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FT 131

JAN 23

FOOD CHEMISTRY

  • - post harvest handling of fresh produce

  • - food formulation

  • - food processing

  • - storage

  • - proper investigation/research: facts derived from the study of one food or model system

Examples:

  • 1. EFFECT OF FOOD ADDITIVE:

    • - antioxidant in preventing rancidity: stability of sweetened-dried young coconut meat with tocopherol randomly selected from 1 batch will enhance our understanding of the stability of the rest of the products and of the future products.

    • - synthetic sweetener as substitute for sugar in polvoron: 60% substitution of sugar significantly reduced calories of the product without affecting sweetness (perceived). Results can be used in modifying similar products or other products.

      • 2. POST-HARVEST CHANGES IN FRUITS

        • - biochemical/chemical changes (ripening) in saba banana at 15C; results will enhance our…

        • - biochemical/chemical changes in solo papaya under MA (Modified Atmosphere) storage

4 COMPONENTS OF ANALYTICAL APPROACH

  • 1. Determination of properties that are important characteristics of safe-high quality foods

  • 2. Determination of chemical and biochemical reactions that influence the loss of quality and/or wholesomeness of foods

  • 3. Integrating approaches 1 and 2 so that one understands how the key chemical and biochemical reactions influence quality and safety of foods

  • 4. Application of the knowledge gained in no. 3 to the various situation encountered during the formulation, processing and storage of foods

SAFE-HIGH QUALITY FOODS

> safety – first requirement to be satisfied by any food (fresh or processed)

> free from hazards (biological/microbiological, chemical, physical) at the time of consumption Examples:

1. canned products, low acid foods, pH>4.5, absence of Clostridium botulinum it toxins 2. drinking water, no heavy metals, e.g. Hg

> quality-sensory and nutritive value: influenced by composition and properties of components > sensory attributes – color, aroma, flavor, taste, texture > chemical and biological reactions: desirable or undesirable Desirable – enhance quality of food; e.g. tenderization of meat during aging Undesirable – deterioration of food; biochemical/chemical reactions resulting to loss of quality attributes specific to the food.

Reactions

Results

Hydrolysis of lipids

ffa react with proteins: change in flavor and texture

Hydrolysis of polysaccharides

sugar released react with protein or aa:

change in texture or flavor

Hydrolysis of pectin

softening, loss of crispness

Association of molecules

change in texture, gel formation

MECHANICAL DAMAGE

  • - physical changes which may induce biochemical/chemical reactions Examples:

    • 1. Bruising of fruits, vegetables, root crops

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2.

Heating

  • 3. Low temperature, refrigeration, freezing

  • 1. Bruising of fruits, vegetables, root crops

    • - may release enzymes which catalyze biochemical changes such as

      • a. browning discoloration – phenolases or polyphenol oxidase (PPO)

      • b. changes in texture – tenderization or softening due to destruction of cell walls, cell membrane by hydrolases

      • c. changes in flavor – loss of formation of flavor compounds

  • 2. Heating

    • - cause protein denaturation: change in texture, flavor, etc.

      • 3. Low temperature, refrigeration, freezing

        • - chilling injury, freezer burn

PRESERVATION OF FOODS

  • A. Short term preservation – extend freshness of fruits, vegetables, seafoods, beef, pork, chicken (poultry), etc.

    • - refrigeration, modified atmosphere packaging (MAP), controlled atmospheric packaging (CAP)

      • B. Long term preservation – canning, drying, freezing

In the selection of the method consider the following: compostion, components of food, post harvest, post mortem biochemical/chemical changes in food (may occur immediately after harvest/death, during processing or storage, thus there is a need to study components of food, macro components, and micro components).

PIGMENTS AND OTHER COLORANTS

Color – one of the most important quality (sensory attribute of food)

  • - influence acceptability; particularly color associated with/to specific food items or quality

  • - influence palatability; index of maturity, e.g. fruits, index of freshness, e.g. meat; influence extent/type of processing

  • - inherent in food

  • - (colorant) added to food to enhance its sensory quality (not to cover faulty processing technique)

Color of food:

  • 1. Pigments inherent in food* - fresh produce

  • 2. Colorants (added) – processed foods

*preserved during processing/preparation?

PIGMENTS

-

normal constituents of cells or tissues of plants or animals that impart color

-

in fruits, leaves, other parts of plants, animal tissues, blood, fish, shellfish, other seafoods

-

in harvested fruits/vegetables and muscle foods, pigments also include compounds/substances formed on heating, storage or processing

-

also synthesized by some microorganisms

JAN 30

COLORANT

-

general term which refers to any chemical compound that imparts color

-

food additives, i.e., intentionally added

-

can be a natural pigment extracted or prepared (e.g. annatto, turmeric, carotenoids, anthocyanins) from a plant source or it can be synthetic

-

regulated particularly the synthetic food colors (subjected to toxicological tests prior approval to use)

OTHER FUNCTIONS OF PIGMENTS

-

in live plant/animals energy receptor – chlorophyll

2

carrier of oxygen protectants against radiation – some carotenoids

  • - post harvest some are considered as neutraceuticals, antioxidants which prevents certain diseases, e.g. cancer

CHLOROPHYLL

  • - belong to the group of tetrapyrrole compounds

  • - all classes of photosynthesized porphyrin pigments

  • - green pigments involved in the photosynthesis of higher plants

TYPES

  • 1. Chlorophyll a – in all plants, algae and cyanobacteria

  • 2. Chlorophyll b – in plants and green algae only

  • 3. Chlorophyll c – in photosynthesis chromista and dinoflagellates

  • 4. Chlorophyll d – in red algae together with type a

QUESTION: What are the types of interest to food chemists?

