J. Dairy Sci.

93:3891–3901
doi:10.3168/jds.2010-3190
© American Dairy Science Association®, 2010.

Invited review: Annatto usage and bleaching in dairy foods

E. J. Kang,* R. E. Campbell,* E. Bastian,† and M. A. Drake*1
*Department of Food, Bioprocessing and Nutrition Sciences, Southeast Dairy Foods Research Center, North Carolina State University,
Raleigh 27695
†Glanbia Nutritionals, Twin Falls, ID 83301

ABSTRACT liano et al., 2003). Prices for annatto seeds depend on

production and are proportional to the content of bixin,
Annatto is a yellow/orange colorant that is widely
the major pigment (Giuliano et al., 2003). The United
used in the food industry, particularly in the dairy
States and Europe are the 2 largest importers of an-
industry. Annatto, consisting of the carotenoids bixin
natto seeds, but the Japanese market has grown rapidly
and norbixin, is most commonly added to produce or-
since the introduction of the colorant in 1963 (Ames
ange cheese, such as Cheddar, to achieve a consistent
and Hofmann, 2001).
color over seasonal changes. This colorant is not all
Annatto as a colorant is not a new concept. The
retained in the cheese, and thus a percentage remains
Aztecs used annatto extract as a dye for textiles, body
in the whey, which is highly undesirable. As a result,
dye (such as in lipsticks), and as a food colorant in the
whey is often bleached. Hydrogen peroxide and benzoyl
drink cacahuatl (Giuliano et al., 2003). In addition to
peroxide are the 2 bleaching agents currently approved
being used to impart color in various items, annatto
for bleaching whey in the United States. Recent stud-
has been used as a spice. Native Americans commonly
ies have highlighted the negative effect of bleaching on
used ground bixa seeds in their cacao beverages to
whey flavor while concurrently there is a dearth of cur-
impart a slight red color and to add a musky flavor
rent studies on bleaching conditions and efficacy. Recent
comparable to that of paprika or saffron (Miksicek et
international mandates have placed additional concern
al., 1981; Norton, 2006). Today, annatto is still used
on the use of benzoyl peroxide as a bleaching agent.
as a spice, especially in Latin American dishes such as
This review discusses the advantages, disadvantages,
cochinita pibil, a pork dish with ground bixa seeds and
regulatory concerns, flavor implications, and optimal
bitter orange juice (Gerlach and Gerlach, 2002). It is
usage conditions of 2 widely used bleaching agents, hy-
also used in sausages, fish, margarine, snacks, dress-
drogen peroxide and benzoyl peroxide, as well as a few
ings, sauces, and confections, but usage varies from
alternative methods including lipoxygenase, peroxidase,
country to country due to different food customs and
and lactoperoxidase systems.
legislations (Ames and Hofmann, 2001; Scotter, 2009).
Key words: annatto, bleach, flavor, whey
The major application of annatto in the United States
is within the dairy industry, where it is used to color
INTRODUCTION cheeses and other dairy products (Emerton, 2008).
Annatto is a yellow/orange carotenoid that is widely
used in the food industry. Annatto comes from seeds of Extraction of Annatto
the tropical tree fruit Bixa orellana, which was named
after Francisco de Orellana, a scientist and explorer Three commercial processes are used to extract the
of the upper Amazon (Giuliano et al., 2003). Clusters carotenoid pigment from dried annatto seeds: direct
of the fruit, which is capsular shaped and covered in extraction into oil, direct extraction into aqueous al-
burrs, grow on this tree and there are about 10 to 50 kali, or indirect extraction with solvents (Preston and
small seeds inside the fruit (Ames and Hofmann, 2001). Rickard, 1980). Extraction with oil or solvent yields a
The seeds are covered in a bright red pulp and this pulp colorant that is mainly bixin (Mortenson et al., 2008).
contains the annatto pigment (Giuliano et al., 2003). Extraction with aqueous alkali (the form used in the
Latin America produces about 60% of the world’s an- dairy industry, e.g., for cheese color) saponifies bixin’s
natto, followed by Africa (27%) and Asia (12%) (Giu- methyl group on bixin, yielding norbixin as the principal
colorant (Giuliano et al., 2003; Mortenson et al., 2008).
Both bixin and norbixin naturally occur in the cis form
Received February 22, 2010.
Accepted May 15, 2010. but can be converted to the trans form by light and
1
Corresponding author: maryanne_drake@ncsu.edu heat (McKeown 1963, 1965). The trans forms of both

3891
3892 KANG ET AL.

