Invited review: Annatto usage and bleaching in dairy foods

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.     

Effect of bleaching whey on sensory and functional properties of 80% whey protein concentrate

Whey is a highly functional food that has found widespread use in a variety of food and beverage applications. A large amount of the whey proteins produced in the United States is derived from annatto-colored Cheddar cheese. Color from annatto is undesirable in whey and must be bleached. The objective of this study was to compare 2 commercially approved bleaching agents, benzoyl peroxide (BP) and hydrogen peroxide (HP), and their effects on the flavor and functionality of 80% whey protein concentrate (WPC80). Colored and uncolored liquid wheys were bleached with BP or HP, and then ultrafiltered, diafiltered, and spray-dried; WPC80 from unbleached colored and uncolored Cheddar whey were manufactured as controls. All treatments were manufactured in triplicate. The WPC80 were then assessed by sensory, instrumental, functionality, color, and proximate analysis techniques. The HP-bleached WPC80 were higher in lipid oxidation compounds (specifically hexanal, heptanal, octanal, nonanal, decanal, dimethyl disulfide, and 1-octen-3-one) and had higher fatty and cardboard flavors compared with the other unbleached and BP-bleached WPC80. The WPC80 bleached with BP had lower norbixin concentrations compared with WPC80 bleached with HP. The WPC powders differed in Hunter color values (L, a, b), with bleached powders being more white, less red, and less yellow than unbleached powders. Bleaching with BP under the conditions used in this study resulted in larger reductions in yellowness of the powders made from whey with annatto color than did bleaching with HP. Functionality testing demonstrated that whey bleached with HP treatments had more soluble protein after 10 min of heating at 90°C at pH 4.6 and pH 7 than the no-bleach and BP treatments, regardless of additional color. Overall, HP bleaching caused more lipid oxidation products and subsequent off-flavors compared with BP bleaching. However, heat stability of WPC80 was enhanced by HP bleaching compared with control or BP-bleached WPC80.     

Colorants in cheese manufacture: Production,chemistry, interactions,and regulation

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.     

The Influence of Bleaching Agent and Temperature on Bleaching Efficacy and Volatile Components of Fluid Whey and Whey Retentate

Fluid whey or retentate are often bleached to remove residual annatto Cheddar cheese colorant, and this process causes off‐flavors in dried whey proteins. This study determined the impact of temperature and bleaching agent on bleaching efficacy and volatile components in fluid whey and fluid whey retentate. Freshly manufactured liquid whey (6.7% solids) or concentrated whey protein (retentate) (12% solids, 80% protein) were bleached using benzoyl peroxide (BP) at 100 mg/kg (w/w) or hydrogen peroxide (HP) at 250 mg/kg (w/w) at 5 °C for 16 h or 50 °CC for 1 h. Unbleached controls were subjected to a similar temperature profile. The experiment was replicated three times. Annatto destruction (bleaching efficacy) among treatments was compared, and volatile compounds were extracted and separated using solid phase microextraction gas chromatography mass spectrometry (SPME GC‐MS). Bleaching efficacy of BP was higher than HP (P < 0.05) for fluid whey at both 5 and 50 °C. HP bleaching efficacy was increased in retentate compared to liquid whey (P < 0.05). In whey retentate, there was no difference between bleaching with HP or BP at 50 or 5 °C (P > 0.05). Retentate bleached with HP at either temperature had higher relative abundances of pentanal, hexanal, heptanal, and octanal than BP bleached retentate (P < 0.05). Liquid wheys generally had lower concentrations of selected volatiles compared to retentates. These results suggest that the highest bleaching efficacy (within the parameters evaluated) in liquid whey is achieved using BP at 5 or 50 °C and at 50 °C with HP or BP in whey protein retentate.     

Influence of storage, heat treatment, and solids composition on the bleaching of whey with hydrogen peroxide