  • - in higher plants ratio of a to b ~ 3:1

STRUCTURE:

  • - porphyrin ring with 4 pyrrole group and Mg

a Mg – chelated tetrapyrrole structure with methyl substituents; with phytol** group esterified to

propionate at the C-7 position **phytol – a 20C monosaturated isoprenoid alcohol

Type

R group*

Chlorophyll a

CH 3 (methyl)

Chlorophyll b

CHO (formyl (aldehyde))

**STRUCTURE

  • - Location: Chloroplasts – plastid bodies where chlorophylls are located Grana – smaller particles with in chloroplasts composed of lamellae where chlorophyll molecules are embedded Stroma – between grana

  • - Occurrence in plants associated with lipids, proteins and lipoproteins bound to these molecules How? Held together in a monolayer by:

    • - mutual attraction

    • - affinity of phytol tail for lipids

    • - affinity of the hydrophobic planar porphyrin ring for proteins

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  • - Properties: Physical and Chemical Physical, solubility in various solvents:

S – Soluble I – Insoluble SltS – Slightly soluble AlmI – Almost Insoluble

 

Alcohol

Ether

Benzene

Acetone

Petroleum

Water

Ether

chlorophyll a

S

S

S

S

SltS

I

pheophytin a

S

S

S

S

SltS

I

chlorophyll b

S

S

S

S

AlmI

I

pheophytin b

S

S

S

S

AlmI

I

Chlorophyllide

 

S

a/b*

 

*In General Insoluble in Oil

 

pheophorbide

 

S

a/b

Chemical Properties

  • - chemical changes in structure of chlorophyll

  • - alterations in the structure are manifested through changes in color of disappearance of color

Factors which influence changes:

  • - temperature (heating, cooking)

  • - pH (acidity)

  • - light (sunlight, artificial light, packaging)

  • - etc.

Alterations/changes in the structure of chlorophyll

  • 1. Isomerization

  • 2. Pheophytinization

  • 3. Removal of phytol group from chlorophyll

  • 4. Removal of phytol group from pheophytin

  • 5. Removal of Mg from chlorophyllide and its replacement with H

  • 6. Formation of pyrophephytin

  • 7. Formation of pyropheophorbide

  • 8. Reaction of side groups Ex. Oxidation of isocyclic ring – allomerized chlorophyll

  • 9. Rupture of tetrapyrrole ring - colorless end products - 2-5 common reactions

Chemical changes

  • 1. Isomerization of chlorophyll by inversion of carbomethoxy group at C10

  • - may occur during heating in the presence of organic solvent Ex. chlorophyll a – chlorophyll a’ These isomers have stronger A on C18 chlorophyll b – chlorophyll b’ Reverse phase HPLC column *observed in spinach leaves when heated at 100C for 10 min, 5-10% conversion

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2.

Pheophytinization – removal of Mg ++ and its replacement with 2H + -Mg ++ , +2H
Pheophytinization – removal of Mg ++ and its replacement with 2H +
-Mg ++ , +2H +
chlorophyll
bright green
pheophytin
dull olive brown

compare susceptibility of chlorophyll a with chlorophyll b to pheophytinization

  • - chlorophyll a is more susceptible; chlorophyll b is more stable

3. Removal of phytol group from chlorophyll

  • chlorophyllide bright green (more intense)

presence of Mg ++ - bright green color

  • 4. Removal of phytol group from pheophytin

-phytol pheophytin pheophorbide olive brown olive brown
-phytol
pheophytin
pheophorbide
olive brown
olive brown
  • 5. Removal of Mg from chlorophyllide and its replacement with H

-Mg ++ , +2H + chlorophyllide pheophorbide bright green olive brown
-Mg ++ , +2H +
chlorophyllide
pheophorbide
bright green
olive brown
  • 6. Formation of pyropheophytin pheophytin *CO 2 CH 3 – carbomethoxy

-CO 2 CH 3 * at C10, +H at C10

2. Pheophytinization – removal of Mg ++ and its replacement with 2H + -Mg ++ ,

pyropheophytin

  • 7. Formation of pyropheophorbide

pheophorbide

-CO 2 CH 3 * at C10, +H at C10

2. Pheophytinization – removal of Mg ++ and its replacement with 2H + -Mg ++ ,

pyropheophorbide

SUMMARY OF CHEMICAL CHANGES

-Mg ++ , +2H + chlorophyll pheophytin (bright green) (olive brown)
-Mg ++ , +2H +
chlorophyll
pheophytin
(bright green)
(olive brown)
2. Pheophytinization – removal of Mg ++ and its replacement with 2H + -Mg ++ ,

pheophorbide

(olive brown)

2. Pheophytinization – removal of Mg ++ and its replacement with 2H + -Mg ++ ,

-phytol

-phytol -Mg ++ , +2H + chlorophyllide (bright green)
-phytol
-Mg ++ , +2H +
chlorophyllide
(bright green)

