Enzyme-assisted extraction of κ/ι-hybrid carrageenan from Mastocarpus stellatus for obtaining bioactive ingredients and their application for edible active film development

N. Blanco-Pascual, A. Alemán, M. C. Gómez-Guillén, M. P. Montero
2014 Food & Function  
14 Two hydrolysates were obtained from dried Mastocarpus stellatus using alcalase. Phenolic 15 content was partially removed from one of them. The phenolic-partially-removed hydrolysate (H) 16 was found to be a potent Angiotensin-I converting enzyme (ACE) inhibitor. However, the 17 phenolic-containing hydrolysate (Hp), showed a higher Folin-reactive substance content and 18 antioxidant capacity (reducing power and radical scavenging capacity). Hp was therefore 19 selected for the development of
more » ... antioxidant Mastocarpus carrageenan-based films. F-Hp0 20 (without hydrolysate), F-Hp15 (with 15% hydrolysate) and F-Hp30 (with 30% hydrolysate) films 21 were developed. κ/ι-hybrid carrageenan was the main film constituent and hydrolysate addition 22 resulted in an increased sulfated proportion, higher protein content and higher number of 23 hydrogen bonds. Therefore interactions between carrageenan helices, plasticizer and peptides 24 in the film-forming solution were enhanced, especially in F-Hp15, and consequently the water 25 vapour permeability (WVP) of the resulting film decreased. Nevertheless, F-Hp30 considerably 26 improved transparency, UV/Vis light barrier, water resistance and elongation at break (EAB). Hp 27 presence increased both puncture force (F) and puncture elongation (E), but not tensile strength 28 (TS) or Young's modulus (Y). The addition of an increased concentration of hydrolysate to the 29 films led to a considerable increase in the Folin-reactive substance content and the antioxidant 30 activity, especially the radical scavenging capacity. 31 (Rhodophyta) are known to have a high protein content, mainly composed of bioactive 43 phycobiliproteins 10 and other wall proteins that might be more efficiently extracted by an 44 enzyme-assisted treatment. 11,12 45 Mastocarpus stellatus is one of the few carrageenophyte species on the Atlantic coast currently 46 harvested for phycocolloid industry purposes, but it is still underutilized. 13 47 Commercial carrageenan is commonly extracted at alkaline conditions (pH 7-9) at temperatures 48 near boiling point (80-110 °C) for 3-4 h, providing yields of 20-40%. 13,14 However, high 49 molecular weight carrageenan can also be extracted at mild temperatures (50 °C) for 1-5 h. 15 50 κ/ι-hybrid carrageenan has been reported to be the main biopolymer structure extracted from M. 51 stellatus, 16 although other components, such as proteins, minerals and polyphenols, are also 52 present in significant amounts. 17 Mastocarpus enzymatic hydrolysis could produce both 53 antihypertensive and antioxidant extracts, as previously reported with another species of the 54 Rhodophyta phylum. 18,19 Protein hydrolysates from different origins have been incorporated in 55 the formulation of protein-based films to improve or confer bioactivity. 20,21 56 Mastocarpus extraction could be maximized by first performing an enzymatic hydrolysis at mild 57 temperatures and alkaline conditions followed by carrageenan precipitation and bioactive 58 compound isolation. The aims of the present study were: (i) to obtain two different potentially bioactive hydrolysates 63 with antioxidant and/or antihypertensive capacities from dried Mastocarpus stellatus, and (ii) to 64 develop antioxidant Mastocarpus carrageenan-based films as an active edible food packaging 65 material. 