bixin and annatto

Colored Cheddar cheeses are prepared by adding an aqueous annatto extract (norbixin) to cheese milk; however, a considerable proportion (∼20%) of such colorant is transferred to whey, which can limit the end use applications of whey products. Different geographical regions have adopted various strategies for handling whey derived from colored cheeses production. For example, in the United States, whey products are treated with oxidizing agents such as hydrogen peroxide and benzoyl peroxide to obtain white and colorless spray-dried products; however, chemical bleaching of whey is prohibited in Europe and China. Fundamental studies have focused on understanding the interactions between colorants molecules and various components of cheese. In addition, the selective delivery of colorants to the cheese curd through approaches such as encapsulated norbixin and microcapsules of bixin or use of alternative colorants, including fat-soluble/emulsified versions of annatto or beta-carotene, has been studied. This review provides a critical analysis of pertinent scientific and patent literature pertaining to colorant delivery in cheese and various types of colorant products on the market for cheese manufacture, and also considers interactions between colorant molecules and cheese components; various strategies for elimination of color transfer to whey during cheese manufacture are also discussed.

Topics: Bixaceae; Carotenoids; Cheese; Food Coloring Agents; Food Handling; Oxidants; Plant Extracts; Whey

Genetic transformation allows for greater bixin or norbixin production in achiote. Knowledge of genes that control the biosynthesis of these important secondary metabolites will allow for targeted amplification in transgenic plants. Annatto is a natural dye or coloring agent derived from the seeds, or their arils, of achiote (Bixa orellana L.), and is commercially known as E160b. The main active component of annatto dye is water-insoluble bixin, although water-soluble norbixin also has commercial applications. Relative to other antioxidants, bixin is light- and temperature stable and is thus safe for human consumption. Bixin is, therefore, widely applied as a dye and as an antioxidant in the medico-pharmaceutical, food, cosmetic, and dye industries. Even though bixin has also been isolated from leaves and bark, yield is lower than from seeds. More biotechnology-based research of this industrial and medicinal plant is needed. Building on provisional genetic transformation studies, it would be advantageous to transform genes that could result in greater bixin or norbixin production. Reliable protocols for the extraction of bixin and norbixin, as well as deeper knowledge of the genes that control the biosynthesis of these important secondary metabolites will allow for targeted amplification in transgenic plants.

Topics: Antioxidants; Biotechnology; Bixaceae; Breeding; Carotenoids; Food Coloring Agents; Humans; Plant Extracts; Plants, Medicinal; Reproduction; Seeds; Transformation, Genetic

Topics: Animals; Antioxidants; Bixaceae; Carotenoids; Disease Models, Animal; DNA Repair; Humans; Keratinocytes; Mice; NF-E2-Related Factor 2; NF-kappa B; Oxidative Stress; Plant Extracts; PPAR alpha; PPAR gamma; Skin; Skin Neoplasms; Sunlight; Toll-Like Receptor 4; Ultraviolet Rays

Topics: Biotechnology; Bixaceae; Carotenoids; Coloring Agents; Plant Extracts

Bixin is a commercially valuable apocarotenoid pigment found in the seed aril of Bixa orellana. The dynamics and regulation of its biosynthesis and accumulation during seed development remain largely unknown. Here, we combined chemical, anatomical, and transcriptomic data to provide stage-specific resolution of the cellular and molecular events occurring during B. orellana seed development. Seeds at five developmental stages (S1-S5) were used for analysis of bixin content and seed anatomy, and three of them (S1, S3, and S4) were selected for Illumina HiSeq sequencing. Bixin accumulated in large quantities in seeds compared with other tissues analyzed, particularly during the S2 stage, peaking at the S4 stage, and then decreasing slightly in the S5 stage. Anatomical analysis revealed that bixin accumulated in the large central vacuole of specialized cells, which were scattered throughout the developing mesotesta at the S2 stage, but enlarged progressively at later stages, until they occupied most of the parenchyma in the aril. A total of 13 million reads were generated and assembled into 73,381 protein-encoding contigs, from which 312 were identified as containing 1-deoxy-D-xylulose-5-phosphate/2-C-methyl-D-erythritol-4-phosphate (DOXP/MEP), carotenoid, and bixin pathways genes. Differential transcriptome expression analysis of these genes revealed that 50 of them were sequentially and differentially expressed through the seed developmental stages analyzed, including seven carotenoid cleavage dioxygenases, eight aldehyde dehydrogenases, and 22 methyltransferases. Taken together, these results show that bixin synthesis and accumulation in seeds of B. orellana are a developmentally regulated process involving the coordinated expression of DOXP/MEP, carotenoid, and bixin biosynthesis genes.

