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' 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 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'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.
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 "clean label." 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.
1. Biomass harvesting and preparation
2. Stock maintenance
3. Cultivation and scale-up
4. Extraction procedure
NOTE: To assess the in vitro cytotoxicity, antioxidant, and antimicrobial properties of G. gracils extracts, its preparation considers two different parameters: the extraction temperature and the type of solvent.
5. Antimicrobial activity
NOTE: The ethanolic and aqueous extracts should be tested individually against Bacillus subtilis subsp. spizizenii (DSM 347), Escherichia coli (DSM 5922) and Listonella anguillarum (DSM 21597). Antimicrobial testing must be performed according to the recommendations of the National Committee for Clinical Laboratory Standards (NCCLS, 2012)22. All cultures were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ). L. anguillarum was grown on tryptic soy broth (TSB) or tryptic soy agar (TSA) supplemented with 1% sodium chloride (NaCl). The remaining two strains were grown on LB medium (VWR Chemicals). Bacillus subtilis subsp. spizizenii (DSM 347) and Listonella anguillarum (DSM 21597) cultures were incubated at 30 °C, while Escherichia coli (DSM 5922) was incubated at 37 °C, according to the supplier's instructions. The broth microdilution method can be used for the determination of antimicrobial activity in a liquid media, and this should be carried out on a microscale, allowing the antimicrobial potential to be determined quickly and efficiently. This low-cost method allows results to be obtained in just 24 h, being therefore suitable for determining, at an early stage, the best extraction conditions that allow, for a given microbial strain, to obtain results in terms of growth inhibitory action. However, the methodology requires the use of sterile microplates with a lid specific for microbial growth, as well as the availability of a microplate reader for the 600 nm wavelength.
6. Antioxidant activity and quantification of total polyphenols
7. Cytotoxicity evaluation in epidermal cells
NOTE: The in vitro cytotoxic effect of the aqueous and ethanol extracts of G. gracilis is evaluated in human keratinocytes (HaCaT cells – 300493) through the MTT colorimetric assay as previously described25. Cells were acquired from Cell Lines Services, Germany (CLS) and the method was performed in compliance with institutional guidelines and CLS instructions.
CAUTION: See the safety data sheet of MTT delivered by the supplier)
8. Food innovation
Antimicrobial activity
When interpreting the results obtained, it should be borne in mind that the higher the percentage of inhibition, the greater the efficacy of the extract in inhibiting the growth of that specific strain and, consequently, the more interesting the extract is as an antimicrobial. Through this methodology, we can rapidly identify which extracts have greater activity on certain bacterial strains, also identifying the most interesting in terms of future use. We can thus have a starting point for further studies on that same extract.
Figure 1 shows the results obtained with aqueous extracts, both in the first and second extractions, immediately after biomass drying (Figure 1A) and in the third and fourth extractions (Figure 1B), obtained after the ethanolic extractions, thus using the biomass integrally. We can see that the most interesting results, corresponding to the third and fourth aqueous extractions, reveal higher antimicrobial activities, particularly in extracts obtained at 70 °C. The concentration of extract in the wells is 5 mg/mL.
Figure 1: Growth inhibition of bacterial species in the presence of aqueous extracts of G. gracilis. Growth inhibition of 3 bacterial species (Bacillus subtilis, Escherichia coli, Listonella anguillarum) after 24 h of growth in a liquid medium, in the presence of aqueous extracts (Aq) of G. gracilis obtained at different temperatures, room temperature (RT), 40 °C (40) and 70 °C (70). The positive control was done with chloramphenicol (CHL), and the results are expressed as mean values (n = 8). Data refer to the 4 sequential extraction steps (1st, 2nd, 3rd, 4th). Please click here to view a larger version of this figure.
The ethanolic extracts seem to be particularly effective in inhibiting the growth of L. anguillarum, as shown in Figure 2. This shows the results obtained with the ethanolic extracts, also in the first and second extractions (Figure 2A) and the third and fourth extractions (Figure 2B), obtained after the first aqueous extraction.
Figure 2: Growth inhibition of bacterial species in the presence of ethanolic extracts of G. gracilis. Growth inhibition of 3 bacterial species (Bacillus subtilis, Escherichia coli, Listonella anguillarum) after 24 h of growth, in a liquid medium, in the presence of ethanolic extracts (Et) of G. gracilis, obtained at different temperatures, room temperature (RT), 40 °C (40) and 70 °C (70). Positive control was done with chloramphenicol (CHL), and results were expressed as mean values (n = 8). Data refer to the 4 sequential extraction steps (1st, 2nd, 3rd, 4th). Please click here to view a larger version of this figure.
