ENZYMES in Food Processing

LTWTHochschule Bremerhaven

SS 2011

What is an enzyme?What is an enzyme?A biological catalyst that

promotes and speeds up a chemical reaction without itself being altered in the process.

Lowers the activation energies of a substance

Energy Profile

reactants

products

H

EA

T.S.

catalyst

Enzymatic ReactionsEnzymatic ReactionsEnzyme combines with a specific

substrate to a form an enzyme-substrate complex in a lock and key concept before forming new products.

Enzyme action

enzyme

substrateproducts

Structure of an enzymeContains both a protein and a

nonprotein.Nonprotein is either a coenzyme

(usually a vitamin) or a cofactor (usually a mineral).

Factors influencing enzyme activity

Operate under optimum conditions of pH and temperature.

Easily inactivated (denatured) in presence of inhibitors.

Enzyme Nomenclature Names usually end in –ase. Usually named after substrates

they act upon e.g. urea --- urease lactose --- lactase or the resulting type of chemical

reaction e.g. hydrolysis --- hydrolases

oxidation --- oxidases This rule does not always apply.

E.g. ficin found in figs and papain in papayas.

Properties of enzymes Control ripening. Cause food spoilage (rotting). Responsible for changes in flavor, color,

texture and nutritional properties. Can be inactivated by heat to extend

storage stability of foods. Control oxidation and spoilage

(bioconservation) Increase nutritive values ( phytase ,

proteases etc.)

Properties Used for fermentation purposes in

foods. Can be immobilized to a surface of a

membrane or other inert object in contact with the food being processed.

Can be extracted and purified to a high degree.

Main Enzyme Classes____________________________________________________Enzyme class Catalyzed reaction____________________________________________________Oxidreductases Oxidation-reduction reaction

Transferases Transfer of functional group

Hydrolases Hydrolytic reactions

Lyases Group elimination (forming double bonds)

Isomerases Isomerizaion reaction

Ligases Bond formation coupled with a triphosphate cleavage____________________________________________________

Enzymes in Industry

Distribution of enzymes by substrate

Protein hydrolyzing 59%

Carbohydrate hydrolyzing 28%

Lipid hydrolyzing 3%

Speciality (analytical, pharma, research) 10%

US Market Bakery US$ 210m 5-7% pa Beverage US$ 130m, 4-6% pa Dairy US$ 195m, 3-5% pa Fats & Oils US$ 30m, 10-12% pa Culinary US$ 30m, 5-6% pa Meat and others incl. food protection US$ 30m, 10-12% pa

A few large competitors

• Novozymes (all applications) • DSM (most applications) • Chr. Hansen (dairy enzymes only) • AB Enzymes (some applications

Industrial Enzyme Market

Annual Sales: $ 1.6 billion

Food and starch processing: 45%Detergents: 34%Textiles: 11%Leather: 3%Pulp and paper: 1.2%

World Market for Some Products of Enzymatic Reactions

High fructose corn syrup: $ 1 billion Aspartame: $ 850 million Acrylamide: $ 300 million

Industrial Enzyme Classes Commodity enzymes

High volume (tonnes p.a) Low purity (but not necessarily so) Low cost (e.g. $5-40 per kg) Low profit margins

Speciality enzymes Low volume (g – kg) High purity High cost ($5 – 10,000 per g) High profit margins

Important Factors in Using Enzymes

• Reactions possible that are not possible using chemistry

• Specificity of reaction including substrate specificity, positional specificity, stereo-specificity

• Allows milder process conditions e.g. temperature, pH, sterility etc.

• Reduces number of process steps required

• Eliminates the need to use organic solvents in processing

• Immobilization of enzyme to allow its reuse or continuous use

• Use of enzymes in combination with other separate chemical steps

• Genetic engineering to improve enzymes

Industrial enzymes Food processing

Amylases in bread-making Lipases in flavor development Proteases in cheese making Pectinases in clarifying fruit juices

Textiles Cellulases in treating denim to generate ‘stone-

washed’ texture/appearance

Grain processing Conversion of corn starch to high fructose syrups

Industrial enzymes Feed enzymes

Waste management

Diagnostic enzymes

Enzymes to assist in the digestibility of animal feeds (cellulase, xylanase, phytase)

