裂殖壶藻(Schizochytrium)是一种单细胞海洋真菌[1]，又称裂殖壶菌，属于网粘菌门、网粘菌纲、破囊壶菌目、破囊壶菌科，单细胞、球形。作为单细胞海洋真菌类异养型生物，其生长过程中可积累大量营养物质，如脂质、蛋白质等。作为一种异养生物，裂殖壶藻可通过改变培养条件的方式，控制其体内营养成分的积累。裂殖壶藻的蛋白质含量相对较高，在蛋白质饲料资源匮乏的情况下，可作为新型的蛋白质饲料资源利用；另外，通过调整培养条件，可使裂殖壶藻油脂含量达到细胞干重的40%以上, 其中二十二碳六烯酸(docosahexaenoic acid, DHA)的含量可达总脂肪酸含量的50%以上，可作为一种功能性添加剂使用。裂殖壶藻除了高DHA含量，还含有虾青素、角鲨烯等活性成分，具有提高动物机体抗氧化[2]、抗炎症[3]等功能。本文结合国内外研究现状，就裂殖壶藻的营养物质、培养和生产条件、主要活性物质及其生理功能进行综述，为裂殖壶藻在畜禽生产中的应用提供参考。
1 裂殖壶藻的主要营养成分裂殖壶藻营养丰富，蛋白质、脂质含量较高，并含有其他一些微量物质，具有较高的饲用价值。作为一种海洋真菌，通过选择底物，筛选培养条件等方式，可使裂殖壶藻的蛋白质含量发生改变，例如使用豆粕水解物作为氮源时，总蛋白含量为9.35%，而使用酵母提取物作为氮源时，总蛋白含量可达42.51%[4]。蛋白质中氨基酸的组成较为稳定，包含10种必需氨基酸和6种非必需氨基酸，但是氨基酸的含量相差较大，与不同的培养底物有关，例如使用豆粕水解物作为氮源时，谷氨酸含量最高，为总氨基酸的20.87%，精氨酸的含量最低，为总氨基酸的0.95%；当使用酵母提取物作为氮源时，蛋氨酸含量最低，为总氨基酸的1.31%，而精氨酸含量相对较高，为总氨基酸的13.35%，谷氨酸可达总氨基酸的33.63%[4]。
裂殖壶藻的油脂含量较高，包括甘油酯、磷脂和不皂化物[5]。甘油酯多为甘油三酯(triglyceride，TG)，磷脂多为磷脂酰胆碱，不皂化物包括烷烃、二丁基羟基甲苯、角鲨烯(含量为0.035~0.162 mg/g)[6]以及胆固醇。此外，裂殖壶藻油脂中还含有强抗氧化物质虾青素[7]。由于裂殖壶藻培养产物的脂肪酸中，DHA占比最高，所以目前裂殖壶藻常被用做功能性添加剂使用。DHA系统名称为全顺式-4，7，10，13，16，19二十二碳六烯酸，其分子结构式中含有6个碳碳双键，并由22碳长链为骨架构成的多不饱和脂肪酸，属于ω-3长链多不饱脂肪酸(omega-3 polyunsaturated fatty acid，ω-3PUFA)中的一类。其相对分子质量为328.49，分子式为C22H32O。DHA具有熔点低、流动性较强的特点，常温下纯净的DHA为浅黄色或无色的澄清油状液体。DHA无色无味，难溶于水，易溶于乙醇、氯仿、乙醚等有机溶剂之中。
裂殖壶藻(培养使用的碳源为葡萄糖，氮源为谷氨酸钠)的主要营养成分见表 1。蛋白质和脂质的含量受培养底物的影响较大，而粗灰分和糖分含量较为稳定。裂殖壶藻的可溶性多糖的单糖主要为半乳糖，其次为甘露糖和鼠李糖，以及少量的木糖和葡萄糖[8]。裂殖壶藻中的多糖主要为硫酸半乳聚糖，其硫酸酯基主要在半乳糖残基的C6位[9]。
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表 1 裂殖壶藻的主要营养成分 Table 1 Main nutrients of Schizochytrium[8] |
