Impact of proteins on food color

Impact of proteins on food color P.L. Dawson, J.C. Acton Clemson University, Clemson, SC, United States

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22.1 Introduction Color is not usually thought of as a functional property of proteins. Therefore, one goal of this chapter will be to convince you that proteins do function in color. Webster's Dictionary (1989) defines function as a special purpose, an action contributing to a larger action. The chapter will attempt to show that proteins act in a manner that contributes to the color properties of food. This action is most often an indirect contribution to the ultimately perceived color.

22.1.1 Properties contributing to color and appearance Color is the perceived description or measurement of an object that is either reflecting or transmitting varying wavelengths of visible light. Proteins within or as part of the object, in this case, a food product, are generally indirectly involved in the color of food as perceived by the human eye over the visible wavelength range of 380–760 nm. Proteins are directly involved in the color if the protein happens to be a pigment and can indirectly affect color as an enzyme. The visible light range is only a small portion of the total electromagnetic energy spectrum which ranges from wavelengths of 60 m for radio waves to 0.0001 nm for gamma waves or rays (Fig. 22.1). The “red” end of the visible spectrum has relatively longer wavelengths of around 700 nm as compared with the “blue-violet” end at around 400 nm. The human eye has maximum sensitivity at 560 nm which is in the yellow-green range. Various color spaces have been developed to represent colors perceived by the human eye. While wavelengths of the electromagnetic spectrum are continuous, the sensitivity of perception by humans is not and varies even between individuals. Due to this asymmetry in the perception of light wavelengths, most developed color spaces used to measure perceived colors have an asymmetric shape. In practice, symmetrical diagrams and spheres are typically used to represent several color spaces and two of those used frequently for food color measures are discussed later.

22.1.1.1 Absorption, transmission, and reflection of light With solutions, transmission and absorption terms at specific wavelengths are used in various ways, and are related using the following: A = log

I0 1 = log I T

where, A = absorbance; T = transmittance. Proteins in Food Processing. https://doi.org/10.1016/B978-0-08-100722-8.00023-1 © 2018 Elsevier Ltd. All rights reserved.

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Proteins in Food Processing Energy-type Invisible(long l) Ohmic Radio Dielectric Microwave Infrared Visible

Red Orange Yellow Green Blue Violet Invisible (short l) Ultraviolet Ultraviolet Ultraviolet Ultraviolet Ionizing radiation X-rays Alpha Beta Gamma Cosmic

Wavelength 10,000,000 m 100–300 m 30 m .3 m 800 nm 800 nm | | | | 400 nm 320–400 nm 280–320 nm 200–280 nm 100 nm

Fluorescent Tanning Max bacteriocidal effect

100–150 nm <100 nm <100 nm <100 nm Very short

Fig. 22.1  Electromagnetic energy spectrum with the visible range.

Given an initial intensity of light of a specific wavelength, any light energy not transmitted is thus absorbed. The term transmission is used in reference to the light energy, and absorption is used in reference to the pigment or light-absorbing substance in the solution. In fact, with any pigment, in solution or as a component of a solid food's surface, light absorption properties are retained. Liquid food materials generally exist visually as transparent, clear solutions, or as translucent, slightly turbid mixtures having suspended particles. Solid foods are generally opaque with reflective-type surfaces but they may also be translucent if they exist in very thin sections. The transparent, translucent, and opaque terms arise from their interaction with light and those designations are used to determine how their respective color characteristics are measured. For example, dilute solutions of egg albumin or myoglobin would be termed “transparent” since light can be transmitted through the solution without any scattering. The Beer-Lambert law would apply to both solutions and the only reduction of light intensity as transmitted from a light source will occur due to the protein's or the pigment's specific absorption characteristics. Since myoglobin is a pigment with defined light absorption characteristics, the amount of absorption occurring at one or more wavelengths would be due to its concentration in a solution. In the case of the solution of egg albumin, no colored “pigment” as such is present yet absorption within the visible wavelength range may still occur due to light absorption characteristics of the amino acids comprising the albumin protein structures. Some proteins absorb light at

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s­ pecific wavelengths and others react with a colored product that can be measured using a spectrophotometer. The absorbance of these solutions can be plotted against concentration to quantify the concentration of a protein. By use of a standard curve of a known compound, colorimetry can be used to create and determine protein solutions. The Beer-Lambert relationship for absorbance at a given wavelength is given by log

I0 = abc I

where, I0, I = light intensities; a = absorbancy coefficient; b = thickness of medium; c = concentration of absorbant. The Beer-Lambert relationship is more often represented as A = e bc ●

●

●

●

A = absorbance (no units, A = log10 I0/I; A = 2 − log %T; ranges from 0 to 2.0) ε = molar absorptivity or extinction coefficient (L mol−1 cm−1) b = path length of the sample (cm) c = concentration of the compound in solution (mol L−1)

This relationship is used to calculate concentrations of protein solutions. Absorbance (A) is accurate and has a linear relationship for readings between 0.05 and 0.70 since A is a log of a ratio. To determine accurately the molar concentration of an unknown protein solution the extinction coefficient (ε) of a standard protein is needed. The ε is a measure of how strongly a compound in solution absorbs light at a particular wavelength. Thus, at equal molar concentrations in solution, a compound that has an ε = 35,000 at the λmax for that compound absorbs light more strongly than another compound with ε = 5000 at its λmax. Extinction coefficients allow researchers to calculate the concentration of a protein in solution (quantitative analysis) by accounting for this difference in absorptivity creating a standard curve based on molar concentration. The most common colorimetric protein assays are: (1) Lowry, (2) Biuret, (3) Bradford, and (4) Colloidal Gold Method. The Biuret method uses cupric ions that interact with peptide bonds under alkaline conditions to produce a violet-purple color. The Lowry method combines the biuret reagent with the Folin-Ciocalteau reagent that reacts with phenol groups found in amino acid residues tyrosine and tryptophan to give a blue color that absorbs in the 500 and 750 nm range. The colloidal gold method is an enzyme-linked assay often used to quantify immunoglobulins in the medical field (Dykman et al., 2005). The Bradford assay is a protein determination method that involves the binding of Coomassie Brilliant Blue G-250 dye to proteins (Bradford, 1976). The dye exists in three forms: cationic (red), neutral (green), and anionic (blue) (Compton and Jones, 1985). Under acidic conditions, the dye is predominantly in the doubly protonated red cationic form (Amax = 470 nm). However, when the dye binds to protein, it is converted to a stable unprotonated blue form (Amax = 595 nm) (Reisner et al., 1975; Fazekas de St Groth et al., 1963). It is this blue protein-dye form that is detected at 595 nm in the assay using a spectrophotometer. The Coomassie Brilliant Blue G-250 dye binds primarily to basic (especially arginine) and aromatic amino acid residues (Compton and Jones, 1985). Research has shown that the extinction coefficient of a dye-albumin complex solution is constant over a 10-fold concentration

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Io

Surface

Subsurface

Fig. 22.2  Surface and subsurface reflectance and scattering of light.

range. Thus, Beer's law may be applied for accurate quantitation of protein by selecting an appropriate ratio of dye volume to sample concentration. If the solution is scanned in a spectrocolorimeter for light transmission over the entire visible wavelength spectrum, it is possible to derive its “color” using data reduction methods to express the solution's color in a three-dimensional color solid or color space. Solid surfaces reflect incident light. The amount of reflection of incident light of specified wavelengths is dependent on whether any absorption by a pigment occurs. Reflectance is therefore a surface property and this term is used in reference to the amount of incident light energy that is not absorbed at the solid's surface. A consumer viewing a meat product forms a color opinion based on integration of the reflected light from the meat surface with learned terminologies. Much like light transmission, if a solid surface is scanned for reflectance over the visible wavelength spectrum, it is possible to derive its “color” using defined color solids or spaces. Since light will be absorbed, scattered, and/or reflected when contacting a solid surface (Fig.  22.2), the amount of light reflection determines both a food's human color perception and its measurement as most colorimeters yield color indices based on the reflected wavelengths of light.

22.1.1.2 Light scattering One protein color function is achromatic which sounds like a contradiction but not when visual perception is considered. Relating this to color measurement, scattering will primarily affect the lightness attribute with lesser effects on saturation (this will be discussed in more detail later). One of the latest food trends is toward achromatic properties with items labeled clear and crystal. Light-colored products have also been perceived as healthier and lower in fat and calories, especially in meat products. “Pork, the other white meat” and “lobster, the ultimate white meat” have been used in product advertisements in the United States. Light scattering can be defined as the nonabsorbent interaction of light with a sample. Scattering technically does not deal with absorption; however, our discussion will touch on the achromatic effect of absorption. Light scattering is a physical

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c­ haracteristic of proteins in suspension when the protein exists in particulate sizes from 0.001 to 1.0 μm in the greatest dimension. Two of the factors affecting light scattering are the particle size and wavelength of the light source or incident light.

