ENZYMES in Food Processing

Präsentation zum Thema: "ENZYMES in Food Processing"—  Präsentation transkript:

1 ENZYMES in Food Processing
LTWT Hochschule Bremerhaven SS 2011

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5 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

6 Energy Profile reactants products H EA T.S. catalyst

7 Enzymatic Reactions Enzyme combines with a specific substrate to a form an enzyme-substrate complex in a lock and key concept before forming new products.

8 Enzyme action products substrate enzyme

9 Structure of an enzyme Contains both a protein and a nonprotein.
Nonprotein is either a coenzyme (usually a vitamin) or a cofactor (usually a mineral).

10 Factors influencing enzyme activity
Operate under optimum conditions of pH and temperature. Easily inactivated (denatured) in presence of inhibitors.

11 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.

12 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.)

13 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.

14 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

15 Enzymes in Industry Distribution of enzymes by substrate
Protein hydrolyzing 59% Carbohydrate hydrolyzing 28% Lipid hydrolyzing % Speciality (analytical, pharma, research) 10%

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

19 A few large competitors
• Novozymes (all applications) • DSM (most applications) • Chr. Hansen (dairy enzymes only) • AB Enzymes (some applications

20 Industrial Enzyme Market
Annual Sales: $ 1.6 billion Food and starch processing: 45% Detergents: % Textiles: % Leather: % Pulp and paper: %

21 World Market for Some Products of Enzymatic Reactions
High fructose corn syrup: $ 1 billion Aspartame: $ 850 million Acrylamide: $ 300 million

22 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

23 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

24 Industrial enzymes Food processing Textiles Grain 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

25 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, b-glucosidase) and diagnostic enzymes (DNA polymerase)

26 Biotechnologisch hergestellte Enzyme (Beispiele)
Produkte | 5 TECHNISCHE ENZYME – MEISTER DER KATALYSE 5-1 Enzyme in der Lebensmittelherstellung Biotechnologisch hergestellte Enzyme (Beispiele) 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 Informationsserie – Biotechnologie

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28 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…

29 Applications Glucose oxidase: Pectidases:
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

30 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

31 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…

32 „Aromaenzyme“ bei Pflanzen

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42 Amylases Alpha – amylase – Beta-amylase – maltose units only
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

43 Amylases

44 Corn starch processing 1
Maize grain Corn steep liquor Germ Edible oil Endosperm Oil meal Hulls Gluten Starch Industrial and food uses Short chain dextrins (foods) Maltose syrups Corn syrups Food additives Ethanol High fructose syrups

45 Production of High Fructose Corn Syrups from Starch
Corn Starch Slurry (30-35% DS( dissolved solids), pH , Ca2+ 50 ppm) Liquefaction Thermostable a-Amylase Gelatinization (105°C, 5 min) Dextrinization (95°C, 2h) Liquefied Starch DE (dextrose equivalent) 10-15 Saccharification Glucoamylase (60°C, pH , h) Glucose Syrups DE 95-96 Isomerization Glucose isomerase (pH , 55-60°C, 5 mM Mg2+) High Fructose Corn Syrups (42% fructose)

46 Generation of Glucose-syrup and Maltose-syrup

47 Production of Glucose from Starch
____________________________________________________________ Liquefaction Saccharification DE Glucose _____________________________________________________________ Acid Acid Acid Glucoamylase Acid/α-amylase Glucoamylase α-Amylase/High pressure Glucoamylase cooking/ α-amylase α-Amylase (thermostable) Glucoamylase α-Amylase (thermostable) Glucoamylase

48 Enzyme step 1: Action of Termamyl® on starch granules
Termamyl® is an a-amylase (cleaves a-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!) Maltose concentration Amylase activity 10 (minutes)

49 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

50 Amylases in Baking and Brewing

51 Different Hydrolases

52 Glucose Isomerase

53 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.

