F.4.1 Distinguish between a dye and a pigment. F.4.2 Explain the occurrence of colour in naturally occurring pigments. F.4.

3 Describe the range of colors and sources of naturally occurring pigments. F.4.5 Describe the factors that affect the color stability of anthocyanins, heme, carotenoids & chlorophyll F.4.5 Discuss the safety issues associated with the use of colorants in food. F.4.6 Compare the processes of non enzymatic browning and caramelisation.

F4 Colour

Dye versus Pigment

Dye
A food grade

Pigment
A naturally occurring

synthetic water soluble colorant that can be added to food to enhance or change its natural color .

colorant found in the cells of plants and animals. Chemical families: porphyrins, carotenoids, flavonoids, chlorophyll, heme and myoglobin.

What causes Colour?

Dyes and Pigment molecules with

conjugated (alternating single and double) carbon-carbon bonds. The electrons of the double bond are delocalized and spread out along the adjacent molecules. Colorless organic molecules do not have conjugated carbon-carbon bonds.

 The delocalized electrons absorb visible light when the energy of the

incoming light photon matches the energy difference between the electrons ground state and excited state. The electron becomes excited and transition from a lower-energy or ground electron configuration to a higher-energy electron configuration. When the electrons drops back down to its ground state it releases/ reflects/transmits visible light with a complementary wavelength. The color we see has a complementary relationship with the color of the visible light absorbed. Molecules that do not absorb light in the 400-700nm visible light range are colorless.

 Pigment molecules differ in color due

to differences in the amount of conjugation. There is a positive relationship between the amount of conjugation and the wavelength of visible light absorbed. As the amount of conjugation increases, the wavelength of light absorbed increases changing the complementary color reflected.

Anthocyanins
Sub-class of flavonoids Orange-red, red-blue colors Foods: strawberries, berries

(blueberries, cherries, blackberries), grapes and some vegetables, such as egg-plant and avocado. Water soluble form hydrogen bonds with water due to the presence of multiple hydroxyl groups. Three phenol groups (benzene ring) with conjugated carboncarbon bonds and varying numbers of hydroxyl groups. Glucose molecules bond to the oxygen atom on the second phenol group. Anthocyanins are found in foods high in sugar content.

Anthocyanin Stability
Unstable with pH, temperature, in the presence of UV light and Fe3+

& Al3+ ions. Most stable at low pH (acid) and low temperature. Equilibrium exists between four different forms. Position of equilibrium affected by adding acid or alkali or by changing the temperature causing different colored forms to predominate. Page 35 of the Data Booklet shows the equilibrium relationship between the four different colored structures at 25C and pH. At high temperature during cooking anthocyanins decompose. The position of equilibrium moves to the right, resulting in a loss of color due to the predominance of the colorless structure. - Blue quinonoidal base (A), stable at pH 6-7 - Red flavylium cation (AH+), stable at pH 1-2 - Colorless carbinol psuedobase (B), stable at pH 4-5 - Colorless chalcone (C), stable at pH > 7 Make good natural acid-base indicators. E.g red cabbage is red in acid and blue and purple in base.

Carotenoids
Color: Yellow to orange-red

pigments Food sources: carrots, oranges, salmon, tomatoes, lobster, yellow peppers Solubility: Fat soluble and insoluble in water. Long conjugated hydrocarbon chain which outweighs the small number of polar hydroxyl groups. Functions: Acts as antioxidants. Can be converted/precursor to Vitamin A (retinol) in the body and so is considered a nutrient. Vitamin A needed for good vision (production of light sensitive cells in the retina) and to hydrate skin cells. Carotenes contribute 30-100% of our Vitamin A requirement.

Color Stability in Carotenoids

Stable in pH 2-7. At low pH color will change from red-

orange to orange-yellow due to the formation of alternate structure. Stable up to 50C and therefore color is not degraded by most forms of food processing. C=C double bonds mean that they are easily oxidized by oxygen in the air causing discoloration, loss of Vitamin A and off odors. Metals from knives and cutting surfaces and UV light also catalyze the oxidation. Food need to be kept wrapped, kept away from light and metal and kept in the refrigerator.