Particle size and scattering Light-scattering results in the reflection of light from particle surfaces throughout a range of angles. The amount of scattered light is maximal at a particle diameter of approximately 0.1 μm (Fig. 22.3). In the case of particles smaller than 0.1 μm, Rayleigh scattering occurs. Particles greater than 1.0 μm (1000 nm) are larger than the wavelengths of visible light and therefore will reflect light. The ratio of the incident light intensity (IO) to scattered light intensity (IS) can be related to the wavelength of the incident light. This relationship further defines Rayleigh scattering: I S / I O = 1 / wavelength 4 IS = intensity of scattered light; IO = intensity of incident light; wavelength = wavelength of incident light. When light of various wavelengths is incident to a particle where the particle diameter is less than the wavelength of visible light, the relative intensity of the scattered light is 1/wavelength4. This condition exists in the atmosphere. The O2 and N2 molecules in the atmosphere have diameters of about 0.2 nm; therefore, the shorter wavelengths of blue light will be scattered more efficiently than the longer wavelengths of red light. Since sunlight contains all the wavelengths of light, the sky appears blue. This also explains why outer space appears black as there is no atmosphere to scatter Reflecting and refracting

Scattering or reflectance

Scattering

0.01

0.1 1 Relative particle diameter (roughly in microns)

10

Fig. 22.3  Effect of particle size on light scattering or reflectance. Modified from Hunter, R.S., Harold, R.W., 1987. The Measurement of Appearance, second ed. John Wiley and Sons, Inc., New York, NY.

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sunlight. Using this relationship, violet light (400 nm) is scattered about 10 times more than red light (710 nm). A particle with a 0.1 μm dimension is equivalent to about 1/4 the wavelength of violet-blue light. Since the violet-blue wavelengths are scattered more than red wavelengths of light, solutions with particles of this size will sometimes have a blue tint even though the effect is achromatic. This is illustrated in foods with the bluish tint such as that associated with scatter (at about a 90 degrees angle) from the surface of skim milk. The low fat content causes a change in particle (micelle) size, thus resulting in a selective wavelength scattering of light. Mie scattering occurs when particles are larger than the wavelength of the incident light and usually results in a white appearance (Nassau, 1983).

22.1.1.3 Lighting properties Since different wavelengths in the visible spectrum are associated with different colors, spectral energy emission of lighting sources is important. An incandescent bulb relies on the heating of a filament and although the lighting produced appears to be white, it is richer in emission in the red region than the blue region. This spectral energy distribution has been termed Illuminant A by the CIE. Fluorescent tubes rely on mercury vapor discharge of visible energy in various spectral lines (the generated energy spikes) through translucent fluorescent coatings on the inside of the tube while the nonvisible, ultraviolet spectral lines excite additional fluorescent powder within the tube r­ esulting in more energy discharge in the visible region. Light-emitting diodes (LEDs) use semiconductors and electroluminescence to create light by exciting electrons. Different semiconductor materials will produce different wavelengths (colors) of light. Spectral emissions from incandescent light emit more red wavelengths than fluorescent which has more blue with red. Various fluorescent lighting sources differ in internal powder compositions and this is needed in some cases to provide “color balance”. Cool white LED produces wavelengths mostly in the blue range while warm LED emits more in the visible green wavelengths. The CIE in 1931 established other illuminants based on spectral emissions of direct sunlight: Illuminant B, and average daylight from the sky, termed Illuminant C. Since average daylight cannot be captured for lighting, Illuminant C is used in color evaluations based on the CIE defining its spectral energy emission in the visible region. We should note that the visual color of an object is based primarily on the wavelengths of light reflected back to the viewer. An object will appear less red if the spectrum of a light source has less “red” compared with when the same object is exposed to a light source containing more red in its spectrum.

22.1.2 Color perception and measurement Before we discuss specific functions of proteins in color, let us briefly look at how the human perception of color relates to how we measure color. This is relevant since the relationship between protein and color properties will determine visual perception. Instrumental color measurement of a food should be related to visual perception of the product for the measurement to have any meaning. Several researchers have discussed the integration of light sources, types of objects, and physical m ­ easurements with the human perception of color (Clydesdale, 1978; Francis and Clydesdale, 1975;

Impact of proteins on food color605

Kropf, 1984; MacAdam, 1985; Puranik et al., 2009; Wyszecki, 1974). From this integration, numerical assignments for color have been achieved. The Commission Internationale de l'Eclairge (CIE) defined color in terms of the tristimulus values X, Y, and Z which denote the mixture of the primary colors red (X), blue (Y), and green (Z). Using visible light transmission/reflection spectrophotometers or tristimulus (filtered light) colorimeters for color measurement, two of the most popular data reporting systems are the Hunter and CIE color spaces, both of which use L, a, and b values. These spatial systems define each color as a point in their respective continuous three-­ dimensional color spaces. Each of the values (L, a, and b) serve as space coordinates and relate to the CIE derivations for X, Y, and Z (Francis and Clydesdale, 1975). The Hunter and CIE color spaces provide a more uniform measure of visual colors and color differences, which is why they are preferred over the CIE X, Y, Z, color diagram which is discontinuous and not amenable to psychophysical color descriptions. The Hunter L, a, b color solid (Fig. 22.4A) shown as a planar cut from the solid is formed with L (lightness) on the vertical axis running from a value of 100 for white to 0 for black. The x horizontal axis ranges from positive values of red to negative values for green. And the y horizontal axis runs from yellow positive to blue negative. Credit L = 100 White Yellow +b

Green

–a

+a

Red

–b Blue Black

(A)

Ye

llo

White L* = 100

w

L=0

Gree

n

L*

–a*

+b* +a* Red

Blu

e

–b*

(B)

Black L* = 0

Fig. 22.4  (A) The hunter color space. (B). The CIE color space.

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Proteins in Food Processing

should be given to Munsell's earlier color ordering system from which many of the present methods for color analysis by spatial designations were derived. The CIE color space (Fig. 22.4B) is almost identical to the Hunter space and uses L*, a*, and b* coordinates to represent lightness, red-green, and yellow-blue, respectively. One important point is that while a location within the three-dimensional color spaces adequately describes color differences among samples being measured, individual coordinates (L, a, b) are not always adequate since each only determines a one-dimensional color property. Additionally, in either color system, tristimulus values and color attributes are illuminant dependent; therefore, the location of a sample in any color space is dependent on the spectral energy profile of the light source.

22.1.2.1 Attributes of color spaces From tristimulus values, which are really coordinates (L, a, b) of a point in color space, three psychophysical attributes can be calculated, each of which highly correlates to visual perception as well as describing separate properties of color. In Hunter space these are hue (h), saturation (S), and lightness (L) and for the CIE space these color attributes are hue (h*), chroma (C*), and lightness (L*) (chroma is identical to saturation). Hue is the angular specification for the color perceived as red, yellow, blue, and green. Hue is the angle which the point with coordinates a and b form from the center core (L). The angles begin at the “red” region with 0 degrees, moving to the “yellow” area at approximately 90 degrees, and continue with ending at the “blue-purplish” region at 360 degrees (Fig. 22.5A). As an angular function in a circular mode, hue measures, while discontinuous numerically, fit the sensory “color” descriptions of light wavelengths starting at red and ending with blue. Chroma (saturation) is the intensity or the amount of hue departure from gray of the same lightness. Thus, a and b alone cannot describe this attribute to what is perceived. The hue intensity is a function of the line connecting the core L coordinate with the points a and b (Fig. 22.5B). Lightness is a scaled proportion of the light energy reflected by or transmitted from the sample relating to achromatic white-to-gray-to-black (Fig. 22.5C). Together with hue and chroma, lightness expresses how light or dark the object appears to the observer. Calculation of the psychophysical attributes for Hunter L, a, b and CIE L*, a*, b* color spaces are given in Table 22.1. For products such as ripened tomatoes and fresh cuts of beef which exhibit a high degree of redness, a redness index has also been found to be useful (Francis and Clydesdale, 1975). Color difference (∆E) is used to quantify the difference between two similar samples or along the path of color change which some products undergo with oxidation over time.

22.2 Role of proteins in color 22.2.1 Scattering properties Two examples of proteins in food affecting scatter are (1) acid precipitation or heat-induced aggregation during gelation and (2) preparation of protein powders. As the discrete aggregates (particles) attain adequate dimensions they begin to scatter

Impact of proteins on food color607

Yellow

Green

0° Red

Blue HUE

(A)

Yellow

Green

Red

Blue

(B)

(C)

SATURATION (CHROMA)

Lightness

Fig. 22.5  Representation of hue, chroma, and L, values as separate parts of the color space: (A) Hue angle as it relates to the CIE color space, (B) chroma (saturation) as it relates to the CIE color space, and (C) L value from the color space.

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Table 22.1  Relationship of Hunter L, a, b; CIE L*, a*, b* to pychophysical attributes Psychophysical color attributes Lightness Saturation (Hunter), Chroma (CIE) Hue Redness index Total color difference

Hunter L, a, b

CIE L*, a*, b*

L (a2 + b2)1/2

L* (a*2 + b*2)1/2

tan–1b/a a/b [(ΔL)2 + (Δa*)2 + (Δb)2]1/2

tan–1b*/a* a*/b* [(ΔL*)2 + (Δa*)2 + (Δb*)2]1/2

Fig. 22.6  Picture of yogurt, small curd, and large cottage cheese.

light and give a white appearance. Two products containing proteins acting in this manner are caseins in cottage cheese and myofibrillar proteins in surimi-derived seafood products. Shown here (Fig. 22.6) are yogurt at the left, small curd cottage cheese in the middle, and large curd cottage cheese at the right. Each of these examples is a milk-based product that has a different appearance at least partially due to protein aggregation. Yogurt is produced via bacterial fermentation where lactic acid production causes aggregation of milk protein (casein). The cottage cheese aggregates are formed from direct acidification of milk with removal of the remaining fluid whey. These are crude illustrations of how particle size differences between the three products can change the visual perception of the product. A second example of protein light scattering is powders produced from protein fractions. Powders will increase the scatter of light as particle size increases and particle sizes greater than 1.0 μm diameter will reflect light as was previously discussed (Fig. 22.3). Sodium caseinate in coffee whiteners is a protein that functions to provide some whitening power in addition to its role in emulsification and flavor effects. During dehydration of coffee whiteners, one goal is to have particles in the 125–150 μm diameter range so that scattered light will appear white (Knightly, 1969).