Lipases as drain-cleaning agents

Reporter enzymes (alkaline phosphatase, glucose oxidase, -glucosidase) and diagnostic enzymes (DNA polymerase)

Informationsserie – Biotechnologie

Produkte | 5 TECHNISCHE ENZYME – MEISTER DER KATALYSE

5-1 Enzyme in der Lebensmittelherstellung

Enzym Wirkung Anwendung

β-Galactosidase Wandelt den Zucker Lactose in Zuckerspezialitäten für den Pharma-, Lactulose um Lebensmittel- und Tierfuttersektor

Aminopeptidasen Spalten einzelne Aminosäuren Änderung des Aromaprofils von Käse, von bestimmten Proteinen ab Fleisch und Gewürzen

Cellulasen Spalten das pflanzliche Getränke- und Spirituosenherstellung (z. B. Heraus- Polysaccharid Zellulose lösen von Gerbsäure aus Traubenschalen)

Glucose-Isomerase Wandelt Traubenzucker (Glukose) Herstellung von Fruktosesirup in Fruchtzucker (Fruktose) um als Süßmittel für Limonade und Colagetränke

Hexoseoxydase (HOX) Wandelt eine Vielzahl von Zuckern z. B. Backindustrie (Steigerung der Teigstabilität,

(z. B. D-Glukose, D-Galaktose Volumenvergrößerung bei Brot) Maltose, Laktose) in Laktone und Wasserstoffperoxid um

Laccase Wandelt Phenole in Chinone und z. B. in Produkten zur Atemerfrischung (Pfefferminz,

Wasser um, wobei Sauerstoff Kaugummi): die gebildeten Chinone reagieren in der

verbraucht wird Mundhöhle mit geruchsbildenden Schwefelverbin-

dungen und neutralisieren diese

Pektinesterasen Spalten eine bestimmte Bindung in der z. B. in der Saftherstellung zur Entfernung

pflanzlichen Gerüstsubstanz Pektin von Trübstoffen oder Erhöhung der Saftausbeute

Biotechnologisch hergestellte Enzyme (Beispiele)

Applications in food industry Carbohydrases:

production of corn syrups from starch (glucoamylase);

conversion of cereal starches into fermentable sugars in malting, brewing, distillery,

baking industry (amylase). Proteases: meat tenderizers (bromelin,

papain, ficin) Lipases: Flavor production in chocolate

and cheese, generation of emulsifier in food –systems…

Applications Glucose oxidase:

desugaring of eggs, flour and potatoes to prevent browning;

preparation of salad dressings. Pectidases:

clarification of fruit juices; increase of yield of juice from grapes and other products;

removal of excess pectin from juices before concentration.

Increasing consistency in fruits and vegetables

Applications contd. Lipoxygenase: bleaching of flours. Phosphatase: quality testing of food

products Phenol oxidase: imparts the characteristic

dark hue to tea, cocoa, coffee and raisins. Renin (chymosin): cheese production Transglutaminase : cross-linking of proteins Phytase : bakeability of rice (gluten free) Asparaginase : avoid or decrease acryamide

Applications Flavorases: restoration and enrichment of

flavor by addition of enzyme preparations to food products e.g. fresh corn enzyme extracts to improve flavor of cannned goods or addition of alliinase to convert alliin of garlic into garlic oil.

Use of e.g. Lysozyme to control bacteria, Chitinases to control fungi, ß-Glucanases to control yeasts

Use of peroxidases (e.g. lacto-peroxidases) instead of preservative agents…

„Aromaenzyme“ bei Pflanzen

Amylases Alpha – amylase –

cuts 1,4 bonds Yields dextrins and oligosaccharides

Beta-amylase – maltose units only Combo - produces almost all maltose Gluco-amylase – glucose Pullunase -

Cuts beta - 1,6 linkages

Amylases

Corn starch processing 1

Maize grain

Endosperm

Starch

Corn syrups

High fructose syrupsEthanol

Food additives

Corn steep liquor

Edible oilOil meal

Hulls

Gluten

Germ

Industrial andfood uses

Short chain dextrins (foods)

Maltose syrups

Corn Starch Slurry (30-35% DS( dissolved solids), pH 6.0-6.5, Ca2+ 50 ppm)

