norbixin 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

Norbixin is the water-soluble carotenoid in annatto extracts used in the cheese industry to color Cheddar cheese. The purpose of norbixin is to provide cheese color, but norbixin is also present in the whey stream and contaminates dried dairy ingredients. Regulatory restrictions dictate that norbixin cannot be present in dairy ingredients destined for infant formula or ingredients entering different international markets. Thus, there is a need for the detection and quantification of norbixin at very low levels in dried dairy ingredients to confirm its absence. A rapid method for norbixin evaluation exists, but it does not have the sensitivity required to confirm norbixin absence at very low levels in compliance with existing regulations. The current method has a limit of detection of 2.7 μg/kg and a limit of quantification of 3.5 μg/kg. The purpose of this study was to develop a method to extract and concentrate norbixin for quantification in dried dairy ingredients below 1 μg/kg (1 ppb). A reverse-phase solid-phase extraction column step was applied in the new method to concentrate and quantify norbixin from liquid and dried WPC80 (whey protein concentrate with 80% protein), WPC34 (WPC, 34% protein), permeate, and lactose. Samples were evaluated by both methods for comparison. The established method was able to quantify norbixin in whey proteins and permeates (9.39 μg/kg to 2.35 mg/kg) but was unable to detect norbixin in suspect powdered lactose samples. The newly developed method had similar performance to the established method for whey proteins and permeates but was also able to detect norbixin in powdered lactose samples. The proposed method had a >90% recovery in lactose samples and a limit of detection of 28 ppt (ng/kg) and a limit of quantification of 94 ppt (ng/kg). The developed method provides detection and quantification of norbixin for dairy ingredients that have a concentration of <1 ppb.

Topics: Animals; Bixaceae; Carotenoids; Food Analysis; Plant Extracts; Sensitivity and Specificity; Solid Phase Extraction; Taste; Whey Proteins

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

Whey protein is a highly functional food ingredient used in a wide variety of applications. A large portion of fluid whey produced in the United States is derived from Cheddar cheese manufacture and contains annatto (norbixin), and therefore must be bleached. The objective of this study was to compare sensory and functionality differences between whey protein isolate (WPI) bleached by benzoyl peroxide (BP) or hydrogen peroxide (HP). HP and BP bleached WPI and unbleached controls were manufactured in triplicate. Descriptive sensory analysis and gas chromatography-mass spectrometry were conducted to determine flavor differences between treatments. Functionality differences were evaluated by measurement of foam stability, protein solubility, SDS-PAGE, and effect of NaCl concentration on gelation relative to an unbleached control. HP bleached WPI had higher concentrations of lipid oxidation and sulfur containing volatile compounds than both BP and unbleached WPI (P < 0.05). HP bleached WPI was characterized by high aroma intensity, cardboard, cabbage, and fatty flavors, while BP bleached WPI was differentiated by low bitter taste. Overrun and yield stress were not different among WPI (P < 0.05). Soluble protein loss at pH 4.6 of WPI decreased by bleaching with either hydrogen peroxide or benzoyl peroxide (P < 0.05), and the heat stability of WPI was also distinct among WPI (P < 0.05). SDS PAGE results suggested that bleaching of whey with either BP or HP resulted in protein degradation, which likely contributed to functionality differences. These results demonstrate that bleaching has flavor effects as well as effects on many of the functionality characteristics of whey proteins.. Whey protein isolate (WPI) is often used for its functional properties, but the effect of oxidative bleaching chemicals on the functional properties of WPI is not known. This study identifies the effects of hydrogen peroxide and benzoyl peroxide on functional and flavor characteristics of WPI bleached by hydrogen and benzoyl peroxide and provides insights for the product applications which may benefit from bleaching.

