Role of Enzymes in food processing.

Food biochemistry and food processing. 2012. Simpson B.K. (Ed.) 2nd edition, John Wiley & Sons, Inc.
 Enzymes in food processing. Advantages.
Enzymes are proteins with powerful catalytic function.
Enzymes have a number of distinct advantages over
conventional chemical catalysts:

➢ High productivity and catalytic efficiency; Active in low

concentrations;
➢ High specificity – able to discriminate between structurally
similar molecules, for example-optical isomers
(stereospecificity). Their action on food components other
than their substrates are negligible, thus resulