Sorbitol Production Research Articles

Transient hyperopic refractive changes in newly diagnosed juvenile diabetes.

To investigate transient hyperopic refractive changes in newly diagnosed juvenile diabetics and to evaluate their clinical course after starting intensive glycemic control of severe hyperglycaemia. 20 hyperopic adolescents with newly diagnosed and uncomplicated type 1 diabetes, selected for this prospective study, were enrolled for a baseline examination and, after starting intensive insulin treatment, followed every two weeks during a four-month follow up. Standardised automated refraction and A-mode ultrasonography were performed. Poor metabolic control was an inclusion criteria. Refractive changes and hyperopic peaks preceded the start of the intensive insulin therapy in all diabetics and, thereafter, refraction decreased gradually with a maximum recovery time of 94 days. A statistically significant positive correlation between refractive changes and magnitude of plasma glucose concentrations as well as HbA1c percentages was observed (P <0.001). No significant modifications in the explored refractive components were recorded. Transient hyperopic changes are highly dependent on the magnitude of plasma glucose concentrations and rapid correction of hyperglycemia is strictly correlated with complete recovery of refraction. To account for this phenomenon, the sorbitol production via the polyol pathway with overhydration of the lens remains the best pathophysiological hypothesis at this time.

Effects of Water Stress on Sorbitol Production and Anatomical Changes in the Nuclei of Leaf and Root Cells of Young Loquat Trees

The effects of water stress on the production of sorbitol in the leaves and roots of one-year-old grafted loquat trees were investigated. Concurrently anatomical changes in the nuclei of the leaves and the root apical meristem were followed by staining with 4'-6-diamidino-2-phenylindole (DAPI) and observing the results by fluorescence microscopy. The degree of water stress was graded into three levels: a) unstressed plots (control), b) moderately stressed plots (m-plots, no irrigation period for 20 days), and c) severely stressed plots (s-plots, no irrigation period for 25 days). The degree of water stress was estimated by the water saturated deficiency (WSD) of the leaves. With an increase in WSD, the sorbitol content in the leaves and roots increased, the accumulation occurring in the roots. Although WSD for the s-plots was higher than that for the m-plots, there was no significant difference in sorbitol content in leaves and roots between the two treatments. After recovery from water stress, the sorbitol content decreased gradually in roots and leaves. Leaves of m-plots abscised little after irrigation was resumed, whereas leaves of s-plots abscised severely; only a few trees died. New roots developed in control plants, but not in stressed-plants. Water stress resulted in an acceleration of nuclear degradation and a decrease in starch accumulation in the mesophyll parenchyma and in the apical meristem from the root tip to 2-3 mm.

Transgenically enhanced sorbitol synthesis facilitates phloem‐boron mobility in rice

The tolerance of crops to a shortage of boron (B) in the soil varies markedly among species. This variation in tolerance is due, in part, to a species ability to form phloem mobile B‐sugar‐alcohol complexes (such as B‐mannitol or B‐sorbitol) which enhance the remobilization of B within the plant. Species lacking the capacity to form B‐sugar alcohol complexes are intolerant of even short‐term deficits in soil B supply. Here we have genetically engineered rice (Oryza sativa L.) cultivar Taipei 309 (TP309) with the sorbitol‐6‐phosphate dehydrogenase (S6PDH) gene, a key enzyme for sorbitol production, and determined the effect of this transformation on the physiology of B remobilization. Sorbitol was detected in the S6PDH transgenic plants as well as in vector‐transformed plants and wild‐type (TP 309) plants, although the concentration of sorbitol in the S6PDH transgenic plants was significantly enhanced. Remobilization of B from mature leaves to flag leaves correlated with increased levels of sorbitol. The presence of sorbitol and detection of B remobilization in the wild‐type and vector‐transformed plants suggests that rice utilizes an unknown pathway for sorbitol synthesis and may partly explain the relative insensitivity of rice to B deficits when compared to other graminaceous crops.

The biotechnological production of sorbitol.

Sorbitol, a polyol found in many fruits, is of increasing industrial interest as a sweetener, humectant, texturizer and softener. At present, it is produced chemically. The bacterium Zymomonas mobilis is able to produce sorbitol and gluconic acid from fructose and glucose, respectively. This is possible in a one-step reaction via a glucose-fructose oxidoreductase so far only known from Z. mobilis. The possibilities for the industrial production of sorbitol by Z. mobilis are discussed, and compared with the current chemical production method as well as other microbiological processes.

