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The Effect of Extraction Conditions on Chemical and Thermal Characteristics of Kappa-Carrageenan Extracted from Hypnea bryoides

The Effect of Extraction Conditions on Chemical and Thermal Characteristics of Kappa-Carrageenan... Hindawi Journal of Marine Biology Volume 2019, Article ID 5183261, 10 pages https://doi.org/10.1155/2019/5183261 Research Article The Effect of Extraction Conditions on Chemical and Thermal Characteristics of Kappa-Carrageenan Extracted from Hypnea bryoides 1,2 2 2 Zainab Mohammed Al-Nahdi, Ahmed Al-Alawi , and Insaaf Al-Marhobi General Directorate of Education in Dhofar, Ministry of Education, Salalah, Oman Department of Food Science and Nutrition, College of Agricultural and Marine Sciences, Sultan Qaboos University, Muscat, Oman Correspondence should be addressed to Ahmed Al-Alawi; ahmed543@squ.edu.om Received 3 April 2019; Revised 10 June 2019; Accepted 11 June 2019; Published 1 August 2019 Academic Editor: Robert A. Patzner Copyright © 2019 Zainab Mohammed Al-Nahdi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Hypnea bryoides collected from the Arabian Sea on the southern coast of Oman was investigated for𝜅 -carrageenan optimal extraction conditions. eTh eeff cts of different conditions of alkali treatment (4, 6, and 8% w/v NaOH), temperatures (70, 75, and 80 C), and time (2, 2.75, 3.5 hours) on carrageenan yield and chemical and thermal properties were evaluated. Yield was significantly aec ff ted by alkaline concentration and temperature, with highest value of 26.74 ± 5.01%. Molecular weights of the extracted carrageenan were significantly reduced by increased temperatures and ranged from 5.95 ± 0.49× 10 Da to 13.90± 0.14× 10 . FTIR showed that samples under all extraction conditions were similar and confirmed the presence of 𝜅 -carrageenan with no traces of𝜇 -precursor. Sulfate content was also significantly reduced by alkaline concentration (from 4% to 6%) and ranged from 7.62± 5.52% to 17.02± 0.14. erm Th al properties showed more sensitivity towards temperature and alkaline strength parameter than time. In addition, melting and gelling temperatures were significantly correlated with the molecular weight, but not sulfate content. In conclusion, mild extraction conditions were found to be more efficient in introducing the intended structural modification while getting the highest yield and quality. 1. Introduction texturing, thickening, suspending, or stabilizing agents in a variety of industrial applications ranging from food products, Among marine macroorganisms, seaweed gained consider- pharmaceutics, and cosmetics to experimental medicine [5– able significance due to its utilizations as a rich raw material 7]. for extraction of various valuable materials, for instance, Rhodophyta of different species, such as Gigartina, Chon- polyphenols, organic pigments, proteins, unsaturated fatty drus crispus, Eucheuma, and Hypnea, have been revealed acids, and polysaccharides [1, 2]. Commercially, the most to be good source of carrageenans [8] and based on the significant seaweed polysaccharide is carrageenan followed source, different types of carrageenans can be acquired. From by agar and alginate. The global market value of carrageenan the view of chemical structure, carrageenans are consisted has improved from US$ 527 million in 2009 to US$ 626 of alternating (1󳨀→ 3)-linked 𝛽 -D-galactose and (1󳨀→ 4)- million in the year 2015 [3]. linked𝛼 -D-galactose, where (1󳨀→ 3)-linked𝛽 -D-galactoses Carrageenans, which are a family of linear sulfated are incompletely sulfated at specific positions of C2 and/or C4, C3, and C6; and the (1󳨀→ 4)-linked𝛼 -D-galactose units polysaccharides, are found and extracted from the cell wall of certain species of red seaweeds (Rhodophyta) [4]. These are infrequently 3,6-anhydrated (3,6AG) [9]. The differences natural polymers are capable of forming thermoreversible in the content and position of sulfate group in galactose gels or viscous solutions when added in small concentration units resulted in different carrageenan structures, which to salt solutions. Therefore, they are commonly used as as a result differ in the rheological features of solutions 2 Journal of Marine Biology and gels. To date, three types of carrageenans have been which then must be neutralized, thereby raising production reported to have commercial significance: kappa ( 𝜅 ), iota costs [18]. (𝜄 ), and lambda (𝜆 ) [5]. The major variation among kappa, Among the recognized seaweeds in Omani coasts, Hyp- nea bryoides was found to have potential to be a good source iota, and lambda carrageenan at the molecular level is the amount along with position of the sulfate ester groups which for carrageenan. Al-Alawi et al. [25] found that Hypnea produces different properties [10]. 𝜆 -carrageenan contains no bryoides gives yield of about 30-32% (d.b.) carrageenan under alkaline condition extraction (6% w/v NaOH, 80-85 hydrophobic 3,6AG bridge but has three hydrophilic sulfate C, 4 h). ester groups, which makes this carrageenan readily water Although the gel gave good property when it was compared soluble under most conditions. 𝜅 -carrageenan possesses a with a reference carrageenan, purity test showed that the 3,6AG bridge and no more than one sulfate ester group, yield contained only 32-36% carrageenan and 55-57% was making this carrageenan less hydrophilic and less soluble salt. No other conditions were tested to see the effect of in water.𝜄 -carrageenan is in-between, with a 3,6AG bridge different conditions on yield and quality. Therefore, the aim and two sulfate ester groups. Accordingly, characterization of this study was to investigate the effect of various conditions of carrageenan is the most crucial for quality control and to (alkali strength, temperature, and duration time) on yield and build up new application supported by their unique intrinsic characteristics (chemical and thermal characteristics) of𝜅 - properties [11]. carrageenan obtained from Hypnea bryoides. As there is little The yield and quality of the extracted carrageenan con- available information on this seaweed in the literature, this firm the commercial significance of both carrageenan and study will help in determining the best possible quality of the mother plant to the industry. However, both yield and carrageenan from Hypnea bryoides which will influence any quality are very much influenced by several factors such future marketing plans. as environmental (salinity, pH, temperatures, light intensity, and water movement), physiological (nutrient uptake, growth 2. Materials and Methods rate, tissue age, stress tolerances, and defences), and extrac- tion conditions [11–16]. The major common and essential Standards, chemicals, and solvents used in this study were step in carrageenan extraction procedures is alkali treatment obtained from Sigma-Aldrich Co. Ltd., and all were of at raised temperature for specific time. This step is crucial analytical grade unless otherwise specified. All solutions were to verify transformation of the biological precursors to the prepared with deionized water. All samples were analyzed at commercial grade carrageenan, (𝜇 -and v-carrageenan into least twice. 𝜅 -and i-carrageenan, respectively) [11]. The treatment with alkali induces desulfation of the polysaccharide, causing .. Sample Collection. Based on our previous work [25], formation of a 3,6AG bridge (between C3 and C6 in the 4- samples of the red seaweed species Hypnea bryoides were har- linked-𝛼 -L-galactose units) which leads to increase in the vested by local specialists from the southern coast of Oman gel strength [17]. The increase in gel strength is attributed to ∘ 󸀠 ∘ 󸀠 from Mirbat city (16 59 28.7 N, 54 41 27.7 E) in November the fact that presence of sulfate at C6 of the𝛼 -L-galactose 2015. The samples were transferred to the laboratory at Sultan residues in the precursor units acts as a “Kink” to prevent Qaboos University in cold boxes via air, washed with running the double helix from forming during gel formation. Closure fresh water, cleaned by hands from foreign matter upon of the ring to form the 3,6AG and elimination of the C- arrival, and then sun-dried under the shade for three days. 6 sulfate group makes the chain straighten and leads to The dried samples were then packed in plastic bags and stored great regularity in the polymer, resulting in enhancing gel in a refrigerator at 4 C until further analysis. strength due to increased capability of forming a double helix [17]. The severity of alkaline treatment (concentration and temperature) as well as duration aec ff ts the yield and quality .. Extraction Method. The alkaline treatment extraction [18] as inadequate treatment will not cause the required was performed according to the method described by Al- transformation. Moreover, existence of substantial amounts Nahdi