bixin and norbixin are more red than their cis forms in the United Kingdom was 50 mg of norbixin/kg of
(Preston and Rickard, 1980; Scotter et al., 1994). cheese and the estimated range of annatto in Red Leic-
ester was 23.7 to 37.5 mg of norbixin/kg of cheese. For
Volatile Compounds ripened orange, yellow, and broken white cheese, and
flavored processed cheese in the United Kingdom, the
Although annatto has been used in foods as a spice maximum level of annatto addition is 15 mg/kg. The
(flavoring), there is little information about how volatile estimated range of annatto is 0.2 to 9.6 mg/kg (n = 16)
compounds of annatto contribute to flavor. Galindo- and 0.2 to 21.4 mg/kg (n = 8) for ripened orange, yel-
Cuspinera et al. (2002) evaluated volatile profiles of oil low, and broken white cheese, and for flavored processed
and water-soluble annatto extracts and reported that cheese, respectively. Currently in the United States, no
β-humulene was the major volatile present. β-Humulene study has been conducted to determine the amounts of
is described as having a woody, spicy aroma and a annatto typically found in cheese. In the United States,
slightly bitter taste (Galindo-Cuspinera et al., 2002). annatto is approved as a color additive in foods at GMP,
Other important volatiles found in annatto extracts meaning that the “Annatto extract may be safely used
that might contribute to flavor were p-xylene, toluene, for coloring foods generally, in amounts consistent with
α- and β-pinene, γ-elemene, and spathulenol. Annatto good manufacturing practice...” (Hallagan et al., 1995;
extraction uses heat (Scotter et al., 2002), which means Giuliano et al., 2003; US FDA, 2009a). Although an-
that different commercial products may experience natto is used to standardize the color of cheese, some
varying degrees of thermal degradation. Further stud- studies have reported the usage of annatto leading to
ies also suggest that heat treatment of annatto in foods pinking or discoloration of cheeses (Hood and White,
may result in additional flavor-contributing volatile 1929; Moir, 1933; Morgan, 1933; Barnicoat, 1937, 1950;
compounds. Scotter (1995, 1998, 2000, 2001) studied Govindarajan and Morris, 1973).
the thermal degradation products of both bixin and Bixin and norbixin have highly conjugated structures
norbixin and found that heat released the aromatic making these compounds susceptible to both oxidation
compounds m-xylene and toluene. As mentioned previ- and reduction. Oxidation is important to the fluid whey
ously, annatto is a major colorant used in the dairy industry because oxidation leads to color loss, which
industry, often in cheese and in processed cheese that is the primary goal of bleaching and will be discussed
involves heat treatment. Volatiles from thermal degra- later in this review. Also important, a study suggested
dation of annatto in actual dairy products (e.g., cheese) that norbixin might be able to bind with proteins
have yet to be studied. Studies conducted by Scotter such as β-casein or β-lactoglobulin and form a stable
et al. (2002) and Galindo-Cuspinera et al. (2002) in- complex that can help prevent oxidation and color loss
dicate that there are numerous odorants in annatto, (Govindarajan and Morris, 1973). This reaction has not
such as β-humulene, p-xylene, toluene, α- and β-pinene, been proven; it may be desirable in some products such
γ-elemene, and sapathulenol, with potential to influ- as cheese but reduce color removal during bleaching of
ence food aroma; however, more studies are needed in whey.
this area.
CAROTENOIDS IN MILK
Discoloration – Oxidation
Carotenoids are lipophilic molecules that are found
Annatto has been used in dairy products since the in milk fat. The diet of the cow can influence the color
1800s to standardize the color of cheese, which varies of cheese (Carpino et al., 2004). A wide variety of caro-
due to seasonal feed variations in the milk. Chr. Hansen tenoids and their degradation products are present in
Inc., a major, modern-day supplier of annatto to the the forage that cows eat, but only a small number of
dairy industry, opened their first factory in 1874 (Kristin different carotenoids can be identified in milk. This is
Schneider, Chr. Hansen Inc., Milwaukee, WI; personal because carotenoid transfer from diet to milk is low and
communication). Over the years, an orange color has carotenoids can be broken down to colorless compounds
become expected in many cheeses, requiring addition of in the gastrointestinal tract (Noziere et al., 2006). Some
annatto to cheesemilk. According to 21CFR73.30 (US of these colorless compounds may be transferred to the
FDA, 2009a), annatto extract may be used for coloring milk and have effects on the sensory profile even though
foods as long as good manufacturing practices (GMP) they do not impart any color to the final product.
are followed. Therefore, in the United States, there is Carotenoids identified in milk include lutein, violaxan-
no “maximum level” of usage for annatto; however, thin, antheraxanthin, zeaxanthin, neoxanthin, all-trans
Scotter et al. (2002) reported that the maximum level β-carotene, α-carotene, and 13-cis β-carotene. Among
of annatto addition in commercial Red Leicester cheese those, lutein and β-carotene are the most quantitatively
Journal of Dairy Science Vol. 93 No. 9, 2010
 INVITED REVIEW: ANNATTO AND BLEACHING IN DAIRY FOODS 3893

