Molecular weight distribution of carrageenans by size exclusion chromatography and low angle laser light scattering

A procedure to determine the absolute weight average molecular weight and molecular weight distribution of carrageenans by high pressure aqueous size exclusion chromatography coupled with low angle laser light scattering is described. Experimental parameters are successively discussed, particular attention being focused on the absence of shear degradation during elution. The distribution curves were highly reproducible in time and weight average molecular weights integrated along the chromatogram were in good agreement with static light scattering results. A large difference in the molecular weight range between native (food-grade) and acidic degraded carrageenan samples was observed. Weight average molecular weights were found to be in good correlation with viscosity values, for degraded as well as undegraded products. It is also shown that the method described can help people using carrageenans in pharmacological studies by providing information on the real molecular weight distribution of the products they are employing.     

Aspects of the farming and processing of Kappaphycus and Eucheuma in Indonesia

Indonesia has become the largest exporter of Eucheuma denticulatum, supplying the foreign demand for iota-carrageenan, and the increasing demand of the Asian food markets. Recent demand fluctuations for Kappaphycus alvarezii and E. denticulatum have resulted in price instability, and slowed the expansion of the less viable raft culture practice. A short 42–45 day harvest interval, propagule selection, and post-harvest treatment remain the causes of poor crop quality and low carrageenan yields.In 1988 the first processing of K. alvarezii commenced. By 1991 there were four factories exporting an alkali-treated chip product, and one producing carrageenan powder. In 1991, 3200t dry weight of the K. alvarezii harvest was processed locally. The viability of future natural food-grade carrageenan production is discussed in relation to new developments in the industry.     

A method for lipase co-precipitation in a biodegradable protein matrix

This article presents a novel method for immobilization of active ingredients. The method is based on CO(2) aided active ingredient co-precipitation with glycinin, a biodegradable protein matrix from edible soybean protein. Glycinin precipitates abundantly under isoelectric conditions and serves as the matrix within which the active substance is trapped during the precipitation process. The enzyme lipase from Candida rugosa was successfully co-precipitated into the protein pellet to prove the principle. It was shown that the lipase within the co-precipitate retained lipase and esterase activity under different pH conditions. In some cases the activity was even higher than the activity of crude lipase, possibly due to the protective role of the matrix protein. Due to the retained lipase activity and food-grade quality of the binary precipitate, it has potential of being used in the food or pharmaceutical industry. Additional quality of the binary precipitate is the potentially significantly reduced downstream processing due to the fact that no organic solvents or precipitants were used in the precipitation process.     

Analysis of the conformational properties of κ‐ and ι‐carrageenan by size‐exclusion chromatography combined with low‐angle laser light scattering

The conformational properties of κ‐carrageenan in 0.2M LiI and ι‐carrageenan in 0.2M LiCl were analyzed by size exclusion chromatography combined with low‐angle laser light scattering. Fractionated samples with narrow molecular weight distributions (M_w/M_n ∼ 1.4) were used, and M_w in the disordered states were 35,000 (κ‐35) and 200,000 (κ‐200) for κ‐carrageenan and 65,000 (ι‐65) and 170,000 (ι‐170) for ι‐carrageenan, respectively. The analyses were performed across a temperature range where the conformational transitions occurred, and at extremely low concentrations (2–50 μg/mL) due to low amounts of samples injected and the subsequent dilution occurring during the separation. The results indicate that a twofold increase of the molecular weight (M_w) occurs for κ‐carrageenan upon inducing the ordered conformation. For ι‐carrageenan an additional increase in M_w may take place, which is attributed to the strong tendency for aggregation of ordered chains especially at high molecular weights. The results thus suggest that both κ‐ and ι‐carrageenan are double (or multiple) stranded in their ordered conformations, within the concentration range studied here. © 1999 John Wiley & Sons, Inc. Biopoly 49: 71–80, 1999     

Double Emulsions Stabilized by Food Biopolymers

Double emulsions of the water-in-oil-in-water (W/O/W) type have application in the formulation of reduced-fat food products and as vehicles for encapsulation and delivery of nutrients during food digestion. Progress in the development of stable double emulsions for food use is dependent on replacing small-molecule emulsifiers and synthetic polymeric stabilizing agents by food-grade ingredients. Of particular value for conferring the required functionality are food proteins and polysaccharides. This review describes how these biopolymers have been successfully incorporated into the internal and external aqueous phases of W/O/W emulsions to improve the stability and yield of model systems. Recent advances in the use of protein–polysaccharide conjugates and complexes for the stabilization of the outer droplets of W/O/W emulsions are highlighted.     

