Hybrid Carrageenan Research Articles

Structural Insights into the Substrate Recognition and Catalytic Mechanism of a GH16 βκ-Carrageenase from Wenyingzhuangia fucanilytica.

Understanding the substrate specificity of carrageenases has long been of interest in biotechnology applications. So far, the structural basis of the βκ-carrageenase that hydrolyzes furcellaran, a major hybrid carrageenan, remains unclear. Here, the crystal structure of Cgbk16A_Wf, as a representative of the βκ-carrageenase from GH16_13, was determined, and the structural characteristics of this subfamily were elucidated for the first time. The substrate binding mode was clarified through a structure analysis of the hexasaccharide-bound complex and molecular docking. The binding pocket involves a conserved catalytic motif and several specific residues associated with substrate recognition. Functions of residues R88, E290, and E184 were validated through site-directed mutagenesis. Comparing βκ-carrageenase with κ-carrageenase, we proposed that their different substrate specificities are partly due to the distinct conformations of subsite -1. This research offers a comprehensive understanding of the recognition mechanism of carrageenases and provides valuable theoretical support for enzyme modification and carrageenan oligosaccharide preparation.

Viscoelastic Reversibility of Carrageenan Hydrogels under Large Amplitude Oscillatory Shear: Hybrid Carrageenans versus Blends.

The viscoelastic response of carrageenan hydrogels to large amplitude oscillatory shear (LAOS) has not received much attention in the literature in spite of its relevance in industrial application. A set of hybrid carrageenans with differing chemical compositions are gelled in the presence of KCl or NaCl, and their nonlinear viscoelastic responses are systematically compared with mixtures of kappa- and iota-carrageenans of equivalent kappa-carrageenan contents. Two categories of LAOS response are identified: strain softening and strain hardening gels. Strain softening gels show LAOS non-reversibility: when entering the nonlinear viscoelastic regime, the shear storage modulus G' decreases with increasing strain, and never recovers its linear value G0 after successive LAOS sweeps. In contrast to this, strain hardening carrageenan gels show a certain amount of LAOS reversibility: when entering the nonlinear regime, G' increases with strain and shows a maximum at strain γH. For strains applied below γH, G0 shows good reversibility and the strain hardening behavior is maintained. For strains larger than γH, G0 decreases significantly indicating an irreversible structural change in the elastic network. Strain hardening and elastic recovery after LAOS prevail for hybrid carrageenan and iota-carrageenan gels, but are only achieved when blends are gelled in NaCl, suggesting a phase separated structure with a certain degree of co-aggregated interface for mixed gels.

Immunomodulation by xylan and carrageenan-type polysaccharides from red seaweeds: Anti-inflammatory, wound healing, cytoprotective, and anticoagulant activities

The immunomodulatory properties of the polysaccharides (carrageenan, xylan) from Chondrus crispus (CC), Ahnfeltiopsis devoniensis (AD), Sarcodiotheca gaudichaudii (SG) and Palmaria palmata (PP) algal species were studied. Using RAW264.7 macrophages, we investigated the proliferation and migration capacity of different extracts along with their immunomodulatory activities, including nitric oxide (NO) production, phagocytosis, and secretion of pro-inflammatory cytokines. Polysaccharides from C. crispus and S. gaudichaudii effectively mitigated inflammation and improved scratch-wound healing. Polysaccharide fractions extracted under cold conditions (25 °C), including CC-1A, SG-1A and SG-1B stimulated cell proliferation, while fractions extracted under hot conditions (95 °C), including CC-3A, CC-2B and A. devoniensis (AD-3A), inhibited cell proliferation after 48 h. Furthermore, RAW264.7 cells treated with the fractions CC-3A, AD-1A, and SG-2A significantly reduced LPS-stimulated NO secretion over 24 h. Phagocytosis was significantly improved by treatment with C. crispus (CC-2B, CC-3B) and A. devoniensis (AD-3A) fractions. RAW264.7 cells treated with the CC-2A and SG-1A fractions showed elevated TGF-β1 expression without affecting TNF-α expression at 24 h. Polysaccharide fractions of A. devoniensis (ι/κ hybrid carrageenan; AD-2A, AD-3A) showed the highest anti-coagulation activity. CC-2A and SG-1A fractions enhanced various bioactivities, suggesting they are candidates for skin-health applications. The carrageenan fractions (CC-3A: λ-, μ-carrageenan, SG-2A: ν-, ι-carrageenan) tested herein showed great potential for developing anti-inflammatory and upscaled skin-health applications.