Chlorophyll
Color: Green to olive green Sources: green leafy plants

(spinach), algae Function: Photosynthesis (food production) in Plants. Solubility: Fat soluble in pure form due to lack of hydroxyl group to hydrogen bond with water. Structure: A mixture of molecules each with a different R group. Ring structure called a porphyrin coordinately bonds 4 nitrogen atoms to a central Mg2+ ion. Square planar shape. Attached to ring IV is a phytol group (C20H39 carbon chain) Structure page 36 of Data Booklet

Colour Stability in Chlorophyll

Unstable - affected by heat,
Olive-brown Pheophytin-a

oxygen gas (in air), UV light and pH. Fresh foods containing chlorophyll should be stored away from heat, light and air so color changes do not occur. Canning and preserving in jars will change color of chlorophyll because processing requires heating to boiling to kill microorganisms.

Bright green chlorophyll-a

 When green leafy plants are exposed to heat, the cell wall

deteriorates releasing H+ ions, lowering the pH. H+ ions displace the Mg2+ ion replacing it with two H atoms forming the olivebrown pheophytin-a pigment. This coloration often occurs in canned and preserved vegetables like peas and beans. Chlorophyll also reacts with OH- ions (source NaOH) to give the bright green chlorophyllin pigment.

Oxygen Transport
The O2 we breath in is not very

soluble in the blood. and can only provide about 1% of the oxygen our cells need. O2 transport in humans is effective when it is carried through the blood to the tissues by a protein molecules called hemoglobin (Hb). When the Hb-O2 complex reaches the tissue, the O2 molecules are transferred to another protein myoglobin (Mb) which transports the oxygen to the individual cells that will use it to make energy during cellular respiration. Both molecules have a heme group

Heme
Function: transport of oxygen for cellular respiration. Structure: Central Fe (II) ion which bonds to 6 other groups (called coordination sites) 4 of the 6 coordination sites occupied by nitrogen atoms from a porphyrin ring. 5th coordination site occupied by the nitrogen atom from a protein molecule. 6th site is available to bind an O2 molecule.

Heme molecule

Hemoglobin vs. Myoglobin

Hemoglobin
Structure: Four protein

Myoglobin
Structure: Single protein

chains and four heme groups that carry oxygen from the lungs to the tissues. Source: red blood cells Function: Carries oxygen in the blood to tissue

chain and one heme group that transports oxygen in tissue - muscle cells (page 35 of Data Booklet) Source: Muscle tissue Function: Carries oxygen in the tissues

Myoglobin One protein chain and one heme group.

Hemoglobin Four protein chains and four heme groups

Colour Stability in Myoglobin

When animal tissue comes in contact with oxygen in the air

the myoglobin exits in three different structures. Myoglobin (Mb) is purple-red with Fe(II) oxidation state. On exposure to oxygen in the air it forms bright red oxymyoglobin (MbO2), also Fe(II). This is the color of freshly cut meat. Mb-Fe2+ + O2 Mb-Fe2+-O2 With extended exposure to oxygen the Fe2+ ion is autooxidized (loses an electron) to form Fe3+ - the metamyoglobin (MMb) structure. MMb has an undesirable brown in color. Mb-Fe2+ Mb-Fe3+ Choose bright red colored meat it is the freshest. To keep meat from forming metamyoglobin store in an oxygen reduced atmosphere low gas permeability film, vacuum packed or replace oxygen with an inert gas. Fe2+ more stable than Fe3+.