et al. [26]. The method involved rehydrating twenty of the natural precursor units in commercial carrageenan grams of the sun-dried samples overnight in 1L of deion- preparations has a critical negative effect on the functional ized water and then depigmentation in 100 ml of ace- (e.g., gelling) properties [19]. tone/methanol (1:1) mixture. The samples were then treated Although the general steps in the extraction process are with alkaline solution (1.5L) of variable strengths (4, 6, and known, the extraction variables do differ [20] as seaweeds 8% w/v of aqueous NaOH solution) and heated at different differ in their composition and conditions and stage of temperatures (70, 75, and 80 C) for different intervals (2, growth [12–16]. Therefore, alkali treatment of each algal 2.75, and 3.5 hours). Occasional and gentle stirring was species must be developed and variables like temperature, employed after the rfi st hour of heating. Then, the algal alkali concentration, and extraction time must be optimized insoluble materials were collected from the solution using to induce as much desulfation as possible, while still avoiding double layers of cheesecloth (coarse lfi tration) and washed the yield losses due to degradation and leaching caused by the three times with deionized water. The retained algal materials treatment [21]. Such loss is frequently associated with elevated were again redissolved in 500 ml of deionized water, pH extraction temperatures and prolonged extraction time [22– adjusted to 7 using 6N HCl, and then heated to 90 Cfor 1h 24]. Furthermore, industrial production of alkaline-treated- with continuous gentle stirring. The insoluble algal materials carrageenan produces considerable pollution in the outflows were separated and discarded from the hot mixture using 󸀠󸀠 󸀠󸀠 Journal of Marine Biology 3 double layers of cheesecloth and Whatman GF/D, GF/C lfi ter Linear Model (GLM) for univariate analysis was employed to paper. Then, the pH of the collected liquid was checked and determine the main and interactive effects of alkali treatment adjusted to 7 and kept overnight (14h) at 4 Cin a refrigerator conditions on carrageenan yield, molecular weight, and to form gel. Volume of the gel was reduced to one-half sulfate content. When significant differences were found, a by evaporation in an air forced oven at 60 Cfor 12h. The multiple post hoc Duncan’s Multiple Range Test was applied. concentrate was dialyzed against deionized water for 72 h Pvalues<0.05 were considered as statistically signica fi nt. using dialysis membrane with 12000-14000 Daltons cut-off (Medicell International Ltd, London) and dried afterwards for 18 h at 40 Cin vacuumoven. 3. Results and Discussion Finally, the dried extracts were milled and then stored in a sealed plastic container at room temperature until further The yield and chemical properties of alkali-treated car- analysis. Two replicates were conducted for each combination rageenan extracted from Hypnea bryoides are summarized in of alkali concentration, temperature, and treatment duration. Table 1. The results demonstrate that the total yield value obtained from different alkali treatments extraction conditions ranged .. FTIR (Fourier Transform Infrared) Analysis. The FTIR between 6.59± 0.09% and 26.74± 5.01%, obtained by the analysis was performed according to the method described treatment with 8% NaOH, 3.5h, 80 Cand 6% NaOH,2.75h, by Al-Alawi et al. [25] using Magna 560 FTIR spectrometer 70 C, respectively. (Thermo Nicolet, USA) equipped with ZeSn ATR cell and In general, some of the carrageenan yields obtained in this DTGS detector. The IR spectrum was collected by averaging −1 study were in the same range of those extracted in aqueous 128 scans at resolutions of 4 cm . media obtained by Yermak et al. [29] from C. pinnulatus (20.5%-18.2), Reis et al. [30] from H. musciformis (48%- .. Sulfate Content Determination. Sulfate content was 21%), and Webber et al. [31] from K. alvarezii (35.8%-18%). determined using the method described by Dodgson [27]. However, all the abovementioned studies were performed In brief the method involved mixing 1% w/v carrageenan without alkaline treatment step and also without dialysis step, solution (0.2 ml) with 4% (w/v) trichloroacetic acid (3.8 ml) which raise questions on purity and quality of the reported and 1 ml of the BaCl -gelatin reagent. The mixture was yield. Therefore, the comparison based on the absolute yield allowed to stand for 15 min at room temperature for color is misleading. development and then absorbance was recorded at 360 nm. For the alkaline treated carrageenan, yields found in this A calibration curve was prepared with solutions of K SO 2 4 study were in the range of those reported for other species 2− containing 20-200𝜇 gof SO ions. such as 26.2% from H. porphyroides (Indian Ocean) [32], 29.1% from H. durvillei (Philippines) [33], 20–32% from K. .. Molecular Weight Analysis. Molecular weight determi- alvarezii (Brazil) [34, 35], 15% from H. durvillei (Madagascar) nation experiment was conducted as described by Al-Nahdi [36], 26.8% from H. floresii (Mexico) ´ [37], and 25.8-37.2% et al. [26]. The analysis was carried out on Agilent 1100 from E. spinosum (Indonesia) [38]. (Agilent, USA) instrument equipped with differential refrac- On the other hand, the values from the present study tive index detector (Agilent RID 1100). The separation was are slightly lower compared to results obtained from other achieved using Waters Ultrahydrogel Linear column (Waters species such as G. skottsbergii, S. crispata, and C. crispus, G. Corporation) and the mobile phase 0.1M NaCl solution with atropurpurea (31-44%) [39], E. isiforme (43.5%, 33.8%) [14], isocratic elution. A sample size of 20𝜇 l of 0.1% w/v of and K. alvarezii (Doty) (30% to 39%) [28]. carrageenan solution was injected in the system at a flow rate The differences in yield between different species is very of 1 ml/min. The temperature of the column compartment much understood due to the morphology differences as and the detector o fl w cell were maintained constant at 40 C. explained above; moreover, difference in the same species grown in different geographical area has been also noticed .. Differential Scanning Calorimeter Measurements. The and it was attributed to differences in the harvest time, growth procedures for Differential Scanning Calorimetry measure- conditions (salinity, deepness, and nutrients), time of growth, ments (DSC) were similar to those used previously by Al- environmental conditions (wind speed, precipitation, cloud Alawi et al. [25] with minor modifications. The thermal cover, insulation, water and air temperature, and length and analysis measurements were carried out using a modulated period of waves), and extraction process and parameters as differential scanning calorimeter (model Q1000, TA Instru- reported by several studies [11–16, 32]. ments, USA). The tested gels were made up of 1.5% (w/v) of In this study, the yield that was obtained by the condition the extracted carrageenan and ionic strength of 30 mM KCl in (6% NaOH, 3.5h, and 80 C) is very low (12.69%) compared an aqueous environment. The samples were scanned during a with the previously reported yield (33.2%) for the same plant ∘ ∘ heating/cooling cycle (25-90-25 C) at a rate of 5 C/min, with collected from the same geographical area and extracted an empty pan as a reference. under nearly the same conditions (6% NaOH, 4 h, and 80- 85 C) [25]. The reason lays behind the fact that in the previous . . Statistical Analysis. The statistical analysis work was study no dialysis step was performed as the case in the performed with the SPSS software version 11.5. The obtained current study. Aeft r correcting for the dialysis step in the data were presented as mean± standard deviation. General previous study (subtracting the salt from the yield), the yield 4 Journal of Marine Biology Table 1: Properties of Carrageenan extracted from Hypnea bryoides under different alkali pre-treatment conditions. Treatment Yield M Sulfate NaOH Time Temp. 1 1 (% w/w) ± SD (Dalton)± SD (% w/w)± SD (w/v) (h) ( C) 70 21.72.