Polysaccharides from red seaweeds: Effect of extraction methods on physicochemical characteristics and antioxidant activities

Seaweed polysaccharides are reported to possess biological and medicinal properties. Specifically, the antioxidant activity of seaweed extracts is the subject of intensive research due to ever-increasing demands from the food and pharmaceutical industries. The structure and composition of polysaccharides (carrageenan, xylan) extracted from four red seaweed species: Chondrus crispus (CC), Ahnfeltiopsis devoniensis (AD), Sarcodiotheca gaudichaudii (SG) and Palmaria palmata (PP) were compared and correlated with their antioxidant activity. Crude polysaccharide extracts were characterized by NMR (1H and 13C), HPLC-SEC, and FTIR spectroscopy. Total phenolic content (TPC), total sugars, sulfate and protein content were determined using spectrophotometric methods. The antioxidant capacity of each fractionated polysaccharide sample was evaluated by the following methods: DPPH (2,2-diphenyl-1-picrylhydrazyl radical), ABTS (2,2-azino-bis-[3-ethylbenzothiazoline-6-sulfonic acid]), superoxide dismutase (SOD), ferric-reducing power in FRAP (Ferric-reducing Antioxidant Power) assays, and the hydroxyl radical (OH) scavenging capacity assay. The highest antioxidant activity was found in extracts from A. devoniensis (ι/κ hybrid carrageenan), while the lowest was in P. palmata (xylans). Moreover, Chondrus crispus extracts did not possess SOD inhibition activity. Polysaccharides evaluated in this study offer high potential for applications in the food, pharmaceutical and biotechnology industries.

Exploring relationships between seaweeds carrageenan contents and extracted hybrid carrageenan properties in wild and cultivated Mastocarpus stellatus, Chondrus crispus and Ahnfeltiopsis devoniensis.

The types and amounts of carrageenans present in a set of Mastocarpus stellatus, Chondrus crispus and Ahnfeltiopsis devoniensis, are assessed by solid-state NMR performed directly on dried and powdered seaweeds. The resulting chemical analysis is compared with the chemical structure and gel elasticity of the hybrid carrageenans extracted from these three carrageenophytes. ν-carrageenan is only found in a single sample of M. stellatus, whereas C. crispus and A. devoniensis essentially contain ι- and μ-carrageenans. A significant amount of κ-carrageenan is found in all samples of M. stellatus, whereas this type of carrageenan was found in a single sample of A. devoniensis. Hybrid carrageenans (HK) made of κ-, ι-, μ- and ν-carrageenan units were found in all hot extracts from the seaweeds, and their respective contents are in line with earlier reports on the HK extracted from these seaweeds. No correlations between the carrageenan contents in eight seaweeds and the chemical structures of their corresponding extracted HK can be found. However, a positive nonlinear correlation between the intrinsic viscosity of extracted HK and their gel elastic modulus is found in a set of 8 carrageenophytes.

Green Extraction of Carrageenans from Mastocarpus stellatus.

The recovery of biopolymers from natural resources using eco-friendly extraction technologies that enhance their mechanical properties has gained attention in recent years. In this context, this work deals with the isolation of hybrid carrageenans from Mastocarpus stellatus red seaweed using subcritical water extraction operating in a wide range of thermal conditions (70–190 °C). The extracted biopolymers were analyzed by means of either Fourier-Transform infrared, nuclear magnetic resonance, rheological or cell viability assays. In parallel, the fundamental chemical composition of the seaweed used as raw material, as well as the main phytochemical properties of the soluble liquid extracts, were also studied. Results indicated that thermal extraction conditions significantly affected the rheological behavior of the recovered hybrid carrageenans. The hybrid carrageenan extraction yields varied, with results between 10.2 and 30.2% being the highest values obtained at hydrothermal treatment of 130 °C. A wide palette of viscous features was identified for recovered hybrid carrageenans, with the strongest rheology properties observed at the same temperature. It should be remarked that the maximum inhibitory effect was also obtained at 130 °C for both the ovarian carcinoma cell line (A2780) (65%, IC50: 0.31 mg/mL) and lung carcinoma cell line (A549) (59%, IC50: 0.41 mg/mL).

