Semi-refined Carrageenan Research Articles

Semi-refined carrageenan induces eryptosis in a Ca<sup>2+</sup>-dependent manner

Background: Semi-refined carrageenan (food additive E407a) is a widely used thickener, which has been reported to exert toxic and pro-inflammatory effects. In particular, there is accumulating evidence that it induces eryptosis, i.e. a programmed cell death of eryptocytes, via ROS-mediated pathways. However, the role of Ca2+-dependent mechanisms in E407a-induced eryptosis is not elucidated. Material and methods: Semi-refined carrageenan at concentrations of 0 mg/ml, 1 mg/ml, 5 mg/ml and 10 mg/ml was incubated with blood of intact female WAG rats (n=9) for 24 h in RPMI and fetal bovine serum. After 24 h, the samples were used to obtain erythrocyte suspensions. The obtained suspensions were stained with a Ca2+-sensitive FLUO4 AM probe (30 min, 2.5 µM). The fluorescence of FLUO4 in erythrocytes was detected by a BD FACSCanto II flow cytometer. Results: The intracellular Ca2+ levels are proportional to the fluorescence of FLUO4. The mean fluorescence intensities (MFI) were compared. Low levels (1 mg/ml) of E407a had no impact on Ca2+ concentrations in erythrocytes (p>0.05). On the contrary, high concentrations (5 mg/ml and 10 mg/ml) of this food additive promoted an increase in the intracellular Ca2+ levels. The MFI values were 2.3- and 2.5-fold higher, respectively (p<0.0001). In addition, the exposure to E407a at concentrations of 5 mg/ml and 10 mg/ml (p<0.0001) increased the percentage of cells with high FLUO4 fluorescence. Conclusion: Food additive E407a induces eryptosis in a Ca2+-dependent manner.

Effects of selected environmental conditions on growth and carrageenan quality of laboratory-cultured Kappaphycus alvarezii (Rhodophyta) in Fiji, South Pacific

The impacts of environmental conditions (temperature, salinity, and nutrients) on growth, semi-refined carrageenan yield, gel strengths, and viscosity were assessed on the red seaweed Kappaphycus alvarezii in laboratory culture. Results showed that K. alvarezii had a higher daily growth rate (0.94% day-1) at higher temperature (30 °C). Its semi-refined carrageenan yield was high (75.79%) at a combination of low salinity (25 ppt) and high temperature (30 °C). Conversely, gel strengths made out of semi-refined carrageenan from K. alvarezii cultured at low temperature (24 °C) demonstrated a higher compression (5.84 N) and high penetration strength (0.29 N). Highest viscosity (289.50 cP) was attained at a combination of high temperature, low salinity, and high nutrient concentration. Gels made from carrageenan of K. alvarezii grown in a higher temperature (30 °C) mixed with pineapple juice showed higher compression strength demonstrating that carrageenan extracted from cultured K. alvarezii is a good thickening agent for fruit jellies or similar food products that do not require refrigeration. This could benefit communities in rural, non-electrified communities/areas of Fiji and other South Pacific Islands that could use K. alvarezii as an alternative gelling agent in the making of food products contributing towards their food and economic security.

Semi-Refined Carrageenan Attenuates Lipopolysaccharide-Induced Reactive Oxygen Species Production in Peripheral Blood Mononuclear Cells and Cell Membrane Alterations in Leukocytes

