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Erythrocytes exhibit high susceptibility to hemolysis in several pathologies due to the oxidation of cellular components. We hypothesized that annatto carotenoids improve the redox status of erythrocyte plasma membranes and promote a consequent increase in human erythrocyte resistance to hemolysis. The objective of this study was to evaluate whether food-grade annatto carotenoids can increase human erythrocyte resistance to hemolysis in vitro and ex vivo. For the in vitro experiment, erythrocytes from healthy volunteers were isolated and coincubated with bixin (BIX) or norbixin (NBIX) and 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), glucose, or sodium nitrite (NaNO 2 ) as hemolysis inducers. In the ex vivo study, healthy volunteers consumed a capsule containing BIX or NBIX (0.05 mg/kg body weight per day) or placebo for 7 days before blood sample collection. Their erythrocytes were isolated and incubated with AAPH, glucose, or NaNO 2 . In both the ex vivo and in vitro studies, erythrocytes were subjected to osmotic fragility tests. The activity of antioxidant enzymes, and reduced glutathione and lipid peroxidation levels in erythrocytes were also evaluated ex vivo. In vitro BIX and NBIX not only reduced erythrocyte membrane fragility induced by AAPH, glucose, or NaNO 2 but also improved basal osmotic resistance in the micromole-per-liter range (P < .05). BIX and NBIX supplementation increased erythrocyte membrane resistance (P < .05), with BIX being more effective. Also, BIX and NBIX protected erythrocytes from lipid peroxidation and improved the cellular redox environment (P < .05). These results support the hypothesis that annatto carotenoids supplementation exerts antihemolytic properties by preventing the oxidative damage of human erythrocytes., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Previous research has shown that bleaching affects flavor and functionality of whey proteins. The role of different bleaching agents on vitamin and carotenoid degradation is unknown. The objective of this study was to determine the effects of bleaching whey with traditional annatto (norbixin) by hydrogen peroxide (HP), benzoyl peroxide (BP), or native lactoperoxidase (LP) on vitamin and carotenoid degradation in spray-dried whey protein concentrate 80% protein (WPC80). An alternative colorant was also evaluated. Cheddar whey colored with annatto (15 mL/454 L of milk) was manufactured, pasteurized, and fat separated and then assigned to bleaching treatments of 250 mg/kg HP, 50 mg/kg BP, or 20 mg/kg HP (LP system) at 50°C for 1 h. In addition to a control (whey with norbixin, whey from cheese milk with an alternative colorant (AltC) was evaluated. The control and AltC wheys were also heated to 50°C for 1 h. Wheys were concentrated to 80% protein by ultrafiltration and spray dried. The experiment was replicated in triplicate. Samples were taken after initial milk pasteurization, initial whey formation, after fat separation, after whey pasteurization, after bleaching, and after spray drying for vitamin and carotenoid analyses. Concentrations of retinol, a-tocopherol, water-soluble vitamins, norbixin, and other carotenoids were determined by HPLC, and volatile compounds were measured by gas chromatography-mass spectrometry. Sensory attributes of the rehydrated WPC80 were documented by a trained panel. After chemical or enzymatic bleaching, WPC80 displayed 7.0 to 33.3% reductions in retinol, β-carotene, ascorbic acid, thiamin, α-carotene, and α-tocopherol. The WPC80 bleached with BP contained significantly less of these compounds than the HP- or LP-bleached WPC80. Riboflavin, pantothenic acid, pyridoxine, nicotinic acid, and cobalamin concentrations in fluid whey were not affected by bleaching. Fat-soluble vitamins were reduced in all wheys by more than 90% following curd formation and fat separation. With the exception of cobalamin and ascorbic acid, water-soluble vitamins were reduced by less than 20% throughout processing. Norbixin destruction, volatile compound, and sensory results were consistent with previous studies on bleached WPC80. The WPC80 colored with AltC had a similar sensory profile, volatile compound profile, and vitamin concentration as the control WPC80., (Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Tocotrienols (T3), the lesser known isomers of vitamin E, have been reported to possess anticancer activity both in in vitro and in vivo experimental models of rodents transplanted with parental tumors or treated with carcinogens. We investigated the effects of dietary supplementation with annatto-T3 (90% δ-T3 and 10% γ-T3) on the spontaneous development of mammary tumors in HER-2/neu transgenic mice. Underlying mechanisms of the antitumor effect were evaluated by studying apoptosis, senescent-like growth arrest, immune modulation, oxidative effect and the expression of HER-2/neu in tumoral mammary glands of transgenic mice and in vitro in human and mice tumor cell lines. Annatto-T3 supplementation delayed the development of mammary tumors, reducing the number and size of mammary tumor masses and those of lung metastases. In annatto-T3-supplemented mice, both apoptosis and senescent-like growth arrest of tumor cells were increased in mammary glands while no immune modulation was observed. In vitro, a dose-dependent inhibition of cell growth, increased apoptosis and senescent-like growth arrest and a time-dependent accumulation of reactive oxygen species were observed in tumor cells treated with annatto-T3 or purified δ-T3. Annatto-T3 reduced both HER-2/neu mRNA and p185(HER-2/neu) protein in tumors and in tumor cell lines. The results show that the antitumor effect of annatto-T3 supplementation in HER-2/neu transgenic mice is mainly related to the direct induction of oxidative stress, senescent-like growth arrest and apoptosis of tumor cells rather than to an immune modulation.