± 3.61 (13.15± 0.64)× 10 07.62± 5.52 75 23.44± 0.01 (11.50± 1.27)× 10 17.42± 2.97 80 14.86± 3.72 (07.00± 7.20)× 10 19.22± 0.14 70 17.62± 3.17 (12.85± 1.30)× 10 15.12± 3.68 4% 2.75 75 18.95± 2.59 (11.30± 0.57)× 10 13.82± 9.19 80 13.24± 3.85 (07.50± 6.51)× 10 20.52± 0.00 70 21.90± 0.05 (13.10± 0.14)× 10 15.92± 0.00 3.5 75 15.12± 2.60 (12.20± 0.00)× 10 18.82± 3.82 80 09.37± 2.37 (12.40± 0.00)× 10 21.22± 1.27 70 23.06± 5.06 (11.80± 0.00)× 10 11.82± 4.38 75 11.87± 1.53 (11.70± 0.71)× 10 16.42± 0.99 80 15.21± 3.62 (11.30± 0.00)× 10 15.82± 3.25 70 16.95± 7.39 (10.20± 0.14)× 10 14.02± 7.78 6% 2.75 75 10.15± 3.73 (10.00± 1.84)× 10 16.72± 0.28 80 12.16± 3.99 (06.65± 5.44)× 10 09.82± 2.40 70 26.74± 5.01 (13.90± 0.14)× 10 17.02± 0.14 3.5 75 11.06± 2.62 (10.90± 1.41)× 10 16.52± 2.26 80 12.69± 6.37 (06.10± 6.93)× 10 15.42± 2.69 70 11.58± 1.79 (12.30± 1.13)× 10 19.62± 0.99 75 11.30± 0.74 (12.35± 0.49)× 10 25.52± 1.70 80 10.24± 1.07 (07.25± 6.15)× 10 21.52± 0.00 70 10.85± 1.97 (07.10± 7.07)× 10 20.92± 1.98 8% 2.75 75 11.06± 0.96 (05.95± 0.49)× 10 25.32± 2.26 80 08.22± 0.34 (07.90± 5.23)× 10 24.72± 1.41 70 17.38± 2.39 (08.05± 7.14)× 10 23.92± 4.81 3.5 75 07.58± 0.77 (11.25± 0.21)× 10 28.12± 0.57 80 06.59± 0.09 (06.75± 6.58)× 10 21.72± 0.85 Dry basis. came down to 10% which is close to the yield extracted The molecular weight (M ) of the extracted carrageenan in this current study under similar conditions and very is shown in Table 1. much lower than the yield (26.74± 5.01% or 23.44± 0.01%) The results demonstrate that the M value obtained obtained at the condition of 6% NaOH, 3.5h, 70 Cand 4%, from different alkali treatments extraction conditions ranged ∘ 5 5 2h, 75 C, respectively. This finding gives significance to the between (5.95± 0.49)× 10 and (13.90± 0.14)× 10 Da at current study to find the optimum conditions for extraction the conditions 8% NaOH, 2.75h, 75 Cand 6% NaOH,2.75h, to maximize the yield with the highest quality. On this 70 C, respectively. The results indicated that the degradation regard, it has been found in this study that carrageenan yield rate was elevated at higher temperatures as lower molecular significantly reduced with the increase in alkali treatment weight carrageenan was obtained (less than 7.00× 10 Da at strength and temperature. Furthermore, statistical analysis 80 C). The effect of NaOH concentration and time on degra- showed that the time parameter insignificantly influenced dation was noticed in this study, but it was not significant carrageenan yield (Figure 1). The interaction between inde- (p> 0.05) (Figure 2). Furthermore, the interaction between pendent variables was found to be nonsignificant. independent variables was also not significant. Studying the This finding was not a surprise, but in contrary it degradation rate during extraction and the possible causes are was expected due to partial degradation of polysaccharide of utmost importance, since functionality of carrageenans in chains by alkali moieties, since alkaline extraction operation most food applications depends on molecular weight and is inevitably involves some degradation of the polysaccharide largely lost if it is below 1.00× 10 Da [41] which was not seen which accelerates at elevated temperatures [4, 11, 31, 37, 40]. in any of the treatments used. Furthermore, degraded, low Furthermore, further increase in the alkali concentration molecular carrageenan was reported to cause inflammation could lead to a sharp decrease in yield [4]. in the colon in rodents, which resembles ulcerative colitis, Journal of Marine Biology 5 0 6 1.5×10 4 6 8 NaOH concentration (% w/v) 4 6 8 NaOH concentration (% w/v) 0 6 1.5×10 10 A 2 2.75 3.5 Time (h) 0 6 2.00 2.75 3.50 1.5×10 Time (h) B 0 C 70 75 80 Temperature ( C) Figure 2: Eec ff ts of alkali treatment on M of carrageenan extracted from H. bryoides (means with the same letter are not significantly dieff rent ( 𝛼 = 0.05)). 70 75 80 Temperature ( C) Figure 1: Eec ff ts of alkali treatment on yield of carrageenan extracted from H. bryoides (means with the same letter are not time compared to this study. Moreover, molecular weights significantly different ( 𝛼 = 0.05)). obtained in this study are in the range of results reported by Jupp [44] from H. bryoides (6.12× 10 Da), Hilliou et al. 5 5 [40] from Mastocarpus stellatus (22× 10 to 4× 10 Da), and Distantina et al. [11] from E. cottonii (10.76× 10 to 5.48× an inflammatory bowel disease [42]. Therefore, low molec- 10 Da). ular weight carrageenans were classiefi d by the European The chemical analyses were further supported by the FT- Scientific Committee [43] and the International Agency for IR analysis. The collected spectra indicated presence of sulfate Research on Cancer as a “possible human carcinogen.” In −1 esters (S=O) with absorption band in the 1255 cm region, this study, any very low molecular weight breakdowns were −1 3,6-anhydro-D-galactose at 925 cm ,and D-galactose-4- eliminated through the dialysis step. −1 Previous study on the same plant [25] gave molecular sulfate at 845 cm . On the other hand, peaks at 830, 820, and 5 −1 weight result of 4.1× 10 Da, which is lower than the current 805 cm , which are corresponding to D-galactose-2-sulfate results, probably due to using higher temperatures and longer (D2S), galactose and D-galactose-6-sulfate (G/D6S), and Yield (%w/w d.b.) Yield (%w/w d.b.) Yield (%w/w d.b.) Molecular weight (Dalton) Molecular weight (Dalton) Molecular weight (Dalton) 6 Journal of Marine Biology Table 2: Peaks assigned to dieff rent carrageenan types. Extracted −1 Wavenumber (cm ) Assignment Found in Carrageenans∗ Carrageenan from H. bryoides −3 1210-1260 Sulfate ester (O-SO )(S) k, i,𝜆 ,𝜇 ,v,𝜃 ,𝜉 Present 928-933, 1070 (shoulder) 3,6-anhydro-D-galactose (DA) k,𝛽 Present 840-850 D-galactose-4-sulfate (G4S) k, i,𝜇 ,v Present 830 D-galactose-2-sulfate (G2S) 𝜆 ,𝜃 ,𝜉 Not found 820, 825 (shoulder) D-galactose-2,6-disulfate (D2S,6S) 𝜆 ,v Not found 810-820, 867 (shoulder) D-galactose-6-sulfate (D6S) 𝜇 Not found 800-805, 905 (shoulder) 3,6-anhydro-D-galactose-2-sulfate (DA2S) i,𝜃 Not found ∗[28]. the mildest extraction conditions (at NaOH concentration of 4%, 2 h, and 70 C) were efficient in modifying the biopolymer chemistry and there is no need for further treatments. This result suggests a main advantage of the proposed procedure which will lead to reducing consumption of solvents and extraction time for industry production. Sulfate is an integral component found in𝜅 -carrageenan used for ionic regulation in the parent plants [46]. Table 1 a presents the effect of different treatment extraction condi- tions on sulfate content. Based on these results, the sulfate percentage of different alkali extraction conditions ranged between 7.62± 5.52% and 28.12± 0.57% at the conditions of ∘ ∘ 4% NaOH, 2 h, 70 Cand 8% NaOH, 3.5h, 75 C, respectively. 1,400 1,300 1,200 1,100 1,000 900 800 700 The standard specifications for carrageenans sulfate content -1 Wavenumber (cm ) are in the range of 15-40% [47]. Figure 3: Infrared spectra of carrageenan extracted from H. These values of H. bryoides are in line with those bryoides at 4% NaOH, 2 h, and 70 C condition (a) compared to determined from alkaline treatment analysis available in kappa (b), iota (c), and kappa/lambda (d) commercial carrageenans. the literature. Hayashi et al. [34, 35] results ranged from 23.08 to 33.48% for carrageenan extracted from K. alvarezii strains from coast of Sao ˜ Paulo state, Brazil. In addition, E. 3,6-anhydro- D-galactose-2-sulfate [11, 31, 37] were absent. isiforme from Nicaragua and E. isiforme from Yucatan [14] −1 were between 26.3% and 19.6%, respectively, E. cottonii from The 805 cm band is characteristic and distinctive of i- Indonesia ranged between 11.45 and 16.15% [11], and H. floresii carrageenan [43] (Figure 3). from Yucatan ´ Peninsula contained 26.8% [37]. The observed absorption bands in all spectra were con- firmatory of 𝜅 -carrageenan. Table 2 gives the major peaks The role of the extraction parameters on sulfate content related to the major functional groups existing on the differ- is illustrated in Figure 4. It is clear that NaOH concentration ent types of carrageenans [45]. Results obtained from raw H. and temperature have significantly aec ff ted sulfate content; in −1 bryoides plant [25] revealed bands at 872 and 842 cm which addition, NaOH concentration/temperature interaction (not is assigned to the presence of C-O-SO group on C6 and C- shown) was also found to have significant effect. With respect O-SO on C4 of galactose, respectively. to time, there is a noticeable trend where the increasing of time caused the values of sulfate content to increase; however This implies existence of 𝜇 -carrageenan in the raw sea- weed, which is𝜅 -carrageenan precursor. The nongelling 𝜇 - this was not significant. carrageenan is the natural precursor that is present in seaweed The results indicated a reduction in sulfate content as that contains𝜅 -carrageenan. The 3,6AG bridges are formed NaOH concentration increased from 4% to 6% w/v. From by the removal of sulfate group from the C-6 sulfate ester literature, there are many reports on decreasing level of of the precursor and formation of the 3,6AG bridge [45]. In sulfate with increasing concentration of alkaline used for −1 our case, the lack of 872 cm signal band indicates the total carrageenan extraction [11]. This is due to the reality that conversion of this precursor to kappa form due to the alkaline formation of 3,6AG bond involves release of sulfate groups; conditions. therefore after alkali treatment the sulfate content should be The almost identical FTIR spectra demonstrate that the