Aim: To assess the effects of semi-refined carrageenan (E407a) on lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) generation in peripheral blood mononuclear cells (PBMCs) and LPS-mediated cell membrane alterations in leukocytes. Methods: Blood samples collected from 8 intact rats were incubated with E407a (10 mg/ml), E407a (50 mg/ml), LPS (1 µg/ml), E407a (10 mg/ml) + LPS (1 µg/ml), E407a (50 mg/ml) + LPS (1 µg/ml) and without those compounds (controls) for 2 h in RPMI 1640 medium enriched with 5% fetal bovine serum. ROS generation in PBMCs obtained from the incubated samples was estimated by flow cytometry using 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) staining. The impact of E407a, LPS and their mixture on leukocyte cell membranes was evaluated spectrofluorimetrically using the fluorescent probe 2-(2¢-hydroxy-phenyl)-5-phenyl-1,3-oxazole. Results: Expectedly, incubation with LPS induced ROS generation in PBMCs and decreased the lipid order of cell membranes in leukocytes. E407a alone was found to alter neither ROS production in PBMCs, nor membrane lipid order in leukocytes. Semi-refined carrageenan partially reduced LPS-mediated ROS overproduction in PBMCs and cell membrane alterations in leukocytes. Conclusion: E407a attenuates LPS-induced alterations of redox homeostasis in rat PBMCs and LPS-mediated modifications of cell membrane lipid order in leukocytes.

Incorporation of Various Nanoclays in Semi-refined Carrageenan Film Composite

In current investigation, various commercial nanoclays with different level of hydrophilicity i.e., hydrophilic bentonite (HB), cloisite 10A, and cloisite® 30B were incorporated to a hydrophilic semi-refined carrageenan (SRC) films to observe their presence in film matrix in regards with the final film’s properties. High shear mixing and sonication were applied to the film preparation to obtain high dispersion of nanoclays in the matrix. As expected, the hydrophilic clay showed better dispersion within the matrix as shown in Energy Dispersive X-Ray Spectroscopy (EDS) image. The more hydrophilic clay inclusion resulted in higher tensile strength, while the more hydrophobic resulted in higher stiffness of the films. The water vapour permeability was decreased in corresponded to the more hydrophobic clay incorporated, and the thermal properties of the films were overall enhanced by the nanoclays reinforcement. In general, nanoclays incorporation in SRC film improved the overall properties of the SRC film.

Effects of E407a on the viability, metabolic and functional activity of dermal fibroblasts

Aim. To evaluate the effects of semi-refined carrageenan (E407a) on the viability, metabolic and functional activity of skin fibroblasts in vitro. Materials and methods. Various concentrations of E407a (0 – 10 mg/ml) were incubated for 24 h with skin fibroblast cultures isolated from rat embryos. The viability of cells exposed to semi-refined carrageenan was assessed by the neutral red uptake assay. MTT test was selected to estimate the metabolic activity of cells. In addition, scratch assay was used to determine the functional activity of dermal fibroblasts. Results. Direct exposure to semi-refined carrageenan did not affect the viability of rat dermal fibroblasts, evidenced by the results of MTT and neutral red uptake assays. However, experimental data of MTT and scratch assays revealed that high concentrations of this food additive increased the metabolic activity of fibroblasts and diminished the motility of these cells, suggesting that E407a is cytotoxic at high concentrations. Conclusion. Food-grade semi-refined carrageenan is cytotoxic towards skin fibroblasts at high concentrations.

Experimental Evaluation of Food-Grade Semi-Refined Carrageenan Toxicity.

The safety of food additives E407 and E407a has raised concerns in the scientific community. Thus, this study aims to assess the local and systemic toxic effects of the common food additive E407a in rats orally exposed to it for two weeks. Complex evaluations of the effects of semi-refined carrageenan (E407a) on rats upon oral exposure were performed. Local effects of E407a on the intestine were analyzed using routine histological stains and CD68 immunostaining. Furthermore, circulating levels of inflammatory markers were assessed. A fluorescent probe O1O (2- (2′-OH-phenyl)-5-phenyl-1,3-oxazole) was used for evaluating the state of leukocyte cell membranes. Cell death modes of leukocytes were analyzed by flow cytometry using Annexin V and 7-aminoactinomycin D staining. Oral administration of the common food additive E407a was found to be associated with altered small and large intestinal morphology, infiltration of the lamina propria in the small intestine with macrophages (CD68+ cells), high systemic levels of inflammation markers, and changes in the lipid order of the phospholipid bilayer in the cell membranes of leukocytes, alongside the activation of their apoptosis. Our findings suggest that oral exposure to E407a through rats results in the development of intestinal inflammation.