important (Noziere et al., 2006) with β-carotene com- Annatto Measurement in Foods
prising about 90% of total carotenoids present in cow’s
milk (Hulshof et al., 2005). Annatto can be found in a wide array of foods ranging
Carotenoids can influence the sensory properties of from dairy products to other naturally colored prod-
milk either indirectly through their antioxidant prop- ucts. A colorimetric method for measuring the amount
erties by stabilizing oxidizable compounds or directly of annatto in commercial bleached and unbleached
through their yellowing properties. β-Carotene can be dry whey powder by extracting the annatto using am-
converted to vitamin A, but incomplete conversion monia, ethanol, and phosphate solution was reported
leaves a yellow color in the milk (Patton et al., 1980). by Hammond et al. (1975). Lancaster and Lawrence
The main components in milk are riboflavin (vitamin (1995) extracted annatto from high-fat dairy products,
B2), a green compound present in the aqueous phase, with good repeatability, although methods were labor
and β-carotene in the lipid phase (Noziere et al., 2006). intensive. Annatto was confirmed and components were
Riboflavin and carotenoid contents vary widely in milk identified via HPLC (Lancaster and Lawrence, 1995).
and are influenced by diet, breed, and season (Schröder, Scotter et al. (2002) extracted and analyzed annatto
2003; Noziere et al., 2006). Croissant et al. (2007) re- in margarine, ice cream, custard powder, breakfast
ported that milk from pasture-fed Holstein and Jersey cereals, cakes, fish, and jellies using various extrac-
cows exhibited more yellow color than that from their tion methods tailored to each food matrix followed
similar counterparts fed a conventional, TMR diet. by HPLC coupled with spectral confirmation. Bareth
These findings were expected because pasture-fed cows et al. (2002) described a rapid, simple extraction and
ingested fresh forage, containing carotenoids, thus rais- detection method specific for annatto in cheese and
ing the concentration of carotenoids in milk fat. Caro- milk products using HPLC and a spectrophotometric
tenoids found in milk also carry over into fluid whey method. Very recently, Croissant et al. (2009) reported
during cheese production, where they may increase a successful method to extract and quantify norbixin
the color of spray-dried whey and require a bleaching (the primary annatto constituent in whey) from liquid
process. whey, liquid retentate, and spray-dried whey protein
concentrate. Analytical methods for annatto and analy-
sis in food have been reviewed by Scotter (2009).
Carotenoid Measurement in Foods

Carotenoid content can be difficult to measure be- BLEACHING

cause carotenoids isomerize rapidly due to their conju-
gated double bonds (de Oliveira and Rodriguez-Amaya, Food is bleached not only to improve color, but in
2007). Isomerization can be promoted by acid, light, some cases to improve its quality such as improving
and heat (de Oliveira and Rodriguez-Amaya, 2007). gluten development in flour. The bleaching process can
As such, precautions must be taken to minimize these be categorized into 3 groups depending on the mecha-
reactions during extraction and measurement. These nism: oxidizing bleach, reducing bleach, and biochemi-
precautions include, but are not limited to, completion cal bleach (Balls et al., 1943; Bottomley et al., 1989;
of analysis within the shortest time possible, protection Roos et al., 2006).
from light, avoiding high temperatures or contact with
acid, and the use of high-purity solvents (de Oliveira Oxidizing Bleach
and Rodriguez-Amaya, 2007). Once carotenoids are
isolated, qualification and quantification are typically The portion of a molecule that emits color is called
performed using the same methods. a chromophore and it is usually the most fragile part
Before quantification, the carotenoid must be posi- of the molecule. Destroying the chromophore will often
tively identified. This is done using chromatography— render a colored molecule colorless. Oxidizing agents
open column, thin layer, or HPLC (Bushway, 1985). (bleach) destroy (oxidize) the double bond(s) within a
The best of these 3 methods, HPLC, saves time and chromophore. This reaction changes the molecule into a
can distinguish between stereoisomers (Bushway, 1985). different substance in which the chromophore does not
The carotenoid can be identified using retention times, exist or a shorter chromophore exists. A shorter chro-
co-chromatography with carotenoid standards, or UV- mophore will absorb light of a shorter wavelength than
visible absorption spectra (de Oliveira and Rodriguez- visible light (UV light) and therefore will not appear
Amaya, 2007). After the carotenoid is positively identi- colored (Winter et al., 2008). Many oxidizing bleaches
fied, it is then quantified by using an external standard are used in industry including chlorine dioxide, which
curve (de Oliveira and Rodriguez-Amaya, 2007). is used for the bleaching of wood pulp, fats and oils,