Topics: Bixaceae; Carotenoids; RNA-Seq; Seeds

To compare the encapsulation of annatto extract by external gelation (EG) and internal gelation (IG) and to maximize process yield (% Y), two central composite designs were proposed. Calcium chloride (CaCl

Topics: Alginates; Bixaceae; Capsules; Carotenoids; Phytochemicals; Plant Extracts; Polyphenols; Seeds

The seeds of Bixa orellana L. is widely used in food industry as a natural colorant. A major technological challenge for its utilization on aqueous food products is the low water solubility. Therefore, the present study aimed to obtain aqueous nanodispersions using this natural raw material by using a simple organic solvent-free and low energy method. Bixin and geranylgeraniol were found on the extract. The nanodispersions were prepared by addition of water on a mixture of extract and surfactant (s). The effect of the surfactants and dilution on the nanodispersions were also evaluated. Best results were achieved using polysorbate 80/sorbitan monooleate (HLB 13) (diluted in water, 1:10). The results shows the potential of this ecofriendly approach, in contrast to common methods that make use of potentially toxic organic solvents and high input of energy, which elevate the costs for further industrialization.

Topics: Bixaceae; Carotenoids; Diterpenes; Gas Chromatography-Mass Spectrometry; Plant Extracts; Seeds; Solvents; Spectrophotometry; Surface-Active Agents; Thermogravimetry; Water

The aim of this study was to evaluate the effect of annatto carotenoids intake associated to a single high-calorie meal (high fat and high carbohydrate) in postprandial biochemical, inflammatory and oxidative stress markers. Twelve healthy subjects (6 men, 6 women) were included in this randomised, controlled crossover study. Baseline blood samples were collected from fasting subjects that immediately received high-calorie meal without carotenoid (placebo) or containing 1.2mg/kg bixin (BIX) or 0.06mg/kg norbixin (NBIX). Blood samples were taken 60, 120 and 240min after meal intake. NBIX intake did not affect biochemical blood markers but reduced the postprandial levels of inflammatory cytokines (IL-1, IL-6 and TNF-α) and lipid oxidation 60-120min after meal. BIX only partially prevented postprandial-induced lipid oxidation. Results indicate that the intake of NBIX may be an alternative to reduce the postprandial inflammatory and oxidative stress responses to high-calorie meals.

Topics: Adult; Bixaceae; Blood Glucose; Blood Pressure; Carotenoids; Cytokines; Diet; Female; Humans; Inflammation; Male; Oxidative Stress; Plant Extracts; Postprandial Period; Young Adult

The effects of annatto seeds and of bixin on the oxidative damage induced by cisplatin in male Wistar rats was evaluated in the present study by way of lipid peroxidation, weight gain, the food efficiency coefficient, fat deposits in the hepatocytes and dosing of the enzymes in this organ. The animals were divided into four groups: control group (CG), cisplatin group (CPG), bixin+cisplatin group (CBG) and annatto+cisplatin group (CUG). Cisplatin (5 mg/kg body weight) was injected intraperitoneally 48 hours before the end of the experiment. The bixin and annatto were administered daily together with the commercial feed. The pre-treatment with annatto and bixin attenuated the cisplatin-induced liver damage and significantly reduced the enzymes AST and ALT. Annatto was shown to be capable of inhibiting lipid peroxidation as determined by TBARS. These results suggest that annatto seeds and bixin could be important agents in the reduction of cisplatin-induced hepatotoxicity.

