Name: 作者聚焦：探索海藻的生物活性化合物以促进工业的可持续创新
Uploaded: 2023-11-21T14:20:00
Description: Watch this Scientific Journal Video about Exploring Seaweed's Bioactive Compounds for Sustainable Innovations in Industries at JoVE.com

这项工作得到了葡萄牙科学技术基金会（FCT）的支持，通过授予MARE-海洋与环境科学中心的战略项目（UIDP/04292/2020和UIDB/04292/2020）和副实验室ARNET（LA/P/0069/2020）。FCT 还资助了授予 Marta V. Freitas （UI/BD/150957/2021） 和 Tatiana Pereira （2021.07791. BD）。这项工作还得到了HP4A--全民健康意大利面项目（共同推广第039952号）的财政支持，该项目由ERDF--欧洲区域发展基金在葡萄牙2020年计划下通过COMPETE 2020--竞争力和国际化运营计划共同资助。

1. 生物质收获和制备
<ol><li>在退潮时收获 G. gracilis 标本，并将它们迅速运送到实验室，装在黑暗、冷却的盒子里，以避免干燥、光线和空气暴露。</li>
	<li>在实验室中，用流动的海水清洗每个菌体并彻底清洁以去除表面的碎屑、坏死部分、附生植物和其他生物。</li>
	<li>将野生生物量保持在恒定充气的海水（31-35 psu）中，在气候室（20±1°C）中，由日光冷白光和荧光灯提供低辐照度，光周?...

利用 Gracilaria gracilis：可持续创新的生物活性潜力

液体介质中的抗菌活性测试用于评估抗菌物质对悬浮在液体介质中的微生物的有效性，通常用于确定物质抑制生长或杀死微生物的能力35,36,37,38.它们用于评估微生物对抗菌剂的敏感性，并在试管或微量滴定板中进行，其中不同浓度的抗微生物物质针对目标微生物的标准化悬浮液进行测试2...

