Development of an eco-friendly agar extraction technique from the red seaweed Gracilaria lemaneiformis

The red seaweed, Gracilaria lemaneiformis growing as an aquaculture bioremediator along the coasts of Liaodong Peninsula, China, was investigated for the agar production. An eco-friendly method called agar photobleaching extraction process was developed for the benefit of workers' health and safety of the environment. The native agar (NA), alkali-modified agar (AA), chemical-bleached agar (CA) and photobleached agar (PA), which were extracted using different processes, were evaluated for their physical and chemical properties. The PA showed most desirable performances in terms of gel strength, gelling temperature, sulfate content and 3,6-anhydro-l-galactose content. Among the different processed agars, PA gel strength was 1913 g/cm2, the highest among the different processed agars, which increased 8.6% on the basis of the AA. Further we applied this new technique to extract agars from Gracilaria asiatica, and similar results were obtained with that of G. lemaneiformis. This indicates that the agar photobleaching extraction process is a feasible method for Gracilaria species and has a potential application. During the whole agar photobleaching extraction process the pigment content of G. lemaneiformis declined gradually and the TOC concentration in photobleaching solution increased along with the increase in the irradiation time. The mechanism of agar photobleaching could be elucidated by the photolysis theory.     

Influence of alkali treatment on agar from Gracilaria cornea from Yucatán, México

The effect of alkali treatments on the yield, rheological and chemical properties of agar from Gracilaria cornea growing along the Yucatáncoast were studied in order to evaluate its potential for industrial use inan attractive economic standpoint. Alkali treatment was carried out with NaOH concentrations of 0.5%, 1%, 3% and 5% in a water bath at 80, 85 and 90 °C. Agar yield, gel strength, gelling and melting temperatures, sulphate, 3,6-anhydro-galactose and ash content weredetermined. The different combinations of NaOH concentration and treatment temperature strongly influenced agar characteristics. There was a variation in the agar content for all NaOH treatments and temperature combinations, ranging between 14.5% to 22.1%. Although the yields obtained for 0.5% NaOH at all temperatures and 1% NaOH at 80 and 85°C were higher than those required by the industry, the physical and chemical characteristics of the agar were similar to those obtained fornative agar from the same species. The gel strengths, sulphate content and gelation hysteresis obtained with agar from the 1% NaOH treatment at 90 °C are in the range required by the food industry. Treatments with 3% and 5% NaOH at all temperatures improved significantly the agar quality giving higher gel strengths (974–1758 g cm ^-2) than those reported for other Gracilaria species. This revised version was published online in August 2006 with corrections to the Cover Date.     

Optimization and scale-up of a new photobleaching agar extraction process from Gracilaria lemaneiformis

An eco-friendly photobleaching extraction process for agar extraction from the red alga Gracilaria lemaneiformis was developed for the benefit of workers’ health and environmental safety. Here we report the optimization of key process parameters (alkali modification concentration, photobleaching duration, algal length and screen filter opening size) in order to scale up this new technique. The optimal conditions were found to be modification by 3–5% NaOH, photobleaching for 5 h, using algal fragments 2 –4 cm in length, and a filter screen with a 6 μm opening. A 20-L agar extraction reactor was thus constructed, and the scale-up of the agar extraction process was tested in six batch experiments. The resulting agar quality was similar to that of the laboratory-scale extraction. In addition, batch-to-batch reproducibility was excellent. The results demonstrate the excellent scale-up ability and potential application of this new photobleaching agar extraction process on a commercial scale. The agar yield and gel strength for 5% NaOH modified agar were 26.8% and 1,897 g cm⁻², while those for 3% NaOH modified agar were 28.2% and 1,287 g cm⁻², respectively. It is clear that the agar yield and quality can be manipulated via alkali modification in this new eco-friendly extraction to meet market demands.     

Highly methylated agar from Gracilaria edulis (Gracilariales, Rhodophyta)

The structure and gelling properties of alkali-modified agar from Gracilaria edulis were investigated. ¹H and ¹³C NMR experiments revealed a basic repeating unit of alternating 3-linked 6- O-methyl-β-D-galactopyranose and 4-linked 3,6-anhydro-α-L-galactopyranose. Partial methylation at O-2 of the anhydrogalactose moiety was also revealed. Meanwhile, the O-4 of the methylated galactose residue was detected to exhibit partial sulfation by NMR and FT-IR spectroscopy. The gel strength and syneresis index of the extracted agar were considerably enhanced by the addition of sodium, potassium, and calcium ions. The ion-driven gelation and peculiar sulfate position conferred the agar's similarity to κ-carrageenan. This revised version was published online in July 2006 with corrections to the Cover Date.     