Topics: Animals; Antineoplastic Agents; Antioxidants; Bixaceae; Carotenoids; Chemical and Drug Induced Liver Injury; Cisplatin; Hepatocytes; Lipid Peroxidation; Male; Malondialdehyde; Oxidation-Reduction; Plant Extracts; Rats, Wistar; Seeds; Thiobarbituric Acid Reactive Substances

Annatto and bixin, the main carotenoid of annatto seeds, were both found to inhibit cholesterol oxidation in minced herring (Clupea harengus) and minced mackerel (Scomber scombrus) during high pressure processing (600 MPa for 10 min) and subsequent chilled storage for 2 wk, a treatment which otherwise increased the content of cholesterol oxidation products above a critical limit for human consumption. Annatto but not bixin reduced the loss of docosahexaenoic acid caused by high pressure processing of herring from 12% to 7%, an effect assigned to antioxidative effects of phenolic compounds in annatto, while bixin as a carotenoid binds to membranes protecting membrane cholesterol.

Topics: Animals; Antioxidants; Bixaceae; Carotenoids; Cholesterol; Docosahexaenoic Acids; Fatty Acids, Unsaturated; Fishes; Food Handling; Humans; Oxidation-Reduction; Perciformes; Phenols; Plant Extracts; Pressure; Seafood

The Cheddar cheese colorant annatto is present in whey and must be removed by bleaching. Chemical bleaching negatively affects the flavor of dried whey ingredients, which has established a need for a better understanding of the primary colorant in annatto, norbixin, along with cheese color alternatives. The objective of this study was to determine norbixin partitioning in cheese and whey from full-fat and fat-free Cheddar cheese and to determine the viability of bixin, the nonpolar form of norbixin, as an alternative Cheddar cheese colorant. Full-fat and fat-free Cheddar cheeses and wheys were manufactured from colored pasteurized milk. Three norbixin (4% wt/vol) levels (7.5, 15, and 30 mL of annatto/454 kg of milk) were used for full-fat Cheddar cheese manufacture, and 1 norbixin level was evaluated in fat-free Cheddar cheese (15 mL of annatto/454 kg of milk). For bixin incorporation, pasteurized whole milk was cooled to 55 °C, and then 60 mL of bixin/454 kg of milk (3.8% wt/vol bixin) was added and the milk homogenized (single stage, 8 MPa). Milk with no colorant and milk with norbixin at 15 mL/454 kg of milk were processed analogously as controls. No difference was found between the norbixin partition levels of full-fat and fat-free cheese and whey (cheese mean: 79%, whey: 11.2%). In contrast to norbixin recovery (9.3% in whey, 80% in cheese), 1.3% of added bixin to cheese milk was recovered in the homogenized, unseparated cheese whey, concurrent with higher recoveries of bixin in cheese (94.5%). These results indicate that fat content has no effect on norbixin binding or entrapment in Cheddar cheese and that bixin may be a viable alternative colorant to norbixin in the dairy industry.

Topics: Animals; Bixaceae; Carotenoids; Cheese; Food Coloring Agents; Food Handling; Plant Extracts; Taste; Whey Proteins

The liver is an important organ for its ability to transform xenobiotics, making the liver tissue a prime target for toxic substances. The carotenoid bixin present in annatto is an antioxidant that can protect cells and tissues against the deleterious effects of free radicals. In this study, we evaluated the protective effect of bixin on liver damage induced by carbon tetrachloride (CCl4) in rats.. The animals were divided into four groups with six rats in each group. CCl4 (0.125 mL kg(-1) body wt.) was injected intraperitoneally, and bixin (5.0 mg kg(-1) body wt.) was given by gavage 7 days before the CCl4 injection. Bixin prevented the liver damage caused by CCl4, as noted by the significant decrease in serum aminotransferases release. Bixin protected the liver against the oxidizing effects of CCl4 by preventing a decrease in glutathione reductase activity and the levels of reduced glutathione and NADPH. The peroxidation of membrane lipids and histopathological damage of the liver was significantly prevented by bixin treatment.. Therefore, we can conclude that the protective effect of bixin against hepatotoxicity induced by CCl4 is related to the antioxidant activity of the compound.