Journal of Dairy Science Vol. 93 No. 9, 2010

3894 KANG ET AL.

cellulose, flour, textiles, and beeswax. Organic perox- Netherlands) is a fungal peroxidase that can purport-
ides, such as hydrogen peroxide and benzoyl peroxide edly be used in place of an oxidizing bleach to bleach
and bromates, are oxidizing bleaches used for whey fluid whey. The enzyme is specific for carotenoids (Zorn
and flour bleaching and as maturing agents. Another et al., 2003), thus yielding whiter whey without the use
application is in the wood and pulp industry to pro- of traditional bleaching agents, and possibly reducing
duce chlorine-free paper for which the industry uses unwanted side effects of traditional bleaching. Like all
peracetic acid, ozone, hydrogen peroxide, and oxygen peroxidases, MaxiBright requires activation with 0.5 to
in bleaching sequences. 1 mM hydrogen peroxide, but the hydrogen peroxide is
consumed and catalase addition is not required. There
Reducing Bleach are currently no available published studies on the use
of MaxiBright with whey or in comparison to tradi-
A reducing bleach works by converting double bonds tional oxidizing bleaches. Food and Drug Administra-
in a chromophore into single bonds. This eliminates the tion’s “generally regarded as safe” (GRAS) status has
ability of the chromophore to absorb visible light. Sun- been filed for MaxiBright, and approval is anticipated
light releases high-energy photons, often in the violet in September 2010.
or UV range, that can disrupt bonds in a chromophore,
rendering the substance colorless. Many colored mole- BLEACHING WHEY
cules are relatively fragile and are damaged by photons
of UV light. Traditionally, exposure to sunlight was the Annatto is used by the dairy industry to color
way to bleach fabrics and make them white (Bloom- cheese. The specific amount of annatto that partitions
field, 2007). Reducing bleaches commonly used in foods into cheese and whey has not, to our knowledge, been
include lemon juice (in combination with sunlight) and directly studied. However, approximately 20% of the
sulfur dioxide. annatto added to cheese milk is estimated to pass into
whey (Barnicoat, 1950). The color from annatto is
Biochemical Bleach highly unfavorable in dried whey products and thus a
decolorizing process must be performed and has been
Enzymes such as lipoxygenase (EC 1.13.11.12; lipoxy- in place in the dairy industry for more than 40 yr (Mc-
dase) can fall in the third category of bleaching agents: Donough et al., 1968). This process involves bleach-
biochemical bleaches. Lipoxygenase was first discov- ing of liquid whey or liquid retentate with hydrogen
ered in 1928 by Bohn and Hass (1953) as a carotene- or benzoyl peroxide. Hydrogen peroxide and benzoyl
destroying enzyme (“carotene oxidase”) in soybeans, peroxide are the only bleaching agents legally allowed
and later designated as lipoxygenase. Acceleration of for treatment of whey in the United States and there
the oxidation of xanthophylls to colorless products has are several restrictions to their use.
long been recognized as a property of soybean lipoxy-
genase used in the bleaching of bread dough (Balls et Hydrogen Peroxide
al., 1943). A patent on biochemical bleaching of dairy
products was introduced in the United States recently Hydrogen peroxide (H2O2) is a clear, colorless liquid
(Roos et al., 2006). Roos et al. (2006) disclosed the with a slightly pungent odor. Hydrogen peroxide de-
use of lipoxygenases to bleach dairy products, includ- composes to oxygen and water during bleaching (Table
ing whey. Only small amounts of lipoxygenases have a 1). Residual hydrogen peroxide must be removed from
whitening effect on dairy products such as milk, cheese, whey and cheese milk physically or by the addition of
butter oil, cream, and whey products. The mechanism catalase according to FDA regulations 184.1366 and
of bleaching by lipoxygenase is based on the oxidative 133.113 (US FDA, 2009l,e). Catalase converts hydro-
transition of double bonds in carotenoids by radicals gen peroxide into oxygen and water (Table 1). Catalase
produced in the reaction of lipoxygenase and linole(n) use must not exceed 20 ppm and must be sufficient
ic acid. Soy lipoxygenase has been used for bleaching to remove any residual hydrogen peroxide (US FDA,
purposes in wheat and maize flour; however, it has not 2009e). As hydrogen peroxide is a GRAS substance, the
been previously applied to bleach whey. Because a more maximum treatment level for bleaching annatto-colored
specific enzymatic reaction is occurring rather than a whey using hydrogen peroxide is 0.05% (<500 ppm)
general oxidation treatment, as is the case with oxidiz- of the whey (US FDA, 2009l). There are no specific
ing bleaches, enzymatic bleaching may reduce off-fla- provisions in European Union regulations regarding the
vors. Recently (November 2009), an external enzymatic use of hydrogen peroxide as a bleaching agent for dairy
(biochemical) bleach was launched for bleaching of fluid products. When no national provisions on processing
whey. MaxiBright (DSM Food Specialties, Delft, the aids exist, their use is controlled by general safety pro-
Journal of Dairy Science Vol. 93 No. 9, 2010
 INVITED REVIEW: ANNATTO AND BLEACHING IN DAIRY FOODS 3895
Table 1. Summary of hydrogen peroxide and benzoyl peroxide for whey bleaching

Bleaching agent

Item Hydrogen peroxide Benzoyl peroxide

Structure

(National Center for Biotechnology Information, 2008b) (National Center for Biotechnology Information, 2008a)

Breakdown 2H2O2 → O2 + 2H2O

(hydrogen peroxide → oxygen + water)
(Smith, 2004)

(Smith, 2004)

Regulation 21CFR184.1366 (US FDA, 2009l) 21CFR184.1157 (US FDA, 2009k)

Pros ČOLWWOHWRQRHIIHFWRQWKHQXWULHQWVSUHVHQW ČHIIHFWLYHDWORZHUXVDJHOHYHOVWKDQK\GURJHQSHUR[LGH

ČPRUHDFFHSWDEOHIRUXVDJHLQRWKHUFRXQWULHV ČGRHVQRWUHTXLUHDFDWDODVHDGGLWLRQWRUHPRYHUHVLGXHV
ČGRHVQRWSLWVWDLQOHVVVWHHOWKHUHIRUHLVOHVVFRUURVLYHWRHTXLSPHQW
ČHIIHFWLYHDFURVVDZLGHUDQJHRIWHPSHUDWXUHV