lower. However, the results of the current study do not totally biopolymers extracted from H. bryoides are essentially made tfi in the inverse relationship mentioned above. This is maybe −1 due to the fact that formation of 3,6AG bonds was achieved of𝜅 -carrageenan (bands at 925 and 845 cm )withno𝜇 - precursor. Hence, it is evident from the FTIR spectra that at the mildest conditions used in this study as it was evident Absorbance Journal of Marine Biology 7 alkali treatment (extraction with 0.3 M NaOH, during 4 h, at 90 C) was found to have no effect on the sulfate content of carrageenan extracted from H. durvillei [36]. According to the results obtained by DSC measurements, melting and gelling temperatures for the carrageenan gel extracted from H. bryoides were in the ranges 47.10± 7.01- ∘ ∘ 55.91± 2.44 C and 30.25 ± 1.07-35.65 ± 4.73 C(Table 2), respectively. The results of melting temperatures were found to be significantly influenced (p < 0.05) by the alkaline concentration (Figure 5(a)) and the interaction of NaOH concentration and time parameters (highest melting temper- ature was at 4% NaOH concentration and 2.75h), whereas the gelling temperatures were significantly influenced by 4 68 temperature parameter (Figure 5(b)). NaOH concentration (% w/v) The finding in this study is consolidating what was reported elsewhere [31] where it was reported that gel thermal properties do not seem to be aec ff ted much by the extraction time compared to temperature and alkaline strength parame- ters. Hence extraction time is not the parameter of choice for modifying the end-product chemical structure. However, the results reported in this study are lower somewhat than those reported elsewhere. For instance, Andrade et al. [50] found that the melting temperatures of gels prepared using 𝜅 -carrageenan polymers extracted from H. musciformis were in the range 74-75 C, and gelation temperature was around 55 C. Additionally,𝜅 -carrageenan extracted from different strains studied by Sahu et al. [28] had melting temperatures that ranged between 70 and 77 C 2 2.75 3.5 and gelling temperatures that ranged between 48 and 54 C. Time (h) Furthermore, the results reported by Al-Alawi et al. [25] on H. bryoides𝜅 -carrageenan gel indicated melting at a midpoint temperature of about 70.9 C. The latter example was of the same seaweed species extract, done under equivalent salt concentration and with same concentration of𝜅 -carrageenan solution. However, the extraction parameters were differ- ent (6% NaOH, 4 h, 80-85 C) and the sample contained higher concentration of salt (55% of the extract was salt, because no dialysis step was performed). Sen and Erboz [51] mentioned that the addition of salt to 𝜅 -carrageenan effectively facilitates the physical gelation. The effectiveness of salts in influencing the phase transition temperatures, gel strength, and the binding state of the strongest junction zones has been shown to play important roles in controlling the 70 75 80 ∘ viscoelasticity, gelation rate, and syneresis [52]. Furthermore, Temperature ( C) in the current study, the samples’ ion content, as determined Figure 4: Eec ff ts of alkali treatment on sulfate content of car- by Inductively Coupled Plasma (ICP) Emission Spectroscopy rageenan extracted from H. bryoides (means with the same letter are (data not shown), was used to adjust the amount needed not significantly different ( 𝛼 = 0.05)). for the proper concentration of the polymer. Therefore, comparison between different studies without standardizing all test parameters such as ionic strength is not possible. In from FTIR spectra. Similar findings were reported recently the current study no attempts were taken to produce gel with by Moses et al. [48], where the sulfate content increased with different salt concentrations. the increase in alkaline concentration which was attributed to Generally, the alkali pretreatment is performed essentially the removal of protein (due to alkaline hydrolysis) and water to improve the gelling properties through lowering sulphate groups in the structure. However, the same treatment may soluble low molecular weight compounds which resulted in concentrating the extract. Other authors also obtained differ- negatively aec ff t the quality features if it is not done properly. ent trends than those found earlier. For instance, carrageenan This is related to the degradation of the polysaccharide aer ft alkali modification which is corroborated by drastic extracted from H. musciformis by NaOH (0.1 N) contained sulfate content in the range of 44.1% [49]. On the contrary, decrease in molecular weight as it was explained above. Sulfate Content (% d.b.) Sulfate Content (% d.b.) Sulfate Content (% d.b.) 8 Journal of Marine Biology B A A A A A A A B A A A 20 20 4 68 2 2.75 3.5 70 75 80 4 68 2 2.75 3.5 70 75 80 ∘ ∘ NaOH (%) Time (h) Temp. ( C) NaOH (%) Time (h) Temp. ( C) (a) (b) Figure 5: Eec ff t of different parameters of alkaline pretreatment on carrageenan melting (a) and gelling temperatures (b). The effect of alkaline treatment on the melting and gelation the intermediate duration time (2h). Nevertheless, higher𝜅 - temperatures of the gel prepared from the extracted 𝜅 - carrageenan gels properties observed in the present study carrageenan as a factor of molecular weight and sulfate were at higher NaOH concentration (8%) which interacted content was tested using Pearson’s correlation analysis. A with lower duration times. Interestingly, comparable results good agreement of significant (P < 0.05) direct relation- can be obtained by lower NaOH concentration (4%). How- ship was found between molecular weight with melting ever, in this case, duration times may increase and tem- (0.46) and gelling temperatures (0.44). On the other hand, perature must be elevated to the maximum. These polysac- weak insignificant correlation was found between sulfate charides, on the other hand, would also be of potential content and melting (0.05) and gelling temperatures (- utility in the applications demanding low gelation or textural 0.19). properties, e.g., in personal care or related domains. These results indicate that gel thermal properties do Moreover, the present study shows promising commercial not seem to be aec ff ted by the extraction time compared potential for H. bryoides plant as a source for𝜅 -carrageenan. to temperature parameter. Similar results were reported by The results obtained from this study, however, may not be Webber et al. [31] who concluded that extraction time is applicable to other Hypnea species and other carrageeno- not the parameter of choice for tuning the end-product phytes due to possible variation of seaweed reaction to the chemical structure of carrageenan and longer extraction treatment. u Th s, optimization of the alkali treatment to other times are only preferred to increase the yield in extracted carrageenan producing seaweeds should be investigated. biopolymers structure. Therefore, extraction temperature, or equally pH, is deemed more efficient parameter in modifying Data Availability the biopolymer chemistry. All data used to support the n fi dings of this study are included within the article. Only data of mineral analysis was not 4. Conclusion provided, but it can be released upon request; however, the The current study investigated the effect of different authors feel it is not of great importance. extraction conditions on the different characteristics of 𝜅 -carrageenan and the optimal conditions that can be Conflicts of Interest applied for carrageenan extraction from H. bryoides grown in Omani coasts to avoid excessive processing (reagents, The authors declare that they have no conflicts of interest. energy, and time) that would result in degradation of carrageenan molecule and compromise its quality and yield. The recommended conditions based on the above discussion Acknowledgments that resulted in a satisfactory yield, M ,and sulfate content were found to be 6% NaOH for 3.5 hours at 70 C. The FTIR Zainab Al-Nahdi would like to thank Sultan Qaboos spectra showed presence of𝜅 -carrageenan, with no extent, University for providing scholarship for her PhD stud- or minor quantities of𝜄 -or𝜇 -carrageenans in all extracts, ies. In addition, financial support provided by Sultan which verify the effectiveness of the mild parameters for total Qaboos University under the strategic research projects conversion achievement. (SR/AGR/FOOD/11/01 and IG/AGR/FOOD/19/01) is greatly In addition, 𝜅 -carrageenan establishing higher melting appreciated and acknowledged. The valuable statistical assis- and gelling temperatures was satisfied at lower NaOH con- tance provided by Prof. Ali Mahdi Kadhim and Dr. Anesh centrations (4%) and lower heating temperature (70 C) with Govender is highly appreciated. Carrageenan Melting Temp. ( C) Carrageenan Gelling Temp. 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The Effect of Extraction Conditions on Chemical and Thermal Characteristics of Kappa-Carrageenan Extracted from Hypnea bryoides

