# Assessment of the Resource Potential of Baltic Sea Macroalgae
## 2 Materials and Methods
### Research Methodology for Assessing the Physicochemical Composition of Macroalgae  
The content of the mass fraction of proteins, fats, and carbohydrates was studied to assess the potential resource attractiveness of drifting algae biomass as a source of nutrients in the feed and food industries. The protein content was determined using two different methods: Bradford and Kjeldahl. The Kjeldahl method [49] consists of mineralizing organic matter with sulfuric acid in the presence of a catalyst, forming ammonium sulfate and destroying ammonium sulfate with alkali, releasing ammonia and removing ammonia with water vapor into a solution of sulfuric or boric acid, which is followed by titration. The mass fraction of nitrogen is calculated and converted into the crude protein content based on the titration results (by multiplication by a factor of 6.25). When using the Bradford method [50], proteins were extracted using distilled water and NaOH solution. A total of 50 mg of dried microalgae biomass were mixed with 4 mL of distilled water and incubated at 4 \({}^{\circ}\)C for 12 h. The algae mixture was then ground in a mortar with a pestle for 5 min before being incubated at 4 \({}^{\circ}\)C for another hour. After grinding, the mixture was centrifuged for 20 min (at 4 \({}^{\circ}\)C, 75,456\(\times g\)). The supernatant was collected and the algae pellet was re-extracted with 1 mL of 0.1 N NaOH with 0.5% \(\beta\)-mercaptoethanol ethanol (\(\
u/v\)). The mixture of algae extract and NaOH solution was kept at room temperature for 1 h, periodically shaking by hand. The mixture was then centrifugedfor 20 min at 21 \({}^{\circ}\)C and 75,456\(\times\)\(g\). The supernatant was combined with the first one. Then, the protein content was determined using the Bradford method [50]. The carbohydrate content of drifting algae biomass was determined in two forms, fiber and reducing sugars, which correspond to the difference in their potential areas of use. The 3,5-dinitrosalicylic acid method was used to determine the content of reducing sugars. When sugars interacted with 3,5-dinitrosalicylic acid, the latter was reduced to 3-amino-5-nitrosalicylic acid. Preliminary extraction was performed according to the method of [PERSON] [51]. For this, 50 mg of microalgae biomass were placed in a 100 mL flask and mixed with 50 mL of 2% H\({}_{2}\)SO\({}_{4}\). The mixture was autoclaved at 121 \({}^{\circ}\)C and 1 bar for 30 min at pH 7. For separation, the resulting mixture was centrifuged at 4 \({}^{\circ}\)C at a speed of 764\(\times\)\(g\) for 10 min. The fiber content in algae samples was estimated by the weight method. The essence of the method is the sequential treatment of a weighed portion of the test sample dried to a constant weight with sulfuric acid and potassium hydroxide solutions, ashing, and the quantitative determination of the organic residue using the gravimetric method [52]. The total lipid content was determined by the Folch method [53]. For this, 20 mL of a solvent (a 2:1 (\(v/v\)) mixture of chloroform and methanol) was added to 1 g of dried biomass and stirred at 11 g for 20 min. Then, the algae biomass cells were destroyed in an ultrasonic disperser, homogenizer, and degasser. Next, the solvent containing the extracted lipids was centrifuged at 56,683\(\times\)\(g\) and filtered under vacuum. The solvent was evaporated to dryness in a drying cabinet at 60 \({}^{\circ}\)C. Total lipids were quantified gravimetrically and expressed as a percentage of dry cell weight. To determine the fatty acid composition, lipids were dissolved in 10 mL of hexane; then, 2 mL of the solution was transferred to a test tube, and 2 mL of a 10% sodium methoxide in methanol solution was added to the lipid solution. After vigorously shaking the mixture for 10 min, the top layer of hexane (1 mL) was removed and transferred to a vial, and the fractional composition was determined using GC/MS on an Agilent 5970B instrument.