Characteristics of Semi-Refined Carrageenan From Kappaphycus Seaweed Farmed in Coastal Waters of Northern Java, Indonesia

Kappaphycus seaweed is the primary source of carrageenan, a polysaccharide widely utilised as a thickening and gelling agent in many commercial products. Unlike pure refined carrageenans, semi-refined carrageenans (SRC) are considered more cost-efficient and easier to produce. This study evaluates the quality of SRC extracted from Kappaphycus alvarezii (Doty) Doty ex Silva 1996, and Kappaphycus striatus (F. Schmitz) Doty ex P.C. Silva 1996, seaweeds farmed in the northern part of Java Island, particularly in Serang, Seribu Islands, and Karimunjawa Islands. Using 8 % KOH as a solvent, SRC was extracted from clean dried seaweed. Physical and chemical characteristics of the SRC were measured by assessing the percentage SRC yield, ash content, moisture, sulphate content, viscosity (cp) and gel strength (g.cm-2). Pearson’s correlation analysis was performed to investigate the relationship between SRC quality and environmental factors. Fourier transform infrared spectroscopy (FTIR) analysis showed that all samples had kappa-carrageenan from peak absorbance of 1218–1224 cm-1 indicating ester sulphate bonds, 924–925 cm-1 indicating 3,6–anhydrogalactose, and 843–844 cm-1 suggesting galactose–4–sulphate groups. Furthermore, ash content, moisture, and viscosity of SRC passed the FAO standard, while the sulphate content was below the threshold limit. Several environmental factors had a significant correlation with SRC quality, such as water pH positively correlated with gel strength, salinity level positively correlated with gel strength, and depth positively correlated with ash content. This study concluded that SRC produced from the sampling sites had varying qualities and may be affected by environmental parameters in their cultivation site.

ВЛИЯНИЕ ПИЩЕВОЙ ДОБАВКИ E407А НА МЕТАБОЛИЧЕСКУЮ АКТИВНОСТЬ КЛЕТОК РАЗЛИЧНЫХ КЛЕТОЧНЫХ КУЛЬТУР

Objectives. To study the effects of various concentrations of the food additive E407a (semi-refined carrageenan) on the metabolic activity of fetal liver cells, splenocytes, and bone marrow cells. Material and methods. Fetal liver, splenocytes and bone marrow cell cultures were incubated with the food additive E407a at concentrations varying from 0 mg/ml to 10 mg/ml for 24 hours (n=8). To analyze the effects of this food additive on the metabolic activity of cells, a colorimetric MTT assay was used. It is based on the ability of viable, metabolically active cells to convert 3 (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide into formazan. The data were statistically processed using Kruskal-Wallis and Dunn’s criteria. Results. The bone marrow cell culture was found to be the most sensitive to carrageenan. More than a twofold statistically significant (p&lt;0.0001) increase in the metabolic activity of bone marrow cells was observed when using E407a from 200 μg/ml and above. The metabolic activity of splenocytes increased approximately 1.5 times and over (p&lt;0.0001) when using carrageenans at the concentration of 500 μg/ml and higher. Fetal liver cells turned out to be the most resistant to the direct toxic effect of the food additive E407a. Conclusions. The food additive E407a is cytotoxic to bone marrow cells and splenocytes at concentrations of 200 μg/ml and 500 μg/ml, respectively.

Food additive E407a stimulates eryptosis in adose-dependent manner.

