Valorization of the Red Seaweed Gracilaria gracilis Through a Biorefinery Approach

Summary

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.

Abstract

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.

Introduction

 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 properties^1,2. 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 methods^6,7. 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 vitamins^2,16. 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.

Protocol

1. Biomass harvesting and preparation

1. Harvest the specimens of G. gracilis during low tide and quickly transport them to the laboratory in dark, cooled boxes to avoid drying, light, and air exposure.
2. In the laboratory, wash each thallus with running seawater and clean thoroughly to remove debris, necrotic parts, epiphytes, and other organisms from the surface.
3. Keep the wild biomass in constantly aerated seawater (31-35 psu) in a climatic room (20 ± 1 °C) with low irradiance provided by daylight cool white and fluorescent lamps and photoperiod set at 16:08 (light: dark) for 7 days. During this period, do not supply any nutrient media; this allows the seaweed to slowly adjust to the new indoor conditions.

2. Stock maintenance

1. Following the acclimatization period, cut healthy tips of seaweed thalli with a sterile blade. Following Redmond et al. (2014)¹⁹ and under sterile conditions, drag each tip through an agar gel previously prepared in Petri dishes (1.0% bacteriological agar, in 1:1 distilled water/seawater ratio) to remove any remaining contaminants. Perform the agar drag three times for each tip and always drag the tip through unused portions of the agar gel.
2. Acid wash glassware in a hydrochloric acid solution (HCl, 15%) and thoroughly rinse with distilled water. Sterilize all tools, glassware, agar, seawater, and distilled water used in the cleaning process by autoclave (121 °C, 15 min).
   CAUTION: See the safety data sheet of HCl delivered by the supplier.
3. Tips grow in sterilized seawater at 35 psu, supplemented with Von Stosch Enriched solution (VSE) modified for red seaweeds, according to Redmond et al. (2014)¹⁹. Add germanium dioxide (GeO₂) to the medium (1 mL/L) to prevent the growth of epiphytic diatoms.
   CAUTION: See the safety data sheet of GeO₂ delivered by the supplier.
   NOTE: Tips that show loss of pigmentation, as observed by partial or total discoloration, are under stress or are already dead and ought to be discarded.

3. Cultivation and scale-up

1. After the acclimatization period, randomly distribute about 8-10 healthy tips into 250 mL flat-bottom flasks in a climatic room set at 20 ± 1 °C with the white cool light of 20 ± 0.5 µmol photons m^-2 s^-1 (1500 lux), a photoperiod set at 16 h: 8 h (light: dark), and sterile seawater enriched with VSE culture media renewed every week.
2. Perform weekly weight measurements, avoiding straining the thalli excessively²⁰. For this, carefully remove the tips from the culture medium, gently rinse, and weigh the milligrams on a laboratory scale.
   NOTE: This procedure can be performed along with the culture media weekly renewal.
3. Thalli may grow in these flasks up to a density of 2 g/L. At this point, perform a recipient scale-up (250 mL, 1 L, and 5 L). Transfer the cultivation to outdoor open white containers of 50 L and larger when the volume reaches 5 L.
4. Calculate relative growth rate (RGR) according to Patarra et al. (2017)²¹ :
   RGR (% fw/day) = ([Ln (fw) - Ln (iw)]/t) x 100
   where iw and fw are the initial and final fresh weight, respectively, expressed in grams, and t is the time in days.
   NOTE: Under this laboratory setup, RGR reaches values up to 21% per day. Biomass harvest can be performed at any time. Biomass must be quickly processed to prevent degradation by either oven-drying, freeze-drying, or simply stored frozen (-20 °C), depending on the intended use. The dried biomass can be preserved at room temperature (RT) or stored frozen as well.

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.