Topics: Animals; Antioxidants; Bixaceae; Carbon Tetrachloride; Carotenoids; Chemical and Drug Induced Liver Injury; Glutathione; Glutathione Reductase; Lipid Peroxidation; Liver; Male; Malondialdehyde; NADP; Oxidative Stress; Plant Extracts; Rats, Wistar; Reactive Oxygen Species; Transaminases

Annatto (Bixa orellana) seeds have been used as a colorant in butter and in a variety of other foods. In this study, we investigated the amelioration of retinal damage by an acetone extract of annatto (A-ext.), bixin (a main component of annatto), and four bixin derivatives (Bx-1, Bx-2, Bx-3, and Bx-4) that we have synthesized.. We used cultured retinal ganglion cells (RGC-5) to examine in vitro effects of A-ext. on stress pathways, focusing on intracellular oxidation induced by reactive oxygen species, expression of endoplasmic reticulum (ER) stress-related proteins, caspase-3 activation, and cell membrane damage. In vivo retinal damage in mice following intravitreous injection of tunicamycin was evaluated by counting the cell numbers in the ganglion cell layer (GCL) and measuring the thickness of outer nuclear layer (ONL). A-ext., bixin, and Bx-1 treatment inhibited both tunicamycin- and H₂O₂-induced cell death. Bixin derivatives also inhibited tunicamycin-induced cell death. Treatment with A-ext., bixin, and Bx-1 reduced tunicamycin-induced caspase-3 activity and inhibited the inversion of phosphatidylserine, an early apoptotic event without antioxidant effect or reduction of ER stress itself. A-ext., bixin, and Bx-1 significantly inhibited the tunicamycin-induced loss of cells from the GCL, and these materials also suppressed the tunicamycin-induced thinning of ONL.. A-ext., its main component bixin, and bixin derivatives may therefore be useful for preventive and therapeutic treatment of retinal-related diseases.

Topics: Animals; Bixaceae; Carotenoids; Caspase 3; Cell Death; Cell Membrane; Cells, Cultured; Drug Evaluation, Preclinical; Endoplasmic Reticulum Stress; Hydrogen Peroxide; Male; Mice; Mice, Inbred Strains; Phosphatidylserines; Plant Extracts; Retinal Degeneration; Retinal Ganglion Cells; Tunicamycin

Populations in the Amazon are exposed to organic mercury via consumption of contaminated foods. These ethnic groups consume a specific plant seed "annatto" which contains certain carotenoids. The aim of this study was to find out if these compounds (bixin, BIX and norbixin, NOR), protect against DNA-damage caused by the metal. Therefore, rats were treated orally with methylmercury (MeHg) and with the carotenoids under conditions that are relevant to humans. The animals were treated either with MeHg (30 μg/kg/bw/day), BIX (0.1-10 mg/kg/bw/day), NOR (0.01-1.0 mg/kg/bw/day) or combinations of the metal compound and the carotenoids consecutively for 45 days. Subsequently, the glutathione levels (GSH) and the activity of catalase were determined, and DNA-damage was measured in hepatocytes and leukocytes using single cell gel electrophoresis assays. Treatment with the metal alone caused a decrease in the GSH levels (35%) and induced DNA damage, which resulted in increased DNA migration after electrophoresis in liver and blood cells, whereas no effects were seen with the carotenoids alone. When BIX or NOR were given in combination with organic mercury, the intermediate and the highest concentrations of the carotenoids (1.0 and 10.0 mg/kg/bw/day BIX and 0.1 and 1.0 mg/kg/bw/day NOR) protected against DNA-damage. Furthermore, we found with both carotenoids, a moderate increase in the GSH levels in both metal-treated and untreated animals, while the activities of catalase remained unchanged. Our results indicate that consumption of BIX and NOR may protect humans against the adverse health effects caused by exposure to organic mercury.

Topics: Analysis of Variance; Animals; Bixaceae; Carotenoids; Catalase; Comet Assay; DNA Damage; Environmental Pollutants; Glutathione; Methylmercury Compounds; Molecular Structure; Oxidation-Reduction; Plant Extracts; Rats; Rats, Wistar

The present studies were performed to examine the contact allergenic effects of an annatto extract (ANT) in female BALB/c mice. ANT at 5-10% induced a greater than threefold increase in lymph node cell proliferation when compared to the control in the LLNA. Moreover, a significant increase in the percent ear swelling at 24h after ANT challenge was observed in the MEST. A significant increase in the percentage of B cells was also observed. To determine which of the two predominant coloring components (norbixin and bixin) in ANT was responsible for the sensitizing effects of ANT, norbixin was subsequently examined, with negative results being observed in both the LLNA and MEST following treatment with norbixin (1-20%). These findings suggested that perhaps bixin was responsible for the positive responses in both the LLNA and MEST following exposure to ANT. Therefore, further studies using a partially purified cis-bixin extract were conducted. Positive responses in both the LLNA and MEST were observed in mice treated with cis-bixin at the concentrations as low as 0.1-0.5%. These results have demonstrated that cis-bixin, but not norbixin, is likely a contact sensitizer and contributes to the contact hypersensitivity effects observed following dermal exposure to ANT in mice.