Concerns about the biosafety of the common food additive E407a have been raised. It has been demonstrated to induce intestinal inflammation, accompanied by activation of apoptosis, upon oral exposure. Thus, it is of interest to investigate how E407a affects eryptosis, asuicidal cell death mode of red blood cells. To evaluate the effects of semi-refined carrageenan (E407a) on eryptosis. Flow cytometry was employed to assess eryptosis in blood exposed to various concentrations of E407a (0 g/L, 1 g/L, 5 g/L, and 10 g/L) during incubation for 24 h by analyzing phosphatidylserine externalization in erythrocytes using annexinV staining and via evaluating reactive oxygen species (ROS) generation using 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA). In addition, the eryptosis indices mentioned above were determined in rats orally administered E407a at adose of 140 mg/kg weight for 2weeks. Confocal scanning laser microscopy was performed to visualize cell membrane scrambling. Oral intake of E407a for 2weeks by rats was not associated with membrane scrambling in erythrocytes. However, ROS overproduction was observed. Meanwhile, incubation of blood with various concentrations of semi-refined carrageenan resulted in adose-dependent promotion of eryptosis, evidenced by the enhanced percentage of annexinV-positive erythrocytes and higher mean fluorescence intensity (MFI) values of annexinV-FITC in all erythrocytes. The highest concentration of E407a promotes astatistically significant increase in ROS generation in erythrocytes, suggesting the role of ROS-mediated induction of eryptosis in this case. Incubation of blood with the food additive E407a leads to the activation of eryptosis in adose-dependent manner. ROS-mediated mechanisms are partially responsible for E407a-induced eryptosis.

The Effect of Semi-Refined Carrageenan and Potassium Chloride Added on Physico-Chemical Characteristics of Instant Grass Jelly

This study focused on the effect of the combination effect of semi-refined carrageenan and potassium chloride on the instant grass jelly formulation. As in the traditional method of grass jelly production, the cornstarch was added to the grass jelly extract to give the gelling texture of grass jelly. Semi-refined carrageenan was added to replace the starch and the physicochemical characteristics and nutrient composition of the product was evaluated. The cincau leaves was boiled in a certain volume of water and the extraction was then converted to powder form via spray dryer. A pre-study has been conducted in determining the best method to produce grass jelly extract which based on the result of production yield, as well as conducted the physic-chemical tests. The finding showed that the extraction of 125g dried leaves of cincau with 4.5 L of water which boiled for 2.5 hours was the best extraction recovery with 75.92 percent. As a conclusion, SRC can be used as a starch replacer in grass jelly production as an instant product and texture is retained as well as nutrient contents of the product is improved

The development of Indonesian seaweed based on innovation cluster model

In 2018, Indonesian production of the seaweeds Eucheuma cottonii and Gracilaria sp. reached 11.08 million tons and 1.55 million tons, respectively. However, the economic value obtained is low, as 85% of the production is still exported as a raw material. The remainder is exported as semi-refined materials, i.e. ATC chips and semi refined carrageenan (SRC), with very little refined carrageenan. There are no companies processing seaweed into end products where seaweed is the main raw material, such as in the food industry, pharmaceuticals, cosmetics, etc. The supply chain from upstream (aquaculture) to intermediate (intermediate products) and downstream industries is not efficient in Indonesia, because almost all intermediate and downstream industries are concentrated in big cities like Jakarta and Surabaya while the bulk of the upstream industry is spread across large expanses of eastern Indonesia. As a result, the bargaining position for upstream products produced by farmers is weak. All of these problems are expected to be minimized by referring to a holistic work plan through Seaweed Innovation Clusters, enhancing communication and collaborative collaboration of Quadruple-Helix ABGC (Academic, Business, Government and Community) and other supporting elements as the main pillar of the innovation cluster. With this synergy, added value, and business continuity, the role of seaweed in raising regional competitiveness can be improved.