The flavor of whey protein can carry over into ingredient applications and negatively influence consumer acceptance. Understanding sources of flavors in whey protein is crucial to minimize flavor. The objective of this study was to evaluate the effect of annatto color and starter culture on the flavor and functionality of whey protein concentrate (WPC). Cheddar cheese whey with and without annatto (15 mL of annatto/454 kg of milk, annatto with 3% wt/vol norbixin content) was manufactured using a mesophilic lactic starter culture or by addition of lactic acid and rennet (rennet set). Pasteurized fat-separated whey was then ultrafiltered and spray dried into WPC. The experiment was replicated 4 times. Flavor of liquid wheys and WPC were evaluated by sensory and instrumental volatile analyses. In addition to flavor evaluations on WPC, color analysis (Hunter Lab and norbixin extraction) and functionality tests (solubility and heat stability) also were performed. Both main effects (annatto, starter) and interactions were investigated. No differences in sensory properties or functionality were observed among WPC. Lipid oxidation compounds were higher in WPC manufactured from whey with starter culture compared with WPC from rennet-set whey. The WPC with annatto had higher concentrations of p-xylene, diacetyl, pentanal, and decanal compared with WPC without annatto. Interactions were observed between starter and annatto for hexanal, suggesting that annatto may have an antioxidant effect when present in whey made with starter culture. Results suggest that annatto has a no effect on whey protein flavor, but that the starter culture has a large influence on the oxidative stability of whey., (Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Annatto is a yellow/orange colorant that is widely used in the food industry, particularly in the dairy industry. Annatto, consisting of the carotenoids bixin and norbixin, is most commonly added to produce orange cheese, such as Cheddar, to achieve a consistent color over seasonal changes. This colorant is not all retained in the cheese, and thus a percentage remains in the whey, which is highly undesirable. As a result, whey is often bleached. Hydrogen peroxide and benzoyl peroxide are the 2 bleaching agents currently approved for bleaching whey in the United States. Recent studies have highlighted the negative effect of bleaching on whey flavor while concurrently there is a dearth of current studies on bleaching conditions and efficacy. Recent international mandates have placed additional concern on the use of benzoyl peroxide as a bleaching agent. This review discusses the advantages, disadvantages, regulatory concerns, flavor implications, and optimal usage conditions of 2 widely used bleaching agents, hydrogen peroxide and benzoyl peroxide, as well as a few alternative methods including lipoxygenase, peroxidase, and lactoperoxidase systems., (Copyright (c) 2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The influence of the food dyes E160e (beta-apo-8'-carotenal in an oil suspension) and E160a (beta-carotene in an oil suspension) on clastogenic effects of cyclophosphamide (CP) and dioxidine (DN) was investigated. Chromosome damage in the bone marrow of C57BL/6 mice was reported. The following protocols were used: (1) simultaneous single administration of the dye and the mutagen and the subsequent animal sacrifice within 24 hr; (2) a 4-day pretreatment with the dye (daily administrations) followed with simultaneous injection of the dye and the mutagen on the 5th day 24 hr before sacrifice; (3) daily co-administration of the dye and the mutagen for 5 days with sacrifice 6 hr after the last administration. CP at a dose of 30 mg/kg and DN at 300 mg/kg were injected intraperitoneally; the dyes at doses of 0.5, 5 and 50 mg/kg were given orally. Under all the protocols applied, E160e at a dose of 50 mg/kg caused a significant reduction of both DN and CP effects. At 5 mg/kg this dye reduced the effects of the mutagens only under the pretreatment regimen. Pretreatment with E160a at doses of 5 and 50 mg/kg resulted in a meaningful reduction of the DN effect. Under the combined treatment with mutagens this dye reduced both CP and DN effects.

Chromosomal aberrations induced by Ponceau 4R (an azo food dye) and beta-carotene (a natural food colour) were studied on bone marrow cells of mice in vivo. The results indicated that Ponceau 4R was more clastogenic than beta-carotene. Ponceau 4R was found to have a minimum effective dose of 4 mg which induced a significant number of chromosome aberrations. This dose is also the recommended 'admissible daily intake'. Chromosome aberrations induced by beta-carotene were not significantly higher than those of the control (olive oil) in the dose range 0.27 to 27 mg/kg body weight. The genotoxicity of these dyes can be attributed to their chemical composition. In so far as genotoxicity is concerned the carotenoid beta-carotene can be safely used as a food colorant whereas Ponceau 4R should be delisted as a food dye.