裂殖壶藻的培养受碳源、氮源、氧气、微量元素等因素影响，并且作为一种海洋真菌，盐度也会影响其脂肪酸组成[10-11]。Song等[12]通过筛选得出裂殖壶藻的最佳培养条件为：温度23.8 ℃，通气量1.48 L/min, 转速250 r/min和接种生长中期的菌种，此条件下，细胞干重(DCW)和DHA含量分别达到最大值24.1和4.7 g/L。不同品种裂殖壶藻的DHA含量不同[13]，通过诱变育种的方法，可以培养出高DHA产量的裂殖壶藻品种[14]。培养底物的改变也显著影响着裂殖壶藻的生长，使用不同的碳源和氮源，产生的蛋白质和脂质也有所不同(表 2)。当使用葡萄糖作为碳源时，培养产物产量最多[15]。同时添加1 g/L苹果酸、0.4 mg/L洛伐他汀和0.3 mg/L生物素等微量成分时可使DHA的产量达到11.55 g/L[16]。此外，非营养物质对裂殖壶藻生长也有影响，如消泡剂的使用，可使培养液与氧气的接触更加充分，使裂殖壶藻的产量提升到48.93 g/L[17]。裂殖壶藻作为一种异养生物，生物量和脂质的积累与碳源有一定的关系，当使用葡萄糖作为碳源时，细胞干重、总脂质含量、DHA含量和DHA生产率分别为72.37、48.86、18.38 g/L和138.8 mg/L[15]。但是，葡萄糖的价格高，投入工业化生产不现实，所以选择价格低，培养效果好的底物是降低成本的关键。目前已经对薯片加工的液体残留[18]、玉米浆[19]、甜高粱汁[20]等进行了研究，但这些原料数量有限，且与传统生产相比，生物量、油脂量和DHA含量还是较低，因而工业生产潜力有限。因此，筛选出廉价、可工业化利用的碳源对裂殖壶藻的培养至关重要。近期研究发现，乙酸可以作为碳源供裂殖壶藻培养使用[21]，乙酸为挥发性脂肪酸中的一种，可通过厌氧生物在有机废物中发酵形成[22-23]。使用乙酰辅酶A合成酶可将乙酸直接转化为乙酰辅酶A，形成脂质合成的中间体[24-28]，可以直接参与脂质合成。并且与葡萄糖和甘油相比，乙酸具有更短的代谢途径[29]，更高的脂质转化率[30]。Shafiq等[31]通过试验证明了使用乙酸作为碳源，生物量达到146 g/L，总脂量为82.3 g/L，DHA产量达到23.0 g/L，并显著降低了成本。此外，有研究表明，裂殖壶藻能利用粗甘油生产DHA，其中75~100 g/L是甘油使用的最佳浓度范围[32]。粗甘油是在工业化生产柴油时，通过TG的酸或碱催化酯交换反应生成的，也包括植物油、动物脂肪和废食用油[33-34]。其产量巨大，经济价值低，且其纯化的成本很高[35]，不恰当的处理还会污染环境，但却可以作为裂殖壶藻培养的有机碳源。Lung等[36]在混合/异养条件下，在20 ℃的培养基中使用3%的粗甘油，可获得最高DHA生产率为233.73 mg/g的生物质。
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表 2 不同培养条件下裂殖壶藻生物量、总脂和DHA的水平 Table 2 Levels of biomass, total fat and DHA of Schizochytrium under different culture conditions |
裂殖壶藻的使用可起到一定抗炎的作用。前列腺素E2(prostaglandin E2，PGE2)参与炎症反应，它是由膜磷脂sn-2位的花生四烯酸(arachidonic acid，AA)分子通过环氧合酶(cyclooxygenase，COX)的作用合成，因此细胞膜中的AA对于PGE2的合成极为重要，通过减少AA的存在可以调节PGE2的合成从而降低炎症反应[46]。AA是ω-6脂肪酸的代谢产物，将ω-3脂肪酸掺入细胞膜时会降低ω-6PUFA/ω-3PUFA的比值，从而减少PGE2的合成，并有利于前列腺素E3(PGE3)的产生，且PGE3所产生的炎症性作用较小[47]。有研究发现，当给绝育后的猫的饲粮中添加4、8、12、16.0 g/kg的裂殖壶藻时，术后猫体内PGE2的水平线性下降，从而减轻了炎症反应，而未添加裂殖壶藻的对照组猫PGE2的水平显著增加，导致炎症反应加强[48]。过氧化物酶体增殖物受体γ(peroxisome proliferator-activated receptor γ，PPARγ)是重要的细胞分化转录因子，在哺乳动物的脂肪组织中有表达，当PPARγ过度表达时，会导致肥胖发生，而肥胖是一种炎症状态。