Antioxidant activity
Regarding the results that highlight the antioxidant potential of the various extracts, namely in the DPPH test, the results expressed in Figure 3 indicate that the temperature of 40 °C was the most effective, resulting in higher inhibition values of the oxidant activity, than those observed in extracts obtained at RT or 70 °C. This holds valid for the G. gracilis samples, and there may be large variations depending on the samples used and the algal growth conditions. Thus, it is recommended that tests be carried out to indicate the best conditions for each specific type of sample.
Figure 3: Inhibition of the DPPH radical (%) in the presence of extracts obtained at different temperatures. Extracts were obtained through ethanolic (Et) or aqueous (Aq) extraction. The 1st and 2nd extractions were made from dry biomass sequentially. The remaining ones (3rd and 4th) were made with biomass previously extracted with the alternative solvent. RT means room temperature; 40, extract obtained at 40 °C; and 70, extract obtained at 70 °C. Please click here to view a larger version of this figure.
Similar results were obtained in terms of total polyphenol quantification (Figure 4), with the temperature of 40 °C being considered the best in terms of antioxidant compound extraction. This shows that the antioxidant activity seems to be correlated with the phenolic components present in the extracts.
Figure 4: Quantification of total polyphenols content (TPC) in extracts obtained at different temperatures. The extracts were obtained by ethanolic (Et) or aqueous (Aq) extraction, where 1st and 2nd extraction were made sequentially from dry algae and the remaining (3rd and 4th) were made with biomass previously extracted with another solvent. RT means room temperature; 40, extract obtained at 40 °C; and 70, extract obtained at 70 °C. Please click here to view a larger version of this figure.
Cytotoxicity in HaCat cells
The first step to assess cosmetic ingredients´ safety is the study of their in vitro cytotoxicity in epidermal and dermal cell lines. As can be seen in Figure 5, no cytotoxic effects were observed on keratinocytes (HaCaT cells), suggesting that, at the maximum assayed concentration (1 mg/mL), both aqueous and ethanol extracts are safe for cutaneous use.
Figure 5: Cytotoxic effect of Gracilaria gracilis extracts (1 mg/mL) on HaCaT cells after 24 h of treatment. Values in each column are expressed as the mean ± standard error of the mean (SEM) of three independent experiments in triplicate. Please click here to view a larger version of this figure.
Food innovation
The final pasta formulations were obtained after numerous trials between theoretical formulation and sensory testing by a semi-trained panel. After this step, the chemical characterization was accessed, including nutritional evaluation, fatty acids and mineral elements profiles. It was possible to build a nutritional characterization (Table 1 and Table 2) and to verify the existence of expected nutritional claims based on the European legislation (REG (EU) No. 1169/2011)32.
Nutrition Facts | by 100 g of Pasta | % RDI | |
Energy | 1478.90 kJ (348.74 kcal) | 17 | |
Lipids | 1.06 ± 0.10 g | 2 | |
From what: | |||
Saturated Fatty Acids | 0.38 ± 0.01 g | 2 | |
Carbohydrates | 72.59± 0.21 g | 28 | |
Fibre | 3.84 ± 0.20 g | ||
Protein | 10.29 ± 0.20 g | 21 | |
Salt | 0.22 ± 0.02 g | 9 |
Table 1: Nutritional facts. Nutritional facts of developed pasta based on chemical analyses (n=3) of energy and carbohydrates obtained by calculation and respective percentage of recommended dose intake (n = 3).
These pasta formulations have been designed to appeal to a target consumer who has a healthier diet in mind, so the amount of fat per 100 g of a product must be as small as possible. Detailed analysis of the fatty acid profile shows a value of 0.38 g saturated fatty acids/100 g, which is well below the limit for low saturated fat products, meeting the initial goal in the production of this pasta. Regarding the fiber content, it was also possible, in accordance with European regulations, to claim this pasta formulation as a source of fiber.
In the mineral element characterization, determined by ICP-OES and presented in Table 2, it is reported the value of about 219 mg/100 g of Na present in 100 g of this product, so it cannot be verified that it is a product with low sodium content (<0.12 g/100 g). Due to the elevated sodium content naturally present in the ingredients, this product may not require the addition of salt in its confection.