Topics: Animals; Bixaceae; Carotenoids; Dermatitis, Contact; Female; Flow Cytometry; Food Coloring Agents; Local Lymph Node Assay; Mice; Mice, Inbred BALB C; Plant Extracts; Random Allocation

A supramolecular solvent (SUPRA) made up of octanoic acid aggregates is proposed for the microextraction of bixin and norbixin, the two major components of the natural food colouring annatto, in food. The procedure involved the extraction of sub-gram quantities (200mg) of homogenized food with 0.8mL of the supramolecular solvent. The overall sample treatment took about 20 min, and several samples could be simultaneously treated using conventional lab equipment. No clean-up or solvent evaporation were required. Extraction efficiencies mainly depended on the major components making up the SUPRAS (i.e. octanoic acid and tetrahydrofuran) and were not affected by the pH or the temperature in the ranges studied (1-4 and 10-80°C, respectively). Bixin and norbixin in the extracts were quantified by liquid chromatography (LC) and diode array detection (DAD). They were separated in a Hypersil C18 column using a mobile phase consisting of 5% acetic acid and methanol (15:85, v/v). The retention times for norbixin and bixin standards were 5.1 and 8.6 min, respectively. Recoveries in samples ranged between about 78% and 113%. The precision of the method, expressed as relative standard deviation, was about 1.5% and the quantitation limits for bixin and norbixin were 0.19 and 0.23 mg kg(-1), respectively, which were far below the maximum limits permitted by the European Union for the level of addition to food. Concentration of norbixin in samples belonging to the five major groups of food commodities defined in the literature, ranged between 3.75 and 43.8 mg kg(-1) whereas bixin was only found in one snack sample (6.6 mg kg(-1)). The method is simple and rapid, while delivering accurate and precise results, and can be used for the routine determination of annatto in food for the control of the compliance of current legislation.

Topics: Bixaceae; Caprylates; Carotenoids; Chromatography, Liquid; Food Analysis; Food Coloring Agents; Furans; Hydrogen-Ion Concentration; Plant Extracts; Reproducibility of Results; Sensitivity and Specificity; Temperature

In pursuit of the anticancer effects of seeds of the rain forest plant Bixa orellana (annatto), we found that its constituent cis-bixin induced cytotoxicity in a wide variety of tumor cell lines (IC(50) values from 10 to 50 microM, 24-h exposures) and, importantly, also selectively killed freshly collected patient multiple myeloma cells and highly drug-resistant multiple myeloma cell lines. Mechanistic studies indicated that cis-bixin-induced cytotoxicity was greatly attenuated by co-treatment with glutathione or N-acetylcysteine (NAC); whereas fluorescence-activated cell sorting (FACS) assays using the cell-permeable dyes 5-(and-6) chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H(2)DCFDA), or dihydroethidium demonstrated that cis-bixin rapidly induced cellular reactive oxygen species (ROS) in dose- and time-dependent fashions, collectively implicating ROS as contributory to cis-bixin-induced cytotoxicity. In pursuit of potential contributors to ROS imposition by cis-bixin, we found that cis-bixin inhibited both thioredoxin (Trx) and thioredoxin reductase (TrxR1) activities at concentrations comparable to those required for cytotoxicity, implicating the inhibition of these redox enzymes as potentially contributing to its ability to impose cellular ROS and to kill cancer cells. Collectively, our studies indicate that the annatto constituent cis-bixin has intriguing selective antimyeloma activity that appears to be mediated through effects on redox signaling.