Agar from the red seaweed,Laurencia flexilis (Ceramiales,Rhodophyta) from northern Philippines

The worldwide production of the gelling agent agar mainly rely on the red algae of the order Gracilariales and Gelidiales for raw material. We investigate here the potential of a species from another red algal order, Ceramiales as an agar source. The agar from Laurencia flexilis collected in northern Philippines was extracted using native and alkali treatment procedures and the properties of the extracts were determined using chemical, spectroscopic and physical methods. The native agar, 26% dry weight basis, forms a gel with moderate gel strength (200 g cm^?2). Alkali‐treatment did not enhance the gel strength, indicating insignificant amounts of galactose‐6‐sulfate residue, the precursor of the gel‐forming 3,6‐anhydrogalactose (3,6‐AG) moieties. Furthermore, the Fourier transform infrared and chemical analysis showed low sulfate and high 3,6‐AG levels, not affected significantly by the alkali treatment. Nuclear magnetic resonance spectroscopic analysis revealed 3‐linked 6‐O‐methyl‐D‐galactose and 4‐linked 3,6‐anhydro‐L‐galactose as the major repeating unit of the native extract, with minor sulfation at 4‐position of the 3‐linked galactose residues. The native and alkali treated agars have comparably high gelling and melting temperatures, whereas the former exhibits higher gel syneresis. Laurencia flexilis could be a good source of agar that possesses physico‐chemical and rheological qualities appropriate for food applications. Due to the inability of alkali treatment to enhance the key gel qualities of the native extract, it is recommended that commercial agar extraction from this seaweed would be done without pursuing this widely‐used industrial procedure.     

Stability of agar in the seaweed Gracilaria eucheumatoides (Gracilariales, Rhodophyta) during postharvest storage

The status of the cell-wall polysaccharide of the red seaweed, Gracilaria eucheumatoides upon postharvest storage was assessed in this study. The yield, chemical composition, physical and textural properties of alkali-treated agar extract was determined at different time intervals within 31 months of storage at dried state after harvest. Minimal fluctuation in agar yield was observed, ranging from 22.9% to 29.0%. The gel strength of agar extracts averaged 318gcm(-2) until the third month of storage but decreased considerably thereafter. The relative viscosity and molecular weight of the extracts varied inversely with storage time. Results indicated that both physical and textural parameters of agar generally decreased with storage time, likely due to depolymerization as indicated by decrease in molecular weight. Agar extracted from seaweeds up to 3 months of storage could be considered to exhibit gel quality suitable for food applications. Prolonged storage of the seaweed harvest is not recommended.     

Seasonal variation of the agar quality and chemical composition of Gracilaria veleroae and Gracilaria vermiculophylla (Rhodophyceae,Gracilariaceae) from Baja California Sur,Mexico

Yield and physico‐chemical properties of agar from Gracilaria veleroae E.Y. Dawson and Gracilaria vermiculophylla (Ohmi) Papenfuss were studied and the chemical composition of the two seaweeds was determined. Samples were collected seasonally from summer 2003 to spring 2005. The agar yield did not vary significantly between seasons for both species. The lowest agar gel strength was obtained from G. veleroae (207.5 g cm^–2) in summer 2003 and the highest from G. vermiculophylla (793.1 g cm^–2) in winter 2004. Melting temperatures and hysteresis were higher in G. vermiculophylla, whereas gelling temperatures and 3,6‐anhydrogalactose content were higher for G. veleroae. Moisture, ash, crude fiber, and ether extract showed no significant seasonal variation for G. veleroae. The chemical composition of G. vermiculophylla showed significant seasonal variation. G. vermiculophylla possesses a better agar quality than G. veleroae and is a species that could be considered as a source of agar for commercial use.     

Gracilaria species (Gracilariaceae, Rhodophyta) from southeastern Australia, including a new species, Gracilaria perplexa sp. nov.: Morphology, molecular relationships and agar content

Select species of the agarophyte Gracilaria were studied from southeastern Australia. The morphology and anatomy of species is described and molecular relations are inferred based on plastid and mitochon‐drial DNA sequence data. Agar yields and qualities are determined for each species. Gracilaria chilensis, found in Tasmania and Victoria, is morphologically and molecularly similar to G. chilensis from New Zealand and Chile and has low agar yields of 11–16%. Gracilaria cliftonii from Victoria, has high crude agar yield (52%) and is molecularly uniform. Gracilaria perplexa sp. nov., known only from Botany Bay, New South Wales, has an agar yield of 39%. The agar of G. perplexa is unusual in requiring the addition of 0.1 mol L^?1 NaCl for alcohol precipitation and is cold‐water (25°C) soluble because of the very high sulfate ester content. Molecular phylogeny shows that G. perplexa is closely related to Gracilaria preissiana from western Australia, but differs from the latter in its reduced branching and narrower more terete axes.     