海藻可以被视为一种有趣的天然原料，可以从制药、食品、饲料和环境部门获利。它们生物合成了一整套分子，其中许多分子在陆地生物中没有发现，具有相关的生物学特性1,2。然而，需要实施海藻优化的养殖方案，以确保大量的生物量储存。
栽培方法必须始终考虑海藻菌体的性质和整体形态。 Gracilaria gracilis 是一个克隆类群，这意味着附着器官产生多个营养轴。因此，实现了通过碎片化（无性繁殖）的繁殖，因为这些轴中的每一个都完全能够采用独立于主菌体3 的生命。克隆类群可以通过简单快速的一步培养方法成功整合，因为通过将菌体分解成小片段来获得大量的生物量，这些碎片可以快速再生并生长成新的、基因相同的个体。在此过程中可以使用单倍体和双倍体。尽管该属表现出复杂的单倍双克隆同构三相生命周期，但很少需要孢子形成，除非需要对种群进行遗传更新以实现改良作物。在这种情况下，四孢子（由减数分裂形成的单倍孢子）和果孢子（由有丝分裂形成的双孢子）都会产生宏观的菌体，然后可以通过无性繁殖生长和繁殖4。生长周期取决于环境条件和个体的生理状态，以及其他生物因素，例如附生植物的出现和其他生物的粘附。因此，优化生长条件对于确保高生产力和生产优质生物质至关重要5.
从海藻中提取生物活性化合物，包括G. gracilis，可以通过各种方法实现6,7。提取方法的选择取决于目标化合物的具体化合物、目标应用和海藻的特性。在这项研究中，我们专注于溶剂萃取，这涉及使用绿色溶剂，如水或乙醇，从海藻生物质中溶解和提取生物活性化合物。萃取可以通过浸渍以通用且有效的方式进行，可用于多种化合物。这是一种简单且广泛使用的方法，涉及将生物质长时间浸泡在溶剂中，通常在室温或略高的温度下。搅拌溶剂以增强提取过程。在所需的提取时间之后，通过过滤或离心将溶剂与固体材料分离。
水是食品应用中常用的溶剂，因为它具有安全性、可用性和与各种食品的相容性。水萃取适用于多糖、肽和某些酚类化合物等极性化合物。然而，它可能无法有效地提取非极性化合物。乙醇也是食品应用中广泛使用的溶剂，可有效提取各种生物活性分子，包括酚类化合物、类黄酮和某些色素。乙醇通常被认为是安全的，可以安全地用于食品中，并且很容易蒸发，留下提取的化合物。值得注意的是，提取方法的选择应考虑效率、选择性、成本效益和环境影响等因素。优化提取参数，如溶剂浓度、提取时间、温度和压力，对于从 G. gracilis 或其他海藻中获得最佳生物活性化合物的产量至关重要。
已发现海藻对多种微生物（包括细菌、真菌和病毒）表现出抗菌活性8.这种活性归因于生物活性成分，包括酚类、多糖、肽和脂肪酸。多项研究表明，它们对大肠杆菌、金黄色葡萄球菌、沙门氏菌属和铜绿假单胞菌等病原体具有功效9。海藻的抗菌活性归因于生物活性化合物的存在，这些化合物会干扰微生物细胞壁、膜、酶和信号通路10。这些化合物可能会破坏微生物生长，抑制生物膜形成，并调节免疫反应。
红海藻，也称为红藻，是一组可以对多种微生物表现出抗菌活性的藻类。在该组中， G. gracilis 含有各种生物活性化合物，可能有助于其报告的抗菌活性。虽然具体分子可能有所不同，但已报道的 G. gracilis 中可能具有抗菌特性的常见类别是多糖、酚类、萜类化合物和色素11。然而，需要注意的是，这些成分的存在和数量可能因海藻收集地点、季节性、海藻的生理条件和环境条件等因素而异。因此， G. gracilis 中抗菌化合物的具体类别和浓度可能会相应变化。
G. gracilis 还被发现具有抗氧化特性，含有各种酚类化合物，已被证明可以清除自由基并减少氧化应激12。抗氧化剂有助于保护细胞免受活性氧造成的损害，并具有潜在的健康益处。抗氧化能力可以通过不同的方法直接评估，包括 2,2-二苯基-1-三硝基肼 （DPPH） 自由基清除活性，以及间接通过总多酚含量 （TPC） 的定量13。
尽管据报道一种成分具有突出的生物活性，但其细胞毒性评估在评估与活细胞或组织接触的天然和合成物质时是必不可少的。有几种测量细胞毒性的方法，每种方法都有优点和局限性。总体而言，它们提供了一系列选择来评估许多物质对细胞的有害影响，同时研究细胞损伤和死亡的机制14。
在这项工作中，我们使用3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴化物（MTT）测定法，这是Mosmann（1983）15引入的比色法。该方法测量代谢活性细胞将四唑盐还原为紫色甲瓒产物的过程。甲臜晶体的量越高，活细胞的数量就越多，从而提供了细胞毒性的间接测量14。由于在这项工作中， G. gracilis 水和乙醇提取物旨在掺入皮肤化妆品配方中，因此在角质形成细胞 （HaCaT） 细胞系中进行 体外 细胞毒性评估。
在食品应用方面，海藻通常热量低，营养丰富，富含膳食纤维、必需元素和氨基酸、多糖、多不饱和脂肪酸、多酚和维生素 2,16。G. gracilis 也不例外，具有有趣的营养价值。Freitas et al. （2021）4 发现，与野生海藻相比，栽培的 G. gracilis 具有更高水平的蛋白质和维生素 C，并维持了总脂质的水平。这可能代表了一种经济和环境优势，因为从营养上讲，生产比开发野生资源更可取。此外，消费者越来越关注他们所吃的食物类型，因此引入新的成分来丰富食物，并使用新的资源来获得可以为产品增加价值并声称“清洁标签”的提取物非常重要。此外，当前市场竞争非常激烈，需要开发新产品和创新策略，以将制造商与竞争对手区分开来17.
将意大利面等营养价值低的产品与海藻等海洋资源进行富集，是一种通过具有不同营养价值的产品将这种资源作为新食品引入的战略和市场差异化战略。另一方面， G. gracilis 是天然红色素的来源，例如藻胆蛋白18，在食品工业中具有很高的应用潜力。这种海藻在多个领域表现出浓厚的兴趣，其应用可以使用整个海藻、提取物和/或剩余的生物质进行。在这项工作中，我们演示了此类应用的一些示例。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Absolute Ethanol</td>
			<td>Aga, Portugal</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Chloride</td>
			<td>PanReac</td>
			<td>12125-02-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Sigma-Aldrich</td>
			<td>1397-89-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical scale balance</td>
			<td>Sartorius, TE124S</td>
			<td>22105307</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacillus subtilis subsp. spizizenii</td>
			<td>German Collection of Microorganisms and Cell Cultures (DSMZ)</td>
			<td>DSM 347</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin</td>
			<td>Panreac AppliChem</td>
			<td>58-85-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf, 5810R</td>
			<td>5811JH490481</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>PanReac</td>
			<td>56-75-7</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Chamber</td>
			<td>Memmert</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Cool White Fluorescent Lamps</td>
			<td>OSRAM Lumilux Skywhite</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Densitometer McFarland</td>
			<td>Grant Instruments</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM medium</td>
			<td>Sigma-Aldrich</td>
			<td>D5796</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>67-68-5</td>
			<td></td>
		</tr>
		<tr>
			<td>DPPH</td>
			<td>Sigma, Steinheim, Germany</td>
			<td>1898-66-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Escherichia coli (DSM 5922)</td>
			<td>German Collection of Microorganisms and Cell Cultures (DSMZ)</td>
			<td>DSM5922</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 96%</td>
			<td>AGA-Portugal</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic Acid Disodium Salt Dihydrate (Na2EDTA)</td>
			<td>J.T.Baker</td>
			<td>6381-92-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Sigma-Aldrich</td>
			<td>F7524</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter Paper (Whatman No.1)</td>
			<td>Whatman</td>
			<td>WHA1001320</td>
			<td></td>
		</tr>
		<tr>