Topics: Acetylcysteine; Antineoplastic Agents; Apoptosis; Bixaceae; Carotenoids; Cell Line, Tumor; Cell Survival; Drug Screening Assays, Antitumor; Glutathione; Humans; Multiple Myeloma; Oxidation-Reduction; Oxidative Stress; Phytotherapy; Plant Extracts; Reactive Oxygen Species; Seeds; Thioredoxin-Disulfide Reductase; Thioredoxins

The development of an analytical method that enables routine analysis of annatto dye, specifically bixin and norbixin, in meat tissue is described. Liquid-solid extraction was carried out using acetonitrile. Analysis was by HPLC with photodiode array detection using two fixed wavelengths (458 and 486 nm). The possibilities of ion trap mass spectrometry (MS) were also assessed. Method performance characteristics, according to Commission Decision 2002/657/EC, were determined, with recoveries between 99 and 102% and calibration curves being linear in the 0.5-10 mg kg(-1) range. The limit of quantification was 0.5 mg kg(-1).

Topics: Animals; Bixaceae; Carotenoids; Chromatography, Liquid; Food Analysis; Food Coloring Agents; Mass Spectrometry; Meat; Plant Extracts; Reproducibility of Results

Annatto or urucum is an orange-yellow dye obtained from Bixa orellana seeds. It has been used as a natural dye in a variety of food products, drugs and cosmetics, and also in Brazilian cuisine as a condiment ('colorau'). Bixin, a carotenoid devoid of provitamin A activity, is the main pigment found in annatto. Some carotenoids (canthaxanthin, astaxanthin and beta-Apo-8'-carotenal) are known to be potent inducers of CYP1A1, a property not shared by others (beta-carotene, lycopene and lutein). Little is known, however, about the CYP1A1-inducing properties of bixin and annatto. The present study was performed to determine the effects of an annatto extract (28% bixin) and bixin (95% pure) on rat liver monooxygenases. Adult female Wistar rats were treated by gavage with daily doses of annatto (250 mg/kg body weight, which contains approximately 70 mg bixin/kg body weight), bixin (250 mg/kg body weight) or the vehicle only (corn oil, 3.75 g/kg body weight) for 5 consecutive days, or were not treated (untreated control). The activities of aniline-4-hydroxylase (A4H), ethoxycoumarin-O-deethylase (ECOD), ethoxy- (EROD), methoxy- (MROD), pentoxy- (PROD) and benzyloxy- (BROD) resorufin-O-dealkylases were measured in liver microsomes. Annatto (250 mg/kg containing 70 mg bixin/kg) induced EROD (3.8x), MROD (4.2x), BROD (3.3x) and PROD (2.4x). Bixin (250 mg/kg) was a weaker inducer of EROD (2.7x), MROD (2.3x) and BROD (1.9x) and did not alter PROD, A4H or ECOD activities. These results suggest that constituents of the extract other than bixin play an important role in the induction of CYP1A and CYP2B observed with annatto food colorings.

Topics: Animals; Bixaceae; Carotenoids; Enzyme Induction; Female; Liver; Microsomes, Liver; Mixed Function Oxygenases; Plant Extracts; Rats; Rats, Wistar

Analytical methods for the determination of the permitted food colouring annatto (E160b) have been developed or refined to encompass the wide range of food commodity types permitted to contain it. Specific solvent extraction regimens have been used depending upon the food commodity analysed and HPLC analysis techniques coupled with spectral confirmation have been used for the determination of the major colouring components. Qualitative and quantitative data on the annatto content of 165 composite and two single retail food samples covering a wide range of foods at levels above the limit of quantification (0.1 mg kg(-1)) is reported. Quantitative results are given for the major colour principals 9'-cis-bixin, 9'-cis-norbixin and trans-bixin. Semi-quantitative results are given for the minor bixin and norbixin isomers monocis- (not 9'-), di-cis- and trans-norbixin, for which authentic reference standards were not available. Repeat analyses (n = 4-9) of 12 different types of food commodity (covering the permitted range) spiked with annatto at levels between 1.7 and 27.7 mg kg(-1) gave mean recoveries between 61 and 96%. The corresponding relative SDs (RSD) were between 2.1 and 7.9%.

Topics: Animals; Bixaceae; Carotenoids; Chromatography, High Pressure Liquid; Fishes; Food Analysis; Food Coloring Agents; Humans; Plant Extracts