Synthetic Colorants in Food

Use of synthetic food colorings/dyes in food is not

regulated by international standards, so countries have their own regulations concerning their use. In the US FD&C numbers are given to approved synthetic food dyes that do not exist in nature. In the European Union, E numbers are used for all additives, both synthetic and natural. Other countries have their own numbering system. The Codex Alimentarius Commission (FAO and WHO) sets food standards, and codes of practice that can be used by international governmental and non-governmental organizations (NGO). Some synthetic food dyes have been linked to health problems hyperactivity in children. Research into the health effects is still in its infancy.

Natural food dyes, commercially produced and allowed in food in the US include: Caramel coloring (E150),used in cola products. Green dye made from chlorella algae (chlorophyll, E140). Cochineal (E120), a red dye derived from the cochineal insect. Betanin extracted from beets. Turmeric (curcuminoids, E100). Saffron (carotenoids, E160a). Paprika (E160c) Often provided in highly purified form, for increased stability. Artificial coloring approved in the US: FD&C Blue No. 1 Brilliant Blue, E133 (Blue) FD&C Blue No. 2 Indigotine, E132 (Dark Blue) FD&C Green No. 3 Fast Green, E143 (Blue-green) FD&C Red No. 40 Allura Red AC, E129 (Red) FD&C Red No. 3 Erythrosine, E127 (Pink) FD&C Yellow No. 5 Tartrazine, E102 (Yellow) FD&C Yellow No. 6 Sunset Yellow, E110 (Orange) Also a list of delisted artificial coloring, no longer permitted. (FD&C Blue No. 1, Red No. 40, Yellow No. 5 and Yellow No. 6 used to color Starbursts)

Browning of Food
Enzymatic Browning Non-Enymatic Browning

Chemical reaction

Browning of food

which occurs in foods containing the enzyme polyphenoloxidase, Results in the production of brown colored pigments.

without enzymes via the Maillard reaction and caramelization.

Maillard Reaction
Food Composition: Food containing protein and carbohydrates (specifically a

reducing sugar). Complex reaction mechanism. The first step involves a condensation reaction between an amino acid and a reducing sugar (glucose, fructose, sucrose, lactose) followed by a series of dehydration, fragmentation and condensation reactions to form brown pigmented products called melanoidins. Also causes flavor and smell changes. Factors effecting rate of browning: pH (4-7 optimum), type of amino acid (lysine most & cysteine least brown), type of reducing sugar, temperature (wide range), time, presence of oxygen and water. E.g. boiling milk, frying meat and foods that involve cooking sugar and protein (often from milk) like fudge, caramel toffee, milk chocolate, baking bread. Features of the Product: Desirable brown color, flavor and smell. Reduces nutritional value of food as amino acids and sugar is lost during reaction. Food affected: Frying meat. Heating sugar and milk to make fudge, caramel toffee, and milk chocolate. Brown crust on baked bread, roasted coffee beans. Browning meat http://www.bbc.co.uk/food/get_cooking/techniques/003001.shtml

What is a Reducing Sugar?

Sugars exist in solution as an equilibrium

mixture of open/straight-chain and closedring (cyclic) structures. In the straight chain form, the C1 atom contains the C=O bond (carbonyl group) either as a ketone or aldehyde. In the ring (cyclic) structure, the carbonyl carbon, C1 is the one which is attached to the O of the ring and an OH group. Straight chained sugars that have an aldehyde or ketone group can act as a reducing agent in a chemical reaction. Reducing agent means the sugar is oxidized gains oxygen atoms & loses electrons in a chemical reaction. Reducing sugars are: glucose, fructose, lactose, maltose (not sucrose)

First Step in Maillard Browning

Condensation of a reducing sugar and the amino group on

an amino acid. One molecule of water formed. Forms an amadori structure that exists in three structural isomers (same molecular formula but different structural formula).

Caramelization
Food Composition: During dry heating and roasting of food

high in sugar and without proteins/amino acids. Complex reaction mechanism. At its most simple the process involves melting sugar at a temperature > 120C in order to dehydrate (remove water) Factors the increase rate of browning: pH <3 & >9, temperature >120C, type of sugar (fructose best). Feature of product: desirable brown color, caramel/ butterscotch smell and flavor. Undesirable effects burned sugar smell and taste, and black color. Occurs when heated for too long at high temperatures causing all the water is removed from the sugar producing carbon. CnH2nOm nC + mH2O Foods affected: roasted vegetables, color and flavor of cola beverages, topping on baked egg dishes.