Recent developments in manufacturing and marketing carrageenan

Carrageenan has annual sales of over US$ 200 million, about 15% of the world use of food hydrocolloids. The market for carrageenan has grown exponentially at 5% per year for at least 25 years: 5 500 metric tons in 1970, and over 20 000 metric tons expected in 1995. The industry has become dominated by very large, multi-product companies with carrageenan factories in Europe and the US, but factories are now springing up in the Philippines and Chile, where red seaweeds grow in abundance. About 80 000 tons of dry red seaweeds are needed to produce 20 000 tons of carrageenan. About 40 000 tons comes from the Philippines, 15 000 tons from Indonesia, 15 000 tons from Chile, and 10 000 tons from elsewhere. Carrageenan growth depends on food fads like the McLean hamburger and food winners like processed pork and turkey. Carrageenan is a regulated food additive, and current health concerns focus on the minimum safe molecular weight for carrageenan when eaten. The most innovative development in carrageenans in recent years has been the introduction of a food grade version of lower cost natural grade carrageenan. Its acceptance, however, has been hampered by strong resistance from conventional carrageenan producers.     

Bacteriocin Production and Different Strategies for Their Recovery and Purification

Bacteriocins from lactic acid bacteria (LAB) are a diverse group of antimicrobial proteins/peptides, offering potential as biopreservatives, and exhibit a broad spectrum of antimicrobial activity at low concentrations along with thermal as well as pH stability in foods. High bacteriocin production usually occurs in complex media. However, such media are expensive for an economical production process. For effective use of bacteriocins as food biopreservatives, there is a need to have heat-stable wide spectrum bacteriocins produced with high-specific activity in food-grade medium. The main hurdles concerning the application of bacteriocins as food biopreservatives is their low yield in food-grade medium and time-consuming, expensive purification processes, which are suitable at laboratory scale but not at industrial scale. So, the present review focuses on the bacteriocins production using complex and food-grade media, which mainly emphasizes on the bacteriocin producer strains, media used, different production systems used and effect of different fermentation conditions on the bacteriocin production. In addition, this review emphasizes the purification processes designed for efficient recovery of bacteriocins at small and large scale.     

Separation and quantification of the cellular thiol pool of pea plants treated with heat, salt and atrazine

A novel procedure for the separation of the cellular thiol pool according to the molecular weight and localization of compounds with sulphydryl groups is presented. This simple and rapid method allows the differentiation of thiols into three major fractions-low molecular weight (LMT, primarily glutathione and free cysteine), protein-bound (TPT) and pellet-bound (PBT, associated with cell walls and broken organelles). Moreover, determination of the ratio between surface (readily reactive) thiols (ATG) and those that are more or less buried in the protein structure (BTG) can be achieved. In intact pea leaves, the amounts of the total thiols (LMT+PBT+TPT) varies from 2.5 to 4.8 micromol/g of fresh material. The data for LMT, PBT and TPT were related to each other in the approximate ratio 1:2:7. Treatments of pea plants with high temperature, salinity and low amounts of atrazine affect these sulphydryl types differently. For a greater understanding of the applicability of this method to physiological research, the main mechanisms leading to alterations in the cellular thiol pool are discussed. Furthermore, it is suggested that the proportion of available to buried thiols (ATG/BTG) in proteins could be used as a convenient marker for stress impacts.     

