ENZYMES in Food Processing

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ENZYMES in Food Processing

• NTRS 525
• H. Singh Ph.D.

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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

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Energy Profile
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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.

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Enzyme action
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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).

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Factors influencing enzyme activity

• Operate under optimum conditions of pH and
  temperature.
• Easily inactivated (denatured) in presence of
  inhibitors.

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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.

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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.

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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.

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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 _________________________________________
___________
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Enzymes in Industry

• Distribution of enzymes by substrate
• Protein hydrolyzing 59
• Carbohydrate hydrolyzing 28
• Lipid hydrolyzing 3
• Speciality (analytical, pharma, research) 10

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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

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A few large competitors

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

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Industrial Enzyme Market Annual
Sales 1.6 billion Food and starch
processing 45 Detergents
34 Textiles
11 Leather
3 Pulp and paper
1.2


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


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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

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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


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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

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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)

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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

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Applications

• Glucose oxidase
• desugaring of eggs, flour and potatoes to
  prevent browning
• preparation of salad dressings.
• Pectinases
• clarification of fruit juices increase of yield
  of juice from grapes and other products
• removal of excess pectin from juices before
  concentration.

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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

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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.

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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

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Corn starch processing 1
Maize grain
Corn steep liquor
Germ
Edible oil
Endosperm
Oil meal
Hulls
Gluten
Industrial and food uses
Starch
Short chain dextrins (foods)
Maltose syrups
Corn syrups
Food additives
Ethanol
High fructose syrups
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Production of High Fructose Corn Syrups from
Starch
Corn Starch Slurry (30-35 DS( dissolved solids),
pH 6.0-6.5, Ca2 50 ppm)
Liquefaction Thermostable a-Amylase
Gelatinization (105C, 5 min) Dextrinization
(95C, 2h)
Liquefied Starch DE (dextrose equivalent) 10-15
Saccharification Glucoamylase (60C, pH
4.0-4.5, 24-72 h)
Glucose Syrups DE 95-96
Isomerization Glucose isomerase (pH
7.5-8.0, 55-60C, 5 mM Mg2)
High Fructose Corn Syrups (42 fructose)
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Production of Glucose from Starch ________________
____________________________________________ Lique
faction Saccharification
DE Glucose _______________________
______________________________________ Acid
Acid 92 85 Acid
Glucoamylase 95
91 Acid/a-amylase
Glucoamylase 96
92 a-Amylase/High pressure Glucoamylase
97 93 cooking/ a-amylase
a-Amylase (thermostable) Glucoamylase
97 94 a-Amylase
(thermostable) Glucoamylase
97-98.5 95-97.5 ______________________________
______________________________


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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
0
10
(minutes)
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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

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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 will convert glucose
  to fructose, in an equilibrium reaction
• Glucose ? Fructose
• A 5050 mixture of glucosefructose is sold as
  high fructose syrup (HFS)
• Xylose (glucose) isomerase is much less
  thermostable, and inhibited by Ca ions.

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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

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Enzymatic browning

• Phenolic substances from brown to black
  pigments
• 1. Enzymes S - Brown color ( melanosis)
• Poluphenol-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).

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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
• Blanching
• Irradiation

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Uses

• In Baking to increase fermentation rate
• Corn syrup
• Liquid center chocolates
• Clarification of Apple juice

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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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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
• They are more heat stable, pH is shifted

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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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Other applications

• Aldehyde dehydrogenase
• Unsaturated FA in Soy produce hexanal (bean like
  flavor)
• Butanediol Dehydrogenase
• Diacetyl formed during beer production
• Transgluaminase
• crosslinking enzyme lysine and glutamic acid
• Naringinase
• hydrolyzes bitter narigin to naringenin