incorporation of seaweed extract in yogurt and color stability evaluation

利用 Gracilaria gracilis：可持续创新的生物活性潜力

&#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

Watch this Scientific Journal Video about Incorporation of Seaweed Extract in Yogurt and Color Stability Evaluation at JoVE.com

Incorporation of Seaweed Extract in Yogurt and Color Stability Evaluation   

酸奶中海藻提取物的加入及颜色稳定性评估 

首先，将 50 毫升磷酸钠缓冲液加入 1 克 Gracilaria gracilis 中。匀浆混合物后，使用研钵和研杵将样品浸渍 10 分钟。将溶液转移到试管中，并以 12298g 离心 20 分钟。合并上清液后，缓慢加入65%硫酸铵，然后离心沉淀沉淀，溶于蒸馏水中。使用管膜对水进行透析24小时，然后冷冻干燥。要掺入提取物，请将其混合在浓度为 0.21% 的酸奶中，将样品储存在 4 摄氏度的单个玻璃烧瓶中直至分析。要检查酸奶中提取物的颜色稳定性，请使用白色陶瓷板校准色度计。用大约 28 克样品填充细胞并分析其颜色。在4摄氏度下评估含色素酸奶的颜色稳定性12天。在酸奶中掺入提取物显示出良好的颜色关注度，贮藏12天后色差为7.01正负2.36。

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Research

JoVE Journal

Biology

Incorporation of Seaweed Extract in Yogurt and Color Stability Evaluation

140 次观看. 首先，将 50 毫升磷酸钠缓冲液加入 1 克 Gracilaria gracilis 中。匀浆混合物后，使用研钵和研杵将样品浸渍 10 分钟。将溶液转移到试管中，并以 12298g 离心 20 分钟。合并上清液后，缓慢加入65%硫酸铵，然后离心沉淀沉淀，溶于蒸馏水中。使用管膜对水进行透析24小时，然后冷冻干燥。要掺入提取物，请将其混合在浓度为 0.21% 的酸奶中，将样品储存在 4 摄氏度的单个玻璃烧?...

视频: Incorporation of Seaweed Extract in Yogurt and Color Stability Evaluation   

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.

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