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22.2.1.1 Subsurface effects: Kubelka-Munk analysis Proteins can have a significant effect on scattering light both at the surface and at subsurface levels of a food product. Food surfaces may appear opaque, translucent, or transparent and their descriptions were noted previously. Although surfaces also have varying degrees of gloss, not many protein-type foods exhibit this property. Internal, subsurface light scattering due to reflection and refraction is important and complicates food color measurement (Clydesdale, 1972). A more complex achromatic component of food color that proteins affect deals with the Kubelka-Munk theory (Francis and Clydesdale, 1975) which is applied to materials which both scatter and absorb light. Light scattering was earlier stated to have an absorption component. The Kubelka-Munk methodology provides a solution for color measurement of problem proteins by taking into account the reflection, transmission, and absorption of light from subsurface layers (Fig. 22.7). The scattering coefficient (S) and the absorption coefficient (K) are related to the amount of light scattered and absorbed per unit length of travel by incident light. By using reflectance spectrophotometry, the K/S ratio has been applied extensively to the measurement of fresh meat color (Francis and Clydesdale, 1975; Hunt, 1980; Kropf, 1984). The Kubelka-Munk transformation was used as part of an invention to measure the oxygenation of blood (hemoglobin) through skin to minimize the light-scattering effect of skin (Parker, 2007). Hughes et al. (2014) postulated that an increase in the lightness of cooked meat was related to myofibrillar and structural protein changes resulting from their spacing or lattice arrangements. This would impact light-scattering properties resulting in visual paleness of a meat sample. The achromatic muscle fibers are composed of overlapping and interlinking proteins having various diameters and some water-soluble proteins present in the water phase of meat that also possess particle dimensions. Therefore, Rayleigh and Mie scattering from the fibrous surfaces of the protein matrix are considered components of the Kubelka-Munk scattering coefficient.

22.2.1.2 Water-binding effects Proteins that are good water binders will result in a more intense color as perceived by the viewer. Loss of color quality in tuna was termed “masking” and was due to

Io

Surface

Fig. 22.7  Subsurface scattering effects.

Subsurface

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the loss of spectral pigment reflectance (Little and MacKinney, 1969). The loss can be attributed to subsurface scattering since the heme-based pigment in tuna exists in an achromatic protein matrix and this color perception could be affected by the ­water-holding properties of the protein matrix. The degree of water binding in meat (a functional property associated with the myofibrillar proteins) at the food surface has a major effect on color since loosely or unbound water will give a pale or translucent appearance and transmit light to subsurface levels where it can be scattered or absorbed. Pale, soft, and exudative (PSE) pork is an example of this phenomenon contrasted with what has been called dark cutters in which the water is tightly bound at the surface (discussed in more detail later). In this schematic, loosely and unbound water at the surface scatters light which is absorbed at subsurface layers.

22.2.2 Absorption characteristics 22.2.2.1 Chromophores The major protein chromophore in meat is myoglobin. The color attributes of meat products are most dependent upon the chemical reactions of the pigments myoglobin of the muscle cell and hemoglobin in the residual blood. Myoglobin is a water-­soluble protein found in the muscle cells and functions to bind oxygen for use in aerobic metabolism. Other nonprotein chromophores exist in food that can be affected by enzymes; they include chlorophyll, carotenoids, anthocyanins, flavonoids, tannins, betalains, quinines, and xanthones.

22.2.2.2 Heme The oxymyoglobin molecule consists of the globin protein and a prosthetic nonprotein heme group composed of a central iron atom covalently bonded with four nitrogen atoms of the tetrapyrole ring of the protoporphyrin IX (Fig. 22.8). The two remaining iron-binding positions are occupied by the histidine of the globin for myoglobin pigments in their native, undenatured state in the fifth position; and various ligands in the sixth position which is oxygen in the case of oxymyoglobin. The specific ligand being bound will also depend upon the state of the iron which is either ferrous (Fe++) or the oxidized ferric (Fe+++). Only native myoglobin, existing in an anaerobic environment, lacks a ligand bound in the sixth position. Characteristics of the major meat pigments for fresh, cooked, and cured meat products are shown with their pathways and respective color (Fig. 22.9). Packaging, processing, and storage conditions have been extensively researched to control the color of meat products and to understand the mechanisms influencing the color of meat. The exclusion of oxygen from myoglobin will result in a predominance of the myoglobin over the oxymyoglobin form. The myoglobin appears darker than the oxymyoglobin in meat that has been exposed to air. How do proteins function with protoporphyrin IX to determine the color of meat? The globin protein is colorless itself yet contributes to the stability of the heme complex through steric and synergistic histidine residue interactions with the heme. Being embedded in the hydrophobic “cleft”, the heme is generally restricted to a

Histidine (Globin origin)

CH3

CH = CH2 H3C

CH = CH2 N

N Fe

N

N CH3

H3C

CH2CH2COO–

–

OOCCH2CH2

O2

Fig. 22.8  Myoglobin (as Oxymyoglobin). Fresh meat Oxymyoglobin Fe2+; red-pink

Oxygenation (+O2) Deoxygenation (–O2)

Oxidation

Reduction

(deoxy)Myoglobin Fe2+; purple-red

Oxidation

Metmyoglobin Fe2+, brown-tan Heat

Cooked meat (denatured globin) Nicotinamide hemichrome Imidazole ferrihematins Fe3+; brown-tan

NO reduction

Cured meat (several steps) Nitrosylmyoglobin Fe2+; red-pink Heat Nitrosylhemichromes Fe2+; red-pink Light, O2 Hemichromes Fe2+; gray-colourless

Fig. 22.9  Pathways of the major meat pigments from fresh meat to coked and cured meat.

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Table 22.2 

Proteins in Food Processing

Major pigments in meat products

Major pigments

Fe state

Ligand

Color

Fe+2 Fe+2 Fe+3 Fe+2 Fe+2 Fe+2

Vacant O2 OH2 CO H2S effects H2O2 effects

Purple Bright red Brown Pink Green Green

Fe+3 Fe+2 Fe+3

Nicotinamide CO Nitrogenous base

Brown Pink Brown

Fe+2

NO

Red-pink

Fe+2 Fe+2 Fe+3 Fe+3

NO 2NO

Pink Pink Gray-colorless Green

Fresh, raw meats (deoxy)Myoglobin Oxymyoglobin Metmyoglobin Carboxymyoglobin Sulphmyoglobin Choleglobin

Fresh, cooked meats Nicotinamide hemichromes Carboxyhemochrome Imidazole ferrihematins

Cured, raw meats Nitrosylmyoglobin

Cured, cooked meats Nitrosylhemochrome Dinitrosylhemochrome Hemichromes Nitrihemin

Excess NO + heat

small group of ligands unless the globin's native structure is altered by heat or acid treatment. The energy of specific wavelengths of light is absorbed by the myoglobin molecule and is transferred to electron clouds of the iron atom and ligand present. The wavelengths of light absorbed are unique for each ligand and iron state combination. Thus when either one or both the iron state and ligand are altered, there will be an associated change in the wavelength (energy) of light absorbed and thus a change in the reflected wavelength and perceived color. The electronic configuration and bonding of various ligands have been reviewed (Giddings, 1977; Livingston and Brown, 1981; Wong, 1989) and the characteristics of major meat pigments are shown in Table 22.2. Total meat pigment concentration will significantly affect a product's color property. Pigment concentration determines the amount of light energy absorbed and reflected and consequently the intensity of color that is viewed and measured. Differences in pigment content can been seen between the light and dark meat of poultry. The intensity of the color is reflected in both the achromatic and chromatic descriptors used by meat scientists such as “light” or “white” and “dark” meat of poultry, tuna and pork, and the “red”, “intermediate”, and “white” fiber types for beef, pork, and poultry. The natural pigment content determines the intensity of the food color; however, this content can be altered by using washing, leaching, or bleaching treatments to reduce the pigmentation of the product. With various washing treatments applied to the ground raw tissues of low economically valued fish during the processing of surimi, fat, heme pigments, and

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other water-soluble constituents are removed creating a lighter colored, higher value protein product useful in further processing (Lanier, 1986). Washing techniques have been applied to poultry and beef yielding tissue strips, flakes, and mechanically deboned tissue having a lighter, less red color. In general, the success of the decolorization treatments of dark poultry meat is dependent on the extent of tissue disruption so that adequate leaching of the water-soluble pigments can occur (Montejano and Ball, 1984; Hernandez et al., 1986; Elkhalifa et al., 1988; Dawson et al., 1989; Bowie et al., 1989). Treatments involving oxidants such as hydrogen peroxide have also been used to “whiten” successfully dark poultry thigh tissue yielding a meat tissue with the appearance near that of raw breast tissue. Removal of myoglobin by leaching or oxidation would account for the decrease in the chromatic property of the meat most often associated with redness. However, the combined effects of reduced pigment content and increased light scatter occurring due to higher moisture contents within the remaining tissue structure would result in an increase in perception and measured lightness. Color also depends upon how light is reflected off the meat. PSE meat has a great amount of extracellular water and hence many reflecting surfaces and limited absorption capabilities. Dark, firm, and dry (DFD) meat has a great amount of tightly bound intracellular water; thus, white light reflection is minimized and color absorption is enhanced. Such a type of meat has a high pH, high rate of oxygen-scavenging enzyme activity, and less oxymyoglobin. Enzyme-reducing systems in meat also help in controlling metmyoglobin formation (Faustman et  al., 1989). Thus, DFD meat will have relatively high levels of bound water, pH, rate of oxygen consumption, and metmyoglobin reducing activity, while PSE meat will have lower values or rates of each of these meat parameters.