Topics: Benzoyl Peroxide; Bixaceae; Bleaching Agents; Carotenoids; Cheese; Color; Electrophoresis, Polyacrylamide Gel; Food Coloring Agents; Functional Food; Gas Chromatography-Mass Spectrometry; Humans; Hydrogen; Hydrogen Peroxide; Lipid Peroxidation; Odorants; Plant Extracts; Proteolysis; Sulfur; Taste; Volatile Organic Compounds; Whey; Whey Proteins

Norbixin is the primary carotenoid in annatto coloring, which imparts the desired orange color in Cheddar cheese. However, a portion of the colorant remains in the cheese whey and is undesirable; therefore, a bleaching step is often applied. Restrictions exist for norbixin concentrations in products destined for infant formula. As such, evaluation of norbixin concentrations in whey and whey ingredients is desirable. Current extraction methods are laborious and require solvents that are banned in many countries. The objective of this study was to develop a fast and inexpensive norbixin extraction and quantitation technique using approved solvents with similar sensitivity to current established methods. Instead of solvent extraction and column purification, acetonitrile was added directly to fluid wheys, retentates, and rehydrated whey protein concentrates. An isocratic mobile phase [70% acetonitrile and 30% water with 0.1% (wt/vol) formic acid] was used and, to increase sensitivity, a large volume (50 μL) was injected onto the column. The column used was a C18 column with a particle size of 2.6 μm and column length of 10 cm. The column inner diameter was 4.6mm and the pore size was 100 Ǻ. All of the previously described conditions allowed the run time to be only 4 min. The sample was sent through a photodiode array detector and quantified at 482 nm. Norbixin was quantified using external standard curves. The developed method had a >90% norbixin recovery in both milk and whey (9.39 μg/L-2.35 mg/L). The limit of detection of norbixin in fluid whey was 2.7 μg/kg and the limit of quantitation was 3.5 μg/kg, both of which are significantly lower than in previously described methods. The extracts were stable over 30 min at 21°C and stable over 24h at 4°C. Repeatability and precision of the method had relative standard deviations of less than 13%. The developed method provides time and cost savings for evaluation of norbixin concentration in whey and whey products.

Topics: Animals; Bixaceae; Carotenoids; Chromatography, High Pressure Liquid; Color; Limit of Detection; Milk; Plant Extracts; Whey

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

Annatto (norbixin) has been used to color cheeses for centuries, but there is very little knowledge about interactions between the pigment and dairy proteins. In this study, binding of norbixin with whey protein isolate (WPI), sodium caseinate (NaCN), and 6 individual dairy proteins was investigated by using fluorescence spectroscopy, Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD) and differential scanning calorimetry (DSC). Norbixin was observed to effectively quench the fluorescence of WPI and NaCN by forming complexes. The binding affinity between NaCN and norbixin was higher than that of WPI-norbixin. For individual proteins, bovine serum albumin had higher binding affinity with norbixin than β-lactoglobulin and α-lactalbumin, while κ-casein bound with norbixin better than α- and β-caseins. Binding changed the conformation of WPI and NaCN, but the extent and trend varied for individual proteins.

Topics: Animals; Bixaceae; Carotenoids; Cattle; Food Coloring Agents; Kinetics; Milk Proteins; Plant Extracts; Protein Binding; Serum Albumin, Bovine; Spectrum Analysis

