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Most common sources of sampling errors @ Uneven distribution of measured constituents in foods — contaminants, fat in meat, proteins in sweetpotato @ Lack of randomness - where and wh

Enzymic analysis

AusAID CARD Project 008/07VIE

Workshop 4, FCRI February 2009

Les Copeland University of Sydney

Outline

Principles of analysis

@ Enzymology

@ Enzymes in analysis

Quality

@ Suitability of a product for a particular purpose

Properties that have usefulness

— processing, nutrition, consumption

@ Influenced by

— genetics and environment (G x E)

— processing and testing technology

@ Quality is different from safety

— freedom from contaminants

@ Quality is assessed by comparing results of chemical

and physical analyses with functional tests relevant to the specific end use

Analytical methods

Chemical analyses

Specific constituents measured

@ Usually well defined and accurate

@ Difficult to relate to chemical properties to

functionality

Functional tests

@ Practical and relevant to applications

@ Measure collective properties of complex materials

@ Difficult to relate cause of properties and effect

Types of samples analyzed

@ Raw materials

— to determine that a delivery conforms to

standards (composition, contaminants)

— to assure consistency of supply

— to evaluate new suppliers

@ Process control samples to monitor operations

@ Finished materials to determine that product

meets standards and claims

@ Complaint samples

Competitors’ samples

Stages of a typical analytical procedure

Verifying the procedure

Appropriateness and cost of methods

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Most common sources of sampling errors

@ Uneven distribution of measured

constituents in foods

— contaminants, fat in meat,

proteins in sweetpotato

@ Lack of randomness - where and

when sample is taken

@ Many sampling protocols are used

- randon, stratified, systematic,

judgment

@ An analytical result can never be

better than the sample

Sample preparation

Sample is ground, milled or blended

— need to avoid of heat generation (loss of moisture and volatiles, chemical reactions)

@ Enzymes are inactivated to prevent loss of labile

compounds

— heating, drying, 80% ethanol, protein denaturants,

— amylases for starch determination

@ Sample is prepared in a way that does not cause loss of the component being analyzed

— addition of reducing agent (eg, ascorbic acid) to

prevent oxidations (eg, unsaturated fatty acids)

@ Chemical knowledge of the analyte is essential

Choice of many methods for measuring stage

Gravimetric (by weight)

Definitions

@ Limit of detection

— the minimum amount that can be detected

— related to the value of the baseline and the

standard deviation of the analytical method

— signal:noise ratio should be > 2:1

@ Sensitivity

— the smallest difference that can be detected

between two measurements

- related to how easy it is to detect a change ina

property

@ Selectivity and specificity

— how well a specifically a component can be

measured in the presence of other components (35

of the sample

Accuracy and Precision

Precision —

¢ reproducibility of measurements within a number of replicate measurements

¢ closeness of agreement between successive analyses

¢ determined by the dispersion of a set of data about the central value (SD)

Accuracy —

¢ closeness of the mean of a set of values to the “true” value

¢ requires comparison of data with standards or reference materials

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Accurate and imprecise

Accurate and precise (random error)

Inaccurate and precise Inaccurate and imprecise

(systematic error)

Accredited methods

@ Recognized by professional societies as giving reliable and

accurate results across a wide range of sample types

— Association of Official Analytical Chemists (AOAC)

— International Organization for Standardization (ISO)

- 157 member countries; 16,500 standards and

procedures

- www.iso.org

— ISO 9001:2000 (ISO 9000) is used widely in the food industry

- set of standards for an organization's quality

management systems

- ISO 9000 compliance gives assurance about products, processes, 4041 assurance, employee competence, resources,

@ Traceability — driven by regulation, QA, certification, marketing,

Analytical results need to be interpreted correctly

@ Concentration that is measured Is not always the

biologically relevant (bioavailable) concentration

the nature and properties of the matrix

pH and redox conditions

adsorption, complexing and precipitation

reactions

Bioavailability - example 1

Turkeys were fed a carefully formulated,

nutritionally balanced diet based on

soybean meal; analysis showed the diet to

contain more than sufficient amounts of all

essential trace metals However, the turkeys

grew poorly Why?

Zn was bound to proteins in the soy meal

and was not available to the animal

Addition of a compound that released Zn

into a bioavailable form improved nutrition

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Bioavailability - example 2

Crab meat was found to contain 1.4 - 13 ppm

of Cd, which is above limits of an average

acceptable daily intake of Cd of 20 - 60 ppb

Should crab meat be removed from sale?

Analysis of the bioavailability of Cd in crab

meat showed that only 2-3% of the metal was

available from the gastrointestinal tract, which

gives the food an acceptable margin of safety

(assuming the consumer's diet is not only crab

meat)

Enzymes are important in food analyses

@ Many enzymes have practical applications

— food and industrial processing

— analytical and diagnostic reagents

@ Knowledge of enzymes comes from

understanding how living cells function

Examples of enzymes used in manufacture of foods

Amylases Starch breakdown in brewing, baking, glucose

syrups, fruit processing

Cellulases, Polysaccharide hydrolysis in cereal and fruit

xylanases, processing, baking, brewing, improve digestibility of

pectinases, feeds

glucanases,

Dextranases Production of glucose syrups

Glucose Glucose to fructose conversion for high fructose

isomerase syrups

Lipases Hydrolysis of fatty acid esters for fat modification,

flavour production, cheese production, emulsifier synthesis

Proteases Protein breakdown in baking, brewing, flavour

enhancement, gluten modification,

B-Galactosidase Lactose breakdown in milk processing

sources of industrial enzymes

@ Production of most commercial enzymes is by microbial

fermentation (bacteria, fungi, molds, yeasts)