22.2.2.3 Flavoproteins and cytochromes Several proteins in the electron transport chain have chromatic characteristics with their prosthetic groups. Flavoproteins and cytochromes are chromophores whose color is determined by the proteins' oxidation state (reduced or oxidized). Flavoproteins act as electron donors and acceptors in the electron transport chain and also catalyze a variety of biochemical reactions. The cytochromes act within the electron transport scheme exclusively. Flavin adenine dinucleoticle (FAD) and flavin mononucleoticle (FMN) exist in three spectrally different oxidation states (Fig. 22.10): the yellow oxidated form, the red or blue one-electron reduced form, and the colorless two-electron reduced form. Nicotinamide adenine dinucleoticle (NAD) is also a flavoprotein, which uses FMN as its prosthetic group and also contains an iron-sulfur protein. The cytochromes are unique to aerobic cells and play a major role in the electron transfer from coenzyme Q to oxygen (Fig. 22.11). At least five different cytochromes are found in higher animal species. Cytochromes b, c, and c1 contain the identical heme configuration found in myoglobin and hemoglobin (iron-protoporphyrin (X) while cytochromes a, and a3 contain a modified heme. The modified heme differs only in the presence of a formyl group where the methyl group is located in the myoglobin version of the heme. Cytochromes c and c1 are the only cytochromes covalently bound to a protein; however, all are closely associated

614

Proteins in Food Processing R CH3

N

CH3

N H Yellow

R N

O NH

CH3

N

CH3

. N+

O

H Blue

H

CH3

N

N

CH3

N H Colorless

O NH

O

lmax = 560 nm

lmax = 450 nm FAD or FMN

R

N

R

O NH

CH3 CH3

N

O NH

. N O

O

FADH2 or FMNH2

N

Red lmax = 490 nm FAD or FMN Semiquinone

Fig. 22.10  Flavin adenine dinucleoticle.

with proteins within the mitochondria. While the chromatic property of the proteins of the electron transport chain has a minimal effect on food color, the application of this chromatic functional property to analysis procedures and research has been significant.

22.2.3 Contribution of proteins to browning reactions 22.2.3.1 Enzymatic reactions Enzymes are complex globular protein catalysts that accelerate reaction rates by factors of 1012 to 1020. For our purposes, we will discuss three types of enzymes, their mechanisms, and how they affect food color. Phenolases are enzymes that catalyze the oxidation of phenols. The reaction begins with a substrate of a phenolic type, often tyrosine, and the phenolase (tyrosinase) catalyzes a two-step reaction (hydroxylation and oxidation) to form dopamine quinine (Fig. 22.12). This is another example of function as defined by Webster in which a protein contributes to a larger action. The color development then occurs after the nonenzymatic continuation of the reaction via oxidation and polymerization (not shown) of indole 5,6 quinone to form brown melanin pigments. Melanins can also combine with other proteins to form colored complexes. Phenolases cause the undesirable browning on the cut surfaces of light-colored fruits and vegetables such as bananas, apples, and potatoes (Fig.  22.13). They are involved to a lesser extent in the desirable brown color development of teas, ciders, cocoa, raisins, figs, dates, and prunes.

Impact of proteins on food color615 NADH (flavin)

NADH dehydrogenase ----------

FMN (Flavin) | Fe-S

Succinate FAD Fe-S (flavin)

Coenzyme Q

Cytochrome b (heme) Coenzyme Q – cytochrome c reductase -----

Fe-S

Cytochrome c1 (heme)

Cytochrome c (heme)

Cytochrome a (heme) Cytochrome a3 (heme)

Fig. 22.11  Electron transport scheme showing flavinproteins and heme-based cytochromes involved in ATP metabolism. HO

CH2 Phenolase | + O2 CHOOH | 2-Steps NH2

HO Diphenol

O= O=

CH2 | CHOOH | NH2

Dopa quinone Condensation/polymerization

Brown melanin brown pigments

Fig. 22.12  The reaction catalyzed by phenolase that leads to browning on cut fruit and vegetables surfaces. Dopa quinone reacts nonenzymatically via oxidation and polymerization to form brown melanin pigments.

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Fig. 22.13  Picture of enzymatic browning on cut potato, apple, and banana.

Lipoxygenases are a diverse group of enzymes that oxygenate polyunsaturated fatty acids or their esters and acylglycerols. The mechanism is the oxygenation between the cis, cis, 1, 4, pentadiene double bond system located between the 6th and 10th carbon from the methyl terminus (Fig. 22.14). The products are optically active hydroperoxide resulting in the undesirable destruction of chlorophyll and carotenoids in vegetables, pasta, and animal feed. One beneficial use of lipoxygenases in food processing is the bleaching of hard wheat flour via carotenoid destruction. Peroxidases are a common group of enzymes found in plants and milk which usually contain a heme prosthetic group. Horseradish peroxidase has been extensively researched while milk is a popular source for peroxidases. Peroxidases catalyze the reaction of an organic hydrogen peroxide with an electron donor, often ascorbic acid, phenols, or amines in the general reaction:

H H H H | | | | CH3-(CH2)4-C=C-CH2-C=C-(CH2)7-COOH Linoleic acid (18:2)

H HH | | | CH3-(CH2)4-C=C-C=C-C-(CH2)7-COOH | | HO O H 9-D-Hydroperoxide (10t,12-c) H O H HH O | | | | CH3-(CH2)4-C-C=C-C=C-(CH2)7-COOH | H 13-L-Hydroperoxide(9-c,11t)

Fig. 22.14  Reaction catalyzed by lipoxygenase on linoleic acid. Lipoxygenases can also catalyze reactions of oxygen with esters and acylglycerols resulting in the destruction of pigments.

Impact of proteins on food color617

ROOH + AH 2 Peroxidase 6H 2 O + ROH + A ROOH = hydrogen peroxide or organic peroxide; AH = electron donor (ascorbate, ­phenols, amines). The oxidation product is often highly colored which is sometimes used as an endpoint for colorimetric analysis. However, peroxidase color effects in food are usually associated with the destruction of pigments such as carotenoids and anthocyanins. There are other specific pigment-degrading enzymes such as anthocyanases and chlorophyllases that exist in plants and which can accelerate the destruction of these pigments in foods. In addition, many enzymes are used in food processes some of which affect the food color perception, especially those used in making cheese and other fermented milk products. Light scatter is the major effect in color perception changes resulting from enzymatic action in many cheeses.

22.2.3.2 Maillard reactions The Maillard reaction is the nonenzymatic reaction of an amino group with a reducing group (often a reducing sugar) leading to the formation of compounds which ultimately polymerize to form brown pigments. The reaction was first found in the writings of French biochemist Louis-Camille Maillard between 1912 and 1916 and is also described as nonenzymatic browning. Of the four browning reaction in foods (enzymatic, Maillard, ascorbic acid oxidation, and carmelization) only enzymatic and Maillard involve proteins and amino acids and three are nonenzymatic. The Maillard reaction involves three stages of seven different chemical reactions (Hodge, 1967) (Fig. 22.15). The most well-defined pathways involve the sugar amine reaction for both aldose and ketose sugars. The first step is the condensation of an amino compound with a reducing compound and in the case of sugars forms a substituted N-substituted glycosylamine (from an aldose such as glucose) or a N-substituted fructosylamine (from a ketose such as fructose) (Fig. 22.16A). This is followed by either Amadori (for aldoses) or Heyn's (for ketoses) rearrangements to form a 1-amino, 2-ketose or 2 amino, 1-aldose (Fig. 22.16A), respectively. The compounds formed in these initial stages are colorless with no UV absorption. The second stage can take several pathways including dehydration, fragmentation, and amino acid degradation with colorless or yellow compounds formed with strong UV absorption. The final stage involves the polymerization of aldehyde-amines, formation of heterocyclic nitrogen compounds, and condensation reactions to form brown melanoidins (Fig. 22.16B). These reactions most often occur between sugars and amines; however, there is much evidence that indicates peroxide formation in oxidizing lipids fosters a protein-lipid reaction similar to those leading to Maillard pigments. In fact, Pokorny et al. (1973a,b) demonstrated that oxidizing lipid peroxides release carbonyl compounds that can enter the Maillard reaction to form colorless or slightly colored intermediates. Intermediate moisture meats are particularly susceptible to Maillard browning via the lipid-protein pathway when heat is used in processing (drying). When heat is used, the browning reaction is accelerated to progress faster than lipid oxidation as the Q10 (change in reaction rate due to a 10°C change in temperature) for lipid oxidation is much lower than for the browning reaction. Maillard

618

Proteins in Food Processing Reducing sugar + Amino group or oxidized lipid

N-substituted glycosylamine or N-substituted fructosylamine Amadori or Heyns’ rearrangement