66 2. Materials and Methods 67 2.1. Seaweed sampling 68 Samples of Mastocarpus stellatus (M), kindly supplied by Porto-Muiños (Cerceda, A Coruña, 69 Spain), were washed several times with running tap water and air-dried at 50 °C for 24-48 h in 70 a ventilated oven. Seaweed samples were stored in sealed plastic bags at 2-4 °C for 1 week 71 until use. 72 2.2. Unrefined biopolymer extraction 73 Dried seaweed was homogenized using an Osterizer blender (Oster, Aravaca, Madrid, Spain) 74 with water in a 1:15 (w:v) proportion and kept for 12 h at 3 ± 2 °C. The seaweed was then 75 filtered and subjected to two consecutive extractions in water at a 1:30 (w:v) proportion, at 91 °C 76 for 2 h during the first step and 1.5 h during the second one. Each extract was centrifuged at 77 3000 rpm for 5 min (Sorvall Evolution RC Centrifuge, Thermo Fisher Scientific Inc., Landsmeer, 78 The Netherlands) and blended. The supernatant was dried in an oven (FD 240 Binder, 79 Tuttlingen, Germany) at 65.0 ± 0.8 °C and this constituted the Mastocarpus biopolymer extract, 80 which was stored at room temperature. 81 2.3. Seaweed hydrolysis 82 Dried seaweed was mixed with 4% distilled water (w/v) and subjected to enzymatic hydrolysis 83 for 3 h, using alcalase 2.4L (EC 3.4.21.14, 2.64 AU/g, Sigma-Aldrich Inc., St. Louis, MO, USA) 84 in optimal conditions for enzymatic activity (pH 8, 50 °C). The enzyme-substrate (seaweed) ratio to the reaction medium using a pH-stat (TIM 856, Radiometer Analytical, Villeurbanne Cedex, 87 France). The enzyme was inactivated by heating at 90 °C for 10 min. The hydrolysate was 88 centrifuged at 7000 g for 15 min. The supernatant was subjected to two consecutive 89 carrageenan extractions by precipitation with ethanol 1:3 (v/v) at 4 °C for 2 h. The precipitated 90 carrageenan was dried at 65 ± 0.8 °C and weighed in order to evaluate extraction yields. The 91 carrageenan-free liquid phase was centrifuged at 13000 g for 5 min. The supernatant was 92 concentrated by rotary evaporation and was subsequently subjected to five organic extractions 93 with ethyl acetate 1:5 (v/v), to remove most of the polyphenols and other compounds such as 94 pigments. After decanting, the successive aqueous phases were concentrated by rotary 95 evaporation. The concentrate was lyophilized, and this constituted the phenolic-partially-96 removed hydrolysate (H). The phenolic-containing hydrolysate (Hp) was obtained under the 97 same conditions as described above, with the exception of the removal of polyphenol 98 compounds with ethyl acetate. The Hp hydrolysate was selected for active film development. 99 2.3.1. Amino acid analysis of hydrolysates 100 The amino acid composition of the hydrolysates (H, Hp) was determined using a Biochrom 20 101 amino acid analyser (Pharmacia, Barcelona, Spain) according to the method described by 102 Alemán et al. 22 The results were expressed as number of amino acid residues per 1000 103 residues. 104 2.3.2. Angiotensin-converting enzyme (ACE) inhibition of hydrolysates 105 Reversed-phase high performance liquid chromatography (RP-HPLC) was used to determine 106 ACE-inhibitory capacity of the hydrolysates (H, Hp), according to the method described by 107 Alemán et al. 22 The IC 50 value was defined as the concentration of hydrolysate (µg/mL) required 108 to inhibit 50% of ACE activity. 109 2.4. Film preparation 110 Three film-forming solutions were prepared to obtain the following films: F-Hp0 (without the 111 addition of hydrolysate), F-Hp15 (with 15% hydrolysate) and F-Hp30 (with 30% hydrolysate). meter series 3 Star Orion with an electrode pH ROSS (Thermo Fisher Scientific Inc., 116 Landsmeer, Netherlands) was used for pH measurements (6.4-6.8). Glycerol (Panreac 117 Química S.A., Barcelona, Spain) was added at 10% (w/w) in relation to the seaweed extract 118 content. The film-forming solutions were centrifuged at 3000 rpm for 3 min to remove air 119 bubbles. Hp was then added at 15 and 30% (w/w) in relation to the seaweed extract content, 120 and was magnetically stirred for 5 minutes. The film-forming solutions were cast into petri dishes 121 and dried in an oven (FD 240 Binder, Tuttlingen, Germany) at 35.0 ± 0.8 °C for 21 h. All the 122 films were conditioned at 58.0 ± 0.2% RH and 22 ± 1 °C for 4 days prior to analysis. 