CIISA - Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, Lisboa, Portugal.
Laboratório Associado para Ciência Animal e Veterinária (AL4AnimalS), Lisboa, Portugal.
PLoS One. 2022 May 19;17(5):e0268565. doi: 10.1371/journal.pone.0268565. eCollection 2022.
Over the last decades, microalgae have gained popularity due to demand for novel environmental green solutions and development of innovative mass-production sources for multiple processes, including animal feed and human diet, turning microalgae into an exquisite candidate for several ecofriendly technologies. Notwithstanding, there is a catch. Most species of microalgae, as the case of common Chlorella vulgaris (C. vulgaris) display a recalcitrant cell wall, characterized by a complex matrix of polysaccharides and glycoproteins, which constitutes a major barrier for monogastric species digestibility and extraction of inner valuable nutritional compounds. To overcome this limitation, the development of feed enzymes, in particular Carbohydrate-Active enZymes (CAZymes) with capacity to disrupt C. vulgaris cell wall may contribute to improve the bioavailability of these microalgae compounds in monogastric diets, namely at high levels of incorporation. In order to disclosure novel combination of feed enzymes to disrupt C. vulgaris cell wall, a lab protocol was implemented by our research team containing the following key steps: after microalgae cultivation and having available a repertoire of two hundred pre-selected CAZymes produced by high-throughput technology, the step 1 is the individual screening of the most functional enzymes on disrupting C. vulgaris cell wall (versus a control, defined as the microalgae suspension incubated with PBS) and the determination of reducing sugars released by the 3,5-dinitrosalicylic acid (DNSA) method; step 2 concerns on finding the best CAZymes cocktail, testing the synergistic effect of enzymes, to disrupt C. vulgaris cell wall (in parallel with running the control) along with characterization of each enzyme thermostability and resistance to proteolytic attack, to which feed enzymes are subjected in the animal gastrointestinal tract; step 3 is the assessment of C. vulgaris cell wall degradation degree by measuring the amount of reducing sugars released by the DNSA method, fatty acid analysis by gas chromatography (GC) with flame ionization detector (FID), oligosaccharides quantification by high performance liquid chromatography (HPLC) equipped with an electrochemical detector (ECD), protein content by the Kjeldahl method, and various pigments (chlorophylls a and b, and total carotenoids) in the supernatant. In the correspondent residue, we also assessed cellular counting using a Neubauer chamber by direct observation on a bright-field microscope and fluorescence intensity, after staining with Calcofluor White for both control and CAZymes cocktail treatments, on a fluorescence microscope. Beyond animal feed industry with impact on human nutrition, our lab protocol may increase the yield in obtaining valued constituents from C. vulgaris microalga for other biotechnological industries.
在过去的几十年中，由于对新型环保绿色解决方案的需求以及对多种工艺（包括动物饲料和人类饮食）的创新大规模生产来源的开发，微藻受到了欢迎，使微藻成为多种环保技术的理想候选对象。然而，这其中存在一个问题。大多数微藻物种，如普通小球藻（Chlorella vulgaris），都具有顽固的细胞壁，其特点是复杂的多糖和糖蛋白基质，这是单胃动物物种消化和提取内部有价值营养化合物的主要障碍。为了克服这一限制，开发能够破坏小球藻细胞壁的饲料酶，特别是碳水化合物活性酶（CAZymes），可能有助于提高这些微藻化合物在单胃动物日粮中的生物利用度，即高水平添加。为了揭示破坏小球藻细胞壁的新型饲料酶组合，我们的研究团队实施了一个实验室方案，其中包含以下关键步骤：在微藻培养完成后，利用高通量技术获得了两百种预先筛选的 CAZymes，步骤 1 是单独筛选对破坏小球藻细胞壁最有效的酶（与对照相比，定义为与 PBS 孵育的微藻悬浮液），并通过 3,5-二硝基水杨酸（DNSA）法测定释放的还原糖；步骤 2 涉及寻找最佳 CAZymes 混合物，测试酶的协同作用，以破坏小球藻细胞壁（同时运行对照），以及对每种酶的热稳定性和抗蛋白水解攻击的特性进行表征，这些都是饲料酶在动物胃肠道中会遇到的情况；步骤 3 是通过 DNSA 法测定释放的还原糖量、气相色谱（GC）-火焰离子化检测器（FID）法测定脂肪酸、高效液相色谱（HPLC）-电化学检测器（ECD）法测定低聚糖、凯氏定氮法测定蛋白质含量以及上清液中的各种色素（叶绿素 a 和 b 以及总类胡萝卜素）来评估小球藻细胞壁的降解程度。在对应的残渣中，我们还使用 Neuber 室通过在明亮场显微镜下直接观察细胞计数，并在荧光显微镜下用 Calcofluor White 对对照和 CAZymes 混合物处理进行荧光强度染色，来评估细胞计数。除了对人类营养有影响的动物饲料行业外，我们的实验室方案还可以提高从小球藻微藻中获得有价值成分的产量，以用于其他生物技术行业。