— use of GMOs is common

@ Enzymes used in processing are not usually pure

— concentrate of a culture supernatant

— typically contain other enzymes (side activities)

@ High purity usually required for enzymes used in analysis

@ Comparing enzyme preparations from different suppliers is a

common problem in industrial enzymology

— no internationally agreed and industrially relevant units

— manufacturers tend to define their own activity units

— enzyme preparations from different sources may give

different results

4 Lyases Cleavage of bonds, not by hydrolysis,

cleaving double bonds or rings; adding

groups to double bonds

5 lsomerases Geometric or structural changes within one

molecule

6 Ligases doining of two molecules, coupled with the

hydrolysis of the pyrophosphate bond ofa} nucleoside triphosphate |

Hexokinase

Enzyme Commission

Transferase

Transferring a phosphorus-containing group

Alcohol group as acceptor

Enzyme number within sub-class

Exo- and endo- enzymes

@ Exoenzymes split terminal bonds in a polymer chain

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@ Endoenzymes split internal bonds in a polymer chain

have a much greater effect on viscosity than

exoenzymes

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e Endo- and exo- glycanases, proteases, nucleases =š

Enzymes are proteins

> Proteins are flexible molecules

> Proteins undergo conformational

changes with consequent change

in activity

- induced by interaction of

protein with other molecules or

surfaces

> Small changes in conformation

may be amplified into large

changes in activity of the protein

- enzyme activation or inhibition

Enzymes are catalysis

Increase the rate of a reaction without

altering its equilibrium position or the

change in free energy

@ Conformation of protein brings functional

groups in the active site into close spatial

proximity for substrate binding and

catalysis

¢ Active site creates a microenvironment

that favours catalysis of the reaction

— exclusion of water for hydrophobic

reactions

— exclusion of O, for reductions

Purification of enzymes

Obtain enzyme in soluble extract from cells/tissue

Precipitate enzyme to separate from low molecular

weight solutes and some unwanted proteins

— (NH,).S0O,, (polyethylene glycol, organic solvents, heat,

low pH)

@ Separation of proteins based on

— size (size exclusion chromatography)

— Charge (ion exchange chromatography)

— biospecific interactions (affinity chromatography,

hydrophobic interaction chromatography)

— electrophoretic mobility

Measuring the rate of enzyme reactions

@ Changes in absorbance or fluorescence

@ Monitoring formation of product by HPLC, GC

Preferable to measure formation of product than

disappearance of substrate

Continuous or stopped assay

Need to optimise assay conditions

Verify dependence of reaction rate on time and

enzyme concentration

@ Stability of enzyme and substrates under assay

conditions

Rates of enzyme reactions are influenced by

Effect of enzyme concentration

Activity

Enzyme concentration

Effect of temperature

Rate of a chemical reaction doubles for every

10°C increase in temperature

But, proteins denature at elevated temperature

@ Temperature optimum depends on enzyme

source

— (37°C for mammalian enzymes, 30°C for plant

enzymes, up to 90°C for enzymes from thermophilic bacteria)

Activity

Temperature

Effect of pH

@ Changing pH alters ionic charges on the enzyme and substrate

Activity

affects binding between the enzyme and substrate

there is a pH at which protein interaction of substrate at the active site is optimum for catalysis

activity declines above and below pH optimum

pH optimum varies depending on enzyme

Effect of substrate concentration

Hyperbolic relationship between initial reaction rate (v) and [S] |

v= Vmax.S/(Km + S)

K,, and V

max

K,, gives a measure of binding affinity

Vinax gives a measure of catalytic power

Enzymes are important analytical and diagnostic tools

+ Foods, feeds and beverages

+ Plants, animals, environmental

@ Clinical

@ Activity of an enzyme is measured as an indicator of

the quality or condition of a material

@ Enzyme is used to convert an analyte to a

measurable product (end-point analysis)

- selectivity and sensitivity of enzymes is used to

measure biological molecules

a-Amylase activity is measured to assess suitability of cereal grains for end use

@ Quality of wheat flour for baking

— adequacy of a-amylase for dough development

— pre-harvest sprouting damage of wheat grains due premature induction of a-

amylase while grain is still attached to the ear

@ Quality of malted barley for brewing

— malting is controlled germination to induce a-amylase and other degradative

enzymes that generate substrates for fermentation

Lipases and lipoxygenases cause off flavours in

dairy products and flour

End-point analysis - the analyte is made the limiting

part of the analysis

Example: determination of glucose (or O.) using

glucose oxidase/peroxidase (GOPOD)

— highly specific for glucose; can be performed in

presence of other sugars

Glucosidase activity is measured using a

NAD a nd NADP are cofactors for many enzymes

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End-point analysis may be linked to change in A340

due to reduction (or reduction) of NAD(P)

NAD(P)* NAD(P)H Change in A340 = [X]

Malo-lactic fermentations are monitored by determination

of L-malate with L-malate dehydrogenase

Oxaloacetate + L-Glutamate ———® |-Aspartate + 2-Oxoglutarate

tại

Glutamate-oxoglutarate aut

transaminase

Potential problems with enzyme analyses

Examples of enzyme based test kits available for analyzing biochemical compounds

ORGANIC ACIDS

Acetic acid

CARBOHYDRATES Ascorbicacdd ALCOHOLS

Sucrose/Fru/Glucose Gluconic acid D-Sorbitol/Xylitol Glucose/Sucrose L-Glutamic acid Glycerol

Lactose/Galactose Hydroxybutyrate

Lactose/Glucose Isocitric acid

Maltose/Glucose D-Lactic acid OTHERS |

Raffinose/Galactose D-Malic acid Ammonia

Cholesterol =;

Succinic acid