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Copyright © 2019 Zainab Mohammed Al-Nahdi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Hindawi Journal of Marine Biology Volume 2019, Article ID 5183261, 10 pages https://doi.org/10.1155/2019/5183261 Research Article The Effect of Extraction Conditions on Chemical and Thermal Characteristics of Kappa-Carrageenan Extracted from Hypnea bryoides 1,2 2 2 Zainab Mohammed Al-Nahdi, Ahmed Al-Alawi , and Insaaf Al-Marhobi General Directorate of Education in Dhofar, Ministry of Education, Salalah, Oman Department of Food Science and Nutrition, College of Agricultural and Marine Sciences, Sultan Qaboos University, Muscat, Oman Correspondence should be addressed to Ahmed Al-Alawi; ahmed543@squ.edu.om Received 3 April 2019; Revised 10 June 2019; Accepted 11 June 2019; Published 1 August 2019 Academic Editor: Robert A. Patzner Copyright © 2019 Zainab Mohammed Al-Nahdi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Hypnea bryoides collected from the Arabian Sea on the southern coast of Oman was investigated for𝜅 -carrageenan optimal extraction conditions. eTh eeff cts of different conditions of alkali treatment (4, 6, and 8% w/v NaOH), temperatures (70, 75, and 80 C), and time (2, 2.75, 3.5 hours) on carrageenan yield and chemical and thermal properties were evaluated. Yield was significantly aec ff ted by alkaline concentration and temperature, with highest value of 26.74 ± 5.01%. Molecular weights of the extracted carrageenan were significantly reduced by increased temperatures and ranged from 5.95 ± 0.49× 10 Da to 13.90± 0.14× 10 . FTIR showed that samples under all extraction conditions were similar and confirmed the presence of 𝜅 -carrageenan with no traces of𝜇 -precursor. Sulfate content was also significantly reduced by alkaline concentration (from 4% to 6%) and ranged from 7.62± 5.52% to 17.02± 0.14. erm Th al properties showed more sensitivity towards temperature and alkaline strength parameter than time. In addition, melting and gelling temperatures were significantly correlated with the molecular weight, but not sulfate content. In conclusion, mild extraction conditions were found to be more efficient in introducing the intended structural modification while getting the highest yield and quality. 1. Introduction texturing, thickening, suspending, or stabilizing agents in a variety of industrial applications ranging from food products, Among marine macroorganisms, seaweed gained consider- pharmaceutics, and cosmetics to experimental medicine [5– able significance due to its utilizations as a rich raw material 7]. for extraction of various valuable materials, for instance, Rhodophyta of different species, such as Gigartina, Chon- polyphenols, organic pigments, proteins, unsaturated fatty drus crispus, Eucheuma, and Hypnea, have been revealed acids, and polysaccharides [1, 2]. Commercially, the most to be good source of carrageenans [8] and based on the significant seaweed polysaccharide is carrageenan followed source, different types of carrageenans can be acquired. From by agar and alginate. The global market value of carrageenan the view of chemical structure, carrageenans are consisted has improved from US$ 527 million in 2009 to US$ 626 of alternating (1󳨀→ 3)-linked 𝛽 -D-galactose and (1󳨀→ 4)- million in the year 2015 [3]. linked𝛼 -D-galactose, where (1󳨀→ 3)-linked𝛽 -D-galactoses Carrageenans, which are a family of linear sulfated are incompletely sulfated at specific positions of C2 and/or C4, C3, and C6; and the (1󳨀→ 4)-linked𝛼 -D-galactose units polysaccharides, are found and extracted from the cell wall of certain species of red seaweeds (Rhodophyta) [4]. These are infrequently 3,6-anhydrated (3,6AG) [9]. The differences natural polymers are capable of forming thermoreversible in the content and position of sulfate group in galactose gels or viscous solutions when added in small concentration units resulted in different carrageenan structures, which to salt solutions. Therefore, they are commonly used as as a result differ in the rheological features of solutions 2 Journal of Marine Biology and gels. To date, three types of carrageenans have been which then must be neutralized, thereby raising production reported to have commercial significance: kappa ( 𝜅 ), iota costs [18]. (𝜄 ), and lambda (𝜆 ) [5]. The major variation among kappa, Among the recognized seaweeds in Omani coasts, Hyp- nea bryoides was found to have potential to be a good source iota, and lambda carrageenan at the molecular level is the amount along with position of the sulfate ester groups which for carrageenan. Al-Alawi et al. [25] found that Hypnea produces different properties [10]. 𝜆 -carrageenan contains no bryoides gives yield of about 30-32% (d.b.) carrageenan under alkaline condition extraction (6% w/v NaOH, 80-85 hydrophobic 3,6AG bridge but has three hydrophilic sulfate C, 4 h). ester groups, which makes this carrageenan readily water Although the gel gave good property when it was compared soluble under most conditions. 𝜅 -carrageenan possesses a with a reference carrageenan, purity test showed that the 3,6AG bridge and no more than one sulfate ester group, yield contained only 32-36% carrageenan and 55-57% was making this carrageenan less hydrophilic and less soluble salt. No other conditions were tested to see the effect of in water.𝜄 -carrageenan is in-between, with a 3,6AG bridge different conditions on yield and quality. Therefore, the aim and two sulfate ester groups. Accordingly, characterization of this study was to investigate the effect of various conditions of carrageenan is the most crucial for quality control and to (alkali strength, temperature, and duration time) on yield and build up new application supported by their unique intrinsic characteristics (chemical and thermal characteristics) of𝜅 - properties [11]. carrageenan obtained from Hypnea bryoides. As there is little The yield and quality of the extracted carrageenan con- available information on this seaweed in the literature, this firm the commercial significance of both carrageenan and study will help in determining the best possible quality of the mother plant to the industry. However, both yield and carrageenan from Hypnea bryoides which will influence any quality are very much influenced by several factors such future marketing plans. as environmental (salinity, pH, temperatures, light intensity, and water movement), physiological (nutrient uptake, growth 2. Materials and Methods rate, tissue age, stress tolerances, and defences), and extrac- tion conditions [11–16]. The major common and essential Standards, chemicals, and solvents used in this study were step in carrageenan extraction procedures is alkali treatment obtained from Sigma-Aldrich Co. Ltd., and all were of at raised temperature for specific time. This step is crucial analytical grade unless otherwise specified. All solutions were to verify transformation of the biological precursors to the prepared with deionized water. All samples were analyzed at commercial grade carrageenan, (𝜇 -and v-carrageenan into least twice. 𝜅 -and i-carrageenan, respectively) [11]. The treatment with alkali induces desulfation of the polysaccharide, causing .. Sample Collection. Based on our previous work [25], formation of a 3,6AG bridge (between C3 and C6 in the 4- samples of the red seaweed species Hypnea bryoides were har- linked-𝛼 -L-galactose units) which leads to increase in the vested by local specialists from the southern coast of Oman gel strength [17]. The increase in gel strength is attributed to ∘ 󸀠 ∘ 󸀠 from Mirbat city (16 59 28.7 N, 54 41 27.7 E) in November the fact that presence of sulfate at C6 of the𝛼 -L-galactose 2015. The samples were transferred to the laboratory at Sultan residues in the precursor units acts as a “Kink” to prevent Qaboos University in cold boxes via air, washed with running the double helix from forming during gel formation. Closure fresh water, cleaned by hands from foreign matter upon of the ring to form the 3,6AG and elimination of the C- arrival, and then sun-dried under the shade for three days. 6 sulfate group makes the chain straighten and leads to The dried samples were then packed in plastic bags and stored great regularity in the polymer, resulting in enhancing gel in a refrigerator at 4 C until further analysis. strength due to increased capability of forming a double helix [17]. The severity of alkaline treatment (concentration and temperature) as well as duration aec ff ts the yield and quality .. Extraction Method. The alkaline treatment extraction [18] as inadequate treatment will not cause the required was performed according to the method described by Al- transformation. Moreover, existence of substantial amounts Nahdi et