1 amino, 2 ketose or 2 amino, 1 ketose

Schiff base of HMF or furfural

Reductones

Fission products (diacetyl, acetol, pyruvaldehyde)

Strecker degradation + aldehydes + Amine

HMF or furfural

Dehydroreductones

+ amine

Aldols Aldimines

Aldimines

+ Amine

Aldimines

Melanoidins (brown nitrogenous polymers or copolymers)

Fig. 22.15  Generalized Maillard reaction.

intermediates are well known for their antioxidative properties; thus, heated lipid oxidation model systems in the absence of protein will oxidize at a faster rate than when adequate amount of protein is present (Morales and Jimenez-Perez, 2001).

lipid

+ HEAT > lipid oxidation > Rancidity

However, in a heated system such as intermediate moisture meats, Maillard intermediates may form, thus preventing rancidity but result in browning. Browning has been shown to be the quality-limiting factor during the storage of intermediate moisture meats.

lipid + protein

+ HEAT > Maillard Reaction (Browning) > blocks lipid oxidation

Impact of proteins on food color619 O OH || | H–C +NHR H – C – NHR | → | H – C – OH H – C – OH | | HO – C – H HO – C – H | | Aldose (C1-C3) N-substituted glycosylamine H H | | H – C – OH +NHR H – C – OH | → | H –C= O H – C – NHR | | HO – C – H HO – C – H | | Ketose (C1-C3) N-substituted fructosylamine

(A)

H | H – C – NHR | C=O | HO – C – H |

→→→

X

(B)

→→

→→

H | H – C – NHR | C=O | HO – C – H | Amadori product (1-amino, 2 ketose) H | C=O | H – C – NHR | HO – C – H | Heyn’s product (2-amino, 1 ketose)

H | H –C–N H | | C ==== N – C – H | | HO – C – H C=O | | HO – C – H |

X

Fig. 22.16  (A) Initial stage of the Maillard reaction for aldose or ketose. (B) Proposed formation of melanoidins (Hayase, 1995).

Another illustration of the lipid-protein Maillard pathway is in smoked meat color development. The carbonyls (aldehydes, ketones) in the smoke and the amino compounds in the meat react to form brown pigments with no reducing sugar present. Furthermore, the volatile acids deposited on the meat surface from the smoke may facilitate the release of amines from the meat. Maillard browning more commonly involves the sugar–amine rather than the lipid– amine source. The free reducing sugar reacts with a free amine group in the first step leading to the development of numerous brown pigments. Browning will occur in many food systems where a reducing sugar and amine are in close proximity, especially during heating. The Maillard reaction is the principal browning mechanism for deep fat-fried foods. The browning of batter and breaded foods is sometimes enhanced by the addition of reducing sugars such as dextrose, maltose, or lactose to the batter. The effect of free amine and reducing sugar concentrations is shown by how the absorption at 375 nm and the degree of browning increases with the amount of reducing

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sugar present in the muscle filtrates from the surface of cooked pork loins. The free amine concentration remains relatively constant. Liu et al. (2008) reported that color development (increase) and antioxidative activity (decrease) followed first-ordered kinetics with galactose concentration and pH decreased logarithmically with galactose concentration. Dehydration of egg, especially egg albumin (white), will result in browning via the Maillard reaction. Eggs contain about 1% carbohydrate, much of which is glucose. During heating, the glucose will react with the free amines to form melanoidins and discolor the egg product. To prevent this, glucose is removed from the egg albumin prior to drying. Glucose was first commercially removed via natural bacterial fermentation in the early 1900s by Chinese processors. Today, glucose is removed from egg albumin enzymatically or by yeast fermentation. Some Maillard reaction products are antioxidants having free radical scavenging capability. Morales and Jimenez-Perez (2001) followed a Maillard controlled reaction and concluded that brown pigment development (420 nm wavelength) was not directly related to antioxidant compound formation and that fluorescence (347 and 415 nm wavelengths) was an effective indicator of free radical scavenger compounds formed during the Maillard reaction. Somewhat conversely, Anese et al. (1999) reported an increase in antioxidant potential with an increase in Maillard browning in dried pasta while prooxidant properties were observed in the early stages of the Maillard reaction.

22.3 Improving protein functionality in color control 22.3.1 Heme pigments of fresh meats Myoglobin, hemoglobin and cytochrome c comprise the heme pigments in fresh meat with myoglobin constituting about 70%–95% of the total pigment in fresh meat (Fox, 1987; Judge et al., 1989; Saffle, 1973). Myoglobin is the aqueous phase heme-containing water-soluble protein present in the sarcoplasmic fluid of muscle cells that is responsible for the color of fresh and cured meats. Myoglobin consists of two main components, the heme ring and globin (protein). The polar amino acid histidine residues of the globin are oriented toward the interior of the myoglobin structure affecting the heme-binding ligand sites (described previously). As a pigment, it has several visual color states depending on the ligand (O2, NO, CO) bound by the central Fe atom of the heme group (protoporphyrin IX). Fresh meat pigment forms are referred to as the “fresh meat color triangle” (Fig. 22.17) that is dependent on the oxidation state of the Fe and whether O2 is or is not present. In fresh meats, consumers typically reject brown discoloration of their expected bright red (beef) or pinkish (pork) appearance. The level of coloration among species is also dependent on the muscle cell's myoglobin content, being very little (0.1–0.4 mg/g) in the case of poultry breast tissue to moderately high (2.5–10 mg/g) among fresh beef muscles. Oxymyoglobin is the desired fresh meat pigment and readily forms when a freshly cut meat surface is exposed to air or is packaged in high oxygen-containing modified atmosphere packages (MAPs). Recently, carbon monoxide (CO) at very

Impact of proteins on food color621

Fresh meat color triangle MMb+ (brown)

Cured meat color development NOMMb+ (brown)

NO

Reduction

MbO2 (red)

Mb (purple)

NO

NOMb (red) Heat

Nitrosylhemochrome (pink) Mb

Fe2+

MYOGLOBIN

MbO2

Fe2+

OXYMYOGLOBIN

MMb+

Fe3+

METMYOGLOBIN

NOMMb+

Fe3+

NITROSYLMETMYOGLOBIN

NOMb

Fe

2+

NITROSYLMYOGLOBIN

Fig. 22.17  Fresh meat pigment triangle.

low processing concentrations was permitted for stabilizing the red color associated with fresh meats. The formed carboxymyoglobin pigment closely resembles that of oxymyoglobin but retains surface redness slightly longer than oxymyoglobin. All retail displayed fresh meats are packaged in oxygen-permeable films. Loss of marketability occurs within a few days due to the slow oxidation of Fe2+ to Fe3+, resulting in the brown appearance at the meat surface. Fresh meat myoglobin and oxymyoglobin pigments will eventually oxidize to form metmyoglobin. This loss is affected by temperature, oxygen, and properties of the meat and can be delayed by changing storage conditions. Jeremiah and Gibson (2001a,b) tested holding temperature effects on the ability of beef steaks to maintain red color over 30 h of retail display. Steaks maintained redness for 30 h in retail display after 17 weeks of being held at −1.5°C but only maintained 30 h color stability in retail display after 8 and 7 weeks at 2°C and 5°C, respectively. Non-UV and UV-filtered fluorescent light effects on the color stability of caseready packages of ground beef was evaluated by Daly and Acton (2004). Ground beef stored in high-oxygen MAP at 0°C before light display lost redness (Hunter + a) slower than packages previously stored at 4.4°C. Panel evaluation of displayed product also confirmed greater retention of redness in lean meat color. They stated that UV-filtering of fluorescent lighting was not necessary for color stability of ground meat during lighted display. Endogenous muscle oxygen scavenging and metmyoglobin reducing enzyme systems also impact fresh meat color. These systems require the presence of NADH,

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which is limited and deplete as time progresses postmortem. The ability of meat to reduce metmyoglobin back to myoglobin via these enzyme systems has a strong influence on fresh meat color and the concentration of NADH may play a significant role in this pathway (Bekhit et al., 2000, 2004).

22.3.2 Myoglobin pigments of cured meats Cured meat products such as typical fresh precooked ham slices have a bright pink color whereas dry country ham slices have a distinct red color. The difference of coloration is dependent on the pigment development sequence. Nitric oxide (NO) from the curing salt, sodium nitrite (NaNO2), reacts primarily with metmyoglobin but may also react directly with native myoglobin leading to the formation of the red nitrosylmyoglobin pigment. If no heat is applied and the product is simply dried, as is done with country hams, some bacon, and dry salamis, the color produced is red. Dehydration simply results in concentrating of the pigment and the globin generally remains a part of the pigment. However, with heating which is typical for precooked luncheon-type products such as frankfurters, bologna, cooked ham, pastrami, and so forth, the globin denatures and the resultant pigment consists simply of the heme with NO, termed nitrosylhemochrome. These pigments are light sensitive and subject to oxidation. Therefore, all cured meats are packaged for lighted display in oxygen-barrier films. Factors affecting the rate of cured meat color loss (also termed “fading”) are, in general, (1) the amount of pigment actually converted to nitrosohemochrome, (2) quantity of oxygen available for reaction with pigments, (3) storage temperature, and (4) intensity of lighting (Kramlich et al., 1973). Color fading, which results in a tan-brown appearance, is thought to occur in a two-step reaction sequence. The first step is a light-accelerated dissociation of nitric oxide from the pigment that is promoted in the presence of oxygen. This is followed by oxidation of the dissociated nitric oxide which then is not available to recombine with the pigment. Color fading or color loss can also occur in the interior of the product although the rate is extremely slow compared to that at the surface. Light appears to also serve as an energy source for the oxidation reaction in addition to the effect of storage temperature.