			<td>Flasks</td>
			<td>VWR International, Alcabideche, Portugal&#160;</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Folin-Ciocalteu</td>
			<td>VWR Chemicals</td>
			<td>31360.264</td>
			<td></td>
		</tr>
		<tr>
			<td>Gallic Acid&#160;</td>
			<td>Merck</td>
			<td>149-91-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Germanium (IV) Oxide, 99.999%</td>
			<td>AlfaAesar</td>
			<td>1310-53-8</td>
			<td></td>
		</tr>
		<tr>
			<td>HaCaT cells &#8211; 300493</td>
			<td>CLS-Cell Lines Services, Germany&#160;</td>
			<td>300493</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot Plate Magnetic Stirrer</td>
			<td>IKA, C-MAG HS7</td>
			<td>06.090564</td>
			<td></td>
		</tr>
		<tr>
			<td>Iron Sulfate</td>
			<td>VWR Chemicals</td>
			<td>10124-49-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar flow hood</td>
			<td>TelStar, Portugal</td>
			<td>526013</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Medium&#160;</td>
			<td>VWR Chemicals</td>
			<td>J106</td>
			<td></td>
		</tr>
		<tr>
			<td>Listonella anguillarum</td>
			<td>German Collection of Microorganisms and Cell Cultures (DSMZ)</td>
			<td>&#160;DSM 21597</td>
			<td></td>
		</tr>
		<tr>
			<td>Manganese Chloride</td>
			<td>VWR Chemicals</td>
			<td>7773.01.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettes&#160;</td>
			<td>Eppendorf, Portugal</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplates</td>
			<td>VWR International, Alcabideche, Portugal&#160;</td>
			<td>10861-666</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplates</td>
			<td>Greiner</td>
			<td>738-0168</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplates (sterile)</td>
			<td>Fisher Scientific</td>
			<td>10022403</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>&#160;Epoch Microplate Spectrophotometer, BioTek, Vermont, USA</td>
			<td>1611151E</td>
			<td></td>
		</tr>
		<tr>
			<td>MTT</td>
			<td>Sigma-Aldrich</td>
			<td>289-93-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Muller-Hinton Broth (MHB)</td>
			<td>VWR Chemicals</td>
			<td>90004-658</td>
			<td></td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>Binder, FD115</td>
			<td>12-04490</td>
			<td></td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>Binder, BD115</td>
			<td>04-62615</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td>Sigma-Aldrich</td>
			<td>1406-05-9</td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter Inolab</td>
			<td>&#160;VWR International, Alcabideche, Portugal&#160;</td>
			<td>15212099</td>
			<td></td>
		</tr>
		<tr>
			<td>Pippete tips</td>
			<td>Eppendorf, Portugal</td>
			<td>5412307</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyrex Bottles Media Storage</td>
			<td>&#160;VWR International, Alcabideche, Portugal&#160;</td>
			<td>16157-169</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary Evaporator</td>
			<td>Heidolph, Laborota 4000</td>
			<td>80409287</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotavapor</td>
			<td>IKA HB10, VWR International, Alcabideche, Portugal</td>
			<td>07.524254</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Carbonate (Na2CO3)</td>
			<td>Chem-Lab</td>
			<td>497-19-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)&#160;</td>
			<td>Normax Chem</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate Dibasic</td>
			<td>Riedel-de Ha&#235;n</td>
			<td>7558-79-4</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraMagic NX</td>
			<td>Konica Minolta, Japan</td>
			<td></td>
			<td>color data analysis software</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Evolution 201, Thermo Scientific, Madison, WI, USA</td>
			<td>5A4T092004</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>57-92-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiamine</td>
			<td>Panreac AppliChem</td>
			<td>59-43-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>T4049</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptic Soy Agar (TSA)</td>
			<td>VWR Chemicals</td>
			<td>ICNA091010617</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptic Soy Broth (TSB)&#160;</td>
			<td>VWR Chemicals</td>
			<td>22091</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure water&#160;</td>
			<td>Advantage A10 Milli-Q lab, Merck, Darmstadt, Germany</td>
			<td>F5HA17360B</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>Buchi, Switzerland</td>
			<td>FIS05-402-103</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitamin B12</td>
			<td>Merck</td>
			<td>68-19-9</td>
			<td></td>
		</tr>
	</tbody>
</table>