123 2.5. Viscoelastic properties of film-forming solutions (FS) 124 A dynamic viscoelastic study of the film-forming solutions was carried out on a Bohlin CVO-100 125 rheometer (Bohlin Instruments Ltd., Gloucestershire, UK) using a cone-plate geometry (cone 126 angle 4°, gap 0.15 mm). A dynamic frequency sweep from 0.1 to 10 Hz took place at auto 127 stress, at a temperature of 10 °C and a target strain of 0.005%. The elastic modulus (G′; Pa) 128 and viscous modulus (G″; Pa) were plotted as functions of the frequency ramp. To characterize 129 the frequency dependence of G′ over the limited frequency range, the following power law was 130 used: 131 G′ = G 0 ′ω n 132 where G 0 ′ is the energy stored and recovered per cycle of sinusoidal shear deformation at an 133 angular frequency of 1 Hz, ω is the angular frequency and n is the power law exponent, which 134 should exhibit an ideal elastic behaviour near zero in gels. At least two determinations were 135 performed for each sample. The experimental error was less than 6% in all cases. 136 2.6. Viscosity 137 A viscosity test for film-forming solutions was performed at 25 °C in the cone-plate cell (cone 138 angle 4°, gap = 150 mm) of the Bohlin rheometer at a constant shear rate of 0.5 s -1 . The results 139 are averages of eight determinations and are expressed as Pa·s. 157 dissolved in distilled water and shaken until they were totally homogeneous. The film solutions 158 were filtered through Whatman No. 1 paper. The method used for the FRAP and ABTS assays 159 was previously described by Alemán et al. 22 Results were expressed as µmoles Fe 2+ 160 equivalents/g for FRAP and mg Vitamin C Equivalent Antioxidant Capacity (VCEAC)/g) for 161 ABTS, based on standard curves of FeSO 4 7H 2 O and vitamin C, respectively. All determinations 162 were performed at least in triplicate. 163 2.10. Folin-reactive substances content of hydrolysates and films 164 Total Folin-reactive substances content was determined according to a modified method by 165 Slinkard and Singleton with the Folin-Ciocalteu reagent. 24 An aliquot of 10 µL of sample was 166 mixed with 750 µL of distilled water and oxidized with 50 µL of Folin-Ciocalteu reagent. The 105°C for 24 h, according to A.O.A.C. (Association of Official Analytical Chemists, 1995). 26 179 Water content was expressed as a percentage of the total weight. 180 2.11.3. Protein content 181 The protein content was determined by a LECO FP-2000 nitrogen/protein analyser (Leco Corp., 182 St. Joseph, MI, USA), according to Dumas (A.O.A.C., 2005) 27 and using a nitrogen-to-protein 183 conversion factor of 6.25. 184 2.11.4. Light absorption and transparency 185 The light barrier properties and transparency of the films were calculated at least in triplicate 186 using a UV-1601 spectrophotometer (model CPS-240, Shimadzu, Kyoto, Japan) at selected 187 wavelengths from 250 to 800 nm following the method described by Pérez-Mateos et al. 25 188 The films were cut into a rectangle piece and directly placed in the spectrophotometer test cell, 189 using an empty test cell as the reference. Transparency was calculated by the equation 190 Transparency = −log(T 600 /x), where T600 is the light transmission (T) at 600 nm, and x is the 235 Software Inc., Chicago, Illinois, USA) for one-way analysis of variance. The variance 236 homogeneity was evaluated using the Levene test, or the Brown-Forsythe when variance 237 conditions were not fulfilled. Paired comparisons were made using the Bonferroni test or the 238 Tamhane test (depending on variance homogeneity), with the significance of the difference set 239 at P ≤ 0.05. 240 3. Results and Discussion 241 3.1. Extraction yield of seaweed hydrolysis 242 Carrageenan extraction yield was 28.65% (dry weight basis) and hydrolysate yields were 243 19.04% for H and 39.17% for Hp (dry weight basis); therefore total seaweed extraction yield by 258 contained 15.02 ± 0.53% of protein. Protein content was concentrated in the hydrolysates up to 259 37.86 ± 1.07% for H and 31.32 ± 0.96% for Hp. 260 The amino acid composition of H and Hp, expressed as residues per 1000 total amino acid 261 residues, is shown in Table 1. As expected, a similar amino acid profile was observed in both 262 hydrolysates. Both H and Hp showed high contents of Ser, Gly, Ala, Asp and Glu, and relatively 263 high contents of Leu, Thr, Val, Pro and Phe. The sum of the aspartic and glutamic acid contents 264 was 192 residues/1000 residues and 198 residues/1000 residues for H and Hp, respectively. 