Agars from three species of Gracilaria (Rhodophyta) from Yucatán Peninsula

Gracilaria cervicornis, Gracilaria blodgettii and Gracilaria crassissima growing along the coasts of Yucatán were investigated for their agar content. The effect of different concentrations of NaOH in the alkali treatment was evaluated. The three species of Gracilaria produced agars, both native and alkali treated, with different properties confirming the heterogeneity of the agar polymers in this genera. G. cervicornis produces agar polymers with an occurrence of methoxylation and sulphation at the C-6 of the beta-D-galactose residues, and with an extra methylation due to the presence of the 4-O-methyl-alpha-L-galactose residue. The presences of these residues is responsible for the extremely poor gelling ability of its agarocolloids, whose commercial value seems to be quite low. Agar extracts from G. blodgettii showed the typical pattern of unsubstituted agar with a very low degree of methylation on both galactose residues. The discrepancy found between sulphate content and NMR data of agar from this species requires a more detailed structural investigation. G. crassissima produces a good quality agar after sulphate precursor sequences have been removed by alkaline treatment, and it may be considered for exploitation as a source of commercial grade agar. Alkali treatment was effective both in removing alkali-labile sulphate and increasing the gel strength in G. crassissima but not in G. cervicornis and G. blodgettii.     

Agars fromGracilaria chilensis (Gracilariales)

The chemical composition of the agars extracted with water at 95 °C fromGracilaria chilensis of Estero de Lenga (43.2% of yield) and of Bahia de Coquimbo (43.4% of yield) were determined. The gel strength, gel melting and gelation temperature of the agars were also measured. Polysaccharides obtained by extraction with cold water and by extraction of the alkali pre-treatedG. chilensis of Bahia de Coquimbo were also analyzed. The physical constants and the contents of sulfate and 3,6-anhydrogalactose of the alkali-modified agar fromG. chilensis of Estero de Lenga were comparable to those of commercial agar. This algae represents an excellent potential commercial resource of agar.G. chilensis of Bahia de Coquimbo gave hydrocolloids with low gel strength values more suitable for use in the food industry. Anion exchange chromatography of the agars obtained by extraction with water at 95 °C indicated that the whole polysaccharides and all of the acidic fractions were not homogeneous. Neutral fractions comprised only about 25% of the agars.     

Chemical characteristics and gelling properties of agar from two Philippine Gracilaria spp. (Gracilariales, Rhodophyta)

The chemical structure of agars extracted from Philippine Gracilaria arcuata and G. tenuistipitata were determined by NMR and infrared spectroscopy. Agar with alternating 3-linked 6-O-methyl-β-D-galactopyranosyl and 4-linked 3,6-anhydro-2- O-methyl-α-L-galactopyranosyl units was isolated from G. arcuata, while the agar from G. tenuistipitata possesses the regular agarobiose repeating unit with partial methylation at the 6-position of the D-galactosyl residues. Both agars exhibit sulphate substitution at varying positions in the polymer. Chemical analyses reveal higher 3,6-anhydrogalactose and lower sulphate contents in alkali-modified than in native agar from both samples. Also, alkali modification enhanced agar gel strength and syneresis. Native G. arcuata agar produces a viscous solution (2000 cP at 75 °C) with a high gelling point (>60 °C) that forms a soft gel even after alkali modification (gel strength: <300 g cm⁻²). On the other hand, the agar from G. tenuistipitata exhibits gel qualities typical of most Gracilaria agars. This revised version was published online in June 2006 with corrections to the Cover Date.     

Superior quality agar from <Emphasis Type="Italic">Gracilaria</Emphasis> species (Gracilariales,Rhodophyta) collected from the Gulf of Mannar,India

Gracilaria edulis, G. crassa, G. foliifera, and G. corticata are naturally occurring agarophytes of Indian waters. These agarophytes were evaluated for their agar contents using an improved process recently reported by us (US Patent 2005/0267296A1). The effect of different concentrations of NaOH in the alkali treatment was studied for optimizing the extraction conditions. These Gracilaria species of Indian waters produced agars, both native and alkali treated, with different properties confirming the heterogeneity of the agar polymers in this genera, as one would expect. Among these, G. edulis and G. crassa produced agar polymers having high gel strengths of 490 ± 8.16 and 800 ± 15.4 g cm⁻², respectively, with 8% NaOH treatment as opposed the low gel strength agars that have been reported in the literature to date.     