-CO 2 CH 3 * at C10, +H at C10

2. Pheophytinization – removal of Mg ++ and its replacement with 2H + -Mg ++ ,

pyropheophytin

CO 2 CH 3 * at C10, +H at C10

pyropheophorbide

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FEB 02 CHLOROPHYLLASE – an esterase; cleavage of phytol from chlorophyll and pheophytin (derivative of chlorophyll)

  • - specific to porphyrins with carbomethoxy groups at C10 and H at C7 and C10

  • - active in solution with watein nr, alcohol, acetone

  • - optimum temperature, 60-82.2C, losses activity at 100C

Temperature in raw fresh vegetable? 28-34C Favorable for chlorophyllase activity? No

In fresh leaves, chlorophyllide is formed only when chlorophyllase is heat activated during post harvest e.g. during blanching

EFFECTS OF FOOD HANDLING, PROCESSING & STORAGE ON CHLOROPHYLL

  • - degradation, rate of extent differ depending on the type/degree of treatment to which the food item was subjected (recall the alterations/changes* in chlorophyll *induced

FACTORS WHICH CAN LEAD TO CHLOROPHYLLL DEGRADATION

  • 1. Heat treatment

  • 2. Exposure to light

  • 3. Enzyme activity

  • 4. Irradiation (before storage)

  • 5. Fermentation

  • 6. Added ingredients

1. HEAT TREATMENT: Blanching, thermal processing

  • - consider stability of a and b (more stable) of chlorophyllase (opt. at 60-82.2C) Blanching: recall temperature and methods Milder compared to thermal processing Temperature: between 60-82.2C will enhance chlorophyllase activity : 100C (short time) loss enzyme activity but reactivated during storage Effect of blanching: green color enhanced or retained Why? Formation of chlorophyllide Prolonged storage: degradation of either frozen products or dried products Higher temperature, longer time – more degradation Kinetic sequence:

Chlorophyll

green

pheophytin olive-brown
pheophytin
olive-brown

Why is chlorophyll b more stable than b?

FEB 02 CHLOROPHYLLASE – an esterase; cleavage of phytol from chlorophyll and pheophytin (derivative of chlorophyll)

pyropheophytin

Stability of chlorophyll b is attributed to the e- with drawing effect of C3 formyl group

(recall structure)

  • - increase in +charge on the 4 pyrrole Ns “reduces the equilibrium constants for the formation of the reaction intermediate” Factors which influence the effect of heating :

1.

pH

  • 2. chloride salt of Na, Mg and Ca

  • 3. detergents – cationic and anionic

pH of the tissue pH 9.0 – very stable during heating pH 7.0 – unstable during heating

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Q.1. What is the pH of green leafy vegetables, green beans and other green vegetables?

  • A. Most leafy vegetables have pH >3.0, hence may remain stable during cooking/processing

Q.2. Why is it that many green leafy vegetables loss their bright green color on cooking, particularly

with longer cooking period?

  • A. There are factors that influence effect of heating which include pH and cooking. Cooking

(long exposure to heat)/processing destroys structure of cells, cell walls, membranes, etc., which can result to: 1) release of acids (inherent) and; 2) formation/production of acid.

Example: spinach, peas, green beans – observed to release/produce 10 acids. Acids implicated:

  • 1. Acetic

  • 2. Pyrrolidone carboxylic acid (major cause)

  • 3. Oxalic acid

  • 4. Malic acid

  • 5. Citric acid

  • 6. Succinic acid

  • 7. Fatty acids from fat hydrolysis

  • 8. Other acids

Chloride salts of Na, Mg and Ca

  • - observed to have reduced chlorophyll degradation.

How?

  • - It is because of electrostatic shielding effects of salts where cations were proposed to neutralize

the negative (-) surface charge of fatty acids and proteins on the membrane of chloroplasts resulting to

the reduction attraction of H + to the membrane surface.

Na + or Ca 2+ or Mg 2+ fa - vs H + H + (from
Na + or Ca 2+ or Mg 2+
fa -
vs
H + H + (from acid)
proteins -
chloroplast

Detergents

  • - cationic detergents (+) repel H + at the membrane surface

  • - limit H + diffusion into the cell

2. EXPOSURE TO LIGHT

  • - cause photodegradation of chlorophyll* : loss of desirable green color *occurs:

1. during senescence

  • 2. during storage

  • 3. when extracted

  • 4. when there is cell damage, e.g. during processing

Occurrence in healthy plant cells? Same degree of degradation?

  • - Minimal or may not occur in healthy plant cells

Healthy plant cells – chlorophyll is protected from destruction by light due to the presence of surrounding carotenoids and other lipids

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SUGGESTED MECHANISM OF PHOTODEGRADATION

tetrapyrrole

SUGGESTED MECHANISM OF PHOTODEGRADATION tetrapyrrole *opening of methine bridges opens* fragments lower mol. wt. compounds oxidized

*opening of methine bridges

opens* fragments
opens* fragments

lower mol. wt. compounds

SUGGESTED MECHANISM OF PHOTODEGRADATION tetrapyrrole *opening of methine bridges opens* fragments lower mol. wt. compounds oxidized

oxidized linear tetrapyrrole

SUGGESTED MECHANISM OF PHOTODEGRADATION tetrapyrrole *opening of methine bridges opens* fragments lower mol. wt. compounds oxidized
SUGGESTED MECHANISM OF PHOTODEGRADATION tetrapyrrole *opening of methine bridges opens* fragments lower mol. wt. compounds oxidized