Physicochemical properties of semi-refined carrageenan by bleaching pretreatment

Judul: Sifat fisikokimia semi-refined carrageenan dengan bleaching pretreatment Title: Physicochemical properties of semi-refined carrageenan by bleaching pretreatment Produk semi refined carrageenan (SRC) merupakan hasil pengekstrakan alga merah dari tumbuhan lontar. Semi refined carrageenan merupakan salah satu unsur dalam Eucheuma cottonii. Karaginan merupakan sulfat galaktan yang berasal dari alga merah (Rhodophyta), tersusun dari D-galaktosa yang terikat secara dalam 5-007-1,3 ikatan β-1,4. Kondisi ekstraksi mempengaruhi kualitas produk karaginan semi murni. Kondisi ekstraksi seperti perlakuan awal, waktu, dan suhu ekstraksi sangat berpengaruh terhadap hasil produk SRC. Penelitian ini bertujuan untuk menyelidiki sifat fisikokimia dari karaginan tyang diekstrak dari rumput laut merah yang telah dilakukan perlakuan awal pemutihan fisik. Hasil terbaik pada variasi waktu ekstraksi dan konsentrasi pelarut adalah pada suhu 80 o C pada 2 jam. Sifat fisikokimia karaginan yang paling baik adalah kekuatan gel 715 gram/cm 2 , kadar air 8%, kadar abu 18%, kadar sulfat 11%, viskositas tujuh cP, titik gel 47,7 o C, titik leleh 57,8 o C, tingkat putih 61%, dan tidak adanya logam berat dalam produk SRC. Analisis FTIR menunjukkan bahwa SRC yang diekstraksi sebagian besar terdiri dari kappa-karaginan. Metode atomic absorption spectrometry (AAS) digunakan untuk menentukan kadar logam substansial dalam SRC, dan hasilnya memenuhi standar FAO. The semi-refined carrageenan (SRC) product is the result of extracting red algae from the palm plant. Semi refined carrageenan is one of the elements in Eucheuma cottonii. Carrageenan is a sulfated galactan derived from red algae (Rhodophyta), composed of D-galactose, which is deeply bonded 5-007-1.3 -1,4 bonds. Extraction conditions affect the quality of semi pure carrageenan products. Extraction conditions such as pretreatment, time, and extraction temperature significantly affect the yield of SRC products. This study aimed to investigate the physicochemical properties of carrageenan extracted from red seaweed, which had been pretreated with physical bleaching. The best results on the variation of extraction time and solvent concentration were at 80 o C at 2 hours. The best physicochemical properties of carrageenan are gel strength 715 gram/cm 2 , water content 8%, ash content 18%, sulfate content 11%, viscosity seven cP, gel point 47.7 o C, melting point 57.8 o C, whiteness 61%, and the absence of heavy metals in SRC products. FTIR analysis showed that the extracted SRC consisted mainly of kappa-carrageenan. The atomic absorption spectrometry (AAS) method was used to determine the actual metal content in SRC, and the results met FAO standards.

The study of phospholipid bilayer of cell membranes in leukocytes incubated with high concentrations of the food additive E407a

The aim of the study was to assess the impact of semi-refined carrageenan (E407a) on the hydrophobic region of phospholipid bilayer in cell membranes of leukocytes exposed to the solution with high concentrations of this food additive. Materials and methods. Fluorescent probe (2-phenyl-phenanthro[9,10-d]-1,3-oxazole) was used to investigate the influence of E407a on the state of lipid bilayer in leukocytes extracted from rats and treated with a 5% solution of the food additive E407a during 4 hours. Results. The shapes of the probe fluorescence spectra did not differ in leukocytes of rats treated with the 5% solution of semi-refined carrageenan and white blood cells of control samples. Such findings suggest that exposure to the E407a solution causes no changes in the proton-donor ability of the media in the lipid membranes of leukocytes in the region where the probe locates. Conclusion. Exposure of white blood cell suspensions to the semi-refined carrageenan solution does not affect the membrane hydration of the hydrophobic region of leukocyte phospholipid bilayer.

Characteristic of semi refined and refined carrageenan flours used in the making of biofilm (bioplastic)

The physical, microbiological, and thermal properties of the raw material, semi refined and refined carrageenan derived from Eucheuma cottonii, were performed in purpose of preliminary research for biofilms (bioplastics) production. The physical properties i.e. particle size, pH, water content, gel strength and viscosity were tested. Meanwhile, the microbiological analysis were performed for the Total Plate Count, mold, and yeast. The thermal properties were performed using Differential Scanning Calorimetry (DSC). The results showed that the Semi Refined Carrageenan (SRC) and Refined Carrageenan (RC) flour have respectively a 60 and 80 mesh of particle size, an 8 and 7 of pH, a 7.9 and 8 % of the moisture content, a 560 and 1140 g / cm2 of gel strength, and an 80 and 35 mPa·s of viscosity. The overall results of the microbiological tested showed that the SRC and RC were recognized as safe to be used for food application in accordance to the regulation, and the thermal analysis showed that the melting points of SRC and RC flour were respectively 168°C and 175°C.