Production of High Fructose Corn Syrupsfrom Starch

Liquefaction Thermostable -Amylase Gelatinization (105°C, 5 min) Dextrinization (95°C, 2h)

Liquefied Starch DE (dextrose equivalent) 10-15

Saccharification Glucoamylase (60°C, pH 4.0-4.5, 24-72 h)

Glucose Syrups DE 95-96

Isomerization Glucose isomerase (pH 7.5-8.0, 55-60°C, 5 mM Mg2+)

High Fructose Corn Syrups (42% fructose)

Generation of Glucose-syrup and Maltose-syrup

Production of Glucose from Starch____________________________________________________________Liquefaction Saccharification DE Glucose_____________________________________________________________Acid Acid 92 85

Acid Glucoamylase 95 91

Acid/α-amylase Glucoamylase 96 92

α-Amylase/High pressure Glucoamylase 97 93

cooking/ α-amylase

α-Amylase (thermostable) Glucoamylase 97 94

α-Amylase (thermostable) Glucoamylase 97-98.5 95-97.5

____________________________________________________________

Enzyme step 1: Action of Termamyl® on starch granules

Termamyl® is an -amylase (cleaves -1-4 glucosidic bonds in starch)

High temperature expands starch granules, making amylose and amylopectin chains more accessible

Termamyl is sufficiently stable at high temperatures if short reaction times are used

Starch hydrolysis is a batch process (the enzyme is not reused!) 0 10(minutes)

Amylase activity

Maltose concentration

Enzyme step 2: Conversion of maltose to glucose Amyloglucosidase is not as thermostable

as Termamyl (temperature must be reduced)

Amyloglucosidase has a pH optimum of 6.5 (Termamyl® operates optimally at 8.5): pH must be reduced

Reaction kinetics are slower Long incubations result in caramelization

of the saccharides - resulting in product loss and increase in impurities

Amylases in Baking and Brewing

Different Hydrolases

Glucose Isomerase

Enzyme step 3: Conversion of glucose to fructose

Fructose is much sweeter than glucose; it can be used as a sweetening agent in foodstuffs, and is more profitable than glucose

The enzyme xylose isomerase (glucose isomerase) will convert glucose to fructose, in an equilibrium reaction

Glucose Fructose A 50:50 mixture of glucose:fructose is sold as high fructose

syrup (HFS) Xylose (glucose) isomerase is much less thermostable, and

inhibited by Ca ions.

Lactase

Abwehrmechanismen gegen Mikroorganismen

Quelle: Mücke, I.: Möglichkeiten der enzymatischen Sauerstoffentfernung und Konservierung im Lebensmittelbereich, Braunschweig, in GFB Monographien (VCH), BD: 11 (1988), S. 189-202

Tab. 1: Abwehrmechanismen gegen Mikroorganismen

Strategie Wirkkomponente Wirkungsweise

-Entfernung Lebenswichtiger Metaboliten

Oxidase/Catalase Transferrine (Lactoferrin/Ovoferrin) Sauerstoffentfernung/Entfernung von Eisen

oder

Ersatz durch Analoga Avidin/SulfonamindeBindung von Biotin, Einbau in Folsäure anstelle von p-Benzoesäure

Bildung von Mikroorganismen toxischen Stoffe Oxidasen Lipasen

Bildung von H2O2, freie Fettsäuren sind toxisch für einige Protozoen, Viren, Bakterien

Lactoperoxidase Bildung von HypothiocyanatMyeloperoxidasen "Xylitolphosphorylase" Xylitol-5-Phosphat ist toxisch für Streptococcus mutans

Zellwandzerstörende Enzyme Lysozyme Zelllyse von Bakterien (gram+)

Chitinasen/ProteasenAktivierung endogener Autolyse-Enzyme, Zellllyse von Pilzen, Zelllyse von Hefen

Manase/ß-Glucanase/Protease

Inhibitoren (Wachstum7Enzyme) Antienzym-Enzyme

Proteasen, SH-Oxidasen, Enzyminhibitoren

Abbau bzw. Hemmung mikrobieller Enzyme, die einen Befall einleiten (Proteasen, Pectinasen)

Bacteriocine (Nisin, Colicilin, Diplococcin) Wachstumsinhibitoren empfindlicher Stämme (Hefen)

Bioconservation :Using Enzymes

Antimicrobial Enzymes

Enzymes as preservatives

Theoretischer Hintergrund

Chitin

bestehend aus β-1,4-N-Acetyl-D-Glukosamin und ist das zweithäufigste Biopolymer der Erde, welches unterschiedlichen Organismen, auch Pilzen, als Stützskelett dient.