Lower molecular weight compounds

  • 1. glycerol – major breakdown product

  • 2. lactic acid

  • 3. citric acid

  • 4. succinic acid

  • 5. malonic acid

  • 6. alanine

  • 7. singlet oxygen

  • 8. hydroxyl radicals

when formed, loss of color, i.e. pigments

can react with other tetrapyrrole chlorophyll or other porphyrin to form peroxides and more free radicals -> total loss of color

Singlet oxygen and hydroxyl radicals can react with other tetrapyrrole chlorophyll or other porphyrin to form peroxides and more free radicals -> total loss of color

Ex. chlorophyll + singlet O 2 peroxides green

SUGGESTED MECHANISM OF PHOTODEGRADATION tetrapyrrole *opening of methine bridges opens* fragments lower mol. wt. compounds oxidized

peroxides + free radicals loss of color

Similar reactions with other porphyrins and hydroxyl radicals

Prevention of the undesirable effect of light on processed foods - proper packaging (clear packaging material vs opaque/dark-colored packaging

  • 3. ENZYME ACTIVITY (BESIDES CHLOROPHYLLASE)

Note: Some enzymes may not directly degrade (act on) chlorophyll but products of reactions that catalyze have been observed to degrade chlorophyll.

Example:

Lipoxygenase – degrade fats/lipids and form free radicals which in turn degrade chlorophyll

lipids

chlorophyll + free radicals

(green)

  • 4. IRRADIATION: GAMMA-IRRADIATION

Fruits/vegetables before storage

  • 5. FERMENTATION

chlorophyll

lipoxygenase

free radicals

  • degradation products (undesirable color formed (brown)

irradiation

pheophytin

Decrease in pH due to acid formed aside from the inherent acids present

fermentation chlorophyll pheophytin, chlorophyllide, normal pH pheophorbide, acidic conditions, lower than normal pH
fermentation
chlorophyll
pheophytin, chlorophyllide,
normal pH
pheophorbide, acidic conditions, lower than normal pH

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FEB 06

6. ADDED INGREDIENTS: ACID, LIME

Acid – pH, reduction - pheophytinization

Q. Why? H + ions from added acid can replace Mg ++ in the porphyrin ring (Note: 2H + /Mg ++

Lime – increase in pH; maintain green color - extreme alkalinity -> degrade chlorophyll, loss of color

Restoration of chlorophyll/green color Can the structure of chlorophyll be fixed or restored? Is it possible to restore the green color in leaves? How?

Metallo Complex Formation in pheophytin and pheophorbide (not in intact chlorophyll) Recall: Mg ++ is readily replaced by H + under acidic conditions

Questions:

  • 1. Can we remove H + and add back Mg ++ ?

  • 2. Can another metal ion replace H + ?

Recall the electrochemical series Metal ions: Cu ++ , Zn ++ Reactions: pheophyrin + Cu ++

FEB 06 6. ADDED INGREDIENTS: ACID, LIME Acid – pH, reduction - pheophytinization Q. Why? H

Cu-pheophyrin* + 2H +

green

favored at low pH *stable at low pH than at alkaline conditions

pheophyrin + Zn ++

Metallo Complex Formation

- pheorphide can undergo similar reaction

  • Zn-pheophyrin* + 2H + green; stable at pH 2.0

Comparison of reactivity of pheophytin and pheophorbide in metallo complex formation:

  • - pheophytin is less reactive than pheophorbide. Why? Size? Structure?

  • - compare structure (pheophytin vs pheophorbide)

  • - the phytol tail in pheophytin causes steric hindrance making it less active than pheophorbide

Recall structure of chlorophyll a and b Comparison of reactivity of chlorophyll a (corresponding derivative) and chlorophyll b (corresponding derivative) in metallo complex formation:

  • - chlorophyll a forms metallic complex with either metal ion faster than chlorophyll b

  • - the formyl (OCH 3 ) group in chlorophyll b has an e- with drawing effect which makes the pyrrole atom becomes more +, therefore less reactive with the metal

PRESERVATION/RESTORATION OF THE GREEN PIGMENT/ COLOR

Can a food technologist prevent chlorophyll degradation? Knowledge about structure and properties of chlorophyll*

DEGRADATION

  • - mechanisms/reactions of degradation

  • - causes, factors which influence (flavor) degradation

*Food technologists/food chemists in controlling/preventing undesirable changes in the pigment

METHODS IN PRESERVATION/RESTORATION OF THE PRESERVATION (recommended)

  • 1. USE OF ALKALINE SALTS (ACID NEUTRALIZATION)

    • - neutralize the medium,e.g. raise pH to 7.0

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-

alkaline salts:

  • a. Ca + NaH 2 PO 4

  • b. MgCO 3 in combination with Na 3 PO 4

 
  • c. Na 2 CO 3

  • d. Ca(OH) 2 + Mg(OH) 2

  • e. Ethyl cellulose + 5% Mg(OH) 2 as interior coating of cans

 

-

a and b are not highly recommended due to softening of tissue and alkaline flavor

-

d [Ca(OH) 2 + Mg(OH) 2 ]

  • è known as “Blair Process”, originally used to preserve texture and crispness of some vegetables

  • è not very successfully commercially for the retention of the desirable green color due loss of color in

less than 2-month storage. Why? Salts were not effective in neutralizing acid in the inner tissues of vegetables at longer period.

