Raw material and biomass preparation
Four different K. alvarezii seaweed strains were used. The strains were obtained from the Fisheries Institute, Ubatuba, São Paulo (SP). The following K. alvarezii strains were used: brown, red, green, and G11. The strains were grown during May and June of 2013. The K. alvarezii strains were grown in the Atlantic Ocean in the experimental field base at Itaguá beach in Ubatuba, SP, Brazil (GPS coordinates 23°27′5,8″S; 45°02′49,3″W). The structure used to grow the seaweed strains consisted of a raft anchored in the bay [31, 32]. Ten shoots of vegetative growth from each strain (approximately 70 g on wet basis) were pre-weighed and bound on a nylon line in a sub-assembly on the surface of the seawater, which provided a cultivation density of 6.7 plants per m2. For cultivation, the strains remained in the structure for 30 days. After 30 days, the strains were weighed again. The wet weight and dry mass from each strain were determined using an average humidity of 35 % (commercial value) [30, 31]. The growth rate was calculated according to the equation: \({\text{Growth}}\,{\text{rate}}\,({\text{percentage}}\,{\text{on}}\,{\text{day}} - 1) = [\left( {w_{t} /w_{0} } \right)1/t - 1]*100\,,\) where w
t
is the final wet mass (g); w
0
is the initial wet mass (g); and t is the cultivation time (30 days) [32, 33]. The productivity was calculated according to the equation: \({\text{Productivity }}({\text{gm}}^{2} \,{\text{day}}^{ - 1} ) \, (w/w,\,\,{\text{dry basis}}) = [({\text{dwtf}} - {\text{dwti}})/t*({\text{dwt}}/{\text{wwt}})]/A,\) where dwtf is the final dry mass (g); dwti is the initial dry mass (g); t is the cultivation time (30 days); dwt = total dry mass; wwt is the total wet mass and A is the total area of cultivation [32, 33]. After collection, the biomass was dried at 25 °C. The biomass was washed with distilled water, with stirring, in a 10 L polypropylene beaker for 45 min at a ratio of 35 g (dry weight) of macroalgae biomass to 1 L of distilled water. After washing, the solution was removed using a sieve with a 1 mm screen. Washing was repeated until the electrical conductivity of the wash solution was similar to that of distilled water (measured with a portable conductivity meter). After washing, the samples were again dried at 25 °C. These materials are hereafter termed ‘untreated material’.
Chemical composition of the samples
Hexane-soluble extractives were determined by extraction with 99 % (v/v) hexane in a Soxhlet apparatus [26]. The samples were air dried, milled, and passed through a 0.84 mm screen. Approximately, 1 g of the milled sample was extracted with 99 % hexane for 8 h in the Soxhlet apparatus. The percentage of lipids was determined based on the dry weights of the extracted and non-extracted milled samples [data provided as mean ± standard deviation (SD)]. This procedure was conducted in triplicate.
The milled samples were hydrolyzed with 72 % (w/w) sulfuric acid at 30 °C for 1 h (3 mL of acid to 300 mg of sample) as described previously [34, 35]. The acid hydrolysate was diluted with 79 mL of distilled water (4 % (w/w) sulfuric acid), and the mixture was autoclaved at 121 °C for 1 h. The residual material was cooled and filtered through a porous glass filter (Scott number 3, Germany). The solids were dried to a constant weight at 105 °C and were assessed as the insoluble aromatics component. The filtrate was further passed through 0.45 µm membranes. The total sugar content in the same solution was determined by the sulfuric acid/phenol method, using sucrose as the calibration standard [36]. The filtrates were evaluated via HPLC/MS analysis (using HPLC Agilent 1200 Series and AB Sciex QTRAP mass spectrometers) to confirm the presence of monomeric sugars. Detection of the monomeric sugars in the soluble fraction was performed using HPX87P columns (Bio-Rad; Hercules, CA, USA) at 80 °C by elution with water at a rate of 0.6 mL min−1. The mass spectrometer was operated using electrospray ionization (ESI) in positive and negative modes. The ionization source parameters in