The residual annatto colorant in liquid whey is bleached to provide a desired neutral color in dried whey ingredients. This study evaluated the influence of starter culture, whey solids and composition, and spray drying on bleaching efficacy. Cheddar cheese whey with annatto was manufactured with starter culture or by addition of lactic acid and rennet. Pasteurized fat-separated whey was ultrafiltered (retentate) and spray dried to 34% whey protein concentrate (WPC34). Aliquots were bleached at 60 °C for 1 h (hydrogen peroxide, 250 ppm), before pasteurization, after pasteurization, after storage at 3 °C and after freezing at -20 °C. Aliquots of retentate were bleached analogously immediately and after storage at 3 or -20 °C. Freshly spray dried WPC34 was rehydrated to 9% (w/w) solids and bleached. In a final experiment, pasteurized fat-separated whey was ultrafiltered and spray dried to WPC34 and WPC80. The WPC34 and WPC80 retentates were diluted to 7 or 9% solids (w/w) and bleached at 50 °C for 1 h. Freshly spray-dried WPC34 and WPC80 were rehydrated to 9 or 12% solids and bleached. Bleaching efficacy was measured by extraction and quantification of norbixin. Each experiment was replicated 3 times. Starter culture, fat separation, or pasteurization did not impact bleaching efficacy (P > 0.05) while cold or frozen storage decreased bleaching efficacy (P < 0.05). Bleaching efficacy of 80% (w/w) protein liquid retentate was higher than liquid whey or 34% (w/w) protein liquid retentate (P < 0.05). Processing steps, particularly holding times and solids composition, influence bleaching efficacy of whey. PRACTICAL APPLICATION: Optimization of whey bleaching conditions is important to reduce the negative effects of bleaching on the flavor of dried whey ingredients. This study established that liquid storage and whey composition are critical processing points that influence bleaching efficacy.     

The effect of bleaching agents on the degradation of vitamins and carotenoids in spray-dried whey protein concentrate

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.     

Bleaching of colored whey and milk by a multiple-enzyme system

Carotenoids are broadly used to enhance the color of cheese types like Cheddar and Gouda. While ～80 % of the colorants are transferred into the cheese, the rest remains in the whey and impedes its commercial utilization. Therefore, cheese whey is currently bleached chemically by addition of either hydrogen peroxide or benzoyl peroxide in the industrial practice. To avoid heat- and peroxide-induced protein denaturation and the formation of off-flavors, an enzymatic bleaching process was developed. The ability of the fungal peroxidase MsP1 to degrade carotenoids was successfully employed for the bleaching of colored whey and milk in two- and three-enzyme systems, respectively. The systems were composed of MsP1, glucose oxidase, and when necessary, acid lactase. The initial step of the three-enzyme system was the lactase-catalyzed hydrolysis of lactose to galactose and glucose. The latter served as a substrate for the enzyme glucose oxidase in the second step which yielded gluconic acid and hydrogen peroxide. Finally, MsP1 oxidatively degraded the carotenoids. The activities of the involved enzymes were fine-tuned to optimize the bleaching process.     

Short communication: Norbixin and bixin partitioning in Cheddar cheese and 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.     

Short communication: Annatto in Cheddar cheese-derived whey protein concentrate is primarily associated with milk fat globule membrane

The yellow color of Cheddar cheese whey arises from a residual amount of annatto that partitions into the whey during Cheddar cheese manufacture. Bleaching of the color using hydrogen peroxide or benzoyl peroxide is often a prerequisite to produce an acceptable neutral-colored whey protein concentrate and isolate. However, the use of these strong oxidizing agents often generates off-flavors as a result of lipid oxidation and results in loss of nutritive value due to protein oxidation. The objective of this study was to determine the extent of partitioning of annatto between protein, milk fat globule membrane (MFGM), and aqueous (serum) phases of cheese whey so that a simple method can be developed to remove annatto from cheese whey. The MFGM was separated from Cheddar cheese whey using a recently developed novel method. Quantitative analysis of the distribution of annatto in the fat-free whey protein isolate (WPI), the MFGM fractions, and the serum phase revealed that annatto was not bound to the protein fraction but was mostly distributed between the serum phase and the MFGM fraction. The results showed that a colorless WPI or whey protein concentrate could be produced from Cheddar cheese whey by separation of MFGM from the whey, followed by diafiltration. This approach will negate the need for using bleaching agents.     