Table 2: Pasta mineral elements profile. Blue – high content of element; Red – source of a certain element (n = 3). Please click here to download this table.
According to the referred European Union regulation and the RDI% for each element, it can be seen that this product has a high content of K, P, Fe, and is also a source of Zn. It has Se, Mg, and Ca present in smaller quantities.
For the pasta acceptance tests, 86 testers were used, of which 63 were female and 23 male, aged over 18 years. The acceptance tests were based on hedonic scales of 9 points, as stipulated in the standards. The Healthy dough scored 5 and 6 on a hedonic scale from 1 to 9 for the parameters "Sea taste" and "Visual appearance", respectively (Figure 6).
Figure 6: Results from sensory consumer acceptance tests. (A) The hedonic choice for visual appearance, color, texture, odor, sea taste, and overall taste. (B) The hedonic choice for overall appreciation and purchase intention. Scale 1-9, where 1 is a poor evaluation, and 9 is a very good evaluation (n = 86). Please click here to view a larger version of this figure.
The pasta scored 5 and 6 on a hedonic scale from 1 to 9 for the parameters "Sea taste" and "Visual appearance", respectively (Figure 6A). This pasta obtained a low value on this scale regarding the texture parameter (considering other formulations already studied), probably because the base ingredient is rice flour; despite this, it obtained values on a scale from 1 to 9, ranging from 4 to 7 which reflects a good acceptance by the average consumer. On a hedonic scale from 1 to 9, the pasta had as most frequent responses the value of 5 for purchase intention and the value of 6 for overall appreciation, as shown in Figure 6B. It was found that about 65% of the tasters chose a response equal to or higher than the score of 6 for the overall assessment of this pasta. The color stability of the yogurts with pigment was evaluated for 12 days at -4 °C, and the results are presented in Figure 7.
Figure 7: Color stability of the yogurts. (left) Control yogurts and (right) yogurts with Gracilaria gracilis pigment during 12 days of storage. The a*, b*, and L* parameters are dimensionless. Please click here to view a larger version of this figure.
The results indicate that the a* and b* parameters were stable over time, while L* values showed higher variance. The lightness demonstrated higher values after 8 days of storage. While some differences were found in the lightness parameters, indicating that the samples were lighter over time, the parameters of redness (a*) and blue (b*) showed good stability. The incorporation of the extracts in yogurts showed good color retention, with a ΔE of 7.01 ± 2.36 after 12 days of storage. Regarding the sensory evaluation of the yogurt, 9 in 13 panelists correctly identified the correct sample in the triangle test, suggesting that there were differences between the yogurts that allowed this distinction. The hedonic tests made to the semi-trained panel showed a good acceptance of the product, reflected in scores above 7 (Figure 8). The mode of the score for any of the three attributes under test was 9, leading to score averages above 8. The best evaluation was made to color, which reflects an excellent acceptance by the panel, a revealing result.
Figure 8: Results of the hedonic sensory evaluation of the yogurt with Gracilaria gracilis pigment. Please click here to view a larger version of this figure.
The antimicrobial activity tests in a liquid medium are used to evaluate the effectiveness of antimicrobial substances against microorganisms suspended in a liquid medium and are usually performed to determine the ability of a substance to inhibit growth or kill microorganisms35,36,37,38. They are used to evaluate the sensitivity of microorganisms to antimicrobial agents and are conducted in test tubes or microtitration plates, where different concentrations of the antimicrobial substance are tested against a standardized suspension of the target microorganism22. Antimicrobial activity is assessed by measuring microbial growth or the presence/absence of turbidity in the culture medium after a proper incubation period.
There are several techniques and methods to perform these tests, such as the broth dilution method, agar diffusion method (such as the disc diffusion test), and the broth microdilution method, which is one of the most used38. Some of the most critical points in these methods include the proper preparation of the inoculum, the choice of the culture medium, the quality of the antimicrobials used, the standardization of the technique, and effective contamination control. The inoculum, which is a standardized suspension of microorganisms, must be prepared correctly to ensure the accuracy of the results. This involves an appropriate selection of microbial culture, a proper culture of pure strains, adjustment of cell concentration, and standardization of the suspension to obtain an appropriate optical density or cell concentration.