265 The high acidic amino acid content is typical of red seaweeds. 19 Nevertheless, some differences 266 between the hydrolysates were noteworthy. Some amino acids (Ser, Thr, Arg, His) were 267 concentrated in the more purified hydrolysate (H), owing to the removal of other amino acids, 268 mainly hydrophobic residues (Ala, Val, Ile, Leu, Pro, Met). These hydrophobic amino acids 269 might have been extracted during the ethyl acetate extraction, suggesting that some of them 270 could be linked to the polyphenols extracted. fully separated with ethyl acetate in the H hydrolysate. Moreover, although the Folin-Ciocalteu 304 assay is a widely used method to determine total phenolic content, additional substances can 305 react with the Folin reagent, including sugars and proteins, and should be taken into account. 40 306 The hydrolysis process would allow an improved extraction of phenolic compounds as well as 307 the release of low molecular weight peptides, 18 which contribute to enhance the antioxidant 308 properties. Hp showed higher antioxidant activity than H (1.4 times higher for reducing power 309 and 2.7 times higher for ABTS radical scavenging), probably owing to a greater presence of 310 phenolic compounds in Hp. The positive correlation between the polyphenolic content of algae 311 and their antioxidant activity has been well documented. 41-45 312 On the other hand, the hydrolysate peptide fraction can also contribute to antioxidant activity. It 313 is well known that biological activities of protein hydrolysates are related to the amino acid 314 composition, sequence, molecular weight and peptide configuration. For example, 315 phosphorylated serine and threonine are known to bind metals, 46 being more hydrophilic and 316 reactive because of their hydroxyl group. Amino acids with non-polar aliphatic groups, such as 317 alanine, leucine or proline, have high reactivity to hydrophobic PUFA radicals, while hydrogen 318 donors such as aspartic and glutamic acids are able to quench unpaired electrons or radicals by 319 supporting protons. 47 The abundance of these amino acids in the peptide sequences of 320 hydrolysates could also be responsible for their antioxidant activity. As previously mentioned, 321 the hydrophobic amino acid content was higher in Hp than in H, which might also have 322 contributed to the higher antioxidant capacity (ABTS and FRAP) of Hp compared with H. 323 Various studies have been carried out to evaluate the antioxidant potential of marine algae 324 hydrolysates. 7,8,18,48-50 However, to our knowledge, no reference has been made in previous 325 studies to the antioxidant or ACE-inhibitory activity of Mastocarpus hydrolysates. 326 Given the Folin-reactive substances content and antioxidant activity results of the hydrolysates, 69 Nevertheless, the higher hydrolysate amount in the F-Hp30 solution 428 considerably reduced the gel-forming capacity with respect to the F-Hp15 solution, with the 429 helical aggregates probably having more difficulty in being created as a result of a carrageenan-430 dilution effect. 70 431 The apparent viscosity of the film-forming solutions, measured at 25 °C and shear rate of 0.5 s -432 1 , was considerably higher in the F-Hp15 solution (14.89 ± 0.53 Pa·s) than in the F-Hp0 and F-433 Hp30 solutions (3.47 ± 0.01 Pa·s and 4.72 ± 0.15 Pa·s, respectively), strongly suggesting 434 effective interactions at the right concentration between carrageenan and other compounds 435 naturally present in the hydrolysate, presumably peptides and phenolic compounds. 