Crystal structure of the NADP(H)-dependent ketose reductase from Bemisia argentifolii at 2.3 Å resolution

Polyhydric alcohols are widely found in nature and can be accumulated to high concentrations as a protection against a variety of environmental stresses. It is only recently, however, that these molecules have been shown to be active in protection against heat stress, specifically in the use of sorbitol by the silverleaf whitefly, Bemisia argentifolii. We have determined the structure of the enzyme responsible for production of sorbitol in Bemisia argentifolii, NADP(H)-dependent ketose reductase (BaKR), to 2.3 Å resolution. The structure was solved by multiwavelength anomalous diffraction (MAD) using the anomalous scattering from two zinc atoms bound in the structure, and was refined to an R factor of 21.9 % (Rfree=25.1 %). BaKR belongs to the medium-chain dehydrogenase family and its structure is the first for the sorbitol dehydrogenase branch of this family. The enzyme is tetrameric, with the monomer having a very similar fold to the alcohol dehydrogenases (ADHs). Although the structure determined is for the apo form, a phosphate ion in the active site marks the likely position for the adenyl phosphate of NADP(H). The catalytic zinc ion is tetrahedrally coordinated to Cys41, His66, Glu67 and a water molecule, in a modification of the zinc site usually found in ADHs. This modified zinc site seems likely to be a conserved feature of the sorbitol dehydrogenase sub-family. Comparisons with other members of the ADH family have also enabled us to model a ternary complex of the enzyme, and suggest how structural differences may influence coenzyme binding and substrate specificity in the reduction of fructose to sorbitol.

Intergeneric protoplast fusion between Kluyveromyces and Saccharomyces cerevisiae - to produce sorbitol from Jerusalem artichokes

The construction of inulin-assimilating and sorbitol-producing fusants was achieved by intergeneric protoplast fusion between Kluyveromyces sp. Y-85 and Saccharomyces cerevisiae E-15. The cells of parental strains were pretreated with 0.1% EDTA (w/v) and 2-mercaptoethanol (0.1%, v/v) and then exposed to 2.0% (w/v) Zymolase at 30 °C for 30–40 min. The optimized fusion condition demonstrated that with the presence of 30% (w/v) polyethylene glycol 6000 (PEG-6000) and 10 mM CaCl2 for 30 min, the fusion frequency reached 2.64 fusants/106 parental cells. The fusants were screened by different characters between two parental strains and further identified by DNA contents, inulinase activity and sorbitol productivity. One of the genetically stable fusants, Strain F27, reached a maximal sorbitol production of 4.87 g/100 ml under optimal fermentation condition.

Catalytic hydrogenation of fine chemicals: sorbitol production

Historically, skeletal nickel is the catalyst of choice in the production of sorbitol on industrial scale. A disadvantage of the use of skeletal nickel in the hydrogenation of glucose containing feedstocks to sorbitol, is the fact that a part of the nickel leaches. As a consequence, purification of the sorbitol is required, which renders this process economically less attractive. To meet the demand for non-leaching catalyst systems, ruthenium on carbon catalyst are presently becoming more important. In this paper, a comparison is made between different 5% Ru on carbon catalysts in the hydrogenation of a dextrose monohydrate and a very high maltose feedstock. The influence of the carbon support, the ruthenium metal location, and the applied ruthenium precursor are investigated. For comparison reasons, both skeletal Ni and Ru catalysts have been applied in the hydrogenation of dextrose to sorbitol. The effect of reaction temperature and pressure during the hydrogenation of a very high maltose containing feedstock is also investigated. It is shown that Ru catalysts are suitable alternatives for skeletal Ni catalysts in respect to re-usability, activity and selectivity.

Glucose transporters control gene expression of aldose reductase, PKCalpha, and GLUT1 in mesangial cells in vitro.