Chitinasen können solche Skelette abbauen. Sie wirken somit als natürliche Fungizide, die keine schädlichen Rückstände hinterlassen.

Das Anwendungspotential von fungiziden Chitinasen ist vielfältig. Die Lebensmitteltechnologie braucht kälteangepasste Chitinasen, die in der Kälte und bei Raumtemperatur aktiv sind.

Erstmals wird es so möglich sein, bei Raumtemperatur und unter Kühlbedingungen im Rahmen des BioControl Konzeptes zu konservieren.

Zellwand-Chitin(P. roqueforti;Calcofluor Färbung)

1,5

2

2,5

3

3,5

4

4,5

5

1 10 20 30 40 50 60

Psychrophil I

Psychrophil II

Mesophil I

Mesophil II

Chitinase-AktivitätIn Abhängigkeit von der Temperatur

Temperatur (°C)

NA

G m

g m

l-1h

-1

Oxidoreductase Glucose oxidase

From Aspergillus niger and P. notatum Used in removal of glucose and oxygen

H2O2 is produced and is destroyed by Catalase

H2O2 is used sometime to pasterurize milk and the excess is removed by Catalase

Glucose-Oxidase

Glucose –Oxidase in apple juice

Glucose-Oxidase in Mayonnaise

Catalase

Lipoxygenase

Ascorbic acid Oxidase

Pectinases

Pectinases

Ananas

Früchte

Waschen

Pine-O-MatRätzmühle

Hammermühle

PressenSchneckenpresse,

Bucherpresse, Dekanter

Pasteurisieren 90°CRückkühlen 50°C

EnzymierungPektinase + „Hemicellulase“ +

Cellulase

Zentrifugieren

Ultrafiltration

SaftbehandlungEntfärbung

Konzentrierung

Klares Ananassaftkonzentrat

Zentrifugieren

Konzentrierung

Trübes Ananassaftkonzentrat

Zentrifugieren

So ungewöhnlich sind Cellulasen und Co. nicht

Exercises Search and identification of

primarily literature in „in food-processings“ as print out….

Topics could be : flavor enhancing, preservation by enzymes, use in food-technologies like cheese-processings, soya- processings, fat and oils (e.g. interesterification), fruit juice technologies, bakery-technology (dough mixing , freshness keeping,convenient products like pizza with fermentation control etc.), enzyme-use for sweeties, meat -processing etc.

Enzymes in Processing and food Storage Polyphenol oxidase – fruit storage Amylase – DE Starches Protease – rennin/chymosin and ficin

( bear clarifier) Lipase – hydrolytic rancidity Lipoxidase – oxidizes fats Muscle tendrizer – bromalin from

pinepapple

Enzymatic browning Phenolic substances – from brown to

black pigments 1. Enzymes + S - Brown color

( melanosis) Polyphenol-oxidase (Cu+ dependent) EC. 1.14.18.1 Need Oxygen and tissue damage Present in foods – Banana, Apples, Pear,

Peaches, Tea leaves, Coffee beans (desirable).

Enzymatic browning reaction (Phenolases)

Ripening and Browning

Browning depends on genetic potential

Inhibition of enzymes Sulfite – reacts with quinone to prevent further chemical

steps pH- vinegar (citric acids) Sodium hexametaphosphate/ascorbate/citrate EDTA (binds copper of PPO) Sugar (limit oxygen diffusion) Vacuum package Cysteine, Chitosan Blanching (inactivate enzymes…) Irradiation

Uses of enzymes In Baking to increase fermentation rate; to

increase dough machinability, to optimize volume and porous structure etc.

Corn syrup to sweeten soft drinks, to produce instant soups , producing sweeties, carrier for flavor etc.