22.4 Applications to maintain color quality There are essentially three methods employed by the food industry to maintain color quality: (1) addition of antioxidants, (2) modified atmosphere packaging, and (3) blanching of plant tissue. Methods to maintain meat color do so by preventing the oxidation of pigments or by promoting the formation of specific pigment forms (example, oxymyoglobin in meat). O'Grady et al. (2001) found a strong relationship between lipid oxidation and oxymyglobin oxidation in bovine muscle. These researchers found that increased lipid oxidation products preceded increased oxymyoglobin oxidation suggesting that free radicals produced during lipid oxidation promoted the oxidation of myoglobin. Gorelik and Kanner (2001) further reported

Impact of proteins on food color623

that oxymyoglobin oxidation was affected by two pathways: (1) active oxygen species such as oxygen radicals, hydrogen peroxide, OH radicals and (2) lipid radicals such as alkyl radicals and hydroperoxides. Thus, the inhibition of myoglobin oxidation to metmyoglobin can only be maximized by blocking both of these free radical reaction pathways. Plant tissue color can be maintained both by inactivating browning and degradative enzymes through blanching and by maintaining slow respiration to sustain natural defenses against enzymatic breakdown of tissues. An interesting use of surface reflectance to evaluate meat quality was reported by Lui and Chen (2001). These researchers used the ratio of metmyoglobin (A485 nm) to oxymyoglobin (A560 nm) wavelengths and the ratio of sulfmyoglobin (A635 nm) to oxymyoglobin wavelengths to classify raw and cooked meat as wholesome or unwholesome. The method was somewhat successful in raw meat but it was unclear what pigments were being measured in cooked meat since the myoglobin molecule is denatured during cooking, leaving hemichromes.

22.4.1 Antioxidants Antioxidants have been added to meat using several different modes of delivery, including through the animal diet and by direct addition to meat. Vitamin E (tocopherol) has been most often used for the incorporation of antioxidants into animal diets for improving meat color stability. Numerous studies have used alpha-tocopherol in cattle diets (Arnold et al., 1992, 1993; Chan et al., 1995, 1996; Faustman et al., 1989; Mitsumoto et al., 1993). Faustman et al. (1998) have reviewed the role of vitamin E in beef color. Some specific examples of feeding antioxidants to animals to improve the color stability in harvested meat include work by Granit et  al. (2001) adding 4000 mg/day of alpha-tocopherol for 90 days to beef cattle diets, King et al. (1995) feeding alpha-tocopherol for 3  weeks to broilers prior to slaughter, Corino et al. (1999) adding 60 mg/kg to feed and 100 mg/L drinking water of alpha-tocopherol to rabbit diets for 15 days, Jensen et al. (1998) adding rapeseed oil (6%) and alpha-­ tocopherol (100–200 mg/kg feed) to pig diets, and Sante and Lacourt (1994) adding 250 mg/kg alpha-tocopherol to turkey diets or spraying the antioxidant on turkey meat, all to improve color stability. Gatellier et  al. (2001) fed Charolais cattle diets with either 75 or 1000 mg alpha-tocopherol/kg feed/day for 111  days prior to slaughter and found meat with higher tocopherol added had lower discoloration scores when packaged in 80%O2/20%CO2. Formanek et al. (2001) added 2000 IU alpha-­tocopherol acetate/kg feed to Fresian steer diets for 50  days prior to slaughter then tested the postslaughter addition of several antioxidants to the meat mince. Only rosemary and BHA/BHT improved meat color stability compared to tocopherol-fed-only meat samples and ­tocopherol-fed samples combined with Duralox added to the meat mince. Montgomery et  al. (2001) supplemented grass-fed beef diets with a seaweed-based product possessing antioxidant activity to improve meat redness retention and reduce browning in steaks. And Stubbs et al. (2002) fed 500 IU/day of alpha-tocopherol to cattle for 124  days and found improved color stability in both top loin steaks and ground chuck patties. Vitamin E supplementation can be used as an efficient, sustainable, and consumer-friendly preharvest strategy to improve beef color stability (Sales

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Proteins in Food Processing

and Koukolova, 2011). Walsh et al. (1998) stabilized low nitrite (60 vs 120 mg/kg)cured turkey meat color by using meat from turkeys fed 600 mg alpha-tocopherol/kg feed compared to 20 mg alpha-tocopherol/kg feed. Direct addition of antioxidants to meat after slaughter has been investigated to a much greater extent. Rosemary extract is commonly added to commercial ground turkey meat as a flavoring agent but also acts to stabilize color. A cross-section of antioxidants have been tested for their ability to stabilize meat color through direct addition. For example, Sanchez-Escalente et al. (2001) found ascorbic acid (500 ppm), taurine (50 mM), carnosine (50 mM), and combinations of ascorbic acid with each of the others had a limited or nil effect on maintaining beef patty color when packaged in 70% oxygen. However, in the same study rosemary (1000 ppm) either alone or with ascorbic acid was highly effective in preventing metmyoglobin formation and lipid oxidation. Phytate and carnosine were tested in a beef model system and found to improve color stability by binding iron to slow lipid oxidation and subsequent metmyoglobin formation (Lee et al., 1998). Aloe vera, fenugreek, ginseng, mustard, sage, soy protein, tea catechins, whey protein concentrate, and BHA/BHT were added to pork patties, with BHA/BHT being most effective in maintaining high Hunter a value but fenugreek, whey protein concentrate, and rosemary also had a color-stabilizing effect (McCarthy et al., 2001). In a separate study, Mansour and Khalil (2000) found freeze-dried extracts from ginger rhizomes and fenugreek seeds were effective in controlling color changes in beef patties compared to samples with potato peel extract. And cooked, vacuum-packaged beef top rounds had higher Hunter a values and lower L and b values when 3% or 4% sodium lactate was added (Maca et al., 1999). Color was stabilized by added a combination of BHT/propyl gallate/citric acid in reduced fat pork sausage products with soy protein concentrate and carrageenan added as fat replacers (Ho et al., 1995). In separate studies, phosphate and tertiary butylhydroquinone (TBHQ) in restructured beef, pork, and turkey (Akamittath et al., 1990) or tocopherol, rosemary, BHA, or BHT in chicken and pork frankfurters (Resurreccion and Reynolds, 1990) or BHA and TBHQ to restructured reindeer steaks (Swanson et al., 1994) all had no positive effect on color stability. Sodium ascorbate at 500 ppm preblended with ground buffalo meat resulted in a redder color and lower metmyoglobin content compared with meat without ascorbate (Sahoo and Anjaneyulu, 1997). Frozen rockfish red color was preserved by the combination of ascorbic acid/tocopherol while tocopherol mixed with BHT or sodium erythorbate was less effective in maintaining red color during 4 months of storage (Wasson et al., 1991). Ghiretti et al. (1997) found the colors of Salame and Mortadella sausages were more stable when 0.05%–0.2% sodium ascorbate was added while phytic acid, sesamol, and catechin did not improve the color stability of the meat. Moore et  al. (2003) incorporated 0.1% BHA, BHT, rosemary extract and δ-­ tocopherol into low-density polyethylene to extend the color stability of fresh beef. All antioxidant-impregnated films maintained higher “a” values of beef over 7 days compared to controls while BHA was more effective than other antioxidant films tested. A pink color defect in cooked, noncured meat has been detected particularly in turkey products. This defect is a food safety and quality concern since the meat interior

Impact of proteins on food color625

appears pink and to be uncooked despite being fully cooked. Nicotinamide hemochrome and nitrosohemochrome are believed to be the cooked pigments that remain pink despite heating. Schwarz et al. (1997) reduced these pigments by the addition of ligands to the meat before cooking. The ligands were chelating agents and one promising compound was calcium reduced nonfat dried milk. Antioxidants have also been used with other food products as color stabilizers including apple slices, potato surfaces, shrimp, dried egg, beverages, and vegetables. Browning of these products is caused by the enzyme polyphenol oxidase (PPO). When fruits and other biological tissues are cut or bruised, PPO is released from the tissue and react with phenolic compounds, also naturally found in the tissue, to form the brown pigment melanin. To prevent browning, the enzyme can be denatured to an inactive form using heat or acid. The enzymatic browning of cut apple and potato surfaces was slowed by coating the surfaces with a cellulose-based film (Nature Seal 1020) containing ascorbic acid (Baldwin et al., 1996). Mastrocola et al. (1996) reduced enzymatic browning in freeze-thawed apple slices by dipping them in an ascorbic acid/ citric acid or sodium chloride solution. More recently, apples have been genetically modified to suppress the gene directing production of PPO resulting in a nonbrowning fruit (USDA, 2015). Lambrecht (1995) prevented a color defect called blackspot in shrimp by applying sodium erythrobate. Blackspot is an enzymatic browning reaction that occurs in crustaceans also resulting in the formation of melanin brown pigments. Sulfites have had widespread use in the food industry to control the enzymatic browning of fruits, vegetables, and beverages but are now being avoided if possible due to allergic reactions in humans. Sulfites prevent enzymatic browning in a different way than antioxidants (which block PPO) by preventing the condensation/polymerization of quinones and not the action of PPO (Fig. 22.12). Due to the allergy concern of sulphites, 4-hexylresorcinol has been used to prevent melanosis in shrimp (Frankos et al., 1991). And the loss of color in spray-dried egg due to oxidation of carotenoids was inhibited by adding 100 and 200 ppm propyl gallate (Guardiola et al., 1997).