-

e.

ethyl cellulose + 5% Mg(OH) 2 as interior coating of cans

 
 

Mg(OH) 2

slow leaching
slow leaching

medium, ~pH 5.0

-

not very successful commercially (partial). Why? 1. increase in pH results to amide hydrolysis

 

glutamine or aspargines -> products with NH 3 -like odor

  • 2. fatty acids* present are oxidized to products associated with rancidity *fatty acids may be formed during heating e.g. blanching at higher pH fatty acids -> peroxides, hydroperoxides, other products

  • 3. struvite* formation - occurs at higher pH, e.g. observed in peas at pH 8.0 *glass-like crystals consisting of Mg and NH 4 phosphate complex (from protein and amino acid)

  • 2. HTST (HIGH TEMPERATURE SHORT TIME) PROCESSING

    • - products with better retention of vitamins, flavor and color

    • - color retention limited to 2 months only, beyond which loss of color was observed

    • - heating particularly within the optimum temperature for chlorophyllase activity favors retention of the green color

      • 3. CONVERSION OF CHLOROPHYLL TO CHLOROPHYLLIDE Recall: chlorophyll

Chlorophyllase

opt. temp 60-82.2C
opt. temp 60-82.2C

chlorophyllide (more stable than chlorophyll)

Blanching – inactivates enzymes if high temperature is used (one of the purposes of blanching) Optimum temperature for chlorophyllase activity

Compare blanching HTST vs LTST

Observations: blanching using LTST

  • 1. 71C <-> 20 min -> better color retention in spinach

  • 2. 65C <-> 45 min -> better color retention in spinach

  • 3. 54C <-> 45 min -> activation of chlorophyllase

Disadvantage: color retention is not sufficient for commercialization

  • 4. APPLICATION OF METALLO COMPLEX Recall: Effect of Cu and Zn ions or chlorophyll derivatives, pheophytin and pheophorbide Use of Copper Ions

    • - allowed in European countries under EEC (European Econom ic Community) to a certain types

of food product; canned foods, candies, soups and dairy are not allowed in US

10

Commercial forms of copper metallo complexes Copper chlorophyll = copper + pheophytin Copper chlorophyllin = copper + pheophorbide

FAO of UN certified the use of copper metallic complexes as safe in foods provided no more than 200 ppm (mg/kg) of free ionizable copper present

“Veri-green” – refers to blanching vegetables in water with Zn or Cu salts to raise tissue concentration of metal to 100-200 ppm

Commercially acceptable vegetables are green Ex. canned green peas, pigments are: Zn pheophytin and Zn pyropheophytin

Direct adding of Zn/Cu ions in canning brine had no significant effect on chlorophyll

  • - best process to retain green color in vegetables

  • - blanch in the presence of Zn ions

  • - increase membrane permeability by heating tissues to 60C before blanching

  • - provide the right pH, i.e., favors metallo complexes use anions to alter surface change of issue

  • - recall effect of surface active detergents

Regreening of Thermal Processed Vegetables

  • - appearance of bright-green areas in the product due to the presence of Cu

process solution

or

Zn

ions in

FEB 09

FLAVONOIDS

  • - subgroup under phenolic compounds

  • - widely distributed in plants

  • - associated with the yellow color in some plants; also responsible for the whiteness in some plants; also impart the following colors: blue, purple, magenta, red, orange, due to anthocyanins

  • - carbon skeleton: basic C 6 C 3 C 6 structure

  • - color depends on the substituents on rings A & B

Basic structure: phenolic compound (s) 3 benzene rings, with OH

Typical Flavonoid Structure

The Structure of a Flavonoid:

Phenolic compounds composed of three benzene rings with hydroxyl (OH) groups Without sugar, molecule called an aglycone

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1.

Remove the OH at (1): flavone

  • 2. Replace OH at (1) with Zn ring: isoflavone

  • 3. Replace the O at (2) with a H: anthocyanin

  • 4. Replace the OH at (3) with glucose; Remove OH at (4) ; Remove OH at (1): glucoside (glycoside if exact sugar is not specified)

ANTHOCYANIN

  • - most prevalent flavonoid

  • - Greek words: anthos – flower; kyanos – blue

  • - Impart a wide range of colors in plants: blue, purp[le, magenta, red, orange

Most notable investigators (Nobel prizes in Chemistry) in part of work in plant pigments

  • 1. Robert Robinson (1886-1975)

  • 2. Richard Willstatter (1872- 1942)

There are 250 anthocyanins in plants, content vary from about 20mg/100g to 600mg/100g fresh weight

Q. Which is responsible for the color in fruits, flowers, leaves (or any plant part)?

A. Distinctive color in the plant part: often due to a combination or a system of pigments* *usually 4-6 pigments, one may predominate Examples:

  • 1. Simple System blackberries – contain primarily cyanidin-3-glucoside

  • 2. Complex System blueberries – contain 15 pigments

Base structure: flavylium cation, a 2-phenylbenzopyrylium of flavylium salt

ANTHOCYANINS IN PLANTS

R 1 , R 3 – H, OH or OCH 3 R 2 – OH R 4 – glycosyl R 5 – H or glycosyl

  • - exist as glycosides of polyhdroxy and/or polymethoxy derivatives of the flavylium salts

  • - differ in the R groups: *no. of hydroxyl or methoxy groups *types, no. and site of attachment of sugars to the molecule *types and no. of aliphatic or aromatic acids attached to the sugars (some contain organic acids & metals Al, Fe, Mg)