The culture medium used should be selected carefully to ensure that it provides the ideal growth conditions for the microorganism under test. Chemical composition, pH, and other factors can affect test results. It is important to follow the manufacturer's recommendations for the preparation of the culture medium. The quality of the antimicrobial agents used as control is fundamental, and it is important to ensure that these are pure, within shelf life, and stored correctly. Labeling and expiry dates should always be consulted, following the manufacturer's instructions. The standardization of the technique is also crucial to ensure the accuracy of the results. This includes the addition of the tested concentrations to the culture medium, as well as the maintenance of aseptic conditions throughout the procedure. Additionally, cross-contamination of microorganisms during testing can lead to false or unreliable results. It is essential to follow strict aseptic practices throughout the procedure, from the manipulation of the inoculum to the incubation of the tubes or plates. The use of clean countertops, proper pipetting techniques, and proper disposal of materials are important measures to avoid contamination.
Attention to detail and strict adherence to established protocols and guidelines are crucial to minimize errors and ensure the reliability of results in antimicrobial activity tests in a liquid medium. Also, the antimicrobial activity tests have some limitations that should be considered37.
Factors such as pH, temperature, presence of blood components, and interactions with the immune system are not considered in these tests, which can limit the ability to predict the effectiveness of an antimicrobial agent in a living organism. Also, tests measure the ability of an antimicrobial substance to inhibit microbial growth or kill microorganisms and do not provide detailed information about the mechanism of action of the antimicrobial or its selectivity against specific microorganisms. There are limitations in detecting resistance, as the methods employed may not allow the detection or prediction of the development of resistance over time. Some microorganisms may develop resistance mechanisms in response to prolonged exposure to an antimicrobial, and these changes may not be easily detected through these tests. Tests of antimicrobial activity in a liquid medium generally do not consider other host factors that may influence the effectiveness of an antimicrobial agent, such as the presence of biofilms or the host's immune response.
It is important to consider these limitations and to complement testing for antimicrobial activity in a liquid media with other methods and approaches, such as in vivo studies, biofilm models, and others39. In the area of bioprospecting for bioactive macroalgae compounds, antimicrobial activity tests can be used to evaluate the antimicrobial potential of algae extracts or isolated compounds, identifying substances with antimicrobial activity against specific microbial pathogens, which can have applications in fields such as the production of medicines, cosmetics, food or agricultural products40.
Antioxidant activity tests are methods used to evaluate the ability of a compound or extract to neutralize free radicals or reduce oxidative stress. These tests are widely used in antioxidant research, both in foods and in natural products such as algae, for example. There are several methods to evaluate the antioxidant activity, and one of the most commonly used is the free radical absorption capacity. In this test, a stable free radical, the 2,2-diphenyl-1-picrylhydrazyl (DPPH), is used to evaluate the ability of a compound to donate an electron and neutralize the free radical. Antioxidant activity is measured by reducing the purple color of DPPH, which occurs when the antioxidant compound donates an electron to the free radical. Other methods include the oxygen radical absorption capacity (ORAC), the iron reduction capacity (FRAP) or the free radical reduction capacity (ABTS)41.
Quantification of total polyphenols (QTP), on the other hand, is not a direct method for determining the antioxidant activity but provides a measure of the total concentration of polyphenols in a sample, although several interfering substances can affect the results. Although many polyphenols are known to have antioxidant properties, the antioxidant activity of a compound is related to several factors, such as its chemical structure, concentration, ability to donate or capture free radicals, and interactions with other cellular components42. The simple quantification of total polyphenols cannot provide an evaluation of the antioxidant activity of the sample. However, it is important to note that the presence of polyphenols in a sample may be associated with a higher likelihood of antioxidant activity since many polyphenols possess antioxidant properties. Thus, QTP can serve as a preliminary indicator of the presence of polyphenols in the sample, but confirmation of antioxidant activity requires antioxidant activity assays. Polyphenols are a class of chemical compounds widely distributed in nature, found in foods, plants, fruits, vegetables, and algae. There are different methods for the quantification of total polyphenols, and the Folin-Ciocalteu method is one of the most used. QTP gives a general measure of the concentration of polyphenols but does not specify the individual polyphenolic compounds present in the sample. Therefore, it is a quantitative but not a qualitative method. Furthermore, it is important to consider that different polyphenols have different antioxidant capacities and biological activities, so QTP does not provide detailed information on the specific functional properties of the polyphenols present.