Effects of sulfate starvation on agar polysaccharides of Gracilaria species (Gracilariaceae,Rhodophyta) from Morib,Malaysia

The effects of sulfate starvation on the agar characteristics of Gracilaria species was investigated by culturing two red algae from Morib, Malaysia, Gracilaria changii and Gracilaria salicornia in sulfate-free artificial seawater for 5 days. The seaweed samples were collected in October 2012 and March 2013, periods which have significant variation in the amount of rainfall. The agar yields were shown to be independent of sulfate availability, with only 0.60–1.20 % increment in treated G. changii and 0.31–1.40 % increment in treated G. salicornia while their gel strengths did not increase significantly (approximately 5–7 %) after sulfate starvation for both species. The gelling and melting temperatures did not vary between control and treated samples from both species, except for the treated G. changii collected in March 2013. The gel syneresis index of G. salicornia collected in March 2013 increased significantly after sulfate deprivation. Sulfate starvation introduced some variations in the content of 3, 6-anhydrogalactose and total sulfate esters, but the changes did not have a pronounced effect on the physical properties of agar.     

Effects of season on the yield and quality of agar from Gracilaria species (Gracilariaceae, Rhodophyta)

The effect of season on yield and physical properties of agars extracted from Gracia gracilis and G. bursa-pastoris were determined. The agar yield from G. gracilis was maximum during spring (30%) and minimum during autumn (19%). In G. bursa-pastoris, the agar yield was greatest in summer (36%) and lowest in winter (23%). Agar yield from G. bursa-pastoris was positively correlated with temperature (r=0.94; P<0.01) and salinity (r=0.97; P<0.01) and negatively with nitrogen content (r=−0.93; P<0.01). Agar gel strengths fluctuated from 229 to 828 g cm⁻² and 23 to 168 g cm⁻² for G. gracilis and G. bursa-pastoris, respectively. The gelling temperature showed significant seasonal variation for both species. Chemical analysis of agar from the two seaweeds indicated variation in 3,6-anhydrogalactose and sulfate content (P<0.01). Furthermore, there was an inverse correlation between the two chemical variables. In general, agar extracted from G. gracilis possessed better qualities than agar extracted from G. bursa-pastoris and can be considered a candidate for industrial use.     

Optimization of protein extraction from Gelidiella acerosa by carbohydrases using response surface methodology

In this study, application of response surface methodology for enzymic pretreatment optimization of Gelidiella acerosa was investigated in order to improve the extraction of algal proteins using Viscozyme L and Celluclast 1.5L. The total protein, soluble proteins and reducing sugar recovery in the water‐soluble fraction were studied in relation to the hydrolysis time, type and concentration of the enzymes. Enzymatic digestion appeared to be an effective treatment for protein extraction. While enzyme hydrolysis by Celluclast 1.5L was able to facilitate the protein extraction, it was a relatively inefficient way to improve protein extraction yield, in comparison with Viscozyme L. The optimum conditions for protein extraction was found to be hydrolysis by 2.8 μL mL^?1 of Viscozyme L for 12 h.     

The yield,physical and chemical properties of agar gel from Gracilaria species (Gracilariales,Rhodophyta) of the Kenya coast

Six species of Gracilaria, G. corticata J. Agardh, G. crassa Harvey, G. millardetii J. Agardh, G. salicornia (J. Ag.) Dawson, G. verrucosa (Huds.) Papenfuss and Gracilaria sp, collected from different stations along the Kenya coast were studied. The yield of hot water native agar extract ranges from 8.1–30% of dry weight, with G. verrucosa and G. salicornia having the greatest and the least yield, respectively. The gel-strength of 1.5% agar solution was highest in G. verrucosa (220 g cm^–2) and lowest in G. corticata (< 60 g cm – 2) whereas the highest gelling temperature was recorded for Gracilaria sp. (40.4 °C) and the lowest in G. verrucosa (28.9 °C). 3,6 anhydrogalactose content was the highest in G. verrucosa and the lowest in G. corticata while sulphate content was higher in the latter.     