2 MAIN COMPONENTS OF ANTHOCYANIN (exist as glycosides in plants)

  • 1. Anthocyanidin (aglycone, aglucone [Whittaker])

  • 2. Sugar(s)

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HYDROLYSIS OF ANTHOCYANINS

SUGARS:

  • 1. glucose

  • 2. galactose

  • 3. arabinose

  • 4. xylose

  • 5. rhamnose

anthocyanin

hydrolysis

HYDROLYSIS OF ANTHOCYANINS SUGARS : 1. glucose 2. galactose 3. arabinose 4. xylose 5. rhamnose anthocyanin

anthocyanidin + sugar

*1-4 most common; *1 or 2 or 3 sugars may be present, homogeneous or heterogeneous

CLASSES OF ANTHOCYANINS BASED ON THE SUGAR PRESENT:

  • 1. monoside

  • 2. bioside

  • 3. trioside

SUGARS MAY BE ACYLATED BY EITHER ALIPHATIC OR AROMATIC ACIDS:

caffeic

malonic

p*-coumaric *para

malic

sinapic

succinic

p-hydroxybenzoic ferulic

acetic

COLOR OF ANTHOCYANINS AND ANTHOCYANIDINS

  • - results from excitation of the molecules by visible light

  • - ease of excitation depends on the electron mobility in the structure

STRUCTURE OF ANTHOCYANINS AND ANTHOCYANIDINS

  • - with several double bonds which are readily (easily) excited, presence of which is important for color

  • - hence, the type of anthocyanidin influence color or deepness of color

SIX COMMONLY OCCURRING ANTHOCYANIDINS IN FOODS

[may vary in hue (color) and intensity (deepness)]

Pelargonidin R 1 , R 3 – H R 2 – OH

Cyanidin R 1 , R 2 – OH R 3 – H

Delphinidin R 1 , R 2 , R 3 - OH

Peonidin R 1 – OCH 3 R 2 – OH

Petunidin R 1 – OCH 3 R 2 , R 1 – OH

Malvidin R 1 , R 2 – OCH 3 R 2 – OH

R 3

H

Anthocyanidin Top to bottom: increasing redness; increasing no. of OCH 3 (methoxy group) *see column 2 & 3 Left to right: increasing blueness; increasing no of OH (hydroxyl group) *see row 1

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FEB 13 Explain observation: Bathochromic changes “Deepening of hue is due to the bathochromic change (i.e., longer wavelength)” – light absorption band in the visible spectrum shifts from violet through red to blue

Q. What brought about this change?

  • A. The auxochrome groups* - groups without chromophoric properties (colorless) but cause deepening of

the hue when attached to a molecule, i.e., anhocyanidin

*e - donating; *has bathochromic effect

AUXOCHROME

  • - (auxanein) “to increase” and (chroma) “colour”

  • - is a group of atoms attached to a chromophore which modifies the ability of that chromophore to absorb light

BATHOCHROMIC CHANGE *e- donating; *has bathochromic effect In anthocyanidins:

R groups: hydroxyl, OH and methoxy, OCH 3 Compare bathochromic effect of OH and OCH 3 OCH3 – has greater e - donating capacity than OH -> cause greater bathochromic effect HYPSOCHROMIC CHANGE – opposite change

PROPERTIES OF ANTHOCYANINS Solubility – water soluble Note: anthocyanidin – less water soluble than anthocyanins Chemical properties – readily undergo changes with pH changes

  • - relatively unstable, degraded during extraction, processing, storage

STABILITY OF ANTHOCYANINS

Recall: structure and substituents (subgroups) on the aglycone (anthocyanidin)

INFLUENCE OF SUBSTITUENTS ON STABILITY :

  • 1. OH – increase hydroxylation, decrease stability

  • 2. OCH 3 – methylation, increase stability

  • 3. Sugars – consider no.4 type of sugar >number of sugars – increase glycosylation, increase stability monoglucoside vs diglucoside

Monoglucoside vs Diglucoside Examples:

Anthocyanin, pH 3.5, 50C

Half-life, hr

Cyanidin-3-rutinoside

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Cyanidin 3-2 glucosylrutinoside

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TYPE OF SUGAR Anthocyanin containing galactose shown to be more stable than anthocyanin containing arabinose in stored cranberry (Starr and Francis, 1968) Note: Substituents (1-3 [OH, OCH 3 , sugar]) do not react but influence marked effect on stability.

INFLUENCE OF THE TYPE OF ANTHOCYANIDIN/AGLYCONES

1. pelargonidin 2. cyaniding 3. delphinidin 4. petunidin (-OCH 3 ) 5. malvidin (2 OCH 3
1.
pelargonidin
2.
cyaniding
3.
delphinidin
4.
petunidin (-OCH 3 )
5.
malvidin (2 OCH 3 )

1-3 anthocyanins are less stable

4-5 anthocyanins are more stable; increase in stability is due to the

blocking of the reactive hydroxyl groups

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FACTORS WHICH AFFECT STABILITY OF ANTHOCYANINS

  • 1. MAJOR FACTORS

    • A. pH Four structural forms

      • 1. blue quinoidal base (A)

      • 2. red flavylium cation (AH+)

      • 3. carbinol pseudobase (B)

      • 4. colorless chalcone (C

A

FACTORS WHICH AFFECT STABILITY OF ANTHOCYANINS 1. MAJOR FACTORS A. pH Four structural forms 1. blue