Cons ČPXVWEHLQDFWLYDWHGZLWKFDWDODVH ČSRVVLEOHIRUPDWLRQRIR[LGL]HGIODYRUV

ČFRXOGSRVVLEO\FDXVHR[LGL]HGIODYRUV ČSRVVLELOLW\WKDWWKHFDUULHUXVHGPD\EHFRQVLGHUHGDQDOOHUJHQ
ČFRUURVLYHWRHTXLSPHQW ČFRQFHUQVIURPRWKHUFRXQWULHVEHFDXVHLWKDVMXVWEHHQUHFHQWO\
ČOHVVHFRQRPLFDOWRXVHEHFDXVHLWUHTXLUHV approved by Codex
much more peroxide for satisfactory bleaching

visions. Therefore, it can be understood that European food ingredient upon the following current GMP condi-
Union regulations are in agreement with FDA regula- tions of use: (1) the ingredient is used as a bleaching
tions for the use of hydrogen peroxide as a bleaching agent in food, (2) the ingredient is used in the following
agent for whey. foods, milk used for production of Asiago fresh and
In addition to being used as a bleaching agent, hydro- Asiago soft cheese (US FDA, 2009b), Blue cheese (US
gen peroxide can be used as an antimicrobial agent in FDA, 2009c), Caciocavallo Siciliano cheese (US FDA,
milk, intended for cheese making at 0.05% (wt/wt) level 2009d), Gorgonzola cheese (US FDA, 2009f), Parmesan
(US FDA, 2009l). Likewise, hydrogen peroxide can be and Reggiano cheese (US FDA, 2009g), Provolone cheese
used as an antimicrobial agent during the preparation (US FDA, 2009h), Romano cheese (US FDA, 2009i),
of modified whey by electrodialysis methods at 0.04% and Swiss and Emmentaler cheese (US FDA, 2009j),
(wt/wt) level (US FDA, 2009l). Hydrogen peroxide can annatto-colored whey, such that the final bleached
be used as a microbial agent in cheese milk and a side product conforms to the descriptions and specifications
effect will be bleaching milk. However, federal food and for whey, concentrated whey, or dried whey, at levels
drug regulations do not support the use of hydrogen not to exceed current GMP. In contrast to hydrogen
peroxide solely for the use of bleaching milk for cheese peroxide, benzoyl peroxide may be used to bleach milk
and related cheese products (US FDA, 2009l). for certain cheese productions as listed above, whereas
hydrogen peroxide may not be used to bleach milk for
Benzoyl Peroxide cheese make in any cheese, but hydrogen peroxide is
permitted to be used at 0.05% in milk for antimicrobial
Benzoyl peroxide (C14H10O4) is a colorless, crystalline purposes. The current GMP is as follows: the weight of
solid permitted for use in removing color in whey prod- the benzoyl peroxide is not more than 0.002% (20 ppm)
ucts that are not used for infant formula. Like hydrogen of the weight of the milk being bleached, and the weight
peroxide, benzoyl peroxide (or a mixture of benzoyl of the potassium alum, calcium sulfate, and magnesium
peroxide with potassium alum, calcium sulfate, and carbonate, singly or combined, should not be more than
magnesium carbonate) is also a GRAS substance and 6 times the weight of the benzoyl peroxide used (US
can be used to bleach dairy ingredients. Unlike hydrogen FDA, 2009b,c,d,f,g,h,i,j). Whey is considered a GRAS
peroxide, benzoyl peroxide has no limitation on usage substance and thus has no standard of identity. This
rates in foods other than current GMP rules (US FDA, means that there is no federal regulation for usage rates
2009k). Benzoyl peroxide is GRAS as a direct human of benzoyl peroxide in whey for bleaching but using
Journal of Dairy Science Vol. 93 No. 9, 2010
3896 KANG ET AL.