1. To carry out the extractions, oven-dry the G. gracilis biomass and grind the biomass (e.g., in a household coffee grinder) until the powder passes through a 200 µm sieve.
2. Weigh the dried biomass (10 g) and dissolve it in 100 mL of solvent (absolute ethanol or sterile water).
3. Stir in a vessel protected from light for 30 min.
4. Perform sequential ethanol > water and water > ethanol extractions at RT, 40 °C, and 70 °C.
5. For each temperature, perform the extractions with ethanol and water separately twice.
6. Separate the liquid extracts from the remaining biomass by filtration through filter paper (Whatman No.1), followed by centrifugation at 8000 x g for 10 min at RT.
7. Re-use the remaining algal biomass for further extraction with the other solvent. If a sample was firstly extracted with ethanol, extract it with water next, and vice-versa.
8. Freeze-dry the aqueous extracts and evaporate the ethanol extracts in a rotary evaporator at 40 °C.
9. Store the dried extracts at 4 °C.
10. Dissolve the extracts at a concentration of 50 mg/mL (antimicrobial assays) or 10 mg/mL (antioxidant assays). Dissolve the aqueous extracts in sterile water and the ethanolic extracts in absolute ethanol.

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.

1. Perform the broth microdilution tests using non-treated and round-bottom 96-well microplates with 170 µL of Muller-Hinton Broth (MHB), inoculated with 10 µL of standardized inoculum (at 0.5 McFarland standard) and 20 µL of each extract (50 mg/mL).
2. Incubate the plates for 24 h at the optimal temperature for each strain.
3. Detect the antimicrobial activity by the reduction of the visible turbidity measured by recording the optical density (OD ₆₀₀) in a microplate spectrophotometer, at 0 h and 24 h.
4. Express the results as a percentage of inhibition:
   
   where Abs. ext is the difference in the absorbance measured, between 0 h and 24 h, in the wells that contain bacterial strain growing in the presence of the extract, and Abs refers to the same measure in wells that contain the bacterial strain and solvent.
5. In this method, include control reactions, with wells containing only culture medium that will be the negative control, but also wells with medium inoculated with the standard strain added to solvent (ethanol or water) and medium with the bacterial strain and the positive control antibiotic (chloramphenicol).

6. Antioxidant activity and quantification of total polyphenols

1. Total polyphenolic content
   ​NOTE: Total polyphenolic content (TPC) is carried out using the Folin-Ciocalteu method²³ and adapted to micro-scale.
   1. Add to each well of a 96-well microplate, protected from light, 158 µL of ultrapure water, 2 µL of the sample, and 10 µL of Folin-Ciocalteu reagent.
      CAUTION: See the safety data sheet of the Folin-Ciocalteu reagent delivered by the supplier.
   2. After 2 min, add 30 µL of Na₂CO₃ (20%).
   3. After incubation in the dark at RT for 1 h, measure the samples spectrophotometrically at 755 nm.
   4. Use gallic acid (which allows the calibration curve to be plotted) or ultrapure water (2 µL) as controls.
   5. Express the results as gallic acid equivalents (mg GAE/g extract).
2. 2,2 diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity
   ​NOTE: The antioxidant activity of the extracts is evaluated as described by Duan et al. (2006)²⁴, adapted to microscale
   1. In a 96-well microplate protected from light, place 2 µL of each sample (at a concentration of 10 mg/mL) and 198 µL of DPPH dissolved in absolute ethanol (0.1 mM).
      ​CAUTION: See the safety data sheet of DPPH delivered by the supplier.
   2. Run the reaction for 30 min at RT in the dark. Measure the absorbance at 517 nm in a microplate spectrophotometer.
   3. Perform a control reaction with 2 µL of absolute ethanol/distilled water and 198 µL of DPPH solution. Perform a blank measurement with 2 µL of extract and 198 µL of absolute ethanol.
   4. Express the results as the percentage of DPPH inhibition using the following equation:
      
      where As is the absorbance of the algal extract, Ab is the absorbance of the blank samples and Ac is the absorbance of the control.