negative mode were: ion spray: −4500 V; curtain gas: 15 psi; temperature: 650 °C; gas 1:50 psi; gas 2:50 psi; and heater interface: on. The ionization source parameters in the positive mode were as follows: ion spray: 5500 V; curtain gas: 15 psi; temperature: 650 °C; gas 1:50 psi; gas 2:50 psi; heater interface: on. The standards were diluted to 1 mg L−1 in water with 0.1 % acetic acid, and the optimization was performed by direct infusion into the automatic flow (10 L min−1) using a syringe. All sugars were detected as water adducts (+18) [M + 18]+ in positive mode. Xylose: (SRM1, Q1 = 168.1, Q3 = 150.0, DwellTime(ms) = 250, SD(V) = 16, EP(V) = 5, CEP(V) = 10, EC(V) = 9 and CXP(V) = 4); (SRM2, Q1 = 168.1, Q3 = 73.2, DwellTime(ms) = 250, SD(V) = 16, EP(V) = 5, CEP(V) = 10, EC(V) = 19 and CXP(V) = 4. Arabinose: (SRM1, Q1 = 168.1, Q3 = 50.1, DwellTime(ms) = 250, SD(V) = 6, EP(V) = 3.5, CEP(V) = 14, EC(V) = 9 and CXP(V) = 4), (SRM2, Q1 = 168.1, Q3 = 73.2, DwellTime(ms) = 250, SD(V) = 16, EP(V) = 5, CEP(V) = 10, EC(V) = 19 and CXP(V) = 4; (SRM2, Q1 = 168.1, Q3 = 73.0, DwellTime(ms) = 250, SD(V) = 16, EP(V) = 3.5, CEP(V) = 14 EC(V) = 21 and CXP(V) = 4 cellobiose: (SRM 1, Q1 = 360.2, Q3 = 163.2, DwellTime(ms) = 250, SD(V) = 21, EP(V) = 4.5, CEP(V) = 16, EC(V) = 17 and CXP(V) = 4), (SRM2, Q1 = 360.2, Q3 = 84.9, DwellTime(ms) = 250, SD(V) = 21, EP(V) = 4.5, CEP(V) = 16, EC(V) = 33 and CXP(V) = 4 Galactose: (SRM1, Q1 = 198.0, Q3 = 163.1, DwellTime(ms) = 250, SD(V) = 21, EP(V) = 6, CEP(V) = 12, EC(V) = 11 and CXP(V) = 4), (SRM2, Q1 = 198.0, Q3 = 91.2, DwellTime(ms) = 250, SD(V) = 21, EP(V) = 6, CEP(V) = 12, EC(V) = 19 and CXP(V) = 4 Glucose: (SRM1, Q1 = 198.1, Q3 = 85.1, DwellTime(ms) = 250, SD(V) = 16, EP(V) = 6.5, CEP(V) = 10, EC(V) = 21 and CXP(V) = 4), (SRM2, Q1 = 198.1, Q3 = 163.2, DwellTime(ms) = 250, SD(V) = 16, EP(V) = 6.5, CEP(V) = 10, EC(V) = 11 and CXP(V) = 4. Mannose: (SRM1, Q1 = 198.1, Q3 = 163.2, DwellTime(ms) = 250, SD(V) = 21, EP(V) = 8, CEP(V) = 12, EC(V) = 11 and CXP(V) = 4); (SRM2, Q1 = 198.1, Q3 = 85, DwellTime(ms) = 250, SD(V) = 21, EP(V) = 8, CEP(V) = 12, EC(V) = 25 and CXP(V) = 4. Rhamnose: (SRM1, Q1 = 198.1, Q3 = 163.2, DwellTime(ms) = 250, SD(V) = 21, EP(V) = 8, CEP(V) = 12, EC(V) = 11 and CXP(V) = 4, (SRM2, Q1 = 198.1, Q3 = 85, DwellTime(ms) = 250, SD(V) = 21, EP(V) = 8, CEP(V) = 12, EC(V) = 25 and CXP(V) = 4. Galacturonic acid was detected as [M–H]− in negative mode. Galacturonic acid: (SRM1, Q1 = 193.02, Q3 = 113, DwellTime(ms) = 2500, SD(V) = −20, EP(V) = −3.5, CEP(V) = −10, EC(V) = −18 and CXP(V) = −2), (SRM2, Q1 = 193.021, Q3 = 59.1, DwellTime(ms) = 2500, SD(V) = −20, EP(V) = −3.5, CEP(V) = −10, EC(V) = −26 and CXP(V) = 0. The concentrations of monomeric sugars in the soluble fraction were determined by HPLC (HPX87P column; Bio-Rad, Hercules, CA, USA) at 80 °C using water as the eluent at a flow rate of 0.6 mL min−1. Sugars were detected using a temperature-controlled refractive index detector at 45 °C. Glucose, xylose, mannose, and galactose were used as external calibration standards. Corrections were performed by considering the anhydrogalactose-degradation reactions that took place during acid hydrolysis. Under the present acid hydrolysis conditions, all the anhydrogalactose present in the sample is degraded [29, 37]. Thus, the anhydrogalactose content in the carrageenans and agars was calculated using the galactose to anhydrogalactose ratio of 1:1.27 [29, 37]. The factor used to convert the sugar monomers to anhydromonomers was 0.9 for glucose and galactose. This procedure was conducted in triplicate. Glucose was reported as glucan and galactose and anhydrogalactose as galactan after correction by the hydrolysis factor. The concentration of hydroxymethylfurfural and furfural in the soluble fractions was determined using an HPLC instrument equipped with a 250 mm long column with an outer diameter of 4 mm (Hypersil; Thermo-Scientific) using acetonitrile:water (1:8) containing 1 % (v/v) acetic acid as an eluent at a flow rate of 0.8 mL min−1. Hydroxymethylfurfural and furfural were detected at 276 nm.