valorization of the red seaweed gracilaria gracilis through a biorefinery approach

人们对海藻作为一种丰富的原料，以获得有价值的多目标生物活性成分的兴趣不断增长。在这项工作中，我们探索了 Gracilaria gracilis的潜力，这是一种可食用的红海藻，因其商业利益而在全球范围内种植，作为化妆品、药理学、食品和饲料应用的琼脂和其他成分的来源。
通过无性繁殖和孢子形成优化了G. gracilis的生长条件，同时操纵了物理化学条件，以实现较大的生物量储量。在海藻生物质上使用乙醇和水进行绿色提取方法。通过一组关于提取物细胞毒性、抗氧化和抗菌特性的体外测定来评估提取物的生物活性潜力。此外，干海藻生物质被纳入意大利面配方中，以增加食物的营养价值。从G. gracilis中提取的色素也已作为天然着色剂掺入酸奶中，并对其稳定性进行了评估。这两款产品都经过了半培训的感官小组的评估，旨在在进入市场之前实现最佳的最终配方。
结果支持 G. gracilis 的多功能性，无论是作为整个生物质，提取物和/或色素使用。通过实施几个优化的协议，这项工作允许开发具有潜在利润的产品，以促进食品、化妆品和水产养殖市场，促进环境可持续性和蓝色循环经济。
此外，根据生物精炼方法，残留的海藻生物质将用作植物生长的生物刺激剂或转化为碳材料，用于葡萄牙莱里亚MARE-Polytechnic内部水产养殖系统的水净化。