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)

1. Cell culture maintenance
   1. Culture HaCaT cells in Dulbecco´s Modified Eagle´s high glucose medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic/antimycotic solution (amphotericin B, 0.25 mM; penicillin, 60 mM; streptomycin, 100 mM).
   2. Use trypsin-EDTA to dissociate the cells.
      NOTE: The sub-culture of HaCaT cells is accomplished after the cells reach total confluence.
   3. Culture the cells in a chamber at 37 °C with 5% CO₂ and 95% humidity.
   4. Subculture the cells according to biobank instructions whenever cultures reach 80%-85% confluence.
2. Cytotoxicity evaluation
   1. After seeding cells in 96-well plates and incubation overnight, treat HaCaT cells (4 x 10⁴ cells/well) with the dried extracts previously dissolved in DMSO (100 mg/mL). Then, add 2 µL of the extract solution to 198 µL of medium and incubate the plates for 24 h.
   2. Remove the culture medium and add 100 µL of MTT (0.5 mg/mL) to the cells. Incubate the cells for 30 min in the dark at regular culture conditions mentioned above.
   3. Remove the MTT solution and solubilize the intracellular formazan crystals with 100 µL of DMSO.
   4. Measure the absorbance at 570 nm using a microplate reader. Express the results as a percentage of control untreated cells.

8. Food innovation

1. New food product: Pasta with seaweed
   1. Selection of ingredients and pasta formulation
      NOTE: The ingredients selection was made in collaboration with a pasta company. The choice of key ingredients (described in section 8.2) was made considering their easy accessibility and compatibility with the existing production lines, using marine resources to obtain pasta with added nutritional value.
      1. After the ingredients' choice, design the formulation following the intended nutritional value (source of fiber, vitamins, and mineral elements, low saturated fats) by analyzing the formulations' theoretical chemical composition using a spreadsheet.
      2. When the theoretical requirements are met, proceed with the laboratory-scale production as described in step 8.1.2.
      3. Perform a sensory test with a semi-trained panel (>10 tasters) to validate the need for reformulation or the acceptance of the formulation for the following steps.
         NOTE: The panel was previously trained for the tasting of pasta and hedonically evaluated the formulations presented regarding attributes such as flavor, taste, odor, texture, and appearance.
   2. Pasta production
      ​NOTE: Produce Chifferi pasta samples using a pasta extruder.
      1. In the equipment, mix previously defined portions of rice flour, G. gracilis, and Chlorella vulgaris, and add about 30% of water to the mixture.
      2. To obtain dry pasta, dry Chifferi at 68 °C for 42 min, followed by 5 h, 30 min at 76 °C, simulating an industrial process.
      3. Finally, pack and vacuum seal the samples and store them in a dark place at RT until further analysis.
   3. Nutritional analysis
      NOTE: For the nutritional profile analyses, use dried and macerated samples in triplicate.
      1. Crude protein content: Perform total protein assay through the Kjeldahl method, adapted from Duarte et al. (2022)²⁶, following steps 8.1.3.2-8.1.3.6.
      2. Accurately weigh 1.0 g of sample (or distilled water for the blank assay) and mix with two Kjeldahl tablets and 25 mL of H₂SO₄ in digestion tubes.
         CAUTION: See the safety data sheet of H₂SO₄ delivered by the supplier.
      3. Perform the digestion of samples in a Kjeldahl digestor at 220 °C for 30 min, followed by 90 min at 400 °C.
      4. After cooling down to RT, add 80 mL of distilled water and distil the ammonia formed into 30 mL of a 4% H₃BO₃ solution containing bromocresol green and methyl red. This step takes place under alkaline conditions (distillation with 40% NaOH using a Kjeldahl distiller.
         CAUTION: See the safety data sheet of the H₃BO₃ solution containing bromocresol green, methyl red and 40% NaOH delivered by the supplier.
      5. Titrate the distilled samples with HCl 0.1 M until a change in color to a greyish pink is observed.
      6. Calculate the crude protein content, represented by the sample's nitrogen content, and express it as g per 100 g using the following equation:
         
         where V_s corresponds to the HCl volume (mL) used in sample titration; V_b corresponds to the volume used in the blank; N corresponds to HCl normality; w corresponds to sample weight (g).
      7. Total fat content: Determine the total fat content using the Folch method, adapted from Folch et al. (1957)²⁷, following steps 8.1.3.8-8.1.3.14.
      8. Prepare Folch reagent by mixing CHCl₃ and MeOH in a proportion of 2:1 (v:v).
         CAUTION: See the Safety Data Sheet of the Folch reagent delivered by the supplier.
      9. To test tubes containing 1 g aliquots of samples, add 5 mL of Folch reagent and 0.8 mL of distilled water. Mix in a vortex for 1 min.
      10. Then, add another 5 mL of Folch reagent and homogenize for 5 min. Add 1.2 mL of 0.8% NaCl solution and homogenize for 2 min_.
      11. Centrifuge the samples at 7000 x g for 10 min. Filter the organic phase (lower phase) through hydrophilic cotton and anhydrous sodium sulfate into a round-bottomed glass flask.
      12. To avoid sample loss, repeat the steps of addition of 5 mL of CHCl₃, homogenization, centrifugation, and filtration under the same conditions.
      13. Remove the organic solvent from the collected organic phases by low-pressure evaporation and leave in the oven at 105 °C for 4 h. Cool down the samples in a desiccator.
      14. Calculate the fat content, expressed as g per 100 g, using the following equation:
          
          where W₁ is the empty round-bottomed glass flask weight; W₂ is the sample's initial weight; W₃ is the round-bottomed glass flask with the sample weight.
      15. Crude fiber content: Determine the crude fiber content using a methodology adapted from ISO 6865 (2000)²⁸, following steps 8.1.3.16-8.1.3.22.
      16. Weigh 1 g of the sample (W0) into a glass crucible with a filter bottom (reference P2) and place it in the fiber analyzer.
      17. The first step is acid hydrolysis: Add 150 mL of 1.25% H₂SO₄, preheated, and 2 mL of anti-foaming agent (n-octanol) to the column of each crucible; heat until boiling and keep for 30 min.
      18. After removal of this solvent, wash three times with deionized water to proceed to the basic hydrolysis. Add 150 mL of 1.25% NaOH, preheated, and 5 mL of anti-foaming agent to the liquid-free column, and perform the same heating procedure as for the acid hydrolysis.
      19. Finally, make a triple wash with 150 mL of acetone for the cold extraction.
      20. After this process, carefully remove the crucibles from the system and place them in an oven at 150 °C for 1 h. Record the final weight (W1).
      21. Place the crucibles in a muffle furnace at 500 °C for 3 h and then record the final weight (W2).
      22. Calculate the crude fiber content and express the results in percentages using the following equation:
          %Crude Fiber = 100 x (W1-W2)/W0
      23. Fatty acid (FA) profile: Determine the fatty acids profile according to Fernández et al.(2015)²⁹, following steps 8.1.3.24-8.1.3.29.
          NOTE: The fatty acids methyl esters (FAMEs) are obtained by direct acid-catalyzed transmethylation of the milled freeze-dried samples. All analyses are done in triplicate.
      24. Add 2 mL of a 2% (v/v) H₂SO₄ solution in methanol to a 50 mg sample and pour the mixture at 80 °C for 2 h with continuous stirring.
          CAUTION: See the safety data sheet of methanol delivered by the supplier.
      25. After cooling to RT, add 1 mL of ultrapure water and 2 mL of n-heptane to each sample, vortex the mixture for 1 min, and centrifuge it for 5 min.
          CAUTION: See the safety data sheet of n-heptane delivered by the supplier.
      26. Recover the upper n-heptane phase (organic) containing the FAMEs and transfer it to gas chromatography (GC) vials.
      27. Analyse in a gas chromatograph equipped with a TR-FAME capillary column (60 m × 0.25 mm ID, 0.25 µm film thickness), an autosampler, and a flame ionization detector (FID).
      28. Set the injector (splitless mode) at 250 °C and the detector at 280 °C. Set the column´ initial temperature at 75 °C and hold for 1 min. Then raise at 5 °C/min to 170 °C and hold for 10 min. Then raise at 5 °C/min to 190 °C and hold another 10 min. Finally, raise at 2 °C/min to 240 °C and hold for 10 min. Use helium as carrier gas at a flow rate of 1.5 mL/min. Supply air and hydrogen at flow rates of 350 and 35 mL/min, respectively.
      29. Determine the FA profile by comparing the resulting retention times with a standard and express the results as a percentage of total fat.
      30. Mineral elements profile: Determine the mineral elements (Ca, P, Mg, Na, K, Fe, Cu, Mn, Zn) analyzed by ICP-OES, following the method adapted from Pinto et al. (2022)³⁰, following steps 8.1.3.31-8.1.3.34.
      31. Accurately weigh about 0.4 g of each dry sample and add 7.5 mL of HNO₃ and 2.5 mL of HCl.
          CAUTION: See the safety data sheet of HNO₃ and HCl delivered by the supplier.
      32. The digestion follows a two-stage process: Increase the temperature from RT to 90 °C in 30 min (and maintain a further 30 min at this temperature) followed by 60 min at 105 °C.
      33. Cool the sample solutions, dilute them to 25 mL, and filter and keep them in labeled tubes. In each digestion, perform the same process with a reference material and a blank. Obtain the concentration of the different elements by ICP-OES.
      34. Express the results in mg per 100 g of fw.
      35. Carbohydrate content: Calculate the carbohydrate content following steps 8.1.3.36-8.1.3.37.
      36. Calculate the available carbohydrates (fiber excluded) content by the difference of the previously determined factors per 100 g, using the following equation, according to the Food and Agriculture Organization (FAO; 2003)³¹
          
      37. Express the results in g per 100 g.
      38. Moisture and ash content: Estimate the moisture and ash contents following steps 8.1.3.39-8.1.3.45.
      39. Incubate porcelain crucibles for 3 h at 105 °C, cool them in a desiccator and weigh them.
      40. Weigh 10 g of the sample into the crucible and place it in a drying oven at 105 °C for 3 h cycles until the values from successive weightings do not differ by more than 10 mg.
      41. Calculate moisture content, expressed as g per 100 g of fw, using the following equation:
          
          where W₁ is the weight of the empty crucible, W₂ is the weight of the crucible with the fresh sample, and W₃ is the weight of the crucible with the dried sample.
      42. After the moisture content assay, place the crucibles with the dried samples in an incinerator at 525 °C for 4 h.
      43. Repeat this procedure until successive weightings do not differ by more than 1 mg.
      44. Cool the samples to RT in a desiccator and then weigh them.
      45. Calculate the ash content, expressed as g per 100 g of fw, using the following equation:
          
          where W₁ is the weight of the empty crucible, W₂ is the weight of the crucible with fresh sample, W₃ is the weight of the crucible with weight.
      46. Energy value: Calculate the energy value following steps 8.1.3.47-8.1.3.48.
      47. Calculate the energetic value of the samples according to the EU regulation:The provision of food information to consumers (Regulation 1169/2011)³², using the equations:
          Energy (kcal/ 100 g) = 4 x (g proteins) + 4 x (g carbohydrates) + 9 x (g fat) + 2 x (g fiber)
          Energy (kJ/ 100 g) = 17 x (g proteins) + 17 x (g carbohydrates) + 37 x (g fat) + 8 x (g fiber)
      48. Express the results in kilocalories per 100 g and kilojoules per 100 g.
   4. Consumer acceptance
      1. Evaluate consumer acceptance using pasta samples cooked in distilled water for 8 min.
      2. Perform consumer acceptance test: Evaluate visual appearance, color, texture, odor, sea taste, overall taste, overall evaluation, and purchase intent of the samples.
         NOTE: The consumer acceptance test is based on hedonic tests evaluating visual appearance, color, texture, odor, sea taste, overall taste, overall evaluation, and purchase intent on a scale of 1-9, where 1 is a poor evaluation, and 9 is a very good evaluation.
      3. Perform sensory tests in individual sensory booths in a sensory analysis laboratory (with temperature and lighting control). Provide cutlery, napkins, and glass cups of mineral water to clean the palate between samples.
         NOTE: Tasters are aged 16-64 from all backgrounds (n > 80).
2. Yogurt
   1. Pigment extraction
      ​NOTE: Perform the pigment extraction through the methodology described in Pereira et al. (2020)¹⁸.
      1. Prepare the extraction solvent, sodium phosphate buffer at 0.1 M, with sodium phosphate dibasic (0.03 M) and sodium phosphate monobasic (0.07 M). Set the pH at pH 6.8 using NaOH or HCl.
      2. Weigh 1 g of G. gracilis and add 50 mL of sodium phosphate buffer (pH 6.8). Homogenize for 10 min, followed by 10 min of maceration with mortar and pestle.
      3. Transfer the solution to a tube and centrifuge for 20 min at 12,298 x g (4 °C).
      4. Pool the supernatant and slowly add 65% ammonium sulfate. When all the ammonium sulfate is dissolved, cover the solution with an aluminum sheet and leave it to precipitate at 4 °C overnight.
         CAUTION: See the safety data sheet of ammonium sulfate delivered by the supplier.
      5. Centrifuge the precipitate for 20 min at 12,298 x g (4 °C). Recover the pellet and dissolve in distilled water (approximately 5 mL).
      6. Perform dialysis of the extract using a tubing membrane (14 kDa) against water for 24 h, followed by freeze-drying. Store the freeze-dried extract protected from light at 4 °C until use.
   2. Yogurt preparation
      1. Prepare natural yogurt by mixing 1 L of pasteurized milk, 120 g of natural yogurt, 20 g of sugar, and 50 g of milk powder in a thermomixer for 5 min, 50 °C, speed 3.
      2. Place the mixture in the thermomixer jar in an incubator at 37 °C for 12 h.
      3. Incorporate the extract by mixing it in yogurt at a concentration of 0.21%. Store the samples in individual glass flasks at 4 °C until analysis.
      4. Store individual portions of yogurt without pigment (control) at 4 °C until analysis.
   3. Color stability
      NOTE: Evaluate the stability of the pigment in the yogurts through color analysis for 12 days. Perform color analysis using a reflectance colorimeter, using a 2-degree standard observer, and a D65 illuminant. The results are presented as CIELab coordinates with L (lightness, black - white, 0 - 100), a* (green - red, -60 - 60), and b* (blue - yellow, -60 - 60) parameters. Parameter a* has positive values for reddish colors and negative values for greenish colors. Parameter b* takes positive values for yellowish colors and negative values for bluish colors. L* is the parameter of luminosity, which is the property according to which each color can be considered equivalent to a member of the greyscale between black and white³³.
      1. Calibrate the colorimeter using a white ceramic plate (L* 88.5, a* 0.32, b* 0.33) provided by the manufacturer.
      2. Fill a cell with approximately 28 g of the sample (or control) and analyze the color using color data analysis software.
         NOTE: The software used for color data analysis was the SpectraMagic NX.
      3. Perform readings 5 times in sample/control triplicates.
   4. Sensory analysis
      ​NOTE: Perform sensory evaluation of yogurts with pigment incorporation using a triangle test (ISO 4120, 2004)³⁴ and a hedonic evaluation of color, taste, and overall appreciation.
      1. For the triangle test, give the panelists three samples (one sample of yogurt with pigment and two samples of control, or two samples of yogurt with pigment and one control) and ask them to choose a different sample based on the aroma, texture, and taste. Provide samples in similar volumes identified with random 3-number codes.
      2. For the hedonic evaluation of the yogurt with pigment, give the panelists a sample of yogurt with pigment and ask them to evaluate the color, taste, and overall appreciation using a 9-point hedonic scale (from extremely dislike to extremely like).

Results

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 strain...

Discussion

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 microorganisms^35,36,37,38. They are used to evaluate the sensitivity of microorganisms to antimicrobial agents and are conducted in te...

Materials

Name	Company	Catalog Number	Comments
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