benzoyl peroxide as a preservative is not acceptable. found in cheeses, but at lower concentrations than in
Should benzoyl peroxide be used at levels >1% (wt/wt) fermented dairy products (Sieber et al., 1995). Sub-
in liquid whey, the manufacturer would need to justify sequently, benzoic acid occurs naturally in whey and
the usage rate. Most additives are used at a rate of 1 to whey protein powders. Adverse reactions to benzoic
3% (wt/wt), but any usage rate over 1% (wt/wt) raises acid-related compounds are rare, and life-threatening
many red flags (N. Ratzlaff, USDA, Lisle, IL; personal reactions are extremely rare (JECFA, 2004). Chang
communication). et al. (1977) found that when benzoyl peroxide was
There are concerns about benzoyl peroxide bleach- added to Edam cheese whey, after it was heated to
ing as it has only been recently approved by Codex 69°C for 6 h and cooled, 91.7% of the benzoyl peroxide
Alimentarius, with the maximum level (100 mg/kg) for used was recovered as benzoic acid, and minor amounts
liquid whey and whey products (excluding infant ap- of hydroxybenzoic acids, phenylbenzoate, phenol, and
plications) being adopted in 2007, and the maximum benzoyl peroxide were found.
level (100 mg/kg) for dried whey and whey products
(excluding infant applications) being adopted in 2005 Lactoperoxidase
(Codex, 2008). The European Commission (EC) allows
both hydrogen peroxide and benzoyl peroxide to be When the lactoperoxidase system is used for antimi-
used as whey bleaching agents (designated as process- crobiological purposes, external addition of thiocyanate
ing aids, not food additives), but individual country or peroxide is required. However, when bleaching whey
provisions may be stricter (Bianca Herr, Leatherhead with hydrogen peroxide, companies often take advan-
Food Institute, Leatherhead, UK; personal communica- tage of the naturally existing lactoperoxidase system
tion). Many Asian and European government regulators in milk in combination with externally added hydrogen
do not like the use of benzoyl peroxide and consider peroxide to obtain maximum bleaching effectiveness
benzoic acid and other breakdown products of benzoyl of fluid whey. No additional enzyme or thiocyanate
peroxide harmful. There is a possibility that the carrier needs to be added for bleaching purposes (E. Bastian;
used for benzoyl peroxide may be considered an aller- unpublished data). Lactoperoxidase (LP; EC 1.11.1.7)
gen. In addition, certain carriers may pose problems is a natural enzyme found in milk and constitutes
with membrane fouling or leave residues of insoluble about 0.5% of the total whey proteins in bovine milk.
carrier on equipment. Benzoyl peroxide reacts with oxi- Lactoperoxidase is the second most abundant enzyme
dizable compounds and is converted into water-soluble in raw milk (Kussendrager and van Hooijdonk, 2000;
benzoic acid following bleaching (Table 1). A petition Fox and Kelly, 2006). The LP system consists of 3
was submitted by the US Dairy Export Council (US- components: lactoperoxidase, thiocyanate (SCN−), and
DEC) to the Taiwan Department of Health, Food Sani- hydrogen peroxide, and the system is only active in the
tation, and Safety for the use of benzoyl peroxide in presence of these 3 components (Seifu et al., 2005). The
whey powder and was approved on December 20, 1999 LP system is a potent bacteriocidal system that has
(Johnson, 2006). Benzoyl peroxide is not an approved been used to preserve raw milk without refrigeration.
bleaching agent for whey in China or Japan and neither Depending on the concentrations of SCN− and hydrogen
country’s regulations allow for residual benzoic acid in peroxide used to activate the LP system, preservation
dried whey products. can vary from 24 h at 35°C to 48 h at 20 to 22°C (Fweja
Because of stricter regulations in China with their et al., 2007). This system is 50 to 100 times more ef-
new food safety law (June 1, 2009), the Chinese gov- fective than hydrogen peroxide alone (Fox and Kelly,
ernment is now routinely testing for the presence of 2006). Lactoperoxidase is relatively heat stable with
benzoic acid (Anonymous, 2009). The safety of benzoic denaturation starting at about 70°C (Kussendrager
acid and the derivative benzoates have been studied and van Hooijdonk, 2000). Heat stability studies were
extensively (Sharratt et al., 1964; Nair, 2001; Qi et al., conducted in milk, whey, permeate, and buffer, and the
2009). Benzoic acid is found naturally in milk at low enzyme was reported to be more stable to heat in whey
concentrations, 0 to 1 mg/kg, and in fermented dairy and milk possibly because of their higher calcium ion
products at higher concentrations, typically about concentration (Kussendrager and van Hooijdonk, 2000).
20 mg/kg but up to 50 mg/kg (Sieber et al., 1995). Below pH 5.3, LP is less heat stable (Kussendrager and
During the fermentation process, lactic acid bacteria van Hooijdonk, 2000) and thus the optimal pH is 5.5
convert hippuric acid, a naturally occurring component to 6.3 (Bottomley et al., 1989), which includes the pH
of milk, to benzoic acid (Sieber et al., 1995). Benzoic range of whey. As such, sufficient amounts of the active
acid can also be added as a preservative to cheese ren- enzyme are present in pasteurized milk and whey.
net and thus be found in cheeses from that route as This system can be applied to bleach fluid whey by
well as from lactic acid fermentations. Benzoic acid is the addition of low concentrations of hydrogen peroxide
Journal of Dairy Science Vol. 93 No. 9, 2010
 INVITED REVIEW: ANNATTO AND BLEACHING IN DAIRY FOODS 3897