&#160;Seaweeds can be regarded as an interesting natural raw material to be profited by the pharmaceutical, food, feed, and environmental sectors. They biosynthesize a panoply of molecules, many not found in terrestrial organisms, with relevant biological properties1,2. However, seaweed-optimized cultivation protocols need to be implemented to ensure a large biomass stock.
Cultivation methods must always consider the nature of the seaweed thalli and overall morphology. Gracilaria gracilis is a clonal taxon, meaning the attachment organ produces multiple vegetative axes. Propagation by fragmentation (vegetative reproduction) is thus achieved, as each of these axes is fully able to adopt an independent life from the main thallus3. Clonal taxa can be successfully integrated with simple and fast one-step cultivation methodologies, as large amounts of biomass are obtained by splitting the thallus into small fragments that quickly regenerate and grow into new, genetically identical individuals. Both haplontic and diplontic thalli may be used in this process. Although the genus exhibits a complex haplo-diplontic isomorphic triphasic life cycle, sporulation is rarely necessary except when genetic renewal of the stocks is required to achieve improved crops. In this case, both tetraspores (haplontic spores formed by meiosis) and carpospores (diplontic spores formed by mitosis) give rise to macroscopic thalli that can then be grown and propagated by vegetative reproduction4. Growth cycles are dictated by environmental conditions and the physiological state of the individuals, among other biological factors such as the emergence of epiphytes and the adhesion of other organisms. Therefore, optimizing growing conditions is crucial to ensure high productivity and produce good quality biomass5.
The extraction of bioactive compounds from seaweed, including G. gracilis, can be achieved through various methods6,7. The choice of the extraction method depends on the specific compounds of interest, the target application, and the characteristics of the seaweed. In this study, we focused on solvent extraction, which involves using green solvents, such as water or ethanol, to dissolve and extract bioactive compounds from the seaweed biomass. The extraction can be carried out through maceration in a versatile and effective way and can be used for a wide range of compounds. It is a simple and widely used method involving soaking biomass in a solvent for an extended period, typically at room or slightly elevated temperatures. The solvent is stirred to enhance the extraction process. After the desired extraction time, the solvent is separated from the solid material by filtration or centrifugation.
Water is a commonly used solvent in food applications due to its safety, availability, and compatibility with a wide range of food products. Water extraction is suitable for polar compounds such as polysaccharides, peptides, and certain phenolics. However, it may not effectively extract non-polar compounds. Ethanol is also a widely used solvent in food applications and can be effective for extracting a variety of bioactive molecules, including phenolic compounds, flavonoids, and certain pigments. Ethanol is generally recognized as safe for use in food and can be easily evaporated, leaving behind the extracted compounds. It is worth noting that the choice of extraction method should consider factors such as efficiency, selectivity, cost-effectiveness, and environmental impact. The optimization of extraction parameters, such as solvent concentration, extraction time, temperature, and pressure, is crucial to achieving optimal yields of bioactive compounds from G. gracilis or other seaweeds.
Seaweeds have been found to exhibit antimicrobial activity against a wide range of microorganisms, including bacteria, fungi, and viruses8. This activity is attributed to bioactive components, including phenolics, polysaccharides, peptides, and fatty acids. Several studies have demonstrated their efficacy against pathogens such as Escherichia coli, Staphylococcus aureus, Salmonella sp., and Pseudomonas aeruginosa, among others9. The antimicrobial activity of seaweeds is attributed to the presence of bioactive compounds that can interfere with microbial cell walls, membranes, enzymes, and signaling pathways10. These compounds may disrupt microbial growth, inhibit biofilm formation, and modulate immune responses.
Red seaweeds, also known as rhodophytes, are a group of algae that can exhibit antimicrobial activity against a variety of microorganisms. Within this group, G. gracilis contains various bioactive compounds that may contribute to its reported antimicrobial activity. While the specific molecules can vary, the common classes that have been reported in G. gracilis and may possess antimicrobial properties are polysaccharides, phenolics, terpenoids, and pigments11. However, it is important to note that the presence and amounts of these components can vary depending on factors such as the location of seaweed collection, seasonality, physiological condition of the thalli, and environmental conditions. Therefore, the specific class and concentration of antimicrobial compounds in G. gracilis may vary accordingly.
G. gracilis has also been found to hold antioxidant properties, containing various phenolic compounds, which have been shown to scavenge free radicals and reduce oxidative stress12. Antioxidants help to protect cells from damage caused by reactive oxygen species and have potential health benefits. Antioxidant capacity can be evaluated directly through different methods, including the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity and, indirectly, through the quantification of total polyphenolic content (TPC)13.
Even though an ingredient is reported to have a prominent bioactivity, its cytotoxicity assessment is indispensable in evaluating natural and synthetic substances to be used in contact with living cells or tissues. There are several methods for measuring cytotoxicity, each one with advantages and limitations. Overall, they offer a range of options to evaluate the harmful effects of many substances on cells and, at the same time, to investigate the mechanisms of cell damage and death14.
In this work, we use the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, a colorimetric method introduced by Mosmann (1983)15. This method measures the reduction of tetrazolium salts to a purple formazan product by metabolically active cells. The higher the amount of formazan crystals, the higher the number of viable cells, thus providing an indirect measure of cytotoxicity14. Since in this work, G. gracilis water and ethanol extracts are intended to be incorporated into dermo-cosmetic formulations, the in vitro cytotoxicity evaluation is performed in a keratinocyte (HaCaT) cell line.
Concerning the food application, seaweeds are generally low in calories and nutritionally rich in dietary fibers, essential elements and amino acids, polysaccharides, polyunsaturated fatty acids, polyphenols, and vitamins2,16. G. gracilis is no exception, having an interesting nutritional value. Freitas et al. (2021)4 found that cultivated G. gracilis had higher levels of protein and vitamin C and maintained the level of total lipids compared to wild seaweed. This may represent an economic and environmental advantage, as nutritionally speaking, production is preferable to the exploitation of wild resources. In addition, consumers are increasingly concerned about the type of food they eat, so it is important to introduce new ingredients for food enrichment and use new resources to obtain extracts that can add value to a product and claim a &#34;clean label.&#34; Besides, the current market is very competitive, requiring the development of new products and innovative strategies to differentiate manufacturers from their competitors17.
The enrichment of products with poor nutritional value, such as pasta, with marine resources, including seaweed, is a strategy to introduce this resource as a new food and a market differentiation strategy through a product with distinct nutritional value. On the other hand, G. gracilis is a source of natural red pigments such as phycobiliproteins18, having high potential for applications in the food industry. This seaweed has shown high interest in several areas, and its application can be made using the whole seaweed, extracts and/or the remaining biomass. In this work, we demonstrate some examples of such applications.