3,6 anhydrogalactose, sulfate and methoxyl contents of commercial agarophytes from different geographical origins

The chemical composition of agars extracted from six economically important species of Gracilaria from different geographical sources (Argentina, Brazil, Indonesia, China and Turkey) and one species of Gracilariopsis (G. lemaneiformis) collected from two different Japanese localities was investigated. Agar was extracted by pretreatment with various concentrations of NaOH (3%, 5%, 7%, 10%) for 2 h at 80°C. The sulfate, 3,6-anhyd, rogalactose and methoxyl contents of each agar extract were analyzed. High sulfate and 3,6 anhydrogalactose contents were found in non-alkali treated agar from Turkish Gracilaria gracilis (3.4%) and from Chilean G. chilensis (54.3%) after alkali treatment concentration of 5% NaOH, respectively. High methoxyl contents (4.9%) were obtained from non-alkali treated agar from Chinese G. tenuistipitata. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of alkali treatment time and extraction time on agar from <Emphasis Type="Italic">Gracilaria vermiculophylla</Emphasis>

The effects of alkali treatment time and extraction time of native agar and alkali treated agar obtained from Gracilaria vermiculophylla were studied. The response characteristics were mainly agar yield and gel strength. Alkali treatment was carried out at 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 h. Agar yield and gel strength decreased with the increase in the time of the alkali treatment. The highest yield (15.3%) and highest gel strength (1,064 g cm⁻²) were obtained at 0.5 h, and therefore this time was used for the next experiment. The extraction of both native and alkali treated agars was carried out at 1.5, 2.0, 2.5, and 3.0 h. The best extraction time for alkali treated agar was 1.5 h, and for native agar 2.5 h. The alkali treated agar obtained with the different alkali treatment and extraction times showed higher melting (92.4–99.7°C) and gelling (35.7–39.6°C) temperatures. Native agar was lower in melting (60.2–64.1°C) and gelling (20.4–23.4°C) temperatures. The 3,6-anhydrogalactose content decreased with increasing alkali treatment time, with the opposite effect during the extraction of native and alkali treated agars.     

Agar Processed from Gracilaria foliifera of Florida

Agar producing a firm gel with specific rheological properties as well as a good commercial value was processed from Gracilaria foliifera (Forsskal) Børgesen, a red alga harvested along the Florida west coast. The alkali pretreatment of the seaweed based on the Funaki-Kojima’s method was applied before the process of agar extraction. The chemical and physical properties of the processed agar were compared with other typical agar experimentally processed.     

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.     

Polysaccharides from the red seaweed Gracilaria dura (Gracilariales, Rhodophyta)

The yield and physical and chemical properties of agars from Gracilaria dura (C. Agardh) J. Agardh, harvested in Thau lagoon (Mediterranean sea, France), were investigated. The agar yield ranged from 32% to 35%. Gel strength of agar ranged from 263 to 600 g cm(-2), with the maximum observed in October. A positive correlation was found between agar yield and gel strength (r = 0.82; P < 0.01). The gelling temperature followed the same pattern of gel strength and also showed higher value in October (43 degrees C). The nitrogen content varied from 1.04+/-0.60% (June) to 4.70+/-0.01% (October). A positive correlation was noted between nitrogen content and gel strength (r = 0.77; P < 0.05). The 3,6-anhydrogalactose content ranged from 0.70 to 0.84 and showed monthly significant differences (P < 0.05). There was a positive correlation between 3,6-anhydrogalactose content and gel strength. The values of sulfate content were relatively constant during the studied period and no significant differences were observed. The relative high gel strength indicates that this species may be considered as source of agar for commercial use.     