The process linking increased glucose utilization and activation of metabolic pathways leading to end-organ damage from diabetes is not known. We have previously described rat mesangial cells that were transduced to constitutively express the facilitative glucose transporter 1 (GLUT1, MCGT1 cells) or bacterial beta-galactosidase (MCLacZ, control cells). Glucose transport was rate limiting for extracellular matrix production in the MCGT1 cells. In the present work, we investigated the effect of GLUT1 overexpression in mesangial cells on aldose reductase (AR), protein kinase Calpha (PKCalpha), and native GLUT1 transcript levels, to determine whether changes in GLUT1 alone could regulate their expression in the absence of high extracellular glucose concentrations. MCGT1 cells grown in normal (8 mM) or elevated (20 mM) glucose had elevated abundance of AR, PKCalpha, and the native GLUT1 transcripts compared with control cells. AR protein levels, AR activity, sorbitol production, and PKCalpha protein content were also greater in the MCGT1 cells than in control cells grown in the same media. This is the first report of the concomitant activation of AR, PKCalpha, and GLUT1 genes by enhanced GLUT1 expression. We conclude that increased GLUT1 expression leads to a positive feedback of greater GLUT1 expression, increased AR expression and activity with polyol accumulation, and increased total and active PKCalpha protein levels, which leads to detrimental stimulation of matrix protein synthesis by diabetic mesangial cells.

Manipulation of in vivo sorbitol production alters boron uptake and transport in tobacco

Recent evidence that some species can retranslocate boron as complexes with sugar alcohols in the phloem suggests a possible mechanism for enhancing boron efficiency. We investigated the relationship between sugar alcohol (sorbitol) content, boron uptake and distribution, and translocation of foliar-applied, isotopically enriched 10B in three lines of tobacco (Nicotiana tabacum) plants differing in sorbitol production. In tobacco line S11, transformed with sorbitol-6-phosphate dehydrogenase, the production of sorbitol was accompanied by an increase in the concentration of boron in plant tissues and an increased uptake of boron compared with either tobacco line A4, transformed with antisense orientation of sorbitol-6-phosphate dehydrogenase, or wild-type tobacco (line SR1, zero-sorbitol producer). Foliar application of 10B to mature leaves was translocated to the meristematic tissues only in line S11. These results demonstrate that the concentration of the boron-complexing sugar alcohol in the plant tissue has a significant effect on boron uptake and distribution in plants, whereas the translocation of the foliar-applied 10B from the mature leaves to the meristematic tissues verifies that boron is mobile in sorbitol-producing plants (S11) as we reported previously. This suggests that selection or transgenic generation of cultivars with an increased sugar alcohol content can result in increased boron uptake, with no apparent negative effects on short-term growth.

Inhibition of aldose reductase in human erythrocytes by vitamin C

Ascorbic acid, or vitamin C, has been reported to lower erythrocyte sorbitol concentrations, and present studies were performed to determine the mechanism of this effect. Incubation of erythrocytes with increasing concentrations of glucose (5–40 mM) progressively increased erythrocyte sorbitol contents, reflecting increased flux through aldose reductase. At extracellular concentrations of 90 μM, both ascorbic acid and its oxidized form, dehydroascorbate, decreased intracellular sorbitol by 25 and 45%, respectively. This inhibition was not dependent on the extracellular glucose concentration, or on erythrocyte contents of free NADPH or GSH. To test for a direct effect of ascorbate on aldose reductase, erythrocyte hemolysates were prepared and supplemented with 100 μM NADPH. Hemolysates reduced glucose to sorbitol in a dose-dependent manner that was inhibited with a K i of 120 μM by the aldose reductase inhibitor tetramethylene glutaric acid. Above 100 μM, ascorbic acid also lowered hemolysate sorbitol generation by about 30%. Studies with ascorbic acid derivatives showed that the reducing capacity of ascorbic acid was not required for inhibition of sorbitol production from glucose in erythrocyte hemolysates. These results show that high, but physiologic, concentrations of ascorbic acid can directly inhibit erythrocyte aldose reductase, and provide a rationale for the use of oral vitamin C supplements in diabetes.

Aldose reductase: a window to the treatment of diabetic complications?