Residual annatto colorant (norbixin) in fluid Cheddar cheese whey can be bleached. The 2 approved chemical bleaching agents for whey, hydrogen peroxide (HP) and benzoyl peroxide (BP), negatively impact the flavor of dried whey protein. The objective of this study was to evaluate alternative methods for bleaching liquid whey: ultraviolet radiation (UV), acid-activated bentonite (BT), and ozone (OZ). Colored Cheddar cheese whey was manufactured followed by pasteurization and fat separation. Liquid whey was subjected to one of 5 treatments: control (CT) (no bleaching; 50 °C, 1 h), HP (250 mg/kg; 50 °C, 1 h), UV (1 min exposure; 50 °C), BT (0.5% w/w; 50 °C, 1 h), or OZ (2.2g/h, 50 °C, 1 h). The treated whey was then ultrafiltered, diafiltered, and spray-dried to 80% whey protein concentrate (WPC80). The entire experiment was replicated 3 times. Color (norbixin extraction and measurement), descriptive sensory, and instrumental volatile analyses were conducted on WPC80. Norbixin elimination was 28%, 79%, 39%, and 15% for HP, BT, UV, and OZ treatments, respectively. WPC80 from bleached whey, regardless of bleaching agent, had lower sweet aromatic and cooked/milky flavors compared to unbleached CT (P < 0.05). The HP and BT WPC80 had higher fatty flavor compared to the CT WPC80 (P < 0.05), and the UV and OZ WPC80 had distinct mushroom/burnt and animal flavors. Volatile compound results were consistent with sensory results and confirmed higher relative abundances of volatile aldehydes in UV, HP, and OZ WPC80 compared to CT and BT WPC80. Based on bleaching efficacy and flavor, BT may be an alternative to chemical bleaching of fluid whey.. The 2 approved chemical bleaching agents for whey, hydrogen peroxide (HP) and benzoyl peroxide (BP), negatively impact flavor of dried whey protein, and restrictions on these agents are increasing. This study evaluated 3 alternatives to chemical bleaching of fluid whey: UV radiation, ozone, and bentonite.

Topics: Benzoyl Peroxide; Bixaceae; Bleaching Agents; Carotenoids; Cheese; Color; Food Handling; Gas Chromatography-Mass Spectrometry; Hydrogen Peroxide; Milk Proteins; Plant Extracts; Taste; Volatile Organic Compounds; Whey Proteins

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

The increasing use and demand for whey protein as an ingredient requires a bland-tasting, neutral-colored final product. The bleaching of colored Cheddar whey is necessary to achieve this goal. Currently, hydrogen peroxide (HP) and benzoyl peroxide (BPO) are utilized for bleaching liquid whey before spray drying. There is no current information on the effect of the bleaching process on the flavor of spray-dried whey protein concentrate (WPC). The objective of this study was to characterize the effect of bleaching on the flavor of liquid and spray-dried Cheddar whey. Cheddar cheeses colored with water-soluble annatto were manufactured in duplicate. Four bleaching treatments (HP, 250 and 500 mg/kg and BPO, 10 and 20 mg/kg) were applied to liquid whey for 1.5 h at 60 degrees C followed by cooling to 5 degrees C. A control whey with no bleach was also evaluated. Flavor of the liquid wheys was evaluated by sensory and instrumental volatile analysis. One HP treatment and one BPO treatment were subsequently selected and incorporated into liquid whey along with an unbleached control that was processed into spray-dried WPC. These trials were conducted in triplicate. The WPC were evaluated by sensory and instrumental analyses as well as color and proximate analyses. The HP-bleached liquid whey and WPC contained higher concentrations of oxidation reaction products, including the compounds heptanal, hexanal, octanal, and nonanal, compared with unbleached or BPO-bleached liquid whey or WPC. The HP products were higher in overall oxidation products compared with BPO samples. The HP liquid whey and WPC were higher in fatty and cardboard flavors compared with the control or BPO samples. Hunter CIE Lab color values (L*, a*, b*) of WPC powders were distinct on all 3 color scale parameters, with HP-bleached WPC having the highest L* values. Hydrogen peroxide resulted in a whiter WPC and higher off-flavor intensities; however, there was no difference in norbixin recovery between HP and BPO. These results indicate that the bleaching of liquid whey may affect the flavor of WPC and that the type of bleaching agent used may affect WPC flavor.