al. [26]. The method involved rehydrating twenty of the natural precursor units in commercial carrageenan grams of the sun-dried samples overnight in 1L of deion- preparations has a critical negative effect on the functional ized water and then depigmentation in 100 ml of ace- (e.g., gelling) properties [19]. tone/methanol (1:1) mixture. The samples were then treated Although the general steps in the extraction process are with alkaline solution (1.5L) of variable strengths (4, 6, and known, the extraction variables do differ [20] as seaweeds 8% w/v of aqueous NaOH solution) and heated at different differ in their composition and conditions and stage of temperatures (70, 75, and 80 C) for different intervals (2, growth [12–16]. Therefore, alkali treatment of each algal 2.75, and 3.5 hours). Occasional and gentle stirring was species must be developed and variables like temperature, employed after the rfi st hour of heating. Then, the algal alkali concentration, and extraction time must be optimized insoluble materials were collected from the solution using to induce as much desulfation as possible, while still avoiding double layers of cheesecloth (coarse lfi tration) and washed the yield losses due to degradation and leaching caused by the three times with deionized water. The retained algal materials treatment [21]. Such loss is frequently associated with elevated were again redissolved in 500 ml of deionized water, pH extraction temperatures and prolonged extraction time [22– adjusted to 7 using 6N HCl, and then heated to 90 Cfor 1h 24]. Furthermore, industrial production of alkaline-treated- with continuous gentle stirring. The insoluble algal materials carrageenan produces considerable pollution in the outflows were separated and discarded from the hot mixture using 󸀠󸀠 󸀠󸀠 Journal of Marine Biology 3 double layers of cheesecloth and Whatman GF/D, GF/C lfi ter Linear Model (GLM) for univariate analysis was employed to paper. Then, the pH of the collected liquid was checked and determine the main and interactive effects of alkali treatment adjusted to 7 and kept overnight (14h) at 4 Cin a refrigerator conditions on carrageenan yield, molecular weight, and to form gel. Volume of the gel was reduced to one-half sulfate content. When significant differences were found, a by evaporation in an air forced oven at 60 Cfor 12h. The multiple post hoc Duncan’s Multiple Range Test was applied. concentrate was dialyzed against deionized water for 72 h Pvalues<0.05 were considered as statistically signica fi nt. using dialysis membrane with 12000-14000 Daltons cut-off (Medicell International Ltd, London) and dried afterwards for 18 h at 40 Cin vacuumoven. 3. Results and Discussion Finally, the dried extracts were milled and then stored in a sealed plastic container at room temperature until further The yield and chemical properties of alkali-treated car- analysis. Two replicates were conducted for each combination rageenan extracted from Hypnea bryoides are summarized in of alkali concentration, temperature, and treatment duration. Table 1. The results demonstrate that the total yield value obtained from different alkali treatments extraction conditions ranged .. FTIR (Fourier Transform Infrared) Analysis. The FTIR between 6.59± 0.09% and 26.74± 5.01%, obtained by the analysis was performed according to the method described treatment with 8% NaOH, 3.5h, 80 Cand 6% NaOH,2.75h, by Al-Alawi et al. [25] using Magna 560 FTIR spectrometer 70 C, respectively. (Thermo Nicolet, USA) equipped with ZeSn ATR cell and In general, some of the carrageenan yields obtained in this DTGS detector. The IR spectrum was collected by averaging −1 study were in the same range of those extracted in aqueous 128 scans at resolutions of 4 cm . media obtained by Yermak et al. [29] from C. pinnulatus (20.5%-18.2), Reis et al. [30] from H. musciformis (48%- .. Sulfate Content Determination. Sulfate content was 21%), and Webber et al. [31] from K. alvarezii (35.8%-18%). determined using the method described by Dodgson [27]. However, all the abovementioned studies were performed In brief the method involved mixing 1% w/v carrageenan without alkaline treatment step and also without dialysis step, solution (0.2 ml) with 4% (w/v) trichloroacetic acid (3.8 ml) which raise questions on purity and quality of the reported and 1 ml of the BaCl -gelatin reagent. The mixture was yield. Therefore, the comparison based on the absolute yield allowed to stand for 15 min at room temperature for color is misleading. development and then absorbance was recorded at 360 nm. For the alkaline treated carrageenan, yields found in this A calibration curve was prepared with solutions of K SO 2 4 study were in the range of those reported for other species 2− containing 20-200𝜇 gof SO ions. such as 26.2% from H. porphyroides (Indian Ocean) [32], 29.1% from H. durvillei (Philippines) [33], 20–32% from K. .. Molecular Weight Analysis. Molecular weight determi- alvarezii (Brazil) [34, 35], 15% from H. durvillei (Madagascar) nation experiment was conducted as described by Al-Nahdi [36], 26.8% from H. floresii (Mexico) ´ [37], and 25.8-37.2% et al. [26]. The analysis was carried out on Agilent 1100 from E. spinosum (Indonesia) [38]. (Agilent, USA) instrument equipped with differential refrac- On the other hand, the values from the present study tive index detector (Agilent RID 1100). The separation was are slightly lower compared to results obtained from other achieved using Waters Ultrahydrogel Linear column (Waters species such as G. skottsbergii, S. crispata, and C. crispus, G. Corporation) and the mobile phase 0.1M NaCl solution with atropurpurea (31-44%) [39], E. isiforme (43.5%, 33.8%) [14], isocratic elution. A sample size of 20𝜇 l of 0.1% w/v of and K. alvarezii (Doty) (30% to 39%) [28]. carrageenan solution was injected in the system at a flow rate The differences in yield between different species is very of 1 ml/min. The temperature of the column compartment much understood due to the morphology differences as and the detector o fl w cell were maintained constant at 40 C. explained above; moreover, difference in the same species grown in different geographical area has been also noticed .. Differential Scanning Calorimeter Measurements. The and it was attributed to differences in the harvest time, growth procedures for Differential Scanning Calorimetry measure- conditions (salinity, deepness, and nutrients), time of growth, ments (DSC) were similar to those used previously by Al- environmental conditions (wind speed, precipitation, cloud Alawi et al. [25] with minor modifications. The thermal cover, insulation, water and air temperature, and length and analysis measurements were carried out using a modulated period of waves), and extraction process and parameters as differential scanning calorimeter (model Q1000, TA Instru- reported by several studies [11–16, 32]. ments, USA). The tested gels were made up of 1.5% (w/v) of In this study, the yield that was obtained by the condition the extracted carrageenan and ionic strength of 30 mM KCl in (6% NaOH, 3.5h, and 80 C) is very low (12.69%) compared an aqueous environment. The samples were scanned during a with the previously reported yield (33.2%) for the same plant ∘ ∘ heating/cooling cycle (25-90-25 C) at a rate of 5 C/min, with collected from the same geographical area and extracted an empty pan as a reference. under nearly the same conditions (6% NaOH, 4 h, and 80- 85 C) [25]. The reason lays behind the fact that in the previous . . Statistical Analysis. The statistical analysis work was study no dialysis step was performed as the case in the performed with the SPSS software version 11.5. The obtained current study. Aeft r correcting for the dialysis step in the data were presented as mean± standard deviation. General previous study (subtracting the salt from the yield), the yield 4 Journal of Marine Biology Table 1: Properties of Carrageenan extracted from Hypnea bryoides under different alkali pre-treatment conditions. Treatment Yield M Sulfate NaOH Time Temp. 1 1 (% w/w) ± SD (Dalton)± SD (% w/w)± SD (w/v) (h) ( C) 70 21.72.