此外，转化生长因子-β1(transforming growth factor-β1，TGF-β1)可以同时作为抗炎和促炎的介质，Jo等[49]研究发现，胃黏膜TGF-β1的水平与炎症的严重程度之间呈负相关。Komprda等[50]研究发现，当给肥胖大鼠的饲粮中添加裂殖壶藻后，相较于饲粮中未添加裂殖壶藻的对照组，添加了裂殖壶藻的试验组，肝细胞中PPARγ蛋白表达量降低，并且血浆中TGF-β1的含量上升。此外，Wang等[51]使用葡聚糖硫酸钠(dextransulfatesodium，DSS)诱导小鼠结肠炎后，给试验组小鼠的饲粮中添加裂殖壶藻藻渣水解物，与对照组相比，试验组小鼠结肠炎中的肿瘤坏死因子-α(tumor necrosis factor-α，TNF-α)、白细胞介素-17(interleukin-17，IL-17)、IL-1β这3种促炎因子的表达水平显著降低，且抗炎因子IL-10的表达水平升高。
3.2 抗氧化氧化还原失衡可在动物体内直接引起多种氧化损伤，DHA属于多不饱和脂肪酸, 有着较强的抗氧化活性, 能有效降低体内氧化活性簇(reactive oxygen species，ROS)含量[2]。Tomaluski等[52]的研究中发现，通过给犊牛补充裂殖壶藻，增加了犊牛体内DHA的含量，从而降低了血清脂质过氧化(lipid peroxidation，LPO)和ROS含量。线粒体中存在大量的抗氧化酶，可保护线粒体的氧化还原平衡，在抗氧化酶中，超氧化物歧化酶(superoxide dismutase，SOD)可与ROS进行反应，它能催化强内源性自由基超氧阴离子的歧化并将其转化为H2O和O2。Alexandros等[53]在山羊饲粮中添加裂殖壶藻浓缩液，与未添加的组相比，添加组山羊血清中SOD和谷胱甘肽巯基转移酶(glutathione S-transferase，GSTs)的活性显著增加。同样，Xiao等[54]通过向镜鲤幼鱼的饲料中分别添加的60、90、120 g/kg的裂殖壶藻，结果发现，镜鲤的肝胰腺和血清中的SOD活性都显著增加，且适宜的添加范围为41.8~60.0 g/kg。丙二醛(malondialdehyde，MDA)是脂质过氧化的主要产物，是氧化应激的主要标志，Long等[55]研究发现，在肉仔鸡的饲粮中添加1%或2%的裂殖壶藻，裂殖壶藻中的DHA可通过激活酶和非酶抗氧化系统来降低MDA含量，以此来有效降低肉中MDA含量，并且提高了肉仔鸡体内SOD活性，使得总抗氧化能力提升。此外，Lv等[56]的研究发现，裂殖壶藻藻油中还存在较高浓度的β-胡萝卜素、维生素E、甾醇、酚类和类黄酮，这些化合物不仅使藻油的内容物更加丰富，也使其具有更高的抗氧化活性。
3.3 调节脂质代谢DHA对脂质代谢有一定的影响，可调节脂质代谢，具有降低血液和肝脏中TG以及胆固醇含量的作用[57]，并且能抑制肝脏脂肪合成[57-58]。血清样淀粉蛋白A(serum amyloid A，SAA)、TNF-α以及IL-6能调节脂肪代谢，DHA能使这些信号蛋白的表达上调[59]。Yu等[60]的试验中，使用高脂饲粮(high fit diet，HFD)诱导高脂模型，向HFD中添加100 mg/kg的裂殖壶藻藻油，通过16周的饲喂，结果发现明显降低了小鼠腹部脂肪堆积，降低了TG、胆固醇和低密度脂蛋白(low density lipoprotein，LDL)的含量，同时抑制了脂肪酸合酶和固醇调节元件结合蛋白-1(SREBP-1)和乙酰辅酶A羧化酶(acetyl CoA carboxylase，ACAC)的基因表达。DHA可以通过降低肝脏中极低密度脂蛋白(very low density lipoprotein，VLDL)的合成来降低TG的含量。TG合成减少进而导致VLDL分泌减少、颗粒更小，更容易转化为高密度脂蛋白(high density lipoprotein，HDL)[61]。LDL可运载胆固醇进入外周组织细胞，而HDL可运载外周组织中胆固醇，通过将胆固醇转化为胆汁酸或直接通过胆汁从肠道排出，因此减少胆固醇的含量可通过降低LDL含量和提高高密度脂蛋白胆固醇(high-density lipoprotein cholesterol，HDL-C)含量来实现[62]。Long等[55]研究发现，饲粮中添加1%或2%的裂殖壶藻促进了HDL-C的合成，并通过促进血清总胆固醇(total cholesterol，TC)、TG、低密度脂蛋白胆固醇(LDL-C)的代谢调节血脂平衡。