作者聚焦：探索海藻的生物活性化合物以促进工业的可持续创新 

通过生物精炼方法对红海藻 gracilaria gracilis 进行增值

Our research is focused on the prospection of new bioactive compounds from seaweed that can be useful in different sectors such as food, cosmetic, and pharmaceutical industries. There is a growing awareness in society about the potential of marine organisms to deliver bioactive compounds. These compounds can be used to produce sustainable and safer functional cosmetics to improve the quality of food products and serve as source of new chemical structures for the development of new pharmaceutical drugs.One of the challenges of using seaweed in the development of new food products is to avoid using high amounts that could lead to unpleasant sensory properties in food. Using seaweed extracts is a way to get around this problem. With this protocol, we were able to develop pasta, wheat, add nutritional value by incorporating whole algae.Usually, the addition of these ingredients provides less common sensory properties. However, with the developed formulation, a consumer acceptance was still achieved.

抗菌活性
在解释获得的结果时，应记住，抑制百分比越高，提取物在抑制该特定菌株生长方面的功效就越大，因此，提取物作为抗菌剂就越有趣。通过这种方法，我们可以快速确定哪些提取物对某些细菌菌株具有更大的活性，并确定未来使用中最有趣的提取物。因此，我们可以为进一步研究同一提取物提供一个起...

Watch this Scientific Journal Video about Exploring Seaweed's Bioactive Compounds for Sustainable Innovations in Industries at JoVE.com

在这里，我们描述了旨在对 Gracilaria gracilis进行综合增值的几种协议：野生物种收获，内部生长和生物活性成分的提取。评估了提取物的抗氧化、抗菌和细胞毒性作用，以及富含全海藻生物质和色素的食物的营养和稳定性评估。

The interest in seaweeds as an abundant feedstock to obtain valuable and multitarget bioactive ingredients is continuously growing. In this work, we ...