Purification and properties of α-1,4-glucan phosphorylase from the red seaweed Gracilaria sordida (Gracilariales)

α-1,4-Glucan phosphorylase (EC 2.4.1.1) from the red seaweed Gracilaria sordida (Harv.) W. Nelson was adsorbed onto starch-Sepharose 6B and Sephacryl S-300 under specified conditions. The algal enzyme was purified to homogeneity by these two steps. A molecular weight of 97.4 kDa was observed on SDS-polyacrylamide gel electrophoresis under reducing conditions, while the native molecular weight was 240 kDa asrevealed by 8-25% native gradient gel electrophoresis or 245 kDa by gel filtration. The pI of the enzyme was 5.4. It had a Km of 227, 264, 285, and 453 μg ml-1, respectively, towards glycogen, amylopectin, amylose, and maltodextrin. The enzyme activity was inhibited by cyclohexaamylose, ADP-glucose, and UDP-glucose. In contrast to other plant sources, cell-free extracts of G. sordida contained only one form of phosphorylase.     

Characterization of an agar fraction extracted from Gracilaria dura (Gracilariales,Rhodophyta)

The evaluation of biopolymers of biotechnological interest has stressed the importance of renewable sources from the northern Adriatic Sea. In the frame of this research program, the red alga Gracilaria cf. dura, has been studied. Agar was obtained from Gracilaria cf. dura by means of alkali treatment and hot water extraction. In order to purify the extracts, amylase and isopropyl alcohol were used. Analysis of sugars was carried out by means of gas chromatography on alditol acetate derivatives; sulfate content was evaluated by means of infrared spectroscopy. An analysis of molecular weight distribution was carried out by means of high performance gel permeation chromatography coupled with a low angle laser light scattering device as detector. Analytical data were examined in terms of specificity of the algal source utilized, and the agar fractions extracted by means of this procedure were compared with a commercial sample.     

Studies on the yield and gel strength of agar from Gracilaria domingensis Sonder ex Kuetzing (Gracilariales,Rhodophyta) following the addition of calcium

Studies were carried out on the seasonal variation in yield and gel strength of agar from Gacilaria domingensis with and without the addition of calcium chloride. Extraction was done with and without treatment with 1% hydrochloric acid. The results showed an increase in yield and gel strength when an alkaline solution of calcium was used, but the gel strength was low. For commercial use, Gracilaria domingensis should be mixed with better quality Gracilaria species because of its low gel strength.     

Comparison of growth rate in culture,dry matter content,agar content and agar quality of two New Zealand red seaweeds,Gracilaria chilensis Bird,McLachlanet Oliveira andGracilaria truncata Kraft

Plant growth rates and agar characteristics were compared for two agarophytes,Gracilaria chilensis (Gracilariales, Rhodophyta) andG. truncata, so that the suitability ofG. truncata for mariculture could be assessed.G. chilensis plants grew steadily in the laboratory culture system at rates of 5–8% day^-1 (mean Relative Growth Rate) throughout the 6 week experimental period, with no decline in health.G. truncata plants grew at rates of 2–4% day^-1 for 5 weeks, but during the sixth week their apical tips became necrotic and growth rates fell to zero. There was no significant difference in the dry matter content (as a percentage of their wet weight) between the two species ofGracilaria, with values falling in the range of 16–19%. Slightly higher agar yields were obtained from alkali-treatedG. chilensis (17–20% dry matter) than from untreatedG. truncata (16–18%) although the agar fromG. truncata formed softer gels from which it was more difficult to recover. The gel strength of untreated agar extracted fromG. chilensis was very low (ca. 100 g cm^-2 for a 1% gel) but when an alkali treatment step was included in the extraction process, it increased to 520 g cm^-2 for a 1% gel. Contrary to an earlier report, untreatedG. truncata agar also had a very low gel strength (ca. 100 g cm^-2 for a 1.5% gel), but it rose to only 167 g cm^-2 after alkaline treatment.     

Optimisation of RNA extraction from Gracilaria changii (Gracilariales, Rhodophyta)

RNA extraction from seaweed tissues is problematic due to the presence of polysaccharides and polyphenolic compounds upon cell disruption. Besides, a successful RNA isolation from seaweed tissues can sometimes be strain- and species-specific. Four different methods were used to extract RNA from Gracilaria changii (Gracilariales, Rhodophyta), collected from the mangrove area at Morib, Selangor, Malaysia. An optimised and modified total RNA extraction method was developed for this recalcitrant species. The use of sand in tissue grinding, and the incorporation of phenol extraction at the initial stage resulted in the highest RNA yield (0.65–1.14 g g^–1 fresh weight) with high quality (A_260:280 ratio 1.80–2.05). The RNA obtained is suitable for cDNA synthesis and future functional genomic studies.