to activate they system. In 1989, a patent was issued advantages to using hydrogen peroxide are that it must
for decolorizing whey products using the LP system be inactivated with catalase, it could cause oxidized
(Bottomley et al., 1989). Lactoperoxidase reacts in flavors, it is corrosive to equipment (Gilliland, 1969),
the presence of hydrogen peroxide to convert SCN− to and it is less economical to use because it requires more
hypothiocyanite (OSCN−). Hypothiocyanite is a strong peroxide for satisfactory bleaching (Chang et al., 1977).
oxidizing agent that reacts with carotenoids such as A current review of prices for hydrogen peroxide and
norbixin and oxidizes the double bonds, removing the benzoyl peroxide suggests that the previous statement
conjugation and thus removing the color of the com- may be dependent on the bleaching conditions and us-
pound. It should be noted that no additional enzyme age rate. Hydrogen peroxide is sold in 53-gal drums for
or thiocyanate needs to be added, nor does catalase $200/drum; the liquid is 34% solids. Benzoyl peroxide
need to be added because the added hydrogen peroxide is sold as a powder and costs $4.62 per kg.
is consumed. The time required can be variable but is Depending on the concentration, temperature, reac-
generally 20 to 45 min at 40°C. The recommended time tion time, and pH, bleaching with hydrogen peroxide
and temperature is 30 min at 40°C; 40°C is acceptable may alter the functionality of total and individual
from a regulatory perspective as long as the whey is whey proteins (Cooney and Morr, 1972; Munyua,
not held at that temperature for more than 2 h. The 1975). Unfortunately, the 2 prominent, albeit dated,
bleaching reaction occurs within 30 min (Bottomley et studies (Cooney and Morr, 1972; Munyua, 1975) on
al., 1989). the functional properties of whey protein bleached with
Lactoperoxidase catalyzes the oxidation of annatto hydrogen peroxide dealt with concentrations above the
but is inactivated by high concentrations of hydrogen legal limit. Because these studies used a level above the
peroxide (Bottomley et al., 1989). As such, hydrogen legal maximum, the effects documented in those studies
peroxide concentrations should be kept at or near 10 may not be representative of the effects of hydrogen
ppm (Bottomley et al., 1989). Additionally, the inac- peroxide used at lower levels. According to Munyua
tivation effect depends on the concentration of hydro- (1975), hydrogen peroxide concentrations greater than
gen peroxide and on the length of time the enzyme 0.1% in fluid whey or milk caused a 5 to 8% decrease in
is exposed. If the concentration of hydrogen peroxide the nonpolar amino acids such as aspartic acid, threo-
present in the whey falls below the inactivating concen- nine, glutamic acid, methionine, trypsin, phenylalanine,
tration before complete inactivation of the enzyme has histidine, lysine, tryptophan (25% decrease), and argin-
occurred, decolorization will still occur but at a slower ine. In contrast, free sulfhydryl groups increased as the
rate (Bottomley et al., 1989). hydrogen peroxide concentration increased. Increasing
exposure time increased the number of free sulfhydryl
ADVANTAGES AND DISADVANTAGES groups up to 24 h. The researchers postulated that
OF BLEACHING hydrogen peroxide reacted first with readily oxidized
amino acids such as methionine. Higher concentrations
Hydrogen Peroxide of hydrogen peroxide, increased temperatures, and
longer holding times all increased the amount of whey
Higher temperatures are generally more effective than protein denaturation.
lower temperatures. Reaction temperatures above 74°C Reaction temperature during bleaching can affect
increased neither the rate nor the extent of color remov- whey protein denaturation. Cooney and Morr (1972)
al but only caused protein denaturation (McDonough demonstrated 4% protein denaturation when whey was
et al., 1968). The oxidized flavors that were apparent treated with 1% (10,000 ppm) hydrogen peroxide for
immediately after bleaching purportedly disappear af- 24 h at 25°C, whereas 28% protein denaturation was
ter evaporation and drying (McDonough et al., 1968), achieved by treating whey for 6 h at 50°C. It should
although a recent study demonstrated that this is not be noted that the hydrogen peroxide concentration
the case (Croissant et al., 2009). In addition, hydrogen range used in this study was above the legal limit for
peroxide has little to no effect on the nutrients present hydrogen peroxide usage. More studies are needed in
(Teply et al., 1958). Teply et al. (1958) analyzed milks this area to determine if these effects are observed at
and subsequent cheese and whey when the milk was lower concentrations. Although the effect of pH was
bleached using 5, 10, and 25 times the normal amount minor compared with other variables, the pH result-
of hydrogen peroxide and found that a strong treat- ing in the greatest amount of protein denaturation
ment may alter proteins and amino acids in milk but depended on the specific whey protein. For instance,
in general there was no effect on the composition or immunoglobulins and bovine serum albumin were more
nutritional value of the milk, cheese, or whey. The dis- readily denatured at lower pH, whereas β-lactoglobulin