Nutraceutical production with food-grade microorganisms

Over the past few years a number of new food ingredients labelled as being nutraceuticals have been launched on the food and pharmaceutical market. These include components that have a proven beneficial effect on human health, such as low-calorie sugars and B vitamins. Lactic acid bacteria, in particular Lactococcus lactis, have been demonstrated to be ideal cell factories for the production of these important nutraceuticals. Developments in the genetic engineering of food-grade microoganisms means that the production of certain nutraceuticals can be enhanced or newly induced through overexpression and/or disruption of relevant metabolic genes.     

The potential of microalgae and their biopolymers as structuring ingredients in food: A review

Microalgae are considered promising functional food ingredients due to their balanced composition, containing multiple nutritional and health-beneficial components. However, their functionality in food products is not limited to health aspects, since microalgae can also play a structuring role in food, for instance as a texturizing ingredient. Photoautotrophic microalgae are actually rich in structural biopolymers such as proteins, storage polysaccharides, and cell wall related polysaccharides, and their presence might possibly alter the rheological properties of the enriched food product. A first approach to benefit from these structural biopolymers consists of isolating the cell wall related polysaccharides for use as food hydrocolloids. The potential of extracted cell wall polysaccharides as food hydrocolloids has only been shown for a few microalgae species, mainly due to an enormous diversity in molecular structure and composition. Nevertheless, with intrinsic viscosities comparable or higher than those of commercial thickening agents, extracellular polysaccharides of red microalgae and cyanobacteria could be a promising source of novel food hydrocolloids. A more sustainable approach would be to incorporate the whole microalgal biomass into food products, to combine health benefits with potential structuring benefits, i.e. providing desired rheological properties of the enriched food product. If microalgal biomass would act as a thickening agent, this would actually reduce the need for additional texturizing ingredients. Even though only limitedly studied so far, food processing operations have been proven successful in establishing desired microstructural and rheological properties. In fact, the use of cell disruption techniques allows the release of intracellular compounds, which become available to create strong particle aggregates resulting in an improved viscosity and network structure. Food processing operations might not only be favorable in terms of rheological properties, but also for enhancing the bioaccessibility of several bioactive compounds. However, this research area is only very scarcely explored, and there is a demand for more standardized research studies to draw conclusions on the effect of processing on the nutritional quality of food products enriched with microalgae. Even though considered as promising food ingredients, some major scientific challenges have been pointed out throughout this review paper for the successful design of microalgal based food products.     

Carrageenan induces interleukin-8 production through distinct Bcl10 pathway in normal human colonic epithelial cells

Carrageenan is a high molecular weight sulfated polygalactan used to improve the texture of commercial food products. Its use increased markedly during the last half century, although carrageenan is known to induce inflammation in rheumatological models and in intestinal models of colitis. We performed studies to determine its direct effects on human intestinal cells, including normal human intestinal epithelial cells from colonic surgeries, the normal intestinal epithelial cell line NCM460, and normal rat ileal epithelial cells. Cells were treated with high molecular weight lambda-carrageenan at a concentration of 1 mug/ml for 1-96 h. IL-8, IL-8 promoter activity, total and nuclear NF-kappaB, IkappaBalpha, phospho-IkappaBalpha, and Bcl10 were assessed by immunohistochemistry, Western blot, ELISA, and cDNA microarray. Increased Bcl10, nuclear and cytoplasmic NF-kappaB, IL-8 promoter activation, and IL-8 secretion were detected following carrageenan exposure. Knockdown of Bcl10 by siRNA markedly reduced the increase in IL-8 that followed carrageenan exposure in the NCM460 cells. These results show, for the first time, that exposure of human intestinal epithelial cells to carrageenan triggers a distinct inflammatory pathway via activation of Bcl10 with NF-kappaB activation and upregulation of IL-8 secretion. Since Bcl10 contains a caspase-recruitment domain, similar to that found in NOD2/CARD15 and associated with genetic predisposition to Crohn's disease, the study findings may represent a link between genetic and environmental etiologies of inflammatory bowel disease. Because of the high use of carrageenan as a food additive in the diet, the findings may have clinical significance.     