22.4.2 Modified atmosphere packaging The effectiveness of modified atmosphere packaging in maintaining fresh meat quality depends upon its ability to slow microbial growth and maintain an appealing appearance. The bright red appearance of meat has long been associated with freshness and quality and results from the predominance of the oxymyoglobin pigment. The use of “aerobic” or nonbarrier films allows pigments at the meat surface to “bloom” since the package headspace is equilibrated with ambient air (~20% oxygen). The permeability of polymer tray over wrap or lid stock films is reported as an oxygen transmission rate (OTR) with various units, a standard one being [mLO2/m2/24 h] or the quantity of oxygen (mLO2) per square meter surface area of film (m2) transferred in 24 h at a specified temperature. Very high (12,000) or low (30) OTR films maintained the hue angle of ground chicken over 14 days compared to ground chicken packaged in 4700 OTR films which lost redness faster than meat packaged in other OTR films (Dawson et al., 1995). This approach has been taken one step further by placing the aerobically packaged meat trays in a “master” or “mother” bulk package. The master package is

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flushed with CO2/N2 to eliminate O2 so the meat pigment will remain in the more stable deoxymyoglobin state until removal from the master pack at which time the meat surface will bloom from exposure to ambient air passing through the aerobic tray film. This was demonstrated by Isdell et al. (1999), who used an O2 scavenger in such a master pack system to further enhance O2 removal since residual O2 will facilitate the formation of metmyoglobin. Freshly cut steaks packed in aerobic film over wrapped trays with an O2 scavenger were held in a master pack flushed with 50% CO2/50% N2 and held at 0°C for 2, 4, or 6 weeks. At each sampling time, individual trays were removed from the master pack and color was monitored for 96 h. In all cases, packaging using the O2 scavenger resulted in blooming of the packaged meat upon exposure to air with high Hunter a values and lower hue angles for meat surface measurements compared to meat packaged without a scavenger. Blooming was not observed for meat packaged without an O2 scavenger. This positive influence of O2 scavengers on color in a master pack system was verified by Tewari et  al. (2001) showing that the improved color stability in beef and increased color stability with use of higher levels of scavengers. Vacuum packaging of meat before oxymyoglobin formation will tend to hold the meat pigment in the (deoxy)myoglobin state. While this packaging option is not preferred for fresh ground beef, it is used for other meat species such as lamb, goat, and ostrich meat. Babji et al. (2000) reported that vacuum packaging maintained minced goat meat sensory color scores and reduced discoloration scores compared with aerobic packaging through 28 days of refrigerated storage. Vacuum packaging or 80% N2/20% CO2 also resulted in greater color stability for ground ostrich meat compared with meat packed in trays with air or 80%O2/20% CO2 (Seydim et al., 2001). Vacuum-packaged pork with a low meat pH bloomed more rapidly when exposed to air than meat with higher pH and longissimus muscle cuts had the highest while the semimembranosus had the lowest change in a* value and hue angle during storage (Zhu et al., 2001). The package atmosphere can also be altered by injecting or replacing a package headspace with various gas mixtures. Vacuum skin packaging is a type of vacuum package that can be used with ­oxygen-impermeable- or oxygen-permeable packaging, the latter making it possible to use with fresh meats to allow oxygen penetration to the meat's surface for oxymyoblobin formation. With less residual space for oxygen, color stability is enhanced as well as purge fluid minimized resulting in attractive packaging for retail display. Vacuum skin packaging has been studied for beef by Li et al. (2012) and Strydom and Hope-Jones (2014). Modified atmosphere packaging generally uses three gases (O2, CO2, and N2), each having a specific function. Oxygen is typically used in fresh meat packaging to form the meat pigment oxymyoglobin in high enough levels at the surface to yield the bright red color. Carbon dioxide is highly soluble in the aqueous phase of foods and acts as a bacteriostatic agent. Nitrogen is an inert filler gas to replace O2 in O2-sensitive foods and also prevents package collapse due to its relatively low water solubility compared to O2 and CO2. Raw ground meat products such as beef, pork, lamb, and ostrich containing high muscle pigment levels are often packaged in a modified ­atmosphere that either excludes oxygen (vacuum packaging) or uses 70%–80% o­ xygen with the residual gas being CO2. The high O2 content in the package headspace (usually a

Impact of proteins on food color627

minimum of a 3:1, gas:meat volume ratio) will diffuse into the meat's aqueous phase where the water-soluble meat pigment myoglobin exists. The high level of dissolved oxygen will bind to the sixth position on the myoglobin molecule resulting in a bright red meat color. While the high oxygen will force the predominance of the oxymyoglobin pigment form, it will also accelerate the oxidation of myoglobin (Fe+2) to metmyoglobin (Fe+3) resulting in an undesirable and nearly irreversible brown-gray appearance. Meat color has a significant effect on consumer perception of freshness; however, color has little effect on eating quality. Carpenter et al. (2001) found that consumers rated preference scores for top round loin and ground beef in order from red > purple > brown. The consumers were served untreated meat but were told it had originated from one of the three color groups. The perception of freshness due to appearance was not transferred to taste; thus, the consumers were not fooled when fed untreated samples presented as being from different appearance groups. In this study, the red-colored group was created by an atmosphere containing carbon monoxide. Carbon monoxide can retard metmyoglobin formation due to the strong association of carbon monoxide to the heme porphyrin ring. Carboxymyoglobin also has a bright red appearance similar to that of oxymyoglobin. The formation of the stable red-pink pigment gives raw meat a fresh appearance but can carry over into cooked products to leave an uncooked perception. This was studied by Cornforth et al. (1998) due to a phenomenon observed in gas ovens related to the surface pinking of cooked beef and turkey. Carbon monoxide, nitric oxide, and/or nitrogen dioxide were believed to be the cause of surface pinking in fully cooked meat. In this case, nitric dioxide in as little as 0.4 ppm nitrogen dioxide caused pinking while up to 149 ppm of carbon monoxide and 5 ppm nitric oxide did not cause pinking. Carbon monoxide has been studied and is now used to stabilize meat color in MAP. The use of CO to stabilize meat color has safety ramifications due to the ingestion of CO since CO binds so tightly to the heme porphyrin ring, easily displacing oxygen. CO binds tightly to both hemoglobin and myoglobin yielding a stable bright red color similar to that of oxymyoglobin. However, the levels needed to stabilize meat color are quite low, usually <1% of the package headspace. This is in contrast to CO levels approved for fresh vegetable packaging which is in the 3%–9% range and is used to regulate plant tissue respiration. Thus from an ingestion standpoint, levels of 1% or less CO used in meat packaging would seem safe. Another safety concern with CO in meat packaging is the safe appearance of meat that is spoiled. Consumers use appearance as one parameter to gauge meat quality and safety; thus, the high stability of carboxymyoglobin in the presence of bacterial growth and oxidation may make differentiation of less than fresh meat from fresh meat difficult from visual inspection. The color-stabilizing effect of CO on beef steaks was demonstrated by Luna et al. (2000) using concentrations from 0.1% to 1.0% CO in a mixture of 25% O2, 50% CO2, and 25%–25.9% N2 all compared with a reference mixture of 70% O2, 20% CO2 and 10% N2. CO concentrations of 0.5%–0.75% delayed metmyoglobin formation (less than 40% of the total myoglobin) through 29 days and maintained no change in a* values and hue angle of the meat surface through 23 days. Luna et al. (1998) previously demonstrated that 1% CO with 70% O2, 20% CO2, 9% N2, or with 24%O2, 50% CO2, and 25% N2 maintained beef steak and ground beef color for 29 days. Jayasingh et al. (2001) stabilized the

628

Proteins in Food Processing

b­ eefsteak and ground beef red color in vacuum packages by pretreating the meat in 5% CO for 24 h or 100% CO for 1 h. Thus, CO is a very effective meat color stabilizer when exposed either prior to or during packaging. Cured meat pigments have previously been described as products from denatured myoglobin hemochromes. The cured meat pigments are stable but will degrade more rapidly in the presence of oxygen, at elevated temperatures, and when exposed to light. Cured meat products are therefore packaged in systems to exclude oxygen (vacuum or MAP). Moller et al. (2000) determined that the critical level of oxygen that was needed to minimize the rate of light-induced discoloration of chilled stored sliced ham was <0.5% and that ham color was stable at 0.1% and 0.2% oxygen. Houben and van Dijk (2001) reported that sliced beef hams in MAP (50%CO2/50%N2) had greater color stability than hams in vacuum packages. Jydegaard et  al. (2004) evaluated semidry fermented sausages to determine whether an Arrhenius-type relationship existed for surface color fading or color loss when packaged slices of product were placed in light display with lighting of different intensities. Rate constants for CIE L* a* b* as well as CIE C*and h* were obtained for slices packaged in a nonoxygen barrier film so that oxygen was not restricted as a reactant in the degradation of the cured pigment, nitrosylhemochrome. When placed in display at 2°C under fluorescent light intensities in the range of 660–3173 lx, decreases in product surface redness and chroma and increases in hue occurred. Plotting first-order rate constants of the color attributes against the reciprocal of light intensity indicated an Arrhenius-type reaction rate fit (Fig. 22.18). This finding thus confirmed that the Arrhenius concept for reaction dependency on temperature is also valid for

Log rate constant (h–1) – CIE h*

–0.90 –1.00 –1.10 –1.20 –1.30 –1.40 –1.50 –1.60 0.0000

0.0005

0.0010

0.0015

0.0020

Reciprocal of light intensity (1/lx)

Fig. 22.18  Arrhenius-type plots showing the dependency of the first-order rate constants (taken from absolute values of slopes) for redness (CIE + a*) decrease and hue (CIE h*) increase as a function of light intensity at the surface of fermented semidry sausage (Jydegaard et al., 2004).