ORGANOLEPTIK MIE BELUT (MONOPTERUS ALBUS ZUIEUW) INSTAN DENGAN PENAMBAHAN SEMI REFINED CARRAGEENAN (SRC) EUCHEUMA COTTONII

The purpose of this research was to study the effect of SRC addition on the organoleptic characteristics of instant eel noodles (Monopterus albus zuieuw) in cup packaging for 30 days of storage. The research was making wet noodles with added crushed meat of eel and then added 2% semi refined carrageenan (SRC). Eel noodles that are still in their wet form are then dried using the frying method. Instant eel noodles are then packaged in paper cups then stored at room temperature for 30 days. Instant noodles were characterized as organoleptic every 10 days. Organoleptic analysis result data appearance, color, taste, smell and texture then analyzed the data using the sign test to determine the differences of each treatment at each storage age. The results of the sign test analysis on the data obtained show that the 2% SRC treatment can maintain organoleptic quality except in texture and taste. The average organoleptic value at each storage level, the addition of SRC 2% i can inhibit the decrease in organoleptic values ​​by an average of 1%.

Analysis on Product Quality of Semi Refined Carrageenan using Six Sigma and Cost of Poor Quality

The study aimed to minimize defective products to improve the production process quality of PT BI by identifying the most types of defects, calculating Defect per Million Opportunities (DPMO) value, suggesting the quality improvement of the Semi Refined Carrageenan (SRC) production process, and calculating Cost of Poor Quality (COPQ) value. The methods used in this research were Six Sigma and the COPQ. The priority improvement based on the Pareto chart was moisture defects with the percentage of damage of 36.9%. The Sigma level of the production process of PT BI was 3.42 with a defect rate of 27,429 DPMO. The analysis on the cause and effect diagram showed that factors affected the occurrence of defective products were error in reading on moisture content, diverse raw material, the wrong method of mixing raw materials prior to production process, and the lack of inspectors of production process. The most influential-dominant factor was the obsolete machine which causes error in reading on moisture content. The company can take preventive and corrective actions to suppress defective products and improve product quality. Based on the calculation of the COPQ, the costs that must be incurred by the company due to defective products was IDR 1,007,690,694.

KARAKTERISTIK KARAGENAN SEMI-MURNI DARI RUMPUT LAUT Kappaphycus striatum DAN Kappaphycus alvarezii

Kappaphycus striatum and Kappaphycus alvarezii are kappa carrageenan-producing seaweeds. K. striatum is also a source of iota carrageenan. The study was aimed to evaluate the effects of extraction time and alkali concentration on the characteristics of semi refined carrageenan (SRC) produced from seaweeds K. striatum and K. alvarezii. The SRC was extracted prepared by cooking dried seaweed samples in KOH solution (ratio seaweed:KOH solution= 1:40) with KOH concentrations of 6, 8 and 10% for 60 and 120 minutes at 70°C. The results showed that the best SRC characteristics produced from K. striatum seaweed were obtained from the extraction process at 6% KOH for 60 minutes which yielded 46.05±0.16%, carrageenan with gel strength of 362,07±21,44 g/cm2 and viscosity at 70°C 74 cP. Meanwhile the best characteristics of SRC produced from K. alvarezii seaweed were obtained from extraction in 10% KOH for 60 minutes having the highest yield of 39,03±0.95%, gel strength 1130.67± 16.34 g/cm2 and viscosity at 70°C of 59 cP. FTIR analysis showed that the functional groups which are kappa carrageenan and iota carrageenan characteristics were found in both samples at wave numbers of 846.97-847.09 and 800.53-801.29 cm-1, respectively.