Mechanical Disintegration and Particle Size Sieving of Chondrus crispus (Irish Moss) Gametophytes and Their Effect on Carrageenan and Phycoerythrin Extraction.

To better understand the migration properties of hybrid carrageenan from the seaweed tissue during carrageenan extraction, the effect of increasing the seaweed surface area by the mechanical disintegration of gametophyte Chondrus crispus chips was studied under various temperature and time extraction conditions. Dried Chondrus crispus seaweed chips were milled by a rotor beater mill and classified into eight different size fractions by sieving with varying mesh sizes from 50 to 2000 μm. During extraction at 22 °C, the red color of the filtrate increased significantly with the decreasing particle size of the fraction, correlating with the increasing phycoerythrin concentration (from 0.26 mg PE/g dry seaweed in the >2000 μm size fraction to 2.30 mg PE/g dry seaweed in the <50 μm size fraction). On the other hand, under the same extraction conditions, only a small increase in carrageenan precipitate was obtained with the decreasing size fractions (from no recovery in the >2000 μm size fraction to 2.1 ± 0.1 g/kg filtrate in the <50 μm size fraction). This yield was significantly lower than the ones from extractions at 45 °C (5.4 ± 0.1 g/kg) or at 90 °C (9.9 ± 2.1 g/kg) for the same particle size and time conditions. It could be concluded that hybrid carrageenan extraction is not surface area dependent, while phycoerythrin is. Therefore, it seems that phycoerythrin and carrageenan extraction follow different mechanisms. This creates potential for the selective extraction of each of those two compounds.

Cloning, Heterologous Expression, and Characterization of a βκ-Carrageenase From Marine Bacterium Wenyingzhuangia funcanilytica: A Specific Enzyme for the Hybrid Carrageenan-Furcellaran.

Carrageenan is a group of important food polysaccharides with high structural heterogeneity. Furcellaran is a typical hybrid carrageenan, which contains the structure consisted of alternative β-carrageenan and κ-carrageenan motifs. Although several furcellaran-hydrolyzing enzymes have been characterized, their specificity for the glycosidic linkage was still unclear. In this study, we cloned, expressed, and characterized a novel GH16_13 furcellaran-hydrolyzing enzyme Cgbk16A_Wf from the marine bacterium Wenyingzhuangia fucanilytica CZ1127. Cgbk16A_Wf exhibited its maximum activity at 50°C and pH 6.0 and showed high thermal stability. The oligosaccharides in enzymatic products were identified by liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) and nuclear magnetic resonance (NMR) spectroscopy. It was confirmed that Cgbk16A_Wf specifically cleaves the β-1,4 linkages between β-carrageenan and κ-carrageenan motifs from non-reducing end to reducing end. Considering the structural heterogeneity of carrageenan and for the unambiguous indication of the specificity, we recommended to name the furcellaran-hydrolyzing activity represented by Cgbk16A as “βκ-carrageenase” instead of “furcellaranase”.

Valorisation of the industrial hybrid carrageenan extraction wastes using eco-friendly treatments

The goal of this paper is the valorisation of the Chondrus crispus (Rhodophyta) wastes generated during the industrial extraction of hybrid carrageenan using microwave technology of a temperature range from 140 to 200 °C, using water as a solvent. Mineral and phenolic content, antioxidant capacity and thermorheological features of the recovered hybrid carrageenans were determined. Results indicated the suitability for the recovery of high valuable compounds from these algae residues, after the optimization of the extraction conditions. At microwave processing conditions (160 °C, 5 min), it is still possible to recover around 10% of semi-refined hybrid carrageenan from the industrial raw material as well as to achieve the maximum value of total phenolic compounds of 12.8 mg GAE/g extract and antioxidant capacity of 51.70 mg Trolox/g extract. Extracted biopolymers exhibited thermomechanical features, in terms of viscoelastic moduli or gelling and melting temperatures, comparable to their commercial counterparts.