Liquid center chocolates Clarification of Apple juice, etc. Makes food-technology possible and

innovative

Enzymes for Baking

Indigenous Enzymes At harvest: plant Amylase

Exogenous Enzymes By microorganisms in situ: lipase, phosphatase, amylases etc.

Produced by Yeast Maltogenase, protease, alcohol dehydrogenase, etc.

Endogenous Enzymes Commercial enzymes added during process: amylases, pentosanases, lipase, proteases etc.

Mechanism of Glucose OxidaseH2O2 oxidizes the Gluten network

H2O2 oxidizes the sulfhydrylgroup (-SH) of the amino acidCysteine from wheat gluten, forming Disulfide bonds within the gluten network. This leads to dough strengthening!

Synergies of Glucose Oxidase with Xylanase and Amylase

Procedure: Straight dough pan bread

Flour: European Flour all doughs contained 40ppm ascorbic acid and the optimal dosage of Amylase and Xylanase

Control HemicellulaseAmylase

AmylaseHemicellulase100 U Glucose Oxidaseper kg of flour

Dosage Response of Maltogenic Amylase onCrumb Texture of White Pan bread

Process: Sponge & Dough

maltogenic alpha-Amylase added on top

Reference350 MANU maltogene Amylase750 MANU maltogene Amylase

Effect of Different Amylases on CrumbSoftness and Elasticity in pan bread

Monoglycerides = 0.5%

Maltogenic alpha-Amylase, 450 U/kg flour

Thermostable Bacterial alpha-Amylase, 1.5 U/kg flour

Sponge & dough procedure using American

flour, differences of loaf volume: max. +/- 3%

Action and deactivation temperatures of differentAmylases during the baking process

A Intact starch granules are inaccessible for enzymes

B Stach granules start to swell

C Amylose starts to leach into intergranular space

D Bulk of starch is gelatinised; optimal temperature for the degradation of amylose and amylopectin

Lipases with different specificity towards native flour lipids

1,3-specific Lipase

• hydrolyzes non-polar lipids f.e. 1,3 ester bonds of triglycerides

Lipase with broad substrate

specificity

• Modifies triglycerides but also polar lipids like f.e. Lecithin by which they become more polar and improve their surface active function.

Effect in bread

• Assures better dough consistency and stability, thereby increasing fermentation tolerance, reduction of dough stickiness

• Increased volume of the baked product with fine, regular crumb structure.

• Mainly the Lipase with broad substrate specificity is an alternative to dough strengthening emulsifiers

Synergy: Combination of Amylase/Xylanase with1,3-specific Lipase

Enzyme: 1,3-specific Lipase combined with Fungal Amylase and Xylanase

• Improved bread volume and bloom

• Uniform and regular crumb structure

• Whiter crumb structure

Combination of Amylase or Xylanase with Lipase inHard Rolls and Pan Bread

Actions towards native flour lipids

HPLC profile of lipids from dough made with and without dual specificity Lipase

Treatment indicates that DGDG and lecithin peaksdecrease, DGMG,lysolecithin and FFA peaksincrease

Lipids extracted from dough using water saturatedbutanol at 25°C

Reducing Acrylamid by Asparaginase

Asparaginase

Lipase and Esterase FFA are produced – may cause rancidity Form mono and diglycerides But in Cheese it is desirable In seeds it is destroyed by heat 1,3 Specific enzymes (position on glycerol) Tailor making of cocoa butter substitute

Lipases

Proteases

Proteases

Proteases

Proteases

Proteases

Immobilized Enzymes Enzyme in solution can be used once It can be fixed on a carrier so can be used

continuously It can be bound, adsorbed, entrapped or

crosslinked (e.g. microencapsulation) They are more heat stable, pH is shifted

Other applications Aldehyde dehydrogenase

Unsaturated FA in Soy produce hexanal (bean like flavor)

Butanediol Dehydrogenase Diacetyl formed during beer production

Transglutaminase crosslinking enzyme – lysine and glutamic acid

Naringinase – hydrolyzes bitter narigin to naringenin

Cross-linking of food-systems

Immobilisation of Enzymes

0

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100

Akti

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Immobilization of Enzymes

Immobilization of Enzymes

Immobilization of Enzymes

New solutions in food processing

New solutions in food processings

Enzymes in Food -Processing