Each method has its advantages and limitations, and the choice of a given test depends on the characteristics of the compound under study and the purpose of the analysis. It is important to follow the specific instructions of each method to ensure reliable and comparable results. When determining the antioxidant capacity, some of the common critical steps include, firstly, the sample preparation. It is important to prepare the samples correctly, ensuring that adequate concentrations are obtained and avoiding contamination. This involves the precise weighing of the compounds or extracts and dissolving them in appropriate solvents. It is also important to consider the stability of the compounds and extracts during the process of preparation, storage, and handling. All the reagents used in the antioxidant activity tests must be properly prepared and standardized to ensure the reproducibility of the results. This involves the correct preparation of the gallic acid standard and the accuracy of the volumes. Reaction time is a critical aspect of getting accurate results in antioxidant tests. It is necessary to optimize the incubation time to ensure a complete reaction between the antioxidant compound and the free radical. Insufficient time can lead to underestimation of antioxidant activity, while excessive time can result in degradation of the compounds or interference from secondary reactions. Also, the temperature during tests must be controlled accurately and consistently. Temperature influences the speed of chemical reactions and can affect results. It is important to follow the temperature conditions recommended by the specific methods and ensure temperature stability throughout the procedure. The inclusion of appropriate controls is essential to validate the results of antioxidant tests. This can include positive controls (compounds known to have antioxidant activity) and negative controls (samples without antioxidant activity). Controls provide a basis for comparison for the interpretation of results and help ensure the accuracy of the data obtained. To obtain reliable results, it is recommended to perform tests of antioxidant activity on replicates and repeat the experiment several times to raise the significance of the data and to obtain more reliable and robust results.
Antioxidant activity testing methods have some limitations that should be considered when interpreting the results, namely, the fact that, in liquid media, they may not fully capture the complexity of biological systems and interactions between different compounds, the wide range of antioxidant reactions that can occur, cellular metabolism, and environmental factors that can influence antioxidant activity. So, different tests should be used. One must also be aware of the lack of direct correlation with health benefits; although antioxidant activity is often associated with health benefits, this does not always translate directly into health benefits in living organisms due to many other factors, such as bioavailability, metabolism, and interactions with other biological systems.
It is important to keep in mind that antioxidant activity tests are useful tools for the initial evaluation of the antioxidant potential of compounds and extracts but should not be considered a definitive indicator of biological effects or health benefits. Additional studies are needed to fully understand the impact of antioxidants on the body.
Although the methods of testing the antioxidant activity in liquid medium are widely used in research and have their advantages and limitations, when compared to the alternatives, these methods can be considered good in terms of practicality, speed, cost, and ease of implementation. One of the main advantages is the simplicity and speed of the procedures and their suitability for initial research, compound screening, and large-scale studies. These methods are relatively quick to perform and can provide results in a short period, in a more affordable way, and require less specialized equipment compared to other methods.
Algae are known for their ability to produce bioactive compounds, including antioxidants, which may have beneficial health properties43. Algae extracts can be prepared from different parts of the algae and can be obtained using different solvents, such as water, ethanol, methanol, or acetone, among others. It is important to remember that the antioxidant activity of algae extracts can vary depending on several factors, such as the species of algae, the extraction methodology, and the concentration of the bioactive compounds present. Therefore, it is recommended to perform antioxidant activity tests on replicates and to perform comparisons with appropriate controls to obtain more reliable results. These methods can be used as an initial tool in the screening and selection of algae extracts with antioxidant potential for further studies.
The MTT assay is a widely used technique for a preliminary evaluation of in vitro cytotoxic effects of substances for human and animal use. However, despite being the most used method for testing cytotoxicity, the conversion of MTT to formazan crystals is influenced by numerous factors like metabolic rate and the number of mitochondria, and it is only applicable to adherent cell targets14. The main critical steps of the protocol described here are related to the eventual occurrence of cell culture contaminations, undesirable HaCaT cells´ growth, and a low percentage of cell confluency. There are other methods, such as lactate dehydrogenase (LDH), adenosine triphosphate (ATP), and colony formation assays for measuring cytotoxicity. However, all of them have advantages and constraints. Ghasemi et al. (2021)44 evaluated the effect of several variables on the MTT assay measurements on a prostate cancer cell line (PC-3). Factors such as cell seeding density, the concentration of MTT, incubation time after MTT addition, serum starvation, the composition of cell culture media, released intracellular contents, and extrusion of formazan to the extracellular space were analyzed. From this study, useful recommendations on how to apply the assay and a perspective on where the assay's utility is a powerful tool, but likewise where it has limitations, were outlined by these authors. Nevertheless, the MTT assay is a rapid, highly sensitive,and easy methodology that can be applied as a preliminary cytotoxicity assessment of many substances with potential application in the therapeutic, food, feed, agriculture, and environmental areas. In particular, the MTT assay here performed evidenced that the ethanol and aqueous extracts from G. gracilis, in the assayed concentrations, did not affect keratinocyte viability and can proceed for in vivo assays to ensure they are completely safe for human cutaneous use.