Kinetic studies on the aldose reductase protein (AR2) have shown that it does not behave as a classical enzyme in relation to ring aldose sugars. These results have been confirmed by X-ray crystallography studies, which have pinpointed binding sites for pharmacological "aklose reductase inhibitors" (ARIs). As with non-enzymic glycation reactions, there is probably a free-radical element involved derived from monosaccharide autoxidation. In the case of AR2, there is free radical oxidation of NADPH by autoxidising monosaccharides, enhanced in the presence of the NADPH-binding protein. Whatever the behaviour of AR2, many studies have showed that sorbitol production is not an initiating aetiological factor in the development of diabetic complications in humans. Vitamin E (alpha-tocopherol), other antioxidants and high fat diets can delay or prevent cataract in diabetic animals even though sorbitol and fructose levels are not modified; vitamin C acts as an AR1 in humans. Protein post-translational modification by glyc-oxidation or other events is probably the key factor in the aetiology of diabetic complications. There is now no need to invoke AR2 in xylitol biosynthesis. Xylitol can be produced in the lens from glucose, via a pathway involving the enzymes myo-inositol-oxygen oxidoreductase, D-glucuronate reductase. L-gulonate NAD(+)-3-oxidoreductase and L-iditol-NAD(+)-5-oxidoreductase, all of which have recently been found in bovine and rat lens. This chapter investigates the molecular events underlying AR2 and its binding and kinetics. Induction of the protein by osmotic response elements is discussed, with detailed analysis of recent in vitro and in vivo experiments on numerous ARIs. These have a number of actions in the cell which are not specific, and which do not involve them binding to AR2. These include peroxy-radical scavenging and recently discovered effects of metal ion chelation. In controlled experiments, it has been found that incubation of rat lens homogenate with glucose and the copper chelator o-phenanthroline abolishes production of sorbitol. Taken together, these results suggest AR2 is a vestigial NADPH-binding protein, perhaps similar in function to a number of non-mammalian crystallins which have been recruited into the lens. There is mounting evidence for the binding of reactive aldehyde moieties to the protein, and the involvement of AR2 either as a 'housekeeping' protein, or in a free-radial-mediated 'catalytic' role. Interfering with the NADPH binding and flux levels--possibly involving free radicals and metal ions--has a deleterious effect. We have yet to determine whether aldose reductase is the black sheep of the aldehyde reductase family, or whether it is a skeleton in the cupboard, waiting to be clothed in the flesh of new revelations in the interactions between proteins, metal ions and redox metabolites.

Simultaneous enzymatic synthesis of gluconic acid and sorbitol

The production of sorbitol and gluconic acid by isolated glucose-fructose oxidoreductase (GFOR) from Zymomonas mobilis has been studied in a convective, 100-mL loop reactor with tangential ultrafiltration. Using a dilution rate of 0.04/h and 5 kU/L GFOR, substrate conversion (3 M sugar) in a single stage was >85%, and productivities of 126 g sorbitol/(L x d) were obtained. At a constant recycle rate (3/min) and a membrane area of 50 cm2, the dilution rates (and thus productivities) were however limited by a more than 30-fold reduction of the permeate flow in the presence of high sugar and protein concentrations (5 g/L). Protein was added, together with 10 mM dithiothreitol, to improve the stability of GFOR during substrate turnover and crossflow filtration, thus leading to a stable operation of the enzyme reactor for at least 5 d.

ENZYMATIC CATALYSIS BY PERMEABILIZED CELLS

This paper presents an enzymatic process for sorbitol and gluconic acid production using cells of Zymomonas mobilis permeabilized with CTAB. Equimolar solutions of glucose and fructose (from 96.0 to 422.2 g/L) were used. In a batch reactor, conversions of 97% were attained after 15 to 20 hours of reaction. The effect of the initial concentration of the substrates was evaluated in experiments using 20% more and 20% less glucose than fructose. It was observed that the reaction performed with more fructose reached completion faster and with a higher value of conversion

Urea and methylamines have similar effects on aldose reductase activity.

The concentration of urea in renal medullary cells is sufficiently high to inhibit activity of many enzymes, yet the cells survive and function. The generally accepted explanation is the counteracting osmolytes hypothesis, which holds that methylamines, such as glycerophosphorylcholine (GPC) and glycine betaine (betaine), found in the renal medulla stabilize biological macromolecules and oppose the effects of urea. The present study tests this hypothesis by determining the effects of urea and methylamines, singly and in combination, on the activity of aldose reductase, an enzyme that is important in renal medullas for catalyzing production of sorbitol from glucose. In apparent contradiction to the counteracting osmolytes hypothesis, urea (1.0 M) and three different methylamines (trimethylamine N-oxide, betaine, and GPC; 0.5 M) all have similar and partially additive inhibitory effects. They all decrease substantially both the Michaelis constant (K(m)) and the maximum velocity (Vmax). Also a high concentration (0.5 M) of other organic osmolytes that are abundant in the renal medulla, namely inositol, sorbitol, or taurine, has a similar but lesser effect. KCl (0.3 M) causes a small increase in activity. We discuss the significance of these findings with regard to function of aldose reductase in the renal medulla and the counteracting osmolytes hypothesis.