STRUCTURAL CHARACTERISTICS AND BIOLOGICAL ACTIVITY OF SULFATED POLYSACCHARIDE FROM RED ALGAE BETAPHYCUS GELATINUS

In this paper, sulfated polysaccharides were extracted from red algae Betaphycus gelatinus species collected at Ninh Thuan sea by using several extraction methods. The structural characteristics of sulfated polysaccharides was investigated by IR and NMR methods. The results indicated that polysaccharide from Betaphycus gelatinus identified as hybrid carrageenan, mainly composed of β-carrageenan, κ-carrageenan and g-carrageenan. Biological activity evaluation showed that the sulfated polysaccharides possessed antioxidant activity with different levels.

The microwave assisted extraction sway on the features of antioxidant compounds and gelling biopolymers from Mastocarpus stellatus

Microwave assisted water extraction of hybrid carrageenan and antioxidant compounds from Mastocarpus stellatus red alga was first proposed to recover biopolymers with specific rheological properties, saving time, avoiding chemicals and enhancing extraction yields when compared with conventional treatments. The hybrid carrageenans, precipitated from the liquid fractions recovered after microwave assisted water extraction were analyzed by Fourier transform infrared attenuated total reflectance, high-performance size-exclusion chromatography and rheology. The corresponding soluble extracts were also characterized by total phenolic content, antioxidant capacity, proteins, sulfate and carbohydrate content. The highest phenolic, antioxidant capacity and sulfate content were observed at 190 °C, without notable effect of heating time between 3 and 6 min. In contrast, a clear impact of both microwave heating temperature and time on the molecular weight distribution of hybrid carrageenans as well as on the viscoelastic behavior of formulated hydrogels was identified, being the strongest hydrogels achieved at 170 °C for 6 min. It should be highlighted that all proposed hydrogels exhibited stable mechanical properties in the absence of syneresis for a month of cold storage, except those made with biopolymers obtained with the mildest extraction conditions (3 min at 70 °C).

A carrageenan/agarose composite sponge and its immunomodulatory activities toward RAW264.7.

A kind of commercial hybrid carrageenan (HC)/agarose composite sponge containing κ-, μ-, ι-, and ν-carrageenan, which could turn into hydrogel and release carrageenan at human body temperature was fabricated for immune stimulation and modulation. Release behavior demonstrated that the hybrid carrageenan contained sponge was mechanically stable and could release carrageenan constantly. RT-PCR and ELISA experiments showed that the leaching liquor of the sponge could stimulate RAW264.7 from M0 state to a polarized state by secreting more anti-inflammatory factor IL-10 than pro-inflammatory ones, such as, IL-6 and TNF-α. Transwell experiments also indicated that the leaching liquor could promote the proliferation of NIH-3T3 by stimulating RAW264.7 of M0 state after 7 days. Results of particle size and intracellular concentration analyses suggested that the released carrageenan might enter into the cellular interior of RAW264.7 in the form of microgels or protein complexes. The sponge would be a promising candidate for skin wound dressing.

Antitumour Potential of Gigartina pistillata Carrageenans against Colorectal Cancer Stem Cell-Enriched Tumourspheres.

Gigartina pistillata is a red seaweed common in Figueira da Foz, Portugal. Here, the antitumour potential of G. pistillata carrageenan, with a known variable of the life cycle, the female gametophyte (FG) and tetrasporophyte (T) was evaluated against colorectal cancer stem cell (CSC) -enriched tumourspheres. FTIR-ATR analysis of G. pistillata carrageenan extracts indicated differences between life cycle phases, being FG a κ/ι hybrid carrageenan and T a ʎ/ξ hybrid. Both carrageenan extracts presented IC50 values inferior to 1 μg/mL in HT29-derived CSC-enriched tumourspheres, as well as reduced tumoursphere area. The two extracts were also effective at reducing cellular viability in SW620- and SW480-derived tumourspheres. These results indicate that carrageenans extracted from two G. pistillata life cycle phases have antitumour potential against colorectal cancer stem-like cells, specially the T carrageenan.