The sulfate group content of the samples was quantified using modified spectrophotometric methods [38–40]. About 0.05 g of the milled sample was weighed and placed in test flasks. One milliliter of 0.5 N HCl was added to each flask, and the flasks were capped with aluminum foil and autoclaved at 120 °C for 1 h at 1 atm. At the end of the reaction, the sample was transferred into 15 mL Falcon tubes. Water was then added to each tube to achieve a volume of 10 mL followed by centrifugation at 7000×g for 10 min. The supernatant (2 mL), 18 mL of distilled water, and 2 mL of 0.5 N HCl were added to the test flasks and stirred for a few seconds. Afterwards, 1 mL of BaCl2 gelatin (Difco-Laboratories, Detroit, EUA) was added. The tubes were kept at 25 °C for 30 min with stirring, and absorbance readings were taken at 550 nm (Genesys 10S, Thermo-Scientific).
The protein content of the samples was quantified using a Kjeldahl digester to determine the total nitrogen. The protein content was calculated using a nitrogen conversion factor of 6.25 [41]. The experiments were performed in triplicate. For quantification of the ash content of the samples [42], approximately 1 g of milled sample was weighed into a porcelain crucible and combusted in a muffle furnace at 575 ± 25 °C for 3 h using a pre-programmed heating ramp. At the end of 3 h, the pots were kept in the oven until the temperature was about 105 °C. The crucibles were cooled and weighed. The experiments were performed in duplicate. The metal content of the samples was also quantified by treating approximately 0.05 g of the milled sample with 1 mL of sulfuric acid and 2 mL of nitric acid in a glass digester at 150 °C until the solutions become clear. These solutions were allowed to swell in a 100 mL volumetric flask and analyzed using a spectrophotometer (ICP Optima Perkin Elmer Model 8000). The following metals were analyzed: manganese, calcium, sodium, copper, silicon, iron, and potassium. The experiments were performed in triplicate.
Carrageenan processing for selected samples
Two strains of previously selected K. alvarezii (brown and red, grown in May 2013) were processed and the semi-refined carrageenan was extracted; the residue from this extraction process was also analyzed. Prior to extraction of the semi-refined carrageenan, “cold” alkali transformation was performed [32, 38]. Briefly, approximately 8 g (dry weight) of macroalgae was soaked in 96 mL of 6 % KOH solution (w/v) for 24 h at 25 °C (“cold” alkali transformation). The material was copiously washed with water, sun bleached for 12 h, and dried at 60 °C until constant weight was achieved. The material was weighed, milled, and passed through a 0.84 mm screen. Approximately, 3 g (dry weight) of the material obtained after alkaline transformation was extracted with 240 mL of distilled water in flasks and incubated at 65 °C for 2 h with rotary agitation at 120 rpm. The solution was then filtered using nylon tissue, and the extract was dried at 60 °C until constant weight was achieved (hereafter referred to as semi-refined carrageenan). The material retained on the nylon tissue after filtering was recovered and dried to constant weight at 60 °C (hereafter referred to as residue). Both the semi-refined carrageenan and the residue were weighed. The yield from the alkali treatment was determined from the difference between the original (untreated material) and final weights (dry weight basis). The partial yield of the semi-refined carrageenan and residue was obtained from the difference between the initial weight [the material treated with 6 % KOH (w/v)] and final weight of semi-refined carrageenan and residue (dry weight basis). The overall yields of the semi-refined carrageenan and residue were obtained from the difference between the original weight (untreated material) and final weight of the semi-refined carrageenan and residue (dry weight basis).
Enzymatic hydrolysis of the selected samples
Enzymatic hydrolysis was performed using commercial enzyme preparations (Cellic CTec II, Novozymes, Denmark) at a dosage of 10 FPU per gram of sample (dry weight basis), corresponding to 200 IU of β-glucosidase. The total cellulases and β-glucosidase activity determined using the Celic CTec II extract were 92 FPU mL−1 and 1800 UI mL−1, respectively. Each hydrolysis experiment was conducted in 50 mL Falcon tubes containing 200 mg of milled sample (dry weight basis) and 10 mL of 50 mM sodium-acetate buffer (pH 4.8) in addition to the enzyme solution. The flasks were incubated at 45 °C with rotary agitation at 120 rpm. The reaction was stopped at defined periods from 4 to 72 h by heating the flask to 100 °C for 5 min, followed by centrifugation of the material at 7000×g for 10 min. The soluble fractions were assayed for glucose using HPLC with an HPX87P column (Bio-Rad) at 45 °C using water as an eluent at an elution rate of 0.6 mL min−1. The sugars were detected using a temperature-controlled infrared detector set at 45 °C. The glucan conversion level reported herein refers to the conversion of the polysaccharides to their monomers. Values (mean ± SD) for the hydrolysis of the samples were estimated from triplicate runs.