我们的研究重点是从海藻中寻找新的生物活性化合物，这些化合物可用于食品、化妆品和制药行业等不同行业。社会越来越意识到海洋生物提供生物活性化合物的潜力。这些化合物可用于生产可持续和更安全的功能性化妆品，以提高食品的质量，并作为开发新药物的新化学结构的来源。在开发新食品时使用海藻的挑战之一是避免使用大量海藻，这可能导致食品中令人不快的感官特性。使用海藻提取物是解决这个问题的一种方法。通过这个协议，我们能够开发面食、小麦，通过加入全藻类来增加营养价值。通常，添加这些成分可提供不太常见的感官特性。然而，通过开发的配方，仍然获得了消费者的接受度。

valorization-red-seaweed-gracilaria-gracilis-through-biorefinery

Research

JoVE Journal

Biology

Valorization of the Red Seaweed Gracilaria gracilis Through a Biorefinery Approach

The interest in seaweeds as an abundant feedstock to obtain valuable and multitarget bioactive ingredients is continuously growing. In this work, we explore the potential of Gracilaria gracilis, an edible red seaweed cultivated worldwide for its commercial interest as a source of agar and other ingredients for cosmetic, pharmacological, food, and feed applications.
G. gracilis growth conditions were optimized through vegetative propagation and sporulation while manipulating the physicochemical conditions to achieve a large biomass stock. Green extraction methodologies with ethanol and water were performed over the seaweed biomass. The bioactive potential of extracts was assessed through a set of in vitro assays concerning their cytotoxicity, antioxidant, and antimicrobial properties. Additionally, dried seaweed biomass was incorporated into pasta formulations to increase food&#39;s nutritional value. Pigments extracted from G. gracilis have also been incorporated into yogurt as a natural colorant, and their stability was evaluated. Both products were submitted to the appreciation of a semi-trained sensorial panel aiming to achieve the best final formulation before reaching the market.
Results support the versatility of G. gracilis whether it is applied as a whole biomass, extracts and/or pigments. Through implementing several optimized protocols, this work allows the development of products with the potential to profit the food, cosmetic, and aquaculture markets, promoting environmental sustainability and a blue circular economy.
Moreover, and in line with a biorefinery approach, the residual seaweed biomass will be used as biostimulant for plant growth or converted to carbon materials to be used in water purification of the in-house aquaculture systems of MARE-Polytechnic of Leiria, Portugal.

Here, we describe several protocols aiming at an integrated valorization of Gracilaria gracilis: wild species harvesting, in-house growth, and extraction of bioactive ingredients. The extracts&#39; antioxidant, antimicrobial, and cytotoxic effects are evaluated, along with the nutritional and stability assessment of food enriched with whole seaweed biomass and pigments.

科研

生物学

Extraction of the Bioactive Ingredients from Gracilaria gracilis

Extraction of the Bioactive Ingredients from Gracilaria gracilis  

To begin, grow Gracilaria gracilis in 250 milliliters of flat bottom flasks in a climatic room. Once the biomass has been scaled up, dried, and ground, weigh the resulting dried biomass. Dissolve it in 100 milliliters of solvent and mix by stirring in a vessel protected from light for 30 minutes.Perform extractions with ethanol and water separately at room temperature. Filter the liquid extracts through filter paper to separate them from the remaining biomass. Centrifuge the extracts at 8, 000 G for 10 minutes, at room temperature.Freeze dry the aqueous extracts and use a rotary evaporator at 40 degrees Celsius to evaporate the ethanol extracts.

从Gracilaria gracilis中提取生物活性成分

Evaluation of the Antioxidant and Cytotoxic Effects of Gracilaria gracilis Extracts

Evaluation of the Antioxidant and Cytotoxic Effects of Gracilaria gracilis Extracts  

To begin, take the aqueous and ethanol extracts obtained from the dried Gracilaria gracilis. In a 96 well microplate, dispense two microliters of each sample and 198 microliters of DPPH dissolved in absolute ethanol. Then, using a microplate spectrophotometer, measure the absorbance at 517 nanometers.Take the human keratinocytes grown in 96 well plates overnight. Add two microliters of the DMSO dissolved extracts to 198 microliters of the medium in microplate wells. Remove the culture medium.Add 100 microliters of MTT to the cells and incubate in the dark for 30 minutes. To solubilize the intracellular formazan crystals, add 100 microliters of DMSO. Using a microplate reader, measure the absorbance at 570 nanometers.The DPPH test demonstrated that the temperature of 40 degrees Celsius showed higher inhibition values of oxidant activity compared to extracts obtained at room temperature or 70 degrees Celsius. No cytotoxic effects were observed on human keratinocytes, suggesting that at the maximum assay concentration, both aqueous and ethanol extracts are safe for cutaneous use.

