激活机体固有的免疫细胞。然而,慢性或过度炎症可出现在慢性疾病过程中,如炎症性肠病、风湿性多肌痛、关节炎、糖尿病、心血管疾病和神经退行性疾病[74]。AOS可通过抑制TLR4/NF-κB和NOD1/NF-κB信号通路,进而提高断奶仔猪的肠道抗炎功能[72]。通过不同的降解方法制备的AOS的抗炎效果明显不同。氧化降解法制备的AOS与使用其他方法制备的AOS相比,具有更强的炎症抑制作用。通过氧化降解法制备的AOS可抑制脂多糖/β-淀粉样蛋白(lipopolysaccharide, LPS/β-amyloid, Aβ) 刺激的小胶质神经炎症,进而促进Aβ小胶质细胞的吞噬能力[7]。氧化降解法制备的AOS通过阻断NF-κB和MAPKs信号通路的激活来抑制LPS活化的RAW264.7巨噬细胞的炎症反应,而通过Pseudoalteromonas sp. strain No. 272褐藻胶裂解酶法和酸降解法制备的AOS没有抗炎活性[75]。AOS通过提高小肠各类细胞簇的比例和数量,增强小肠细胞的功能和发育,进而缓解白消安诱导的小肠黏膜炎的细胞损伤,并在IPEC-J2细胞中进一步研究发现AOS可通过甘露糖受体信号通路发挥作用[76]。此外,GAOS和MAOS同样具有较好的抗炎活性,可作为有效的抗炎药物。研究表明,30 mg/kg GAOS在佐剂诱导的关节炎、实验性自身免疫性脑脊髓炎、肾病综合征和肾小球肾炎等多种试验模型中具有免疫抑制作用[77],GAOS和MAOS的抗炎活性与炎症过程中TLR4信号通路的调节有关[78-80]。
2.5 益生元功效及抑菌活性益生元是一些不被宿主消化吸收但可选择性地促进体内如双歧杆菌Bifidobacterium和乳酸菌Lactobacillus等有益菌的代谢和增殖,并产生生物活性代谢物,从而改善宿主健康的有机物质。通过体外培养试验发现,AOS可刺激Bifidobacterium和Lactobacillus的生长[6]。在小鼠日粮中添加2.5% AOS时,其可选择地增加小鼠粪便中Bifidobacterium和Lactobacillus的数量,并降低肠杆菌科(Enterobacteriaceae)和肠球菌(Enterococci)的数量[6]。当AOS浓度为0.625 g/L–1.25 g/L时,可以明显地缩短Bifidobacterium生长的适应期,使其大量生长[81]。AOS可提高断奶仔猪回肠中Bifidobacterium和Lactobacillus的数量,降低盲肠和结肠中的大肠杆菌(Escherichia coli, E. coli) 的数量[59],并提高了盲肠食糜中乙酸、丙酸和丁酸的浓度以及结肠中丁酸的浓度。因此,AOS是一种有效的Bifidobacterium的增殖因子,为其开发为益生元提供了一定的科学依据。
AOS可抑制革兰氏阴性和阳性细菌的生长,表现出广谱抗菌活性,具有潜在应用价值。其抑菌的作用可能是AOS先与细菌表面结合,调节细菌表面电荷,诱导微生物聚集,进而抑制细胞的运动性,破坏生物膜并增强抗生素的作用[82]。氧化降解法制备的AOS可抑制金黄色葡萄球菌Staphylococcus aureus、E. coli及藤黄微球菌Micrococcus luteus的生长,但对真菌没有抑制作用[83]。Han等[84]采用厌氧发酵系统,研究了AOS对猪粪便微生物的发酵能力及影响。发酵24 h后,AOS显著增加了Dysgonomonas的相对丰度,并显著降低了埃希菌属Escherichia、志贺氏菌属Shigella和嗜胨菌属Peptoniphilus的相对丰度,同时提高了丁酸和戊酸的浓度。AOS可减轻肠炎沙门氏菌对肉鸡生产性能的负面影响,通过促进Lactobacillus生长、调节黏膜细胞因子表达和抗体产生,并抑制沙门氏菌在盲肠的定殖[85]。此外,Zhu等[73]也证明了MAOS可降低肉鸡盲肠中E. coli的数量,并提高乙酸的浓度。
2.6 抗糖尿病活性糖尿病是常见的代谢性疾病,发达国家和发展中国家糖尿病的发病率都在不断上升。近年来,人们对多种多糖和寡糖进行了研究,并证实了其抗糖尿病活性。其中,AOS和硫酸酸化AOS具有强大的降糖作用[86]。AOS和甘露酸盐-铬复合物可用于辅助治疗Ⅱ型糖尿病,其可增强胰岛素敏感性,毒性比常用的二甲双胍低[77]。Yang等[87]研究发现,AOS在体外和体内对HepG2细胞均有较强的抗糖尿病活性,其通过调节JNK-IRS1/PI3K信号通路抑制葡萄糖水平的升高,防止体重增加,同时刺激胰岛素的分泌,提高葡萄糖耐量。
2.7 其他活性除了上述生物学活性外,AOS还具有降血压、抗哮喘、抗肥胖和神经保护活性。此外,AOS还可调节细胞生长及胆固醇代谢,防护射线辐射等[21]。
3 总结与展望AOS是一种有开发前途的天然添加剂,阐明AOS的结构与生物活性之间的关系有助于靶向制备具有特定组成和结构特征的AOS[88],从而应用于食品、药品和饲料添加剂等领域。AOS的生物活性由制备方法、M/G比例、分子量、空间构象及其衍生物等多种因素决定。与物理降解法、化学降解法和有机合成法相比,酶降解法具有明显的优势。首先,酶促反应是安全的,因为反应过程中几乎不需要化学试剂,并在温和的条件下制备AOS,可避免辐射、消耗较少的能源并有较高的收率[4]。其次,酶降解法制备的AOS是不饱和褐藻寡糖,可调节机体免疫和抗氧化功能。氧化降解法制备的AOS由于末端存在羧基具有较好的抗炎功能; 酸水解制备通过非选择性地断裂分子间的糖苷键[77],所得AOS保留了褐藻胶的大分子结构,形成饱和寡糖产物,生物活性较低。此外,AOS通过硫酸化、醇化等不同化学结构的修饰也可调节机体的生物活性[38]。但是,目前对AOS的结构与生物活性之间关系的研究还不够深入,大部分研究集中在AOS混合物的生物活性上,并多以体外模型研究为主,从而限制了AOS的进一步开发和利用。
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