Impact of proteins on food color629

dependency on light intensity, another form of energy that can negatively affect the color stability of cured meats. Packaging has also been used to stabilize the color of nonmeat products including fresh-cut fruit and vegetable surfaces. Broccoli packed in glass jars with oil-coated corn zein films sealed across the jar top maintained green color longer than other treatments and the zein film allowed the headspace gases to modify, thereby maintaining broccoli freshness (Rakotonirainy et  al., 2001). McHugh and Senesi (2000) concluded that natural wraps made from apple puree containing various levels of fatty acids, fatty alcohols, beeswax, and vegetable oil were effective in preserving freshly cut apple flesh for 12 days stored at 5°C and were significantly more effective than coatings. Several researchers have modified gas package headspace to stabilize vegetable and fruit color. For example, Drosinas et al. (2000) found an improved color stability of Greek-style tomato salad using 5%CO2/95%N2 compared to salad packaged with air. Soliva-Fortuny et  al. (2001) reduced polyphenol oxidase activity by 62% and maintained higher L values on fresh-cut Golden Delicious apples using a MAP of 90.5%N2/7%CO2/2.5%O2 compared with slices packed in air. The enzymatic browning in pears was inhibited with paper wraps treated with oil and ethoxyquin compared with pears in nontreated paper when stored in a controlled atmosphere room (1.5%O2/3%CO2/95.5%N2) (Drake et al., 2001).

22.4.3 Ionizing radiation effects Ionizing radiation can affect the color of both raw and cooked meats with the raw meat often being redder and the cooked meat being more or less red when irradiated. The mechanism of color change due to irradiation is similar to mechanisms found in nonirradiated meats but the color change depends on animal species among other factors. In general, light (low myoglobin content) meats appear pink while darker meat species turn brown after irradiation (Nam and Ahn, 2003). Sommers et al. (2001) found the irradiation gave beef bologna a less red appearance while raw turkey (Nam and Ahn, 2002) and precooked turkey (Nam et  al., 2002) were both redder when irradiated. Increased redness of irradiated raw meat was also reported by Bagaoragoza et al. (2001) for turkey breast irradiated at 2.4–2.9 kGy and for pork loins (Lacroix et al., 2000; Nam et al., 2001) when meat was irradiated under vacuum as opposed to aerobically. The reddening effect of irradiation can be mitigated by the addition of antioxidants. For instance, irradiation (1.5 and 3.0 kGy) induced a redder color in precooked turkey roll slices; however, redness was reduced by the addition of gallic acid, rosemary extract, or sesamol (Du and Ahn, 2002) and irradiated (3.0 kGy) raw chicken also had a redder appearance which dissipated with aerobic storage but remained prevalent in vacuum-packaged chicken (Du et al., 2002). A reduction of the oxidation-reduction potential of the meat has also been detected due to irradiation. The more red color of raw and cooked turkey was attributed to the formation of carbon monoxide myoglobin from carbon monoxide generated in the meat during irradiation and was facilitated by the decrease in oxidation-reduction potential. Irradiation (2.5 and 5.0 kGy) also decreased the redness of egg yolk powder (Du and Ahn, 2000).

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Proteins in Food Processing

22.4.4 Meat processing effects on color How the animal is processed as well as genetic predisposition to certain processing conditions can affect the appearance and color of the meat after processing. A recent study used enzyme inhibitors to inhibit overall protein phosphorylation to stabilize ground pork meat implying that fresh meat color stability is partly regulated by the redox state of myoglobin and postmortem glycolysis (Li et al., 2017). Two conditions that can impact meat protein color are PSE and DFD) quality defects. These defects, PSE in particular, were initially associated with swine but are now found in multiple species including turkey (McKee and Sams, 1997), chickens (Barbut, 1997), ostrich (Van Schalkwyk et al., 2000), and cattle (Aalhus et al., 1998). The quality defects result in significant financial loss to the meat industry (Cassell et al., 1991), partly due to consumer dissatisfaction with meat appearance (Fig. 22.19). The cause of PSE and DFD defects is generally considered to be due to how the animal is handled prior to slaughter although as stated earlier, some animals are genetically predisposed for these defects. The biochemical cause of both of these defects is how the resolution of rigor mortis proceeds in the tissue postmortem. In the normal tissue, there is adequate energy (ATP) and other necessary muscle components to continue anaerobic respiration, producing lactic acid and lowering the pH to between ~5.5 and 6.1, depending on the species. The pH drop normally occurs slowly and does not reach a point that can denature protein until the meat temperature has been dropped to avoid this phenomenon. If the pH drops too quickly while the meat temperature is too high, PSE meat results due to muscle protein denaturation, which reduces the protein's ability to bind water giving the tissue a pale, less pigmented appearance. Furthermore, the final pH is closer to the isoelectric point of meat proteins, resulting in less charge and lower water-binding ability. The

PSE

Normal

A rapid drop in meat pH while meat temperature is still high usually causing a breakdown of muscle protein and the meat becoming very pale with pronounced acidity (pH values of 5.4–5.6 immediately after slaughter) and poor flavor. This type of meat is difficult to use or cannot be used at all by butchers or meat processors and is wasted in extreme cases.

Muscle glycogen is not depleted prior to slaughter and a slow drop in pH occurs postmortem as glycogen is metabolized into lactic acid. The pH eventually drops 6.0–6.2 (depending upon the meat species) which is the optimal range for water binding.

DFD The muscle glycogen has been used up during the preslaughter period, thus after slaughter, there is little lactic acid production, which results in DFD meat. This meat is of inferior quality as the less pronounced taste and the dark colour is less acceptable to the consumer and has a shorter shelf life due to the abnormally high pH-value of the meat (6.4–6.8).

Fig. 22.19  Effect of pale, soft, and exudative (PSE) and dark, firm, and dry (DFD) conditions on appearance of pork. (Pictures of the National Pork Board).

Speceis

Parameter

DFD

Normal

PSE

Reference

Turkey

L pH Expressible moisture (%) Drip loss (%) L pH Expressible moisture (%) Drip loss (%) L

– – – – – – – – 35.1–41.7

49.0 6.09 23.4 0.7 52.2 6.07 25.2 3.3 44.0–46.5

54.7 <5.72 32.3 2.5 59.8 <5.76 30.6 4.4 54.0–57.1

pH

>6.40

5.62–5.82

<5.5

Drip loss (%)

0.4–0.9

2.1–4.0

5.23–5.54

L pH Dip loss (%)

<37

38–40 5.69 13.0

42.0 5.63 35.8

Owens et al., 2000 Owens et al., 2000 Owens et al., 2000 Owens et al., 2000 Woelfel et al., 2002 Woelfel et al., 2002 Woelfel et al., 2002 Woelfel et al., 2002 Chmieil et al., 2011; Pospiech, 2000; van der Wal et al., 1988 Chmieil et al., 2011; Pospiech, 2000; van der Wal et al., 1988 Chmieil et al., 2011; Pospiech, 2000; van der Wal et al., 1988 Muchenje et al., 2009; Aalhus et al., 1998 Aalhus et al., 1998 Aalhus et al., 1998

Chicken

Pork

Beef

Impact of proteins on food color631

Table 22.3  Effect of postmortem processing defects (dry-firm-dark and pale-soft-exudative) on lightness (L), pH, expressible moisture and drip loss

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Proteins in Food Processing

DFD condition can arise if the animal is physically stressed for a relatively long time prior to slaughter using up the reserve energy sources in the muscle such that anaerobic respiration cannot proceed to lower the pH via production of lactic acid. The high pH (>6.0) causes excessive water binding as this pH is higher than the normal meat pH and farther away from the muscle protein's isoelectric point. The excessive water binding by the protein gives the meat a dark appearance and dry texture. The effect of PSE and DFD on meat color (lightness), pH and water binding is shown in Table 22.3.

22.5 Future trends One predicted future trend for preserving color is the increased use of modified atmosphere packaging replacing overwrap-type systems. New gases will also be utilized including the increased use of carbon monoxide to stabilize pigments. Argon has also been investigated as a packaging gas. Antioxidant films as part of the active packaging revolution are likely to find their way into the meat case. These films are being investigated as slow-release antioxidant carriers to maintain fresh meat color and may also have applications for cured meats.

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Further reading Hettiarachchy, N.S., Ziegler, G.R., 1994. Protein Functionality in Food Systems. Marcel Dekker, New York, NY. Hunter, R.S., 1975. The Measurement of Appearance. John Wiley and Sons, Inc., New York, NY. Hunter, R.S., Harold, R.W., 1987. The Measurement of Appearance, second ed. John Wiley and Sons, Inc., New York, NY. Hutchings, J.B., 1994. Food Colour and Appearance. Blackie Academic & Professional, an imprint of Chapman and Hall, Glasgow. Mancini, R.A., Hunt, M.C., 2005. Current research in meat colour. Meat Sci. 71, 100–121.