The use of marine resources in food products has, once again, shown its potential not only to obtain products with added nutritional value but also to find cleaner-label products. The addition of whole G. gracilis (pasta) or extract (as food coloring) shows market potential, and its application could be one of the strategies for companies to stand out in the market, satisfy consumers' nutritional needs, and follow their market trends.
When seaweed was added to the pasta formulations, it was initially found that the structure was altered and that it was not possible to obtain the desired dough shapes. We had the challenge of adjusting quantities and adding other ingredients not previously foreseen, aiming to maintain the texture of the pasta. Besides the appropriate amount of each ingredient, the extrusion method was adapted throughout the development of the pasta. Apart from this difficulty, the development of new products is based on three fundamental principles: the product must be sensorial appealing (texture, flavor, smell); the product must have added nutritional value; and it must make maximum use of sustainable ingredients and methodologies. In this sense, another major challenge is the use of the sensory panel to achieve the most appealing formulation. Most of the physicochemical analyses carried out on pasta were already optimized for food matrices; however, in this work, the sample preparation was optimized so that it could be efficiently applied to all the analysis methods.
Regarding the use of G. gracilis pigment as a colorant, a refrigerated product was chosen due to the thermal sensitivity of this type of molecule45. Since yogurt preparation involves thermal processing, the pigment was added to the final product. Mixing the pigment in the yogurt resulted in a product slightly more fluid than the control without pigment. In fact, at the end of the triangle test, some panelists commented that the only difference between samples was the texture. This is a good result since the main purpose of the triangle test was to verify if there were noticeable differences between yogurt with and without pigment, especially differences in taste and smell, as seaweed extracts may confer unpleasant taste/smell to food products. This was a preliminary study involving a small semi-trained panel. In further studies, a larger number of tasters should be considered to achieve more reliable market results. As for the evaluation of pigment stability in yogurt, further studies could include the evaluation of other physicochemical properties of the yogurt with pigment over time, such as pH, water activity (aw), and texture. Sensory evaluation over time would also be desirable.
In conclusion, the protocols described here highlight the potential of the red seaweed G. gracilis as a source of ingredients to develop novel products with potential applications in the pharmaceutical, dermocosmetic, and food industries. Moreover, the post-extracted residual biomass remains a valuable material to be applied as a plant growth biostimulant, soil enrichment, fish feeding, or feedstock to obtain biochar and/or functionalized carbons for water purification purposes. The biorefinery approach described here can be applied to other seaweed species, promoting a blue circular economy and environmental sustainability.
The authors have nothing to disclose.
This work was supported by the Portuguese Foundation for Science and Technology (FCT) through the Strategic Projects granted to MARE-Marine and Environmental Sciences Centre (UIDP/04292/2020 and UIDB/04292/2020), and Associate Laboratory ARNET (LA/P/0069/2020). FCT also funded the individual doctoral grants awarded to Marta V. Freitas (UI/BD/150957/2021) and Tatiana Pereira (2021. 07791. BD). This work was also financially supported by the project HP4A – HEALTHY PASTA FOR ALL (co-promotion no. 039952), co-funded by ERDF – European Regional Development Fund, under the Portugal 2020 Programme, through COMPETE 2020 – Competitiveness and Internationalisation Operational Programme.