Journal of Dairy Science Vol. 93 No. 9, 2010

3898 KANG ET AL.

denaturation was enhanced at pH closer to neutral. al., 2005; Gallardo-Escamilla et al., 2005; Drake et
α-Lactalbumin exhibited much less denaturation than al., 2009; Wright et al., 2009). Kuramoto and Jezeski
β-lactoglobulin under the same conditions (Law and (1954) studied the bleaching effects of benzoyl peroxide
Leaver, 2000). in cream (30% fat) for blue cheese manufacture at tem-
peratures ranging from 52 to 85°C for periods of up to
Benzoyl Peroxide 4 h with concentrations of benzoyl peroxide of 4.5, 9,
and 18 mg/kg. They found that flavor problems, such
The effectiveness of benzoyl peroxide for removing as tallow or oxidized flavors, were more apparent with
color in whey depends on the amount used, how it is increasing temperature, contact time, and benzoyl per-
applied, the whey components present, and the expo- oxide concentration. McDonough et al. (1968) reported
sure time and temperature. McDonough et al. (1968) that oxidized flavors present in fluid whey bleached
reported that both benzoyl peroxide and hydrogen per- with benzoyl peroxide at 20 and 10 mg/kg at 52°C and
oxide were effective bleaching agents, but that benzoyl 63°C for 1.5 h or hydrogen peroxide at 500 and 300
peroxide was more effective at all temperatures. The mg/kg at 52°C and 63°C for 1.5 h were not detected
rate and extent of decolorization by hydrogen peroxide in the dried whey powder. However, Croissant et al.
and benzoyl peroxide for annatto in Cheddar cheese (2009) conducted a controlled study with hot bleaching
whey increased as the temperature was increased from of liquid whey, with either hydrogen peroxide at 250 or
32.2 to 63°C. However, no additional increase with ei- 500 mg/kg at 60°C for 90 min or benzoyl peroxide at 10
ther agent was seen at 74°C. The advantages to using or 20 mg/kg at 60°C for 90 min, and then manufactured
benzoyl peroxide are that it is effective at lower us- WPC from those wheys. They demonstrated sensory
age levels than hydrogen peroxide, it does not require effects and volatile compound changes in WPC from
addition of catalase to remove residues, and it does hydrogen peroxide or benzoyl peroxide bleached whey
not pit stainless steel, and therefore is less corrosive compared with unbleached whey.
to equipment (Chang et al., 1977). As with reports of Whey with higher total solids, such as condensed
off-flavors from hydrogen peroxide, oxidized flavors in whey, needs greater amounts of peroxide to remove
Cheddar cheese whey were strong immediately after color (JECFA, 2004). The most effective conditions
treatment with benzoyl peroxide; however, off-flavors are 60°C for 15 min at pH 6 to 7 (El-Samragy, 2004).
purportedly dissipated following evaporation and dry- Longer holding times are required if lower tempera-
ing (McDonough et al., 1968), an effect not observed in tures are used (McDonough et al., 1968; El-Samragy,
a more recent study (Croissant et al., 2009). 2004). Once whey has been dried, the annatto becomes
Contrary to the findings of McDonough et al. (1968), highly resistant to bleaching. It is important to note
our recent unpublished research has reported that hy- that much of the above discussion regarding the use of
drogen peroxide is more temperature-dependent than hydrogen peroxide and benzoyl peroxide for bleaching
benzoyl peroxide. McDonough et al. (1968) recom- whey originates from old literature or unpublished data
mended bleaching temperatures in the range of 55 to and has not recently been thoroughly evaluated.
65°C. Benzoyl peroxide reacts quickly to remove color
and additional time will not increase color removal. At REGULATORY CONCERNS
30°C, benzoyl peroxide may never completely bleach
whey (Roos et al., 2006). Regulatory concerns focus on the use of either hydro-
Peroxide bleaching (hydrogen or benzoyl) may have gen or benzoyl peroxide for preservation of whey rather
an effect on flavor in whey proteins, although few stud- than bleaching. That is, these agents are approved for
ies have been conducted. Mortenson et al. (2008) stud- bleaching, not for maintenance of membrane flux during
ied the flavor of whey protein concentrates (WPC) processing or for microbial control. The use of peroxide
and whey protein isolates. Contrary to expectations, for preservation of whey during any process other than
they found that flavor of WPC34 (WPC with 34–36% electrodialysis is prohibited. When bleaching is applied,
protein) and whey protein isolate were not affected the agent, concentration, time, and temperature vary
by instantizing, ion exchange, or bleaching. However, widely within the industry depending on the existing
lack of strict experimental controls and other process- facility and its specific process regimen. Regulatory
ing variables suggest confounding factors in this study. agencies typically use the point of peroxide addition
Other recent studies have unequivocally established in the process to determine if the purpose of peroxide
sensory and volatile compound differences associated is bleaching or preservation (USDA/AMS/Dairy Divi-
with whey sourced from different cheeses, agglom- sion, 2008). Bleaching whey is usually applied at 1 of 2
eration and instantization (Carunchia Whetstine et possible steps during the whey production process. Per-

Journal of Dairy Science Vol. 93 No. 9, 2010

 INVITED REVIEW: ANNATTO AND BLEACHING IN DAIRY FOODS 3899

oxide can be added to fluid whey after pasteurization, greatly evolved, emphasizing a need to scientifically
before or after fat separation, as it is pumped into a evaluate bleaching and its effect on whey protein. Only
storage tank, or when whey retentate is in the hot well two agents, hydrogen peroxide and benzoyl peroxide,
of the evaporator. Bleaching is also conducted under a are currently approved for bleaching. Of these two,
wide range of temperatures from 5 to 70°C. The USDA the latter is viewed negatively in many countries and
cites 2 specific cases where use of peroxide would be some regulations prevent its use. Both bleaching agents
assumed to be for preservation purposes (Hammond can negatively affect whey protein flavor. More precise
et al., 1975; USDA/AMS/Dairy Division, 2008). The application of currently approved bleach agents (e.g.,
first case is addition of peroxide before the separator minimum concentrations, optimal time/temperature
or any point in the process before preheating for the exposure) or development of bleaching alternatives may
evaporator. Fluid whey can legally be bleached follow- facilitate enhanced whey protein flavor.
ing pasteurization if the preheated whey goes into a
storage tank for bleaching followed by fat separation. In ACKNOWLEDGMENTS
this situation, the plant typically alternates between 2
Funding was provided in part by Dairy Management
tanks. Alternating between 2 tanks for bleaching must
Inc. (Rosemont, IL). Paper FSR 10-16 of the Depart-
be completed within 4 h for microbiological reasons.
ment of Food, Bioprocessing and Nutritional Sciences
In addition, legal bleaching may also be carried out if
at North Carolina State University. The use of trade
the bleach is added to the hot well before condensing.
names does not imply endorsement nor lack of endorse-
The second situation generating concern is addition of
ment of those not mentioned.
peroxide before holding the whey for more than 2 h at
temperatures between 7 and 63°C (USDA/AMS/Dairy
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Journal of Dairy Science Vol. 93 No. 9, 2010