AH +

FACTORS WHICH AFFECT STABILITY OF ANTHOCYANINS 1. MAJOR FACTORS A. pH Four structural forms 1. blue

B

one form may predominate over the others depending

on the pH C
on the pH
C

(Assumption: equilibrium exists)

low pH

pH 4-6

One may predominate over the others, depending on the pH Example:

Malvidin-3-glucoside, pH 0.6 pH 4-6: carbinol pseudobase dominates; colorless pH <4: plavylium cation dominates; red

  • B. TEMPERATURE

    • - rate of degradation is influenced by pH, O 2 and structural conformation

    • - structural features which increase pH stability, also increase thermal stability

highly hydroxylated anthocyanidins

vs

methylated, glycosylated or acylated anthocyanidins

Example:

more stable

Half-life of the following at pH 2.8

 

Anthocyanins

Half-life (days)

3,4,5,5’,7-pentahydroxyflavylium

0.5

3,4’,5,5’,7-pentamethoxyflavylium

6.0

cyanidin-3-rutinoside

6.5

cyanidin

0.5 (12hr)

  • C. OXYGEN

    • - anthocyanins are unsaturated, readily undergo oxidation

    • - manifestations: browning of some fruits or its products

*Influence of Aw on stability against oxidation Aw = 0.63 – 0.79 -> anthocyanins are more stable

*Aw of dried fruits/vegetables, if there is minimal degradation of anthocyanins during drying, such will be preserved, hence color will be maintained

Prevention of Oxidation:

  • - exclusion or removal of oxygen during drying: vacuum drying, freeze drying

*Processing:

1. proper exhausting before sealing of canned/bottled products

  • 2. processing under nitrogen atmosphere or under vacuum

  • 3. packaging in nitrogen atmosphere (e.g. powdered grape juice)

  • 4. vacuum packaging

  • D. LIGHT

    • - accelerates anthocyanin degradation

Photodegradation – adverse effect observed in fruit juices and wines

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Comparison of anthocyanin stability in the presence of light

acylated,methylated non-acylated

diglucosides

digluclosides

monoglucosides

------------------------------decreasing stability towards photodegradation--------------------------------->

  • E. CONCENTRATION

FEB 27

  • 2. MINOR FACTORS

    • A. DEGRADATIVE ENZYMES Two groups implicated

      • 1. Glycosidases

      • 2. Polyphenol oxidases

Comparison of anthocyanin stability in the presence of light acylated,methylated non-acylated diglucosides digluclosides monoglucosides ------------------------------decreasing stability

generally referred to as anthocyanases however substrates acted are not

limited anthocyanins hence use of term is not encouraged

Glycosidases – hydrolyze glycosidic linkage

anthocyanins

*results from:

glycosidase

Comparison of anthocyanin stability in the presence of light acylated,methylated non-acylated diglucosides digluclosides monoglucosides ------------------------------decreasing stability
  • 1. Formation of colorless products

sugar + anthocyaninidin

Comparison of anthocyanin stability in the presence of light acylated,methylated non-acylated diglucosides digluclosides monoglucosides ------------------------------decreasing stability

loss of color intensity*

  • 2. Loss of solubility of anthocyanidin -> polyphenol oxidase (PPO) – oxidize/degrades anthocyanins in the presence of o-diphenol and oxygen -> not directly act/oxidize anthocyanins; oxidize o-diphenol and product formed reacts with anthocyanins

Steps:

1. o-diphenol + O 2

PPO
PPO
  • 2. o-benzoquinone + anthocyanin

o-benzoquinone

Comparison of anthocyanin stability in the presence of light acylated,methylated non-acylated diglucosides digluclosides monoglucosides ------------------------------decreasing stability

oxidized anthocyanin + degradation products brown products

1 – enzymatic

2 – chemical

Catechol oxidase – copper oxidase, catalyzes oxidation of ortho-diphenol to ortho-quinones

  • B. ASCORBIC ACID - degradative motion on anthocyanins is indirect; it undergoes oxidation and products degrade anthocyanins Cu ++ ascorbic acid ------- H 2 O 2 cleaves pyryllium ring by a nucleophilic attack at C 2 anthocyanin + H 2 O 2 ---------> colorless esters + coumarin derivatives ---> brown precipitate* *occurs in some juices

  • C. SULFUR DIOXIDE - used in food processing* *as preservative: 500 ppm to 2000 ppm *as bleaching agent: higher concentration; 0.8-15% Ex. production of maraschino or candied & glace cherries

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Effect of SO 2 – loss of color

Reversible or irreversible? Reversible: color can be restored through desulfuring , i.e., wash off SO 2 before further processing

Reaction:

anthocyanin + SO 2 ------> complex formation (colorless) Suggested reaction: SO 2 attacks C 4

Note:

  • 1. Some anthocyanins are resistant to bleaching; C 4 is blocked or exists as dimmers linked through C 4

  • 2. Irreversible in maraschino or candied cherries

  • D. METAL IONS Anthocyanin + metal

Effect of SO – loss of color Reversible or irreversible? Reversible: color can be restored through

complex

Nature: metal complexes of anthocyanins give characteristics colors to flowers

In food processing, e.g., canning, metal – anthocyanin complex formation may occur* *desirable or undesirable