Topics: Adult; Benzoyl Peroxide; Bixaceae; Carotenoids; Color; Female; Food Technology; Humans; Hydrogen Peroxide; Male; Middle Aged; Milk Proteins; Plant Extracts; Principal Component Analysis; Taste; Volatile Organic Compounds; Whey Proteins

Annatto preparations based on extracts of the seed of tropical bush Bixa orellana L consist of carotenoid-type pigments. Previous reports indicate that commercial annatto extracts have biological activities against microorganisms of significance to food fermentation, preservation, and safety. The objective of this study was to separate and identify the compound(s) responsible for the antimicrobial activity of annatto preparations. Commercial water-soluble annatto extracts were screened by thin-layer chromatography and bioautography followed by liquid chromatography/photodiode array/mass spectrometry (LC/PDA/MS) analysis of active fractions. Bioautography revealed two fractions with antimicrobial activity against Staphylococcus aureus. LC/PDA/MS analysis of both fractions revealed 9'-cis-norbixin (UV(max) 460 and 489 nm) and all-trans-norbixin (UV(max) 287, 470, and 494 nm) as the major components. Structure confirmation was achieved by (1)H NMR spectroscopy. Results indicate that 9'-cis-norbixin and all-trans-norbixin are responsible for the antimicrobial properties of annatto.

Topics: Anti-Infective Agents; Bacteria; Bixaceae; Carotenoids; Chromatography, Liquid; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; Mass Spectrometry; Plant Extracts; Solubility; Water

There is considerable interest in determining the added levels of the natural dye annatto in foods like snack products, particularly because they are mostly consumed by young people. The objective was to use response surface methodology to develop a new method to analyse annatto in extruded snacks. A pretreatment of the samples was necessary, digesting the ground sample with alpha-amylase at room temperature. The pigment was extracted by shaking with ethyl acetate at room temperature, eight extractions being necessary for completion extract the pigment. Lipids were removed by alkaline saponification. Under these conditions, 100% of the bixin was converted into norbixin, which was then quantified by high-performance liquid chromatography. The method had a mean recovery of 97% and a coefficient of variation for duplicate analysis of 1%. Using this method, of the 13 commercial samples analysed, a parmesan cheese-flavoured snack product showed the highest level of dye expressed as norbixin (15.5 mg kg(-1)), whilst other brands of onion-flavoured snack products had the lowest levels (0.7 and 0.4 mg kg(-1), respectively).

Topics: Bixaceae; Carotenoids; Chromatography, High Pressure Liquid; Food Analysis; Food Coloring Agents; Humans; Hydrolysis; Plant Extracts; Zea mays

A subchronic oral toxicity study of annatto extract (norbixin), a natural food color, was conducted. Groups of 10 male and 10 female Sprague-Dawley rats were fed annatto extract at dietary levels of 0, 0.1, 0.3 and 0.9% for 13 weeks. There were no treatment-related adverse effects on body weight, food and water consumption, ophthalmology and hematology data. Blood biochemical analysis revealed changes in rats of both sexes confined to the 0.9% and 0.3% groups, including increased alkaline phosphatase, phospholipid, total protein, albumin and albumin/globulin ratio. Marked elevation in absolute and relative liver weights was also found in both sexes of the 0.9% and 0.3% groups, but not the 0.1% group. Hepatocyte hypertrophy was evident and an additional electron microscopic examination demonstrated this to be linked to abundant mitochondria after exposure to a dietary level of 0.9% annatto extract for 2 weeks. Thus, the No-Observed-Adverse-Effect-Level (NOAEL) was judged to be a dietary level of 0.1% (69 mg/kg body weight/day for males, 76 mg/kg body weight/day for females) of annatto extract (norbixin) under the present experimental conditions.

Topics: Administration, Oral; Alkaline Phosphatase; Animals; Bixaceae; Blood Proteins; Carotenoids; Dose-Response Relationship, Drug; Hepatocytes; Hypertrophy; Liver; Male; No-Observed-Adverse-Effect Level; Organ Size; Plant Extracts; Rats; Rats, Sprague-Dawley; Seeds

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