Impact of counterions on the thermo-rheological features of hybrid carrageenan systems isolated from red seaweed Gigartina skottsbergii

The effect of different counterions (Na+, K+ and Ca2+) on the sol-gel transition diagrams of kappa/iota-hybrid carrageenan isolated from Gigartina skottsbergii, together with the thermo-rheological features of the obtained gels are presented. Extraction yield (47.8 ± 3.5 g/100 g), molar fractions of kappa (62 ± 2) and iota (38 ± 2) and polymers with molecular masses between 7.5 104 and 7 103 g/mol were also determined for G. skottsbergii extracts. Gels formed in NaCl exhibited stable and weak gel properties. Gels made in KCl and CaCl2 featured stable and intermediate gel characteristics, except those formulated at the largest carrageenan and salt content that can be considered strong. All gels achieved stability after 15 min of maturation. No water syneresis was identified in tested gels. Gel setting and melting temperatures depended on the biopolymer and ionic contents. Gels formed in CaCl2 at the largest biopolymer content and ionic concentration showed thermo-reversible features.

A Novel Enzyme Portfolio for Red Algal Polysaccharide Degradation in the Marine Bacterium Paraglaciecola hydrolytica S66T Encoded in a Sizeable Polysaccharide Utilization Locus.

Marine microbes are a rich source of enzymes for the degradation of diverse polysaccharides. Paraglaciecola hydrolytica S66T is a marine bacterium capable of hydrolyzing polysaccharides found in the cell wall of red macroalgae. In this study, we applied an approach combining genomic mining with functional analysis to uncover the potential of this bacterium to produce enzymes for the hydrolysis of complex marine polysaccharides. A special feature of P. hydrolytica S66T is the presence of a large genomic region harboring an array of carbohydrate-active enzymes (CAZymes) notably agarases and carrageenases. Based on a first functional characterization combined with a comparative sequence analysis, we confirmed the enzymatic activities of several enzymes required for red algal polysaccharide degradation by the bacterium. In particular, we report for the first time, the discovery of novel enzyme activities targeting furcellaran, a hybrid carrageenan containing both β-carrageenan and κ/β-carrageenan motifs. Some of these enzymes represent a new subfamily within the CAZy classification. From the combined analyses, we propose models for the complete degradation of agar and κ/β-type carrageenan by P. hydrolytica S66T. The novel enzymes described here may find value in new bio-based industries and advance our understanding of the mechanisms responsible for recycling of red algal polysaccharides in marine ecosystems.

Hybrid carrageenans as beer wort fining agents

Pure Na+ forms of furcellaran (β/κ-hybrid carrageenan), κ-carrageenan, κ/ι-hybrid carrageenan, ι-carrageenan and a hybrid carrageenan mix (made up of κ-, ι-, λ- and ν-carrageenans) were tested as beer wort fining agents. To find out which of the used carrageenans are the best beer wort clarifiers and whether they are prone to overdosage, polysaccharides with varying concentrations and molecular weights were tested. The polysaccharides were characterized by 1H NMR spectroscopy and size-exclusion chromatography. The fining efficiency of the tested polysaccharides was measured nephelometrically. Ultrasonicated and alkali treated carrageenan preparations were tested as beer wort fining agents and compared to the native samples. Undegraded furcellaran, κ-carrageenan and κ/ι-hybrids were efficient in lowering the turbidity of various beer worts, but the addition of ι-carrageenan and the carrageenan mix caused the turbidity to rise above the unfined sample value. Compared to the degraded samples, native galactans were more effective in lowering the beer wort turbidity. Higher concentrations of ultrasonicated carrageenans are needed to clarify the beer wort to similar turbidity values. Alkali treated carrageenans were more efficient beer wort fining agents compared to the native samples.

Effect of pre-extraction alkali treatment on the chemical structure and gelling properties of extracted hybrid carrageenan from Chondrus crispus and Ahnfeltiopsis devoniensis

Different alkali pre-treatments were applied to Chondrus crispus and Ahnfeltiopsis devoniensis seaweeds to investigate the effect of alkali type, concentration and treatment duration on the chemical structure and the gelling properties of extracted hybrid carrageenan (HK). In both seaweeds, increasing the KOH content and the pre-treatment duration gives HK with lower amounts of ν-carrageenan units, which have a direct impact on the improved gelling properties. Nevertheless, HK with smaller molecular mass and depressed gel properties are found when excessive KOH content and pre-treatment time are employed. NaOH is more efficient in converting ν-carrageenan to ι-carrageenan for extracts from C. crispus, whereas NaOH and KOH showed similar results for extracts from A. devoniensis. No correlation between the chemical structure and gel properties was found. A set of HK with 75–80 and 45–50 mol% of κ-carrageenan for C. crispus and A. devoniensis, respectively, and tailored gelling properties for a wide range of applications is presented.

