Valorization of the Red Seaweed Gracilaria gracilis Through a Biorefinery Approach

Our research is focused on the prospection of new bioactive compounds from seaweed that can be useful in different sectors, such as food, cosmetic and pharmaceutical industries. There is a growing awareness in society about the potential of marine organisms to deliver bioactive compounds. These compounds can be used to produce sustainable and safer functional cosmetics to improve the quality of food products and serve as source of new chemical structures for the development of new pharmaceutical drugs.

One of the challenges of using seaweed in the development of new food products is to avoid using high amounts that could lead to unpleasant sensory properties in food. Using seaweed extracts is a way to get around this problem. With this protocol, we were able to develop pasta which are nutritional value by incorporating all algae.

Usually the addiction of this ingredients provides less common sensory properties. However, with the developed formulation, a consumer acceptance was still achieved. To begin, grow gracilaria gracilis in 250 milliliters of flat bottom flasks in a climatic room.

Once the biomass has been scaled up, dried and ground, weigh the resulting dried biomass. Dissolve it in 100 milliliters of solvent, and mix by stirring in a vessel protected from light for 30 minutes. Perform extractions with ethanol and water separately at room temperature.

Filter the liquid extracts through filter paper to separate them from the remaining biomass. Centrifuge the extracts at 8, 000G for 10 minutes at room temperature. Freeze dry the aqueous extracts and use a rotary evaporator at 40 degrees Celsius to evaporate the ethanol extracts.

To begin, take the aqueous and ethanol extracts obtained from the dried gracilaria gracilis. In a 96 well microplate, dispense two microliters of each sample, and 198 microliters of DPPH dissolved in absolute ethanol. Then, using a microplate spectrophotometer, measure the absorbance at 517 nanometers.

Take the human keratinocytes grown in 96 well plates overnight. Add two microliters of the DMSO dissolved extracts to 198 microliters of the medium in microplate wells. Remove the culture medium.

Add 100 microliters of MTT to the cells, and incubate in the dark for 30 minutes. To solubilize the intracellular formazan crystals, add 100 microliters of DMSO. Using a microplate reader, measure the absorbance at 570 nanometers.

The DPPH test demonstrated that the temperature of 40 degrees Celsius showed higher inhibition values of oxidant activity compared to extracts obtained at room temperature or 70 degrees Celsius. No cytotoxic effects were observed on human keratinocytes, suggesting that at the maximum acid concentration, both aqueous and ethanol extracts are safe for cutaneous use. To begin, combine predefined portions of rice flour, gracilaria gracilis, and chlorella vulgaris in the equipment, and add approximately 30%water to the mixture.

After a few minutes of mixing, followed by extruding the pasta using the equipment, cut it manually. To produce chifferi, dry the pasta at 68 degrees Celsius for 42 minutes, followed by an additional five hours and 30 minutes at 76 degrees Celsius, simulating an industrial process. Finally, vacuum seal the samples and store them in a dark place at room temperature.

Take one gram sample in a glass crucible with a filter bottom and position it in the fiber analyzer. Add 150 milliliters of preheated 1.25%sulfuric acid and two milliliters of anti foaming agent to the column of each crucible. Remove the solvent and rinse the crucible three times with deionized water to prepare the sample for the basic hydrolysis.

After washing the crucibles three times with 150 milliliters of acetone, carefully transfer those to an oven set at 150 degrees Celsius for one hour. Record the final weight. Transfer the crucibles to a muffle furnace set at 500 degrees Celsius for three hours, and then record the final weight.

To determine the fatty acids profile, add two milliliters of 2%sulfuric acid solution in methanol to a 50 milligram sample. Heat the mixture at 80 degrees Celsius for two hours. Then cool the mixture to room temperature, and add one milliliter of ultrapure water and two milliliters of N heptane to each sample.

After vortexing the mixture for one minute, centrifuge it for five minutes. Later, recover the upper N heptane phase containing the fatty acid methyl esters, and transfer it to gas chromatography vials. Analyze the samples in a glass chromatograph equipped with a TRFAME capillary column, an auto sampler, and a flame ionization detector.

Determine the fatty acid profile by comparing the resulting retention times with a standard. Incubate porcelain crucibles for three hours at 105 degrees Celsius. Allow them to cool in a desiccater, and then record their weights.

After incubating 10 grams of the sample as demonstrated previously, calculate its moisture content using the displayed equation. Then transfer the crucibles with the dried samples to an incinerator heated to 525 degrees Celsius for four hours. After cooling the samples, record their weight.

To assess consumer acceptance, use chifferi pasta samples cooked in distilled water for eight minutes. Conduct sensory tests in individual sensory booths located in a sensory analysis laboratory. Evaluate the visual appearance;color, texture, odor, sea taste, overall taste, overall evaluation, and purchase intent of the samples.

The pasta received ratings of five and six on a hedonic scale of one to nine for sea taste and visual appearance respectively. The pasta had a purchase intention score of five, and an overall appreciation score of six. To begin, add 50 milliliters of sodium phosphate buffer to one gram of gracilaria gracilis.

After homogenizing the mixture, use a mortar and pestle to macerate the sample for 10 minutes. Transfer the solution to a tube, and centrifuge it for 20 minutes at 12, 298G. After combining the supernatant, slowly add 65%ammonium sulfate.

Then pellet the precipitate by centrifugation, and dissolve it in distilled water. Perform dialysis of the extract using a tubing membrane against water for 24 hours, followed by freeze drying. To incorporate the extract, mix it in yogurt at a concentration of 0.21%Store the samples in individual glass flasks at four degrees Celsius until analysis.

To check the color stability of the extract in yogurt, calibrate the colorimeter using a white ceramic plate. Fill a cell with approximately 28 grams of the sample, and analyze its color. The color stability of the yogurts with pigment was evaluated for 12 days at minus four degrees celsius.

The incorporation of the extracts in yogurts showed good color retention, with a color difference of 7.01, plus or minus 2.36 after 12 days of storage.