Effect of temperature and bleaching agent on bleaching of liquid Cheddar whey

The use of whey protein as an ingredient in foods and beverages is increasing, and thus demand for colorless and mild-tasting whey protein is rising. Bleaching is commonly applied to fluid colored cheese whey to decrease color, and different temperatures and bleach concentrations are used. The objectives of this study were to compare the effects of hot and cold bleaching, the point of bleaching (before or after fat separation), and bleaching agent on bleaching efficacy and volatile components of liquid colored and uncolored Cheddar whey. First, Cheddar whey was manufactured, pasteurized, fat-separated, and subjected to one of a number of hot (68°C) or cold (4°C) bleaching applications [hydrogen peroxide (HP) 50 to 500 mg/kg; benzoyl peroxide (BP) 25 to 100 mg/kg] followed by measurement of residual norbixin and color by reflectance. Bleaching agent concentrations were then selected for the second trial. Liquid colored Cheddar whey was manufactured in triplicate and pasteurized. Part of the whey was collected (no separation, NSE) and the rest was subjected to fat separation (FSE). The NSE and FSE wheys were then subdivided and bleaching treatments (BP 50 or 100 mg/kg and HP 250 or 500 mg/kg) at 68°C for 30 min or 4°C for 16 h were applied. Control NSE and FSE with no added bleach were also subjected to each time-temperature combination. Volatile compounds from wheys were evaluated by gas chromatography-mass spectrometry, and norbixin (annatto) was extracted and quantified to compare bleaching efficacy. Proximate analysis, including total solids, protein, and fat contents, was also conducted. Liquid whey subjected to hot bleaching at both concentrations of HP or at 100mg/kg BP had greater lipid oxidation products (aldehydes) compared with unbleached wheys, 50mg/kg BP hot-bleached whey, or cold-bleached wheys. No effect was detected between NSE and FSE liquid Cheddar whey on the relative abundance of volatile lipid oxidation products. Wheys bleached with BP had lower norbixin content compared with wheys bleached with HP. Bleaching efficacy of HP was decreased at 4°C compared with 68°C, whereas that of BP was not affected by temperature. These results suggest that fat separation of liquid Cheddar whey has no effect on bleaching efficacy or lipid oxidation and that hot bleaching may result in increased lipid oxidation in fluid whey.     

The Effect of Feed Solids Concentration and Inlet Temperature on the Flavor of Spray Dried Whey Protein Concentrate

Previous research has demonstrated that unit operations in whey protein manufacture promote off‐flavor production in whey protein. The objective of this study was to determine the effects of feed solids concentration in liquid retentate and spray drier inlet temperature on the flavor of dried whey protein concentrate (WPC). Cheddar cheese whey was manufactured, fat‐separated, pasteurized, bleached (250 ppm hydrogen peroxide), and ultrafiltered (UF) to obtain WPC80 retentate (25% solids, wt/wt). The liquid retentate was then diluted with deionized water to the following solids concentrations: 25%, 18%, and 10%. Each of the treatments was then spray dried at the following temperatures: 180 °C, 200 °C, and 220 °C. The experiment was replicated 3 times. Flavor of the WPC80 was evaluated by sensory and instrumental analyses. Particle size and surface free fat were also analyzed. Both main effects (solids concentration and inlet temperature) and interactions were investigated. WPC80 spray dried at 10% feed solids concentration had increased surface free fat, increased intensities of overall aroma, cabbage and cardboard flavors and increased concentrations of pentanal, hexanal, heptanal, decanal, (E)2‐decenal, DMTS, DMDS, and 2,4‐decadienal (P < 0.05) compared to WPC80 spray dried at 25% feed solids. Product spray dried at lower inlet temperature also had increased surface free fat and increased intensity of cardboard flavor and increased concentrations of pentanal, (Z)4‐heptenal, nonanal, decanal, 2,4‐nonadienal, 2,4‐decadienal, and 2‐ and 3‐methyl butanal (P < 0.05) compared to product spray dried at higher inlet temperature. Particle size was higher for powders from increased feed solids concentration and increased inlet temperature (P < 0.05). An increase in feed solids concentration in the liquid retentate and inlet temperature within the parameters evaluated decreased off‐flavor intensity in the resulting WPC80.     

The effect of bleaching agent on the flavor of liquid whey and whey protein concentrate

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°C followed by cooling to 5°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.     

Flavor of Cheddar Cheese: A Chemical and Sensory Perspective

Considerable knowledge has been accumulated on the biochemical processes occurring during ripening of Cheddar cheese, which in turn has major consequences on flavor and texture development. The present review outlines major metabolic pathways and agents involved in the modification of milk constituents in Cheddar cheese ripening. Mechanisms of volatile flavor and off‐flavor production and recent developments in the analysis, both sensory and instrumental, of Cheddar flavor and flavor compounds are also detailed here.     

Incorporation of Ultrafiltration Concentrated Whey Solids into Cheddar Cheese for Increased Yield

A process for incorporating whey solids into Cheddar cheese was evaluated. Whey was concentrated by ultrafiltration to between 9.8 and 20.3% solids (4.3 to 7.1% protein) and then heated at 75°C for 30 min. Return of this concentrate to cheese milk increased average yield 4.0% at constant cheese moisture. Cheese made by this procedure was lower in fat than control cheese and had a higher moisture content. Setting time was shorter, and acid development was faster. The pH was lower than that of the control cheese. Specific body, texture, and flavor characteristics were identified. Acid was the only flavor defect more prominent in experimental than in control cheese. None of the specific body or texture characteristics was significantly different.