Hybrid carrageenan‐based formulations for edible film preparation: Benchmarking with kappa carrageenan

ABSTRACTAn intense search for new renewable sources to produce natural polymers for edible and biodegradable packaging is observed as they offer lower environmental costs. The objective of this work is to investigate the use of hybrid carrageenan, extracted from Mastocarpus stellatus seaweeds, as an alternative to commercial kappa carrageenan in new edible film formulations. To this end, the production and characterization of biodegradable films obtained with mixtures of rice starch and hybrid carrageenan or commercial kappa carrageenan were carried out. Thin, flexible, and transparent films with attractive functional properties were obtained from rice starch–carrageenan mixtures. Films forming solutions produced with hybrid carrageenan showed rheological properties comparable to commercial kappa carrageenan‐based solutions. Films formulated with hybrid carrageenan show significantly enhanced UV barrier, oxygen barrier, and hydrophobic properties. Thus, hybrid carrageenan proved to be a promising material for the production of edible coatings and biodegradable films and a good alternative to kappa carrageenan for such application. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016, 133, 42263.

Culture of the red alga Sarconema filiforme in open waters and hybrid carrageenan from the cultivated seaweeds

This is a first time report on the successful cultivation of the red alga Sarconema filiforme, employing floating rafts along the south-east coast of India, with an associated study of carrageenan content. Experiments were carried out on seasonality of growth rate and biomass of S. filiforme for 30 months, from August 2010 to January 2013. Harvest was done at the end of 25-day growth. In addition, experiments were also carried out optimizing harvests at the end of different duration of culture period (i.e. 20-, 25- and 30-day culture), and measurements were made at different localities (e.g. Ervadi and Thonithurai). Maximum biomass density (2.28 ± 0.03 kg fresh wt. m−2) and daily growth rates (DGR; 11.63 ± 0.06 %) were found from August to September each year, during the study, and these values were significantly different. Harvesting at the end of 25-day duration of culture period resulted in a maximum biomass (i.e. 4.24 ± 0.95 kg fresh wt. m−2), while 20-day duration of culture period showed higher DGR i.e. 13.20 ± 0.20 % which were significantly different from those plants harvested after 30 days. The range of biomass (i.e. 2.22 to 6.46 kg fresh wt. m−2) and DGR (i.e. 5.0 to 10.91 %) was higher at Ervadi than at Thonithurai site and was significantly different (P < 0.001). Physico-chemical analyses, infra-red and 13C nuclear magnetic resonance (NMR) spectral analyses of extracted carrageenan revealed the presence of hybrid lambda and iota carrageenan. The carrageenan yield from the cultivated material was higher than that of the wild stock of S. filiforme of Indian waters. The results of the study revealed that the peak growth period of S. filiforme was between September and December. Harvesting at the end of the 25-day growth period was more ideal. This study further revealed that production of better strains having hybrid, sulphated galactan, with higher yield and rheological properties the same as the wild stocks, could be possible through cultivation.

Natural Giesekus fluids: Shear and extensional behavior of food gum solutions in the semidilute regime

The shear and extensional behavior of two aqueous gum solutions, namely (1) 1–20 g/L guar gum (Torres et al., Food Hydrocolloids. 2014;40:85–95) and (2) κ/ι‐hybrid carrageenan solutions (5–20 g/L), are shown to exhibit Giesekus‐fluid behavior when in the semidilute regime. In this regime, a common set of Giesekus fluid parameters described both shear and extensional behavior. A new analytical result describing the extension of a Giesekus fluid in the filament stretching geometry is presented. This also gave reasonable predictions of the Trouton ratio. Higher concentration guar solutions, in the entangled regime, yielded different Giesekus fluid parameters for extension to those for simple shear. The extensional data for all concentrations of both gums collapsed to a common functional form, similar to that reported for cake batters (Chesterton et al., J Food Eng. 2011;105(2):332–342); the limits of the new filament thinning expression provide insight into this behavior. © 2014 American Institute of Chemical Engineers AIChE J, 60: 3902–3915, 2014