Observations: effect of complexation

  • 1. Complexation stabilizes (metal protects) some anthocyanins – beneficial Ca, Fe, Al, Sn, in cranberry juice

  • 2. Complexation results to discoloration

  • 3. “Pinking” occurs in pears, peaches & lychees; undesirable

Mechanism (in complexation):

Vicinal phenolic hydroxyl groups (in some anthocyanins) sequester multivalent metal* * Al 3+ , Ca 2+ , Fe 2+ , Sn 2+

Note:

Not all anthocyanins have vicinal phenolic hydroxyl groups, hence metal complexation can be used to differentiate groups of anthocyanins

Mechanism (in complexation):

Complexation, an analytical tool to differentiate groups of anthocyanins Reagent: aluminum chloride

Anthocyanins which can complex with metals** Cyanidin

Pelargonidin

Petunidin

Peonidin

Delphinidin

Malvidin

**with vicinal phenolic OH, recall R 1 , R 2 , and R 3 groups

Mechanism in pinking:

Proanthocyanidin

(colorless)

heat; acidic conditions

Effect of SO – loss of color Reversible or irreversible? Reversible: color can be restored through

anthocyanins

metal

Effect of SO – loss of color Reversible or irreversible? Reversible: color can be restored through

metal-anthocyanin complex (pink)

  • E. SUGARS Effect on anthocyanins depends on sugar concentration; beneficial or not High sugar concentration, i.e., fruit preserves, stabilize anthocyanins Stabilizing effect is due to the lowering of Aw Low sugar concentrations, i.e., at concentration with little or negligible effect on Aw -> accelerate anthocyanin degradation

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Types of sugar -> varying effect

Fructose

Arabinose

Lactose

Sorbose

Glucose

Sucrose

Maltose

  • FALSor – greater degradation

  • MGSuc (or MSG) – less degradation

Mechanism: (not direct interaction between sugar and anthocyanin) degraded

sugar furfural O 2 anthocyanins + furfural condensation
sugar
furfural
O 2
anthocyanins + furfural
condensation

brown compounds (discoloration)

EFFECT OF COPIGMENTATION

  • - either accelerate or retard depending on the situation Protective effect of some flavonoids:

    • 1. polyhydroxylated flavone sulfonate*

    • 2. polyhydroxylated isoflavone sulfonate*

    • 3. polyhydroxylated aurone sulfonates*

*protective effect against photodegradation

  • - the negatively charged sulfonates and the positively charged flavylium cations form intermolecular ring interactions

EFFECT OF SUBSTITUTIONS OF OH GROUP

o-diphenol more susceptible to photodegradation

  • - unsubstituted or monosubstituted anthocyanins -> prone to nucleophilic attack at the C 2 or C 4 positions

PREVENTION OF PHOTODEGRADATION:

*Proper packaging, e.g. dark colored bottles or plastic films Ionizing radiation – degrades anthocyanins

OTHER FLAVONOIDS

  • - non-anthocyanin type flavonoids

  • - impart a range of color to plants and food yellow: yellow flavonoids xanthos – yellow

  • - whiteness of some plant materials

  • - brown or black due to the oxidation products of phenolic groups (occur in nature) >>classes of non-anthocyanin type flavonoids (differences is based on the state of oxidation of the 3 - carbon link)

PROPERTIES

  • - with light absorption characteristics

  • - differ due to differences in degree of unsaturation

  • - also influenced by the acylation and/or glycosylation

  • - involved in copigmentation

  • - metal chelators chelation or reaction with metal; influence color formation Ex. Luteolin + Al -------> yellow complex

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IMPORTANCE IN FOODS:

  • 1. Color, particularly in copigmentation Ex. >whiteness: in onions, cauliflower, potato >greenish: black discoloration in canned asparagus Rutin** + Fe +++ **Rutinoside quercetin

IMPORTANCE IN FOODS: 1. Color, particularly in copigmentation Ex. >whiteness: in onions, cauliflower, potato >greenish: black

Fe – rutin complex Control: add EDTA, metal chelator

>yellow color in wax bean Rutin + Sn ++

IMPORTANCE IN FOODS: 1. Color, particularly in copigmentation Ex. >whiteness: in onions, cauliflower, potato >greenish: black

Sn – rutin complex (more stable)

>color of black olives – due to the oxidation of flavonoids Reaction occurs during fermentation and storage Ex. luteolin 7-glucoside -------> oxidation products

  • 2. Antioxidant properties - ability to sequester metal

  • 3. Contribute to flavor, e.g., bitterness , astringency Ex. tea – astringent >contains quercetin kaempherol myricetin glycosides

IMPORTANCE IN FOODS: 1. Color, particularly in copigmentation Ex. >whiteness: in onions, cauliflower, potato >greenish: black

MAR 02

  • 4. Potential sweetener >citrus, has naringen* bitter *a flavanone – potential sweetener

  • 5. Deodorizer

contribute to astringency

>tannins and flavonoids from chestnut inner shell, shown to have antioxidant and deodorizing activities

  • 6. Health factor – citrus flavonoids; with bioflavonoid (Vit P) synergistic activity - citrus flavonoids with bioflavonoid (Vit P) synergistic activity

COMPOUNDS WITH COMPLEX SUBSTITUENTS WITH DEEPER COLORS

>Napthoquinone and anthraquinone – deep purple to black

>Xanthone – yellow

Ex. Xanthone mangiferine – glucoside in mango

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FIRST LONG EXAM

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