± 3.61 (13.15± 0.64)× 10 07.62± 5.52 75 23.44± 0.01 (11.50± 1.27)× 10 17.42± 2.97 80 14.86± 3.72 (07.00± 7.20)× 10 19.22± 0.14 70 17.62± 3.17 (12.85± 1.30)× 10 15.12± 3.68 4% 2.75 75 18.95± 2.59 (11.30± 0.57)× 10 13.82± 9.19 80 13.24± 3.85 (07.50± 6.51)× 10 20.52± 0.00 70 21.90± 0.05 (13.10± 0.14)× 10 15.92± 0.00 3.5 75 15.12± 2.60 (12.20± 0.00)× 10 18.82± 3.82 80 09.37± 2.37 (12.40± 0.00)× 10 21.22± 1.27 70 23.06± 5.06 (11.80± 0.00)× 10 11.82± 4.38 75 11.87± 1.53 (11.70± 0.71)× 10 16.42± 0.99 80 15.21± 3.62 (11.30± 0.00)× 10 15.82± 3.25 70 16.95± 7.39 (10.20± 0.14)× 10 14.02± 7.78 6% 2.75 75 10.15± 3.73 (10.00± 1.84)× 10 16.72± 0.28 80 12.16± 3.99 (06.65± 5.44)× 10 09.82± 2.40 70 26.74± 5.01 (13.90± 0.14)× 10 17.02± 0.14 3.5 75 11.06± 2.62 (10.90± 1.41)× 10 16.52± 2.26 80 12.69± 6.37 (06.10± 6.93)× 10 15.42± 2.69 70 11.58± 1.79 (12.30± 1.13)× 10 19.62± 0.99 75 11.30± 0.74 (12.35± 0.49)× 10 25.52± 1.70 80 10.24± 1.07 (07.25± 6.15)× 10 21.52± 0.00 70 10.85± 1.97 (07.10± 7.07)× 10 20.92± 1.98 8% 2.75 75 11.06± 0.96 (05.95± 0.49)× 10 25.32± 2.26 80 08.22± 0.34 (07.90± 5.23)× 10 24.72± 1.41 70 17.38± 2.39 (08.05± 7.14)× 10 23.92± 4.81 3.5 75 07.58± 0.77 (11.25± 0.21)× 10 28.12± 0.57 80 06.59± 0.09 (06.75± 6.58)× 10 21.72± 0.85 Dry basis. came down to 10% which is close to the yield extracted The molecular weight (M ) of the extracted carrageenan in this current study under similar conditions and very is shown in Table 1. much lower than the yield (26.74± 5.01% or 23.44± 0.01%) The results demonstrate that the M value obtained obtained at the condition of 6% NaOH, 3.5h, 70 Cand 4%, from different alkali treatments extraction conditions ranged ∘ 5 5 2h, 75 C, respectively. This finding gives significance to the between (5.95± 0.49)× 10 and (13.90± 0.14)× 10 Da at current study to find the optimum conditions for extraction the conditions 8% NaOH, 2.75h, 75 Cand 6% NaOH,2.75h, to maximize the yield with the highest quality. On this 70 C, respectively. The results indicated that the degradation regard, it has been found in this study that carrageenan yield rate was elevated at higher temperatures as lower molecular significantly reduced with the increase in alkali treatment weight carrageenan was obtained (less than 7.00× 10 Da at strength and temperature. Furthermore, statistical analysis 80 C). The effect of NaOH concentration and time on degra- showed that the time parameter insignificantly influenced dation was noticed in this study, but it was not significant carrageenan yield (Figure 1). The interaction between inde- (p> 0.05) (Figure 2). Furthermore, the interaction between pendent variables was found to be nonsignificant. independent variables was also not significant. Studying the This finding was not a surprise, but in contrary it degradation rate during extraction and the possible causes are was expected due to partial degradation of polysaccharide of utmost importance, since functionality of carrageenans in chains by alkali moieties, since alkaline extraction operation most food applications depends on molecular weight and is inevitably involves some degradation of the polysaccharide largely lost if it is below 1.00× 10 Da [41] which was not seen which accelerates at elevated temperatures [4, 11, 31, 37, 40]. in any of the treatments used. Furthermore, degraded, low Furthermore, further increase in the alkali concentration molecular carrageenan was reported to cause inflammation could lead to a sharp decrease in yield [4]. in the colon in rodents, which resembles ulcerative colitis, Journal of Marine Biology 5 0 6 1.5×10 4 6 8 NaOH concentration (% w/v) 4 6 8 NaOH concentration (% w/v) 0 6 1.5×10 10 A 2 2.75 3.5 Time (h) 0 6 2.00 2.75 3.50 1.5×10 Time (h) B 0 C 70 75 80 Temperature ( C) Figure 2: Eec ff ts of alkali treatment on M of carrageenan extracted from H. bryoides (means with the same letter are not significantly dieff rent ( 𝛼 = 0.05)). 70 75 80 Temperature ( C) Figure 1: Eec ff ts of alkali treatment on yield of carrageenan extracted from H. bryoides (means with the same letter are not time compared to this study. Moreover, molecular weights significantly different ( 𝛼 = 0.05)). obtained in this study are in the range of results reported by Jupp [44] from H. bryoides (6.12× 10 Da), Hilliou et al. 5 5 [40] from Mastocarpus stellatus (22× 10 to 4× 10 Da), and Distantina et al. [11] from E. cottonii (10.76× 10 to 5.48× an inflammatory bowel disease [42]. Therefore, low molec- 10 Da). ular weight carrageenans were classiefi d by the European The chemical analyses were further supported by the FT- Scientific Committee [43] and the International Agency for IR analysis. The collected spectra indicated presence of sulfate Research on Cancer as a “possible human carcinogen.” In −1 esters (S=O) with absorption band in the 1255 cm region, this study, any very low molecular weight breakdowns were −1 3,6-anhydro-D-galactose at 925 cm ,and D-galactose-4- eliminated through the dialysis step. −1 Previous study on the same plant [25] gave molecular sulfate at 845 cm . On the other hand, peaks at 830, 820, and 5 −1 weight result of 4.1× 10 Da, which is lower than the current 805 cm , which are corresponding to D-galactose-2-sulfate results, probably due to using higher temperatures and longer (D2S), galactose and D-galactose-6-sulfate (G/D6S), and Yield (%w/w d.b.) Yield (%w/w d.b.) Yield (%w/w d.b.) Molecular weight (Dalton) Molecular weight (Dalton) Molecular weight (Dalton) 6 Journal of Marine Biology Table 2: Peaks assigned to dieff rent carrageenan types. Extracted −1 Wavenumber (cm ) Assignment Found in Carrageenans∗ Carrageenan from H. bryoides −3 1210-1260 Sulfate ester (O-SO )(S) k, i,𝜆 ,𝜇 ,v,𝜃 ,𝜉 Present 928-933, 1070 (shoulder) 3,6-anhydro-D-galactose (DA) k,𝛽 Present 840-850 D-galactose-4-sulfate (G4S) k, i,𝜇 ,v Present 830 D-galactose-2-sulfate (G2S) 𝜆 ,𝜃 ,𝜉 Not found 820, 825 (shoulder) D-galactose-2,6-disulfate (D2S,6S) 𝜆 ,v Not found 810-820, 867 (shoulder) D-galactose-6-sulfate (D6S) 𝜇 Not found 800-805, 905 (shoulder) 3,6-anhydro-D-galactose-2-sulfate (DA2S) i,𝜃 Not found ∗[28]. the mildest extraction conditions (at NaOH concentration of 4%, 2 h, and 70 C) were efficient in modifying the biopolymer chemistry and there is no need for further treatments. This result suggests a main advantage of the proposed procedure which will lead to reducing consumption of solvents and extraction time for industry production. Sulfate is an integral component found in𝜅 -carrageenan used for ionic regulation in the parent plants [46]. Table 1 a presents the effect of different treatment extraction condi- tions on sulfate content. Based on these results, the sulfate percentage of different alkali extraction conditions ranged between 7.62± 5.52% and 28.12± 0.57% at the conditions of ∘ ∘ 4% NaOH, 2 h, 70 Cand 8% NaOH, 3.5h, 75 C, respectively. 1,400 1,300 1,200 1,100 1,000 900 800 700 The standard specifications for carrageenans sulfate content -1 Wavenumber (cm ) are in the range of 15-40% [47]. Figure 3: Infrared spectra of carrageenan extracted from H. These values of H. bryoides are in line with those bryoides at 4% NaOH, 2 h, and 70 C condition (a) compared to determined from alkaline treatment analysis available in kappa (b), iota (c), and kappa/lambda (d) commercial carrageenans. the literature. Hayashi et al. [34, 35] results ranged from 23.08 to 33.48% for carrageenan extracted from K. alvarezii strains from coast of Sao ˜ Paulo state, Brazil. In addition, E. 3,6-anhydro- D-galactose-2-sulfate [11, 31, 37] were absent. isiforme from Nicaragua and E. isiforme from Yucatan [14] −1 were between 26.3% and 19.6%, respectively, E. cottonii from The 805 cm band is characteristic and distinctive of i- Indonesia ranged between 11.45 and 16.15% [11], and H. floresii carrageenan [43] (Figure 3). from Yucatan ´ Peninsula contained 26.8% [37]. The observed absorption bands in all spectra were con- firmatory of 𝜅 -carrageenan. Table 2 gives the major peaks The role of the extraction parameters on sulfate content related to the major functional groups existing on the differ- is illustrated in Figure 4. It is clear that NaOH concentration ent types of carrageenans [45]. Results obtained from raw H. and temperature have significantly aec ff ted sulfate content; in −1 bryoides plant [25] revealed bands at 872 and 842 cm which addition, NaOH concentration/temperature interaction (not is assigned to the presence of C-O-SO group on C6 and C- shown) was also found to have significant effect. With respect O-SO on C4 of galactose, respectively. to time, there is a noticeable trend where the increasing of time caused the values of sulfate content to increase; however This implies existence of 𝜇 -carrageenan in the raw sea- weed, which is𝜅 -carrageenan precursor. The nongelling 𝜇 - this was not significant. carrageenan is the natural precursor that is present in seaweed The results indicated a reduction in sulfate content as that contains𝜅 -carrageenan. The 3,6AG bridges are formed NaOH concentration increased from 4% to 6% w/v. From by the removal of sulfate group from the C-6 sulfate ester literature, there are many reports on decreasing level of of the precursor and formation of the 3,6AG bridge [45]. In sulfate with increasing concentration of alkaline used for −1 our case, the lack of 872 cm signal band indicates the total carrageenan extraction [11]. This is due to the reality that conversion of this precursor to kappa form due to the alkaline formation of 3,6AG bond involves release of sulfate groups; conditions. therefore after alkali treatment the sulfate content should be The almost identical FTIR spectra demonstrate that the