Gracilaria gracilis提取物的抗氧化和细胞毒作用评价

Pasta Production Using Gracilaria gracilis, Nutritional Evaluation, and Sensory Evaluation

Pasta Production Using Gracilaria gracilis, Nutritional Evaluation, and Sensory Evaluation  

To begin, combine predefined portions of rice flour, gracilaria gracilis, and chlorella vulgaris in the equipment, and add approximately 30%water to the mixture. After a few minutes of mixing, followed by extruding the pasta, using the equipment, cut it manually. To produce chifferi, dry the pasta at 68 degrees Celsius for 42 minutes, followed by an additional five hours and 30 minutes at 76 degrees Celsius, simulating an industrial process.Finally, vacuum seal the samples and store them in a dark place at room temperature. Take one gram sample in a glass crucible with a filter bottom and position it in the fiber analyzer. Add 150 milliliters of preheated 1.25%sulfuric acid, and two milliliters of anti-foaming agent to the column of each crucible.Remove the solvent and rinse the crucible three times with deionized water to prepare the sample for the basic hydrolysis. After washing the crucibles three times with 150 milliliters of acetone, carefully transfer those to an oven set at 150 degrees Celsius for one hour. Record the final weight.Transfer the crucibles to a muffle furnace, set at 500 degrees Celsius for three hours, and then record the final weight. To determine the fatty acids profile, add two milliliters of 2%sulfuric acid solution in methanol to a 50 milligram sample. Heat the mixture at 80 degrees Celsius for two hours.Then, cool the mixture to room temperature and add one milliliter of ultrapure water and two milliliters of N-heptane to each sample. After vortexing the mixture for one minute, centrifuge it for five minutes. Later, recover the upper N-heptane phase containing the fatty acid methyl esters and transfer it to gas chromatography vials.Analyze the samples in a gas chromatograph equipped with a TR-FAME capillary column, an auto sampler, and a flame ionization detector. Determine the fatty acid profile by comparing the resulting retention times with a standard. Incubate porcelain crucibles for three hours at 105 degrees Celsius.Allow them to cool in a desiccator, and then record their weights. After incubating 10 grams of the sample, as demonstrated previously, calculate its moisture content using the displayed equation. Then, transfer the crucibles with the dried samples to an incinerator heated to 525 degrees Celsius for four hours.After cooling the samples, record their weight. To assess consumer acceptance, use chifferi pasta samples cooked in distilled water for eight minutes. Conduct sensory tests in individual sensory booths located in a sensory analysis laboratory.Evaluate the visual appearance, color, texture, odor, sea taste, overall taste, overall evaluation, and purchase intent of the samples. The pasta received ratings of five and six on a hedonic scale of one to nine for sea taste and visual appearance, respectively. The pasta had a purchase intention score of five and an overall appreciation score of six.

使用 Gracilaria gracilis生产意大利面，营养评估和感官评估

Incorporation of Seaweed Extract in Yogurt and Color Stability Evaluation 

To begin, add 50 milliliters of sodium phosphate buffer to one gram of Gracilaria gracilis. After homogenizing the mixture, use a mortar and pestle to macerate the sample for 10 minutes. Transfer the solution to a tube and centrifuge it for 20 minutes at 12298g.After combining the supernatant, slowly add 65%ammonium sulfate, then pellet the precipitate by centrifugation and dissolve it in distilled water. Perform dialysis of the extract using a tubing membrane against water for 24 hours, followed by freeze drying. To incorporate the extract, mix it in yogurt at a concentration of 0.21%Store the samples in individual glass flasks at 4 degrees Celsius until analysis.To check the color stability of the extract in yogurt, calibrate the colorimeter using a white ceramic plate. Fill a cell with approximately 28 grams of the sample and analyze its color. The color stability of the yogurts with pigment was evaluated for 12 days at 4 degrees celsius.The incorporation of the extracts in yogurts showed good color attention with a color difference of 7.01 plus or minus 2.36 after 12 days of storage.