Absolute Ethanol | Aga, Portugal | 64-17-5 | |
Ammonium Chloride | PanReac | 12125-02-9 | |
Amphotericin B | Sigma-Aldrich | 1397-89-3 | |
Analytical scale balance | Sartorius, TE124S | 22105307 | |
Bacillus subtilis subsp. spizizenii | German Collection of Microorganisms and Cell Cultures (DSMZ) | DSM 347 | |
Biotin | Panreac AppliChem | 58-85-5 | |
Centrifuge | Eppendorf, 5810R | 5811JH490481 | |
Chloramphenicol | PanReac | 56-75-7 | |
CO2 Chamber | Memmert | N/A | |
Cool White Fluorescent Lamps | OSRAM Lumilux Skywhite | N/A | |
Densitometer McFarland | Grant Instruments | N/A | |
DMEM medium | Sigma-Aldrich | D5796 | |
DMSO | Sigma-Aldrich | 67-68-5 | |
DPPH | Sigma, Steinheim, Germany | 1898-66-4 | |
Escherichia coli (DSM 5922) | German Collection of Microorganisms and Cell Cultures (DSMZ) | DSM5922 | |
Ethanol 96% | AGA-Portugal | 64-17-5 | |
Ethylenediaminetetraacetic Acid Disodium Salt Dihydrate (Na2EDTA) | J.T.Baker | 6381-92-6 | |
Fetal Bovine Serum (FBS) | Sigma-Aldrich | F7524 | |
Filter Paper (Whatman No.1) | Whatman | WHA1001320 | |
Flasks | VWR International, Alcabideche, Portugal | N/A | |
Folin-Ciocalteu | VWR Chemicals | 31360.264 | |
Gallic Acid | Merck | 149-91-7 | |
Germanium (IV) Oxide, 99.999% | AlfaAesar | 1310-53-8 | |
HaCaT cells – 300493 | CLS-Cell Lines Services, Germany | 300493 | |
Hot Plate Magnetic Stirrer | IKA, C-MAG HS7 | 06.090564 | |
Iron Sulfate | VWR Chemicals | 10124-49-9 | |
Laminar flow hood | TelStar, Portugal | 526013 | |
LB Medium | VWR Chemicals | J106 | |
Listonella anguillarum | German Collection of Microorganisms and Cell Cultures (DSMZ) | DSM 21597 | |
Manganese Chloride | VWR Chemicals | 7773.01.5 | |
Micropipettes | Eppendorf, Portugal | N/A | |
Microplates | VWR International, Alcabideche, Portugal | 10861-666 | |
Microplates | Greiner | 738-0168 | |
Microplates (sterile) | Fisher Scientific | 10022403 | |
Microplate reader | Epoch Microplate Spectrophotometer, BioTek, Vermont, USA | 1611151E | |
MTT | Sigma-Aldrich | 289-93-1 | |
Muller-Hinton Broth (MHB) | VWR Chemicals | 90004-658 | |
Oven | Binder, FD115 | 12-04490 | |
Oven | Binder, BD115 | 04-62615 | |
Penicillin | Sigma-Aldrich | 1406-05-9 | |
pH meter Inolab | VWR International, Alcabideche, Portugal | 15212099 | |
Pippete tips | Eppendorf, Portugal | 5412307 | |
Pyrex Bottles Media Storage | VWR International, Alcabideche, Portugal | 16157-169 | |
Rotary Evaporator | Heidolph, Laborota 4000 | 80409287 | |
Rotavapor | IKA HB10, VWR International, Alcabideche, Portugal | 07.524254 | |
Sodium Carbonate (Na2CO3) | Chem-Lab | 497-19-8 | |
Sodium Chloride (NaCl) | Normax Chem | 7647-14-5 | |
Sodium Phosphate Dibasic | Riedel-de Haën | 7558-79-4 | |
SpectraMagic NX | Konica Minolta, Japan | color data analysis software | |
Spectrophotometer | Evolution 201, Thermo Scientific, Madison, WI, USA | 5A4T092004 | |
Streptomycin | Sigma-Aldrich | 57-92-1 | |
Thiamine | Panreac AppliChem | 59-43-8 | |
Trypsin-EDTA | Sigma-Aldrich | T4049 | |
Tryptic Soy Agar (TSA) | VWR Chemicals | ICNA091010617 | |
Tryptic Soy Broth (TSB) | VWR Chemicals | 22091 | |
Ultrapure water | Advantage A10 Milli-Q lab, Merck, Darmstadt, Germany | F5HA17360B | |
Vacuum pump | Buchi, Switzerland | FIS05-402-103 | |
Vitamin B12 | Merck | 68-19-9 |