Molecular weight of scleroglucan and other extracellular microbial polysaccharides by size-exclusion chromatography and low angle laser light scattering

High-performance aqueous size-exclusion chromatography coupled to a low angle laser light scattering detector has been applied to the analysis of scleroglucan and various other extracellular microbial polysaccharides. Emphasis has been focused on three main findings. (1) The molecular weight of these macromolecules is not very sensitive to changes in fermentation conditions. This is specially true in the case of scleroglucan and related (1 → 3)-β- -glucans including schizophyllan, which all exhibited a constant weight-average molecular weight of 5·7×10⁶±5%. (2) In contrast to plant polysaccharides, polydispersity is very low, usually near unity. (3) The molecular weight levels off quickly during biosynthesis since the molecular weight is constant from the middle of fermentation, if not before.     

Purification and characterisation of a fructosyltransferase from Rhodotorula sp

The present work was devoted to investigations concerning the purification and characterisation of the fructooligosaccharide (FOS)-producing extracellular enzyme of Rhodotorula sp. LEB-V10. FOS are functional food ingredients showing prebiotic properties, meaning that it could stimulate selectively the growth and/or activity of probiotic bacteria in the gut. The purification of the enzyme was carried out according to the following sequential procedure: cell separation by centrifugation, recovering by ethanol precipitation and purification by anion exchange chromatography. The molecular weight was estimated to be 170 kDa by preparative gel filtration and 77 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, signifying that the native enzyme exists as a dimer. With sucrose as substrate, the data failed to fit the Michaelis-Menten behaviour, rather showing a sigmoid shape similar to that of the allosteric enzymes (cooperative behaviour), requiring high sucrose concentrations to obtain high reaction rates. The enzyme showed both fructofuranosidase (FA) and fructosyl-transferase (FTA) activities. The optimum pH and temperature for FA activity were found to be around 4.0 and 72-75 degrees C, respectively, while FTA showed optimum activity at pH 4.5 and 65-70 degrees C. Both activities were very stable at temperatures below 66 degrees C, while for FA, the enzyme was more stable at pH 4.0 and for FTA at pH 5.0.     

Nisin-inducible secretion of a biologically active single-chain insulin analog by Lactococcus lactis NZ9000

Oral delivery of insulin to diabetic patients is highly desirable because it would be non-invasive and more closely mimic normal physiology, but this route of administration typically results in low bioavailability due to low pH and enzymatic degradation along the gastrointestinal tract. To explore an alternative approach that may mitigate these obstacles and also facilitate local synthesis of new therapeutic protein molecules in the small intestine, we engineered the food-grade bacterium Lactococcus lactis (NZ9000) for nisin-inducible expression and secretion of a bioactive single-chain insulin (SCI) analog, SCI-57. We show that the addition of nisin during early-log phase has a modest inhibitory effect on cell growth but induction during mid-log phase has a negligible impact on proliferation, suggesting a tradeoff between cell growth rate and duration of induction. We find that a signal peptide such as usp45 is necessary for secretion of SCI-57 into the medium; furthermore, we demonstrate that this secreted SCI-57 is biologically active, as assessed by the ability of conditioned L. lactis medium to stimulate Akt signaling in differentiated 3T3-L1 adipocytes. Finally, we show that the biological activity of SCI-57 was enhanced by near-neutral or slightly alkaline pH during induction, which is comparable to the pH in the small intestine, and by removal of a C-terminal purification tag. This study demonstrates that food-grade bacteria can be engineered to secrete bioactive insulin analogs and opens up the possibility of oral insulin delivery using live microorganisms.     