Recombinant insulin-like growth factor I normalizes expression of renal glucose transporters in diabetic rats.

By immunocytochemistry we have studied the effect of recombinant human insulin-like growth factor I (rhIGF-I) on expression of renal GLUT-1, -2, and -5 in rats with streptozotocin (STZ)-induced diabetes. In the renal tubules of these rats, expression of GLUT-1 was reduced and that of GLUT-2 was increased. GLUT-1 expression was restored, and GLUT-2 expression was normalized by 2-wk administration of rhIGF-I. We have shown that GLUT-5 was expressed at the brush-border membrane of the proximal convoluted tubules (PCT) of the cortex and at the glomerular mesangial cells (GMC) in normal rat kidney. In the diabetic rats, GLUT-5 expression was increased at both sites, along with an increase of GLUT-2 expression at the basolateral membrane of PCT, and was decreased to normal level at both sites by treatment with rhIGF-I. Thus, like GLUT-2, GLUT-5 is suggested to regulate glucose reabsorption in PCT. The relationship between overexpression of GLUT-5 in GMC and accumulation of sorbitol and advanced glycosylation end products are discussed. Regulation of GLUT expression may play an important role on renal glucose homeostasis.

Improved operational stability of cell-free glucose-fructose oxidoreductase fromZymomonas mobilis for the efficient synthesis of sorbitol and gluconic acid in a continuous ultrafiltration membrane reactor

For the continuous, enzymatic synthesis of sorbitol and gluconic acid by cell-free glucose-fructose oxidoreductase (GFOR) from Zymomonas mobilis, the principal determinants of productivity have been identified. Most important, the rapid inactivation of the soluble enzyme during substrate conversion can be avoided almost completely when weak bases such as tris(hydroxymethyl)aminomethan or imidazol are used for the titration of the produced gluconic acid and when 5-10 mM dithiothreitol are added to prevent thiol oxidations. With regard to a long-term operational stability of the enzyme for continuous syntheses, thermal deactivation becomes significant at reaction temperatures above 30 degrees C. Without any additional purification being required, the crude cell extract of Z. mobilis can be employed in a continuous ultrafiltration membrane reactor over a time period of more than 250 h without significant decrease in substrate conversion or enzyme activity. The use of soluble GFOR thus appears to be an interesting alternative to employing permeabilized cells of Zymomonas for the production of sorbitol and gluconic acid and may be superior with regard to reactor productivities, at comparable operational stabilities.

Evaluation of the flocculent strain Zymomonas mobilis Z1‐81 for the production of sorbitol and gluconic acid

AbstractThe biological production of sorbitol and gluconic acid from glucose and fructose is normally performed with previously grown and concentrated Zymomonas mobilis suspensions, in reactions catalyzed by the enzymes glucose‐fructose oxidoreductase (GFOR) and glucono‐δ‐lactonase (GL). By using the flocculent strain Z. mobilis Z1‐81, mechanical methods for cell concentration are unnecessary. Flocculation of Z. mobilis Z1‐81 is seriously affected by both stirring and initial glucose concentration (S0). Under mild agitation conditions, with S0 = 100 g · 1−1, up to 76% of the final biomass was recovered by settling. At any impeller speed tested, similar specific GFOR/GL activities (8.4 to 9.5 U · g−1 cell) were measured. When S0 was increased to 144 and 210 g · 1−1, however, enzyme activities above 14 U · g−1 were found. Ideal pH and temperature values for GFOR/GL, in whole Z. mobilis Z1‐81 cells, are 6.4 and 39 °C, respectively. The Km and the theoretical Vmax are 250 g glucose · 1−1 and 26.6 g gluconic acid · g−1 cell · h−1. With the settled biomass, bioconversion of 300 g · 1−1 each of glucose and fructose led to gluconic acid and sorbitol yields between 91 and 92% of the theoretical maximum, with specific productivities of 1.09 and 1.00 g product · g−1 cell · h−1.