4 裂殖壶藻在畜牧生产中的应用随着生活水平的提升，人们对畜禽产品的需求已经开始从量向质发生转变。裂殖壶藻含有丰富的DHA等多不饱和脂肪酸，具有改善畜禽产品品质的作用。Gładkowski等[63]在鹌鹑的饲粮中添加0.5%的裂殖壶藻粉, 发现其蛋黄中的DHA含量显著提高。陈秀丽等[64]在海兰褐鸡饲粮中添加2%裂殖壶藻粉，饲喂到第15天时显著提高了蛋黄DHA含量。王浩等[65]在京红蛋鸡饲粮中添加0.5%的裂殖壶藻油，发现显著提高了蛋黄中ω-3PUFA的含量，并降低了蛋黄中ω-6PUFA的含量以及ω-6PUFA/ω-3PUFA的比例。李浩洋等[66]在杏花鸡饲粮中分别添加1%、2%和3%的含有15% DHA的裂殖壶藻粉，结果发现鸡蛋油脂中DHA含量由1.50%分别提高至3.95%、4.60%和5.25%，且蛋黄中的胆固醇含量下降，但料蛋比、平均日采食量、蛋壳厚度、平均蛋重无显著变化。
反刍动物对裂殖壶藻营养成分具有较高的利用效率。Tomaluski等[52]的研究中发现，通过将裂殖壶藻粉稀释至牛奶中，增加了犊牛的裂殖壶藻摄入量，从而起到增加断奶胸围的作用，并降低了血清ROS和脂质过氧化物(LPO)的含量，减少氧化应激。Franklin等[67]通过向奶牛饲粮中添加裂殖壶藻发现，可以使乳脂中DHA的含量显著提高。Alexandros等[53]在山羊饲粮中添加裂殖壶藻，提高了山羊奶中ω-3PUFA的含量。裂殖壶藻可以作为蛋白质和能量的来源添加在饲粮之中，且由于其DHA含量高，可以改变动物机体脂肪酸的组成。在肉羊饲粮中加入裂殖壶藻，提高了DHA、二十碳五烯酸(eicosapentaenoic acid，EPA)、共轭亚麻油酸(conjugated linoleic acid，CLA)的含量，降低了肉类胆固醇的含量，具有改善胴体品质，脂肪组织和肌肉脂肪酸比例的作用[68-70]。使用大肠杆菌K99攻毒哺乳犊牛后，饲喂裂殖壶藻，结果发现过氧化氢酶(catalase，CAT)、SOD活性显著提高，说明添加裂殖壶藻具有促进肠道健康、降低腹泻和提高抗氧化能力的作用[71]。
饲粮添加裂殖壶藻粉还具有改善猪肉品质和生长性能的作用，Sardi等[72]和Vossen等[73]分别对公猪和母猪进行研究发现，通过添加适量的裂殖壶藻粉可显著提高猪肉产品中的DHA含量。Kalbe等[74]研究也表明，饲粮添加裂殖壶藻可提高长白猪肌肉中DHA含量。此外，Kibria等[75]研究发现，断奶仔猪饲粮中添加1.0%裂殖壶藻显著提高了仔猪生长速度，并且能提高断奶仔猪对干物质和氮的表观总消化率和回肠养分消化率。
5 小结在全面禁抗和饲料资源缺乏的背景下，开发新的饲料资源成为畜牧领域的研究热点。裂殖壶藻富含蛋白质、脂质、氨基酸和糖类等，且其培养具有低污染、低消耗、可循环等优势，具有良好的开发前景。但目前培养裂殖壶藻所选用的底物价格较高，致使生产成本高。因此，开发廉价、可大规模获取的工业及农业副产物作为培养底物，并通过优化培养工艺降低成本是裂殖壶藻作为饲料资源大规模应用的前提。此外，多不饱和脂肪酸具有改善畜禽健康和畜禽产品品质的作用，但当其含量过高会造成肉品质变差，氧化过程加快，因此探究裂殖壶藻在饲粮中的合理使用同样对其在畜牧生产中的应用具有重要的意义。
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