POTENTIAL TOOLS FOR TRACKING OCEAN CLIMATE: VARIABILITY IN STABLE ISOTOPES IN LIVING CORALLINE ALGAE

A phylogenetic model for the selection of commercial resources using the cladistic method is proposed. The group selected as an example was the marine agarophyte red algal genus Gracilaria Greville. We suggest the use of the cladistic principle of evolutionary transformational series in order to test the quality of agars instead of the assay-herror traditional method that consumes time and budget. If we asume that the "good quality of agar" in extant taxa is a sinapomorphic character (but not a reliable taxonomic one), then taxa included in the same monophiletic clade in which the species with "good quality of agar" are, has a high evolutionary posibility to share that character. In order to do this we have to incorporate to the set of available specific characters, those of the taxa actually used as a agar source but not present in the area under scope. A complete set of the basic cladistic data required for run the most popular program currently in use (PAUP) are provided. We applied the model to the Mexican Atlantic species and found that, using Gracilaria chilensis and G. cornea as "indicator taxa," and found Mexican populations of G. crassissima , G. caudata , G. cervicornis and Gracilariopsis lemaneiformis are candidates for a study of yield and agar properties.     

Agar from some Hawaiian red algae

From describing the agars of Gelidiella acerosa Forssk., Gelidium pluma Loomis, G. pusillum (Stackh.) Lejolis, Gracilaria abbottiana Hoyle, G. bursapastoris (Gmelin) Silva, G. canaliculata (Kützing) Sonder, G. coronopifolia J.Ag., G. epihippisora Hoyle, Pterocladia caerulescens (Kützing) Santelices and P. capillacea (Gmelin) Born. & Thur. as found in Hawaiian samples of these species, it is concluded that the species of Gelidium and especially Pterocladia and Gelidiella may merit more consideration for usage due to their agar gel strengths.The nature of the gel from Gracilaria abbottiana suggests the generic status might well be reexamined. The agars from the Gelidiella and the other Gracilaria species should be studied further for their prospective values to the food industry other than gel strength. Mixtures of the agars from G. bursapastoris and G. coronopifolia would merit attention for the taste texture of their mixtures.     

Seasonal variation in the physical properties of agar and biomass of Gracilaria sp. (chorda type) from Tosa Bay,southern Japan

Plants of Gracilaria sp.(chorda type), which grow along the coast of Uranouchi Inlet in Tosa Bay, southern Japan, showed the highest biomass in the summer (26 °C to 31 °C) and spring season (15.1 °C to 24.9 °C). Maximum biomass was 6952 g m^–2 in July, but gradually decreased in the autumn (30.5 °C in September to 20 °C in November) and winter (19.5 °C in December to 14.9 °C in February). Variation in yields and gel strength of the agars, were shown to depend on the time in the season. After alkali treatment (5% NaOH, 2 h) at three different temperatures (70, 80, and 90 °C), the agars showed gel strengths essentially that of commercial grade agars, with the best gel obtained at 80 °C. Maximum gel strength (1455 g cm^–2 of 1.5% agar gel) occurred in winter when the biomass and agar yield were low. Minimum gel strength was in spring. Gel strength was inversely correlated with agar yield, but was positively correlated with apparent viscosity. Maximum viscosity was 40 cP. in December. Gelling temperatures, pH of 1.5% agar gel, and moisture content in agars showed little variation.     

Influence of nitrogen availability on agar-polysaccharides fromGracilaria verrucosa strain G-16: structural analysis by NMR spectroscopy

The influence of nitrogen availability on the chemical structure of agar polymers isolated fromGracilaria verrucosa strain G-16 was ascertained by sequential solvent extraction and carbon 13 nuclear magnetic resonance (¹³C NMR) spectroscopy. Agar isolated fromG. verrucosa strain G-16 cultured under nitrogen limited conditions showed large non-polar components but produced spectra indicative of only minor amounts of methylation. These agars also produced spectra suggesting the presence of floridean starch. The nitrogen supplemented cultures ofG. verrucosa strain G-16 produced agars of a more polar nature (hot-water soluble) and contained little detectable starch. The data suggest that the higher gelling temperatures of agar from nitrogen limited plants is not due to markedly higher methylation. These data also suggest that nitrogen effects on agar content may not be as significant as previously thought.     