436 3.3.4. Light barrier properties 437 Colour parameters, L* (lightness), a* (reddish/greenish) and b* (yellowish/bluish), are shown in 438 Table 4. All the films were quite similar, having low lightness (28-29) and slightly greenish and 439 yellowish tendencies. The F-Hp30 film exhibited the highest (P≤0.05) lightness and greenish 440 colouration, and lowest (P≤0.05) yellowish tendency. Changes in L*a*b* values, however, did 441 not correlate with increasing amounts of added hydrolysate in the film, which could be due to a 442 different degree of interactions between protein pigments and carrageenan. Comparing these 443 results with previously developed commercial κ-carrageenan films, the present M. stellatus films 444 presented considerably lower lightness and more red tendency, owing to the concomitant 445 extraction of non-carrageenan compounds. 71-73 446 In general, the films exhibited low light transmission in the UV range (250-300 nm) (0-1.12%) 447 (Figure 4), as compared to commercial κ-carrageenan films, 74 with F-Hp0 providing the least 448 efficient UV barrier. Two absorption peaks were defined in all the films in the ranges 400-450 449 nm and 600-700 nm, which might be associated with the presence of pigments, such as 450 carotenoids and chlorophyll, which absorb at 400-450 (violet-blue-green colours), and 451 phycoerythrin and phycocyanin at 600 nm (red colour). 75 In the visible range, the light 452 transmission was significantly (P≤0.05) lower in F-Hp30, especially in the wavelength range 453 between 350 and 700 nm, which might be largely due to the increase in thickness associated 454 with the hydrolysate addition, as Table 5 shows. The hydrolysate contained small molecules 455 (mainly peptides and oligosaccharides) that might have interfered in carrageenan helix 456 aggregation during the film drying process. This interference might have caused a plasticizing 457 effect with an increase in free volume that would have resulted in thicker films. 458 3.3.5. Physico-chemical properties 459 Slight variations in moisture content were observed among the three film formulations (Table 5) , 460 with F-Hp30 showing slightly higher values, which could be related to its increased thickness. 461 The protein content in the films increased significantly (P≤0.05) with the addition of increasing 462 amounts of hydrolysate (Table 5). 463 3.3.5.1. Water barrier 464 No significant differences in film water solubility were found in M. stellatus films with either 15 or 465 30% added hydrolysate (Table 5). A similar finding was reported earlier in gelatin films 466 incorporating different percentages of gelatin hydrolysate. 76 Although the solubility values were 467 not high, the films totally lost their original structure, becoming a very viscous solution with 468 gelling tendency at low temperatures. Solubility was similar to previous results obtained in 469 commercial carrageenan films. 73 3.3.5.2. Mechanical properties 484 F-Hp0 had the significantly highest (P≤0.05) tensile strength (TS) (Table 5), which was lowest in 485 F-Hp30. The opposite behaviour was found regarding the elongation at break (EAB) values, 486 confirming the hydrolysate-induced plasticizing effect in the film. The TS and EAB values in the 487 three M. stellatus films studied were, respectively, higher and lower than the results reported 488 with commercial κ-carrageenan or ι-carrageenan films, 73,79 suggesting a reinforcement effect 489 caused by the presence of other non-carrageenan components. As far as Young's modulus (Y) 490 is concerned (Table 5), the highest stiffness also corresponded to F-Hp0 (P≤0.05), decreasing 491 with increasing amount of Hp. The small molecules (mainly peptides and oligosaccharides) that 492 form part of the hydrolysate have been proved to act as film plasticizers by preventing 493 carrageenan helix associations and increasing the molecular mobility of polymer chains, which 494 in the case of F-Hp30 was favoured by the increased water plasticizing effect. Similarly, 495 Salgado et al. also observed a reduction in TS and Y and an increase in EAB in protein films 496 with added hydrolysate, which, in view of the lack of film moisture increase, was attributed to 497 interferences in protein cross-linking. 21
doi:10.1039/c3fo60310e pmid:24337179 fatcat:biscv4dyb5adjbym457hhejgxa