lower. However, the results of the current study do not totally biopolymers extracted from H. bryoides are essentially made tfi in the inverse relationship mentioned above. This is maybe −1 due to the fact that formation of 3,6AG bonds was achieved of𝜅 -carrageenan (bands at 925 and 845 cm )withno𝜇 - precursor. Hence, it is evident from the FTIR spectra that at the mildest conditions used in this study as it was evident Absorbance Journal of Marine Biology 7 alkali treatment (extraction with 0.3 M NaOH, during 4 h, at 90 C) was found to have no effect on the sulfate content of carrageenan extracted from H. durvillei [36]. According to the results obtained by DSC measurements, melting and gelling temperatures for the carrageenan gel extracted from H. bryoides were in the ranges 47.10± 7.01- ∘ ∘ 55.91± 2.44 C and 30.25 ± 1.07-35.65 ± 4.73 C(Table 2), respectively. The results of melting temperatures were found to be significantly influenced (p < 0.05) by the alkaline concentration (Figure 5(a)) and the interaction of NaOH concentration and time parameters (highest melting temper- ature was at 4% NaOH concentration and 2.75h), whereas the gelling temperatures were significantly influenced by 4 68 temperature parameter (Figure 5(b)). NaOH concentration (% w/v) The finding in this study is consolidating what was reported elsewhere [31] where it was reported that gel thermal properties do not seem to be aec ff ted much by the extraction time compared to temperature and alkaline strength parame- ters. Hence extraction time is not the parameter of choice for modifying the end-product chemical structure. However, the results reported in this study are lower somewhat than those reported elsewhere. For instance, Andrade et al. [50] found that the melting temperatures of gels prepared using 𝜅 -carrageenan polymers extracted from H. musciformis were in the range 74-75 C, and gelation temperature was around 55 C. Additionally,𝜅 -carrageenan extracted from different strains studied by Sahu et al. [28] had melting temperatures that ranged between 70 and 77 C 2 2.75 3.5 and gelling temperatures that ranged between 48 and 54 C. Time (h) Furthermore, the results reported by Al-Alawi et al. [25] on H. bryoides𝜅 -carrageenan gel indicated melting at a midpoint temperature of about 70.9 C. The latter example was of the same seaweed species extract, done under equivalent salt concentration and with same concentration of𝜅 -carrageenan solution. However, the extraction parameters were differ- ent (6% NaOH, 4 h, 80-85 C) and the sample contained higher concentration of salt (55% of the extract was salt, because no dialysis step was performed). Sen and Erboz [51] mentioned that the addition of salt to 𝜅 -carrageenan effectively facilitates the physical gelation. The effectiveness of salts in influencing the phase transition temperatures, gel strength, and the binding state of the strongest junction zones has been shown to play important roles in controlling the 70 75 80 ∘ viscoelasticity, gelation rate, and syneresis [52]. Furthermore, Temperature ( C) in the current study, the samples’ ion content, as determined Figure 4: Eec ff ts of alkali treatment on sulfate content of car- by Inductively Coupled Plasma (ICP) Emission Spectroscopy rageenan extracted from H. bryoides (means with the same letter are (data not shown), was used to adjust the amount needed not significantly different ( 𝛼 = 0.05)). for the proper concentration of the polymer. Therefore, comparison between different studies without standardizing all test parameters such as ionic strength is not possible. In from FTIR spectra. Similar findings were reported recently the current study no attempts were taken to produce gel with by Moses et al. [48], where the sulfate content increased with different salt concentrations. the increase in alkaline concentration which was attributed to Generally, the alkali pretreatment is performed essentially the removal of protein (due to alkaline hydrolysis) and water to improve the gelling properties through lowering sulphate groups in the structure. However, the same treatment may soluble low molecular weight compounds which resulted in concentrating the extract. Other authors also obtained differ- negatively aec ff t the quality features if it is not done properly. ent trends than those found earlier. For instance, carrageenan This is related to the degradation of the polysaccharide aer ft alkali modification which is corroborated by drastic extracted from H. musciformis by NaOH (0.1 N) contained sulfate content in the range of 44.1% [49]. On the contrary, decrease in molecular weight as it was explained above. Sulfate Content (% d.b.) Sulfate Content (% d.b.) Sulfate Content (% d.b.) 8 Journal of Marine Biology B A A A A A A A B A A A 20 20 4 68 2 2.75 3.5 70 75 80 4 68 2 2.75 3.5 70 75 80 ∘ ∘ NaOH (%) Time (h) Temp. ( C) NaOH (%) Time (h) Temp. ( C) (a) (b) Figure 5: Eec ff t of different parameters of alkaline pretreatment on carrageenan melting (a) and gelling temperatures (b). The effect of alkaline treatment on the melting and gelation the intermediate duration time (2h). Nevertheless, higher𝜅 - temperatures of the gel prepared from the extracted 𝜅 - carrageenan gels properties observed in the present study carrageenan as a factor of molecular weight and sulfate were at higher NaOH concentration (8%) which interacted content was tested using Pearson’s correlation analysis. A with lower duration times. Interestingly, comparable results good agreement of significant (P < 0.05) direct relation- can be obtained by lower NaOH concentration (4%). How- ship was found between molecular weight with melting ever, in this case, duration times may increase and tem- (0.46) and gelling temperatures (0.44). On the other hand, perature must be elevated to the maximum. These polysac- weak insignificant correlation was found between sulfate charides, on the other hand, would also be of potential content and melting (0.05) and gelling temperatures (- utility in the applications demanding low gelation or textural 0.19). properties, e.g., in personal care or related domains. These results indicate that gel thermal properties do Moreover, the present study shows promising commercial not seem to be aec ff ted by the extraction time compared potential for H. bryoides plant as a source for𝜅 -carrageenan. to temperature parameter. Similar results were reported by The results obtained from this study, however, may not be Webber et al. [31] who concluded that extraction time is applicable to other Hypnea species and other carrageeno- not the parameter of choice for tuning the end-product phytes due to possible variation of seaweed reaction to the chemical structure of carrageenan and longer extraction treatment. u Th s, optimization of the alkali treatment to other times are only preferred to increase the yield in extracted carrageenan producing seaweeds should be investigated. biopolymers structure. Therefore, extraction temperature, or equally pH, is deemed more efficient parameter in modifying Data Availability the biopolymer chemistry. All data used to support the n fi dings of this study are included within the article. Only data of mineral analysis was not 4. Conclusion provided, but it can be released upon request; however, the The current study investigated the effect of different authors feel it is not of great importance. extraction conditions on the different characteristics of 𝜅 -carrageenan and the optimal conditions that can be Conflicts of Interest applied for carrageenan extraction from H. bryoides grown in Omani coasts to avoid excessive processing (reagents, The authors declare that they have no conflicts of interest. energy, and time) that would result in degradation of carrageenan molecule and compromise its quality and yield. The recommended conditions based on the above discussion Acknowledgments that resulted in a satisfactory yield, M ,and sulfate content were found to be 6% NaOH for 3.5 hours at 70 C. The FTIR Zainab Al-Nahdi would like to thank Sultan Qaboos spectra showed presence of𝜅 -carrageenan, with no extent, University for providing scholarship for her PhD stud- or minor quantities of𝜄 -or𝜇 -carrageenans in all extracts, ies. In addition, financial support provided by Sultan which verify the effectiveness of the mild parameters for total Qaboos University under the strategic research projects conversion achievement. (SR/AGR/FOOD/11/01 and IG/AGR/FOOD/19/01) is greatly In addition, 𝜅 -carrageenan establishing higher melting appreciated and acknowledged. The valuable statistical assis- and gelling temperatures was satisfied at lower NaOH con- tance provided by Prof. Ali Mahdi Kadhim and Dr. Anesh centrations (4%) and lower heating temperature (70 C) with Govender is highly appreciated. Carrageenan Melting Temp. ( C) Carrageenan Gelling Temp. 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