Comparison of three gracilarioids: growth rate, agar content and quality

Three gracilarioid species, Gracilariopsis bailiniae and Gracilaria tenuistipitata from Vietnam and Gracilaria gracilis from Russia, were studied in order to determine whether Gracilaria gracilis might be a superior species for cultivation in brackish-water ponds for agar production compared with the Vietnamese species. The effects of different salinity levels on the growth rate and agar production as well as agar properties of three gracilarioid species were compared in controlled laboratory experiments. Gracilaria tenuistipitata and G. gracilis were tolerant to low salinity (∼10‰), whereas Gp. bailiniae died under these conditions. G. tenuistipitata showed superior growth among the three species examined. Gracilaria gracilis had the highest agar content [36.8–46.6% dry weight (dw)]. Agar yield from Vietnamese gracilarioids did not exceed 30% dw. Gel strength of native agar from Gracilaria gracilis was two-fold higher that from Vietnamese species (278 g cm⁻² vs 130 g cm⁻²). Alkali pretreatment increased gel strength significantly for Gracilaria gracilis (1.4-fold), and G. tenuistipitata and Gp. bailiniae (2.3-fold) compared with native agar. The results suggest that Gracilaria gracilis may be a suitable species for production of reasonably good quality agar.     

Comparison of the photosynthetic efficiency,agar yield,and properties of <Emphasis Type="Italic">Gracilaria salicornia</Emphasis> (Gracilariales,Rhodophyta) with and without adelphoparasite

The red seaweed Gracilaria salicornia is widely distributed along the Gulf of Thailand and Andaman sea coast. In nature, the adelphoparasite Gracilaria babae is mostly found to grow on this alga, and it appears as a gall with dark green or yellowish orange color. This study was conducted to examine the photosynthetic efficiency, agar yield, and properties of G. salicornia in plants with (PGs) and without the adelphoparasite (Gs). The results showed that the photosynthetic efficiency of the PGs and Gs was influenced by environmental factors. The agar yield extracted from the Gs was mostly lower than that from the PGs. The gelling temperature of agar solution from the PGs was lower than in the Gs but was not significantly different (p > 0.05). The gel melting point and the viscosity of the agar solution of the PGs was significantly higher than those of the Gs (p < 0.05). The gel strength and total carbohydrate content of the PGs agar were higher than those obtained from the Gs agar, except for the sample collected in June. The content of 3,6-anhydrogalactose was higher in the PGs than in the Gs collected in February and August, when the PGs agar had greater gel strength. Similarly, the PGs agar had a higher sulfate content than did the Gs agar. This study showed that changes in the photosynthetic efficiency, yield, and properties of agar extracted from G. salicornia were influenced by the environmental factors in association with the presence of the adelphoparasite G. babae.     

Agar as a gelling agent: chemical and physical analysis

Agars with different performance in bioassays were analysed for physical and chemical properties. Agars with the highest gel strength had the best performance. Good performance was also related to a low pH of a suspension of agars and to a low sulphur content. The diffusion rate of ions in gels differed between agars, but could not explain differences in agar performance. The time of autoclaving had a marked effect on the gel strength, however, without affecting the performance. Chemical analysis revealed large differences between agars. The best agars had a relatively low salt content. One of the best agars had an extremely high content of trace elements. Agar impurities, especially of the trace elements were tightly bound to the agars. Up to 30% of the Murashige and Skoog (MS) salts were also immobilized in the gel. At pH 4.2, less nitrogen and phosphate were available from the MS medium than at pH 5.7. Chlorine could be washed out completely and appeared to be a good marker for agar quality and purity. With AgNO₃, chlorine contamination could easily be visualized. Received: 28 April 1996 / Revision received: 15 December 1996 / Accepted: 20 January 1997     

In Vitro Anti-influenza Virus Activities of Sulfated Polysaccharide Fractions from Gracilaria lemaneiformis