Influence of intra- and extracellular sucrases of Zymomonas mobilis on the ethanol production and by-product formation

A spontaneous mutant of Zymomonas mobilis LS1A lacking intracellular sucrase SacA was isolated from a levan-sucrase mutant of Z. mobilis LS1. The intracellular sucrase SacA does not have a role in sucrose hydrolysis and fermentation. The amount of the extracellular levansucrase SacB produced by the strain B-806 was about one third of the total sucrase activity. In the absence of the SacB, the strains LS1 and LS1A did not produce levan during sucrose fermentation. The extracellular sucrase SacC was sufficient for the complete hydrolysis of sucrose for fermen-tation. The low hydrolysis rate of sucrose was responsible for the increased amount of ethanol production (37.5 g/l to 44.2 g/l) and the decreased amount of sorbitol production (4.5 g/l to 1.2 g/l) by the strains LS1 and LS1A.

Characterization of the osmotic response element of the human aldose reductase gene promoter.

Aldose reductase (EC 1.1.1.21) catalyzes the NADPH-mediated conversion of glucose to sorbitol. The hyperglycemia of diabetes increases sorbitol production primarily through substrate availability and is thought to contribute to the pathogenesis of many diabetic complications. Increased sorbitol production can also occur at normoglycemic levels via rapid increases in aldose reductase transcription and expression, which have been shown to occur upon exposure of many cell types to hyperosmotic conditions. The induction of aldose reductase transcription and the accumulation of sorbitol, an organic osmolyte, have been shown to be part of the physiological osmoregulatory mechanism whereby renal tubular cells adjust to the intraluminal hyperosmolality during urinary concentration. Previously, to explore the mechanism regulating aldose reductase levels, we partially characterized the human aldose reductase gene promoter present in a 4.2-kb fragment upstream of the transcription initiation start site. A fragment (-192 to +31 bp) was shown to contain several elements that control the basal expression of the enzyme. In this study, we examined the entire 4.2-kb human AR gene promoter fragment by deletion mutagenesis and transfection studies for the presence of osmotic response enhancer elements. An 11-bp nucleotide sequence (TGGAAAATTAC) was located 3.7 kb upstream of the transcription initiation site that mediates hypertonicity-responsive enhancer activity. This osmotic response element (ORE) increased the expression of the chloramphenicol acetyltransferase reporter gene product 2-fold in transfected HepG2 cells exposed to hypertonic NaCl media as compared with isoosmotic media. A more distal homologous sequence is also described; however, this sequence has no osmotic enhancer activity in transfected cells. Specific ORE mutant constructs, gel shift, and DNA fragment competition studies confirm the nature of the element and identify specific nucleotides essential for enhancer activity. A plasmid construct containing three repeat OREs and a heterologous promoter increased expression 8-fold in isoosmotic media and an additional 4-fold when the transfected cells are subjected to hyperosmotic stress (total approximately 30-fold). These findings will permit future studies to identify the transcription factors involved in the normal regulatory response mechanism to hypertonicity and to identify whether and how this response is altered in a variety of pathologic states, including diabetes.

Improvement of the process for sorbitol production with Zymomonas mobilis immobilised in κ-carrageenan

The production of sorbitol by permeabilised cells of Zymomonas mobilis immobilised in κ-carrageenan has been investigated. Permeabilised cells were treated with cetyltrimethylammoniumbromide (CTAB) prior to immobilisation to cross-link the enzymes of the glucose-fructose oxidoreductase (GFOR). Rigidity of the immobilised beads was increased two-fold with 90% conversion efficiency by addition of 40% (w/v) polyols (glycerol 25 g + propylene glycol 5 g) to 60% (w/v) distilled water containing 2·5% (w/v) κ-carrageenan as final concentration, prior to immobilisation, κ-Carrageenan beads entrapping permeabilised cells were dried to improve bead rigidity and storage stability. During a semi-batch process for 72 h with dry beads, there was an improvement of the loss of enzyme activity (less than 10%). In a batch process, the K m, fructose value for the free cells, wet beads and dry κ-carrageenan beads were 71·7, 72·4 and 116·7 g litre −1, respectively. Higher productivity was obtained with a two-stage continuous packed bed reactor with both wet and dry κ-carrageenan beads.