Oral Intake of Semi-refined Carrageenan by Rats Affects Apoptosis of Lymphocytes

Introduction. The safety of generally recognized as safe food additives E407 and E407a is under rigorous debate. The aim of our research was to evaluate the effects of the orally administered food additive E407a (semi-refined carrageenan) on the viability of lymphocytes and their cell death modes. Methods. Totally, 16 adult WAG rats were divided into two equal groups (experimental – oral intake of 140 mg/kg of E407a during 2 weeks; control – oral consumption of drinking water instead). Blood samples were used to obtain leukocyte suspensions stained with Annexin V-FITC and 7-aminoactinomycin D (7-AAD). The region of lymphocytes was analyzed after collecting data using a BD FACSCanto™ II flow cytometer. Results. Oral administration of E407a led to a decrease in the amount of viable (Annexin V-, 7-AAD-) circulating lymphocytes. Furthermore, exposure to semi-refined carrageenan resulted in an elevated number of early apoptotic (Annexin V+, 7-AAD-) lymphocytes. Their percentage was approximately 4 times higher in rats exposed to E407a compared with the control group. Conclusion. Our findings indicate that dietary intake of E407a promotes apoptosis of circulating lymphocytes. Keywords: processed Eucheuma seaweed, annexin V, 7-aminoactinomycin D, flow cytometry.

Semi-refined carrageenan promotes generation of reactive oxygen species in leukocytes of rats upon oral exposure but not in vitro.

To assess the ability of the common food additive E407a (semi-refined carrageenan) to enter leukocytes in vitro and generate reactive oxygen species (ROS) in leukocytes as awhole and granulocytes in particular, both during incubation and in experimental animals. ROS production was assessed in leukocytes incubated with E407afor 2 h at the final concentrations of 5 and 10 g/L using the dye 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), as well as in cells isolated from rats orally exposed to E407a (140 mg/kg of weight) during 2weeks (n = 8) and control rats (n = 8), by flow cytometry. Carrageenan uptake by leukocytes was estimated by confocal microscopy using incubation of rhodamineB isothiocyanate-labelled carrageenan with leukocyte suspensions. Uptake of carrageenan by viable neutrophils, monocytes, and lymphocytes was confirmed. Oral administration of the food additive E407awas associated with excessive ROS formation by viable leukocytes (CD45+, 7‑aminoactinomycin D- cells) and especially in granulocytes. Unexpectedly, adirect impact of semi-refined carrageenan during incubation for 2 h did not affect ROS production in leukocytes, evidenced by statistically insignificant differences in mean fluorescence intensity values of 2',7'-dichlorofluorescein, which is aROS-sensitive product of intracellular H2DCFDA conversion. Oral intake of E407a and direct exposure of leukocyte suspensions to it decreased the viability of leukocytes. Food-grade carrageenan can enter leukocytes without affecting ROS generation as aresult of incubation for 2 h with leukocyte suspensions. On the contrary, oral exposure to E407a is accompanied by ROS overproduction by white blood cells, suggesting an indirect mechanism for the stimulation of ROS synthesis in vivo. E407a promotes cell death of leukocytes both in vivo and in vitro.

Effects of semi-refined carrageenan (food additive E407a) on cell membranes of leukocytes assessed in vivo and in vitro.

Aim To assess the impact of semi-refined carrageenan (E407a) on hydrophobic regions of phosphololipid bilayer in cell membranes of leukocytes collected from rats orally administered this food additive and white blood cells incubated with E407a. Methods Fluorescent probes (ortho-hydroxy derivatives of 2,5-diaryl-1,3-oxazole) were used to estimate the state of lipid bilayer in leukocytes obtained from rats orally exposed to the food additive E407a and in white blood cells incubated with E407a. Results No noticeable changes in the physico-chemical properties were observed in the lipid membranes of leukocytes in the region where the probes locate in response to oral intake of semi-refined carrageenan. Incubation of leukocytes with E407a solutions resulted in a decrease in polarity and proton-donor ability of leukocytes in the area of carbonyl groups of phospholipids and in the area of hydrocarbon chains of phospholipids near the polar region of the bilayer. Conclusion Membrane fluidity abnormalities found in cells exposed to E407a are similar to those observed in patients with IBD suggesting that contribution of carrageenan to IBD development may be partially explained by leukocyte membrane modifications.