54 Lactase

55 Abwehrmechanismen gegen Mikroorganismen
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/Sulfonaminde Bindung 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 Hypothiocyanat Myeloperoxidasen "Xylitolphosphorylase" Xylitol-5-Phosphat ist toxisch für Streptococcus mutans Zellwandzerstörende Enzyme Lysozyme Zelllyse von Bakterien (gram+) Chitinasen/Proteasen Aktivierung 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) Quelle: Mücke, I.: Möglichkeiten der enzymatischen Sauerstoffentfernung und Konservierung im Lebensmittelbereich, Braunschweig, in GFB Monographien (VCH), BD: 11 (1988), S

56 Bioconservation :Using Enzymes

57 Antimicrobial Enzymes

58 Enzymes as preservatives

59 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. Zellwand-Chitin (P. roqueforti; Calcofluor Färbung) Chitinasen können solche Skelette abbauen. Sie wirken somit als natürliche Fungizide, die keine schädlichen Rückstände hinterlassen. Chitinase-Aktivität In Abhängigkeit von der Temperatur Temperatur (°C) NAG mg ml-1h-1 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.

60 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

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62 Glucose-Oxidase

63 Glucose –Oxidase in apple juice

64 Glucose-Oxidase in Mayonnaise

65 Catalase

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67 Lipoxygenase

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70 Ascorbic acid Oxidase

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72 Pectinases

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74 Pectinases

75 Ananas So ungewöhnlich sind Cellulasen und Co. nicht

76 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.

77 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

78 Enzymatic browning Phenolic substances – from brown to black pigments
1. Enzymes + S - Brown color ( melanosis) Polyphenol-oxidase (Cu+ dependent) EC Need Oxygen and tissue damage Present in foods – Banana, Apples, Pear, Peaches, Tea leaves, Coffee beans (desirable).

79 Enzymatic browning reaction (Phenolases)

80 Ripening and Browning

81 Browning depends on genetic potential

82 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

83 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

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86 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.

87 Mechanism of Glucose Oxidase H2O2 oxidizes the Gluten network
H2O2 oxidizes the sulfhydrylgroup (-SH) of the amino acid Cysteine from wheat gluten, forming Disulfide bonds within the gluten network. This leads to dough strengthening!

88 Synergies of Glucose Oxidase with Xylanase and Amylase
Hemicellulase Amylase Amylase Hemicellulase 100 U Glucose Oxidase per kg of flour Control Procedure: Straight dough pan bread Flour: European Flour all doughs contained 40ppm ascorbic acid and the optimal dosage of Amylase and Xylanase

89 Reference 350 MANU maltogene Amylase 750 MANU maltogene Amylase
Dosage Response of Maltogenic Amylase on Crumb Texture of White Pan bread Reference 350 MANU maltogene Amylase 750 MANU maltogene Amylase Process: Sponge & Dough maltogenic alpha-Amylase added on top

90 Effect of Different Amylases on Crumb Softness 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%

91 Action and deactivation temperatures of different Amylases 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

92 Lipases with different specificity towards native flour lipids
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 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.

93 Synergy: Combination of Amylase/Xylanase with 1,3-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

94 Combination of Amylase or Xylanase with Lipase in Hard Rolls and Pan Bread

95 Actions towards native flour lipids

96 HPLC profile of lipids from dough made with and without dual specificity Lipase
Treatment indicates that DGDG and lecithin peaks decrease, DGMG, lysolecithin and FFA peaks increase Lipids extracted from dough using water saturated butanol at 25°C

97 Reducing Acrylamid by Asparaginase

98 Asparaginase

99 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

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102 Proteases

103 Proteases

104 Proteases

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107 Proteases

108 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

109 Other applications Aldehyde dehydrogenase Butanediol 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

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111 Cross-linking of food-systems

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113 Immobilisation of Enzymes

114 Immobilization of Enzymes

115 Immobilization of Enzymes

116 Immobilization of Enzymes

117 New solutions in food processing

118 New solutions in food processings

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121 Enzymes in Food -Processing