Encapsulation of tannase for the hydrolysis of tea tannins

Tannase was encapsulated in alginate, chitosan, carrageenan or pectin gel matrices, and in the case of alginate, coated with high or low molecular weight chitosan to reduce enzyme release. Cross-linking with glutaraldehyde also improved enzyme retention. Active enzyme preparations were obtained, although carrageenan gels were unstable in tea. Tannase activity was evaluated by reduction in centrifugable (flocculated) tea solids, and a reduction in tea cream measured turbidimetrically after removal of flocculated solids. Tannin interactions with the polysaccharide gels increased the level of centrifugable solids (flocculent) in the tea. An optimum bead formulation consisted of an alginate core, coated with chitosan and cross-linked with glutaraldehyde. Both core and coating materials contained active enzyme. Beads were prepared in a single step procedure involving extrusion of alginate/tannase solution into a hardening bath containing tannase-loaded, chitosan solution. Tannase retained hydrolytic activity through three successive batch cycles, for a total period of 39h processing, and tea cream was visibly removed by treatment with the immobilized tannase. Activity remained stable during 1-month bead storage under refrigeration.     

Molecular characterisation of kappa- and lambda-carrageenan by gel permeation chromatography, light scattering, sedimentation analysis and osmometry.

Gel permeation chromatography, in conjunction with a double detection system involving a low angle laser light scattering apparatus (LALLS) and a refractive index monitoring device (RI), has been used to obtain both the molecular weight and the molecular weight distribution of sodium salts of kappa-carrageenan and lambda-carrageenan in saline solutions. The results, Mw and Mn, are in excellent agreement with independent determinations of molar mass based on static light scattering experiments, sedimentation-diffusion analysis and osmometry. The relevance of the data is discussed with respect to current problems in carrageenan research.     

Electrodissociation of high molecular weight complexes of interferon alpha during recycling isoelectric focusing

Natural human interferon alpha has been separated by selective ultrafiltration into low molecular weight components and the molecules exceeding 100K daltons. Interferon associated with a higher molecular weight fraction showed partial pH sensitivity and resisted dissociation after treatment with urea, mercaptoethanol, sodium chloride or significant changes in pH. However, interferon activity was released from high molecular weight components during recycling isoelectric focusing. Electrodissociation was carried out in 1% ampholytes for 574 watt-hours. The interferon activity was concentrated in a pH range of 6-6.5, whereas, the majority of proteins were generally found in a more acidic position. The dissociated interferon was neutralized by polyclonal antibody to human interferon alpha (IFN alpha) and showed no presence of pH labile form. A pH sensitivity of high molecular weight interferon (HMW-IFN) may reflect an aggregation phenomenon rather than intrinsic structural differences.     

Crystallization for the Downstream Processing of Proteins

Protein crystallization offers great potential in downstream processing of pharmaceutical protein active ingredients. The advantages, which are well known and widely utilized in low‐molecular weight crystallization, can also be expected to be found to some extent in protein crystallization. However, there is still a marked need for improvement in two main areas of protein processing, namely, in crystallization from impure solutions and scale‐up.     

Interaction of degraded iota carrageenans with plasma membranes: Sedimentation of erythrocytes of different species

The sedimentation of erythrocytes by polydisperse fractions of iota carrageenan has been studied. For human erythrocytes, it is found that their sedimentation is dependent on the number average molecular weight of the carrageenan fraction, the sedimentation rate increasing generally as the molecular weight of the fraction increases until a maximum value is reached, after which the sedimentation rate decreases. The increase in sedimentation of erythrocytes can be reversed by removal of the carrageenan. Addition of EDTA to the carrageenan-erythrocyte medium does not effect the increase in erythrocyte sedimentation rate by carrageenan. The carrageenan-facilitated sedimentation of erythrocytes exhibits wide species differences. The mechanism by which iota carrageenans enhance erythrocyte sedimentation is discussed.     

Reply to comments regarding “The Carrageenan Controversy”

Carrageenan has been used in the laboratory for decades because it reliably causes inflammation. Coincident with its use in the laboratory, carrageenan has been used in an ever-increasing list of processed foods, as well as in infant formula, pharmaceuticals, cosmetics, toothpaste, room air deodorizers, and other products. The biological reactivity of carrageenan makes it a useful substance to improve the texture and solubility of ingredients in processed foods. Individuals may consume several grams of carrageenan on a daily basis. However, the potential contribution of carrageenan exposure to chronic human diseases, due to its activation of immune responses and its potent inflammatory properties, must be recognized.