In this paper, in vitro anti-influenza virus activities of sulfated polysaccharide fractions from Gracilaria lemaneiformis were investigated. Cytotoxicities and antiviral activities of Gracilaria lemaneiformis polysaccharides (PGL), Gracilaria lemaneiformis polysaccharide fraction-1 (GL-1), Gracilaria lemaneiformis polysaccharide fraction-2 (GL-2) and Gracilaria lemaneiformis polysaccharide fraction-3 (GL-3) were studied by the Methyl thiazolyl tetrazolium (MTT) method, and the inhibitory effect against Human influenza virus H1-364 induced cytopathic effect (CPE) on MDCK cells were observed by the CPE method. In addition, the antiviral mechanism of PGL was explored by Plaque forming unit (PFU), MTT and CPE methods. The results showed: i) Cytotoxicities were not significantly revealed, and H1-364 induced CPE was also reduced treated with sulfated polysaccharide fractions from Gracilaria lemaneiformis; ii) Antiviral activities were associated with the mass percentage content of sulfate groups in polysaccharide fractions, which was about 13%, in polysaccharides (PGL and GL-2) both of which exhibited higher antiviral activity; iii) A potential antiviral mechanism to explain these observations is that viral adsorption and replication on host cells were inhibited by sulfated polysaccharides from Gracilaria lemaneiformis. In conclusion, Anti-influenza virus activities of sulfated polysaccharide fractions from Gracilaria lemaneiformis were revealed, and the antiviral activities were associated with content of sulfate groups in polysaccharide fractions.     

Effects of phosphate and nitrate supply on productivity,agar content and physical properties of agar ofGracilaria strain G-16S

Gracilaria strain G-16S was cultured in various phosphorus (P) supply rates with low or high nitrogen (N) supply to determine the effects of nutrient supply on its productivity, agar content and physical properties of the agar. Productivity was reduced after four weeks of growth in zero P supply as plants reached 0.07% P tissue content (critical level), with fragmentation of these plants by six weeks (0.05% P; minimum viable level). Native agar content was higher in low P and high N, or low N conditions. Agar content appeared to increase with decreasing P under high N supply. This increase was not apparent with alkali treatment prior to extraction. Agar gel strength was greatly increased by alkali treatment. The highest gel strengths were obtained under high N supply at all P supply rates except zero P, and under low N supply at 12 M P week^–1. Native agar gel strengths showed a similar pattern on a lower scale. Melting temperatures were higher in agars with higher gel strengths. Dynamic gelling temperatures were generally high for alkali-treated agar, with agar from plants grown in zero P supply showing a slightly elevated gelling temperature. Melting and gelling temperatures of native agars with the highest gel strengths were in the same range as bacteriological agar. These results show that P and N supply affects productivity, agar content and agar physical properties, but the tradeoffs between a slightly higher agar quantity under nutrient limitation and higher agar quality under nutrient-replete conditions seem to favor the latter.     

Cultivation of Gracilaria verrucosa (Gracilariales,Rhodophyta) Strain G-16 for agar

The agarophyte, Gracilaria verrucosa Strain G-16, has been grown in sustained cultivation over a period of five years. During this period, a number of experiments were conducted to examine the productivity, agar yield and agar gel strength of this strain. Productivities range from 3–31 g dry wt m^–2 d^–1 and are generally highest in the summer when annual water temperatures and daily irradiances are highest. In the summer months agar yield from Strain G-16 appears to be lowest whereas the gel strength of the agars was highest (> 750 g cm^–2).     

Preparation of superior quality products from two Indian agarophytes

The effect of alkali pre-treatments on the properties of agar obtained from Gelidiella acerosa and Gelidium pusillum growing along the West and South coast of India was studied in order to evaluate its potential for industrial use. Agar samples were extracted by hot water extraction and termed native agar, and alkali pre-treatment was carried out with various NaOH concentrations e.g. 4%, 6%, 8%, 10% and 15%, to optimise the alkali concentration for preparation of alkali-treated agar. Gelidiella acerosa and Gelidium pusillum produced superior quality agar polymers having greater gel strengths 2,000 ± 50 and 1,400 ± 50 g cm⁻² and lower sulphate contents ≤0.30% and ≤0.50%, respectively, as opposed the low gel strength and high sulphate content agars reported in the literature. Furthermore, the physicochemical properties of agars were identical with the commercially available agars. The results of this study revealed that Indian agarophytes may be used for production of superior quality agar and agaroses, which may be used for food and biological applications.