Activity 1
When hydrogen is added across the double bond of

lycopene (the yellow/orange) pigment found in tomatoes, the number of C=C double bonds decreases. Predict and explain the color change that would be observed in the tomato.

Activity 2
1. The color of Cherry Kool-Aid is the result of added an artificial dye. Explain why makes cherry Kool-Aid, appear red to your eye? 2. Using structures on Pages 37-38 of the Data Booklet predict whether -carotene and lutein would appear orange or red.

Activity 3
1. What color are

anthocyanins at pH 0-6 and pH 7-14. 2. Using Bronsted-Lowry theory and the equation on page 35 of the Data Booklet explain how that addition of ethanoic acid and sodium bicarbonate changes the color of red cabbage. Account for the purple color.

Activity 4
and -carotene absorb

visible light in the 430-490nm range and reflect/transmit yelloworange light. The carotenoid found in lobster shell is called astaxanthin (page 38 of Data Booklet). Suggest a reason why lobsters are blue-green when live and orange-red when cooked.

Activity 5
Astaxanthin is responsible for

the natural pink color of shrimp. It contains C: 80.54%, H: 8.72%, and O: 10.74% by mass. 1. Distinguish between a pigment and a dye 2. Calculate the empirical formula for Astaxanthin 3. Given that the Molar Mass of Astaxanthin was found to be 620.00 gmol-1 determine the molecular formula. 4. How are the empirical and molecular formula related?

Activity 6
A UV-Visible spectrometer/

spectrophotometer measures the amount of visible light absorbed by a colored pigment. This can be read as Absorbance versus wavelength. Maximum absorbance corresponds to the wavelength of light absorbed. The color seen / reflected is its complementary color. Beers Law can be used to determine the concentration.

The absorbance spectrum of chlorophyll-a taken from a spectrophotometer is shown alongside. 1. Determine max .What does it represent? 2. Explain why chlorophyll appears green.

Activity 7
Normally metal ions form ionic compounds with

non-metal ions. Complex ions are an exception to this rule. Chlorophyll is a complex ion because each of the N atoms on the four rings of the porphyrin donates its lone pair of electrons to the central Mg2+ ion forming a coordinate covalent bond. The porphyrin rings are called ligands. 1. Define a coordinate bond and ligand. 2. Name another molecule with this structure.

Activity 8
1. Compare the structural features of chlorophyll and

heme. 2. Deduce and explain their solubility in water.

Activity 9
Erythrosine is a cherry-pink/

red synthetic dye used to color foods like cherries, candy bars, and baked goods. It has been linked to hyperactive behavioral disorders in children, thyroid cancer in rats. Banned in in Norway. Identified using the following coding. * FD&C Red No. 3 * E127 (Food Red 14) * Indian Standards No. 1697 Outline some of the issues related to the use of this dye.

Activity 10
1.

2.

3.

4.

Write an equation to show the condensation reaction that occurs between glucose and cysteine. Give the structure of the three possible amadori compounds formed. Distinguish between the type of browning that occurs in bread and bananas. Explain each process. Compare the Maillard browning and caramelization in terms of the chemical composition of the food and the products formed. Write an equation for the burning of sucrose during caramelization.

Activity 11
Deduce with a reason the type of browning: 1. Caramel candy or milk candy is a made by boiling milk, sugar, butter, vanilla essence, water, and glucose or corn syrup. It is heated in a pot up to 120 C. 2. Many cultures have a baked egg desert made with cream, eggs and sugar (crme brulee, crme caramel, flan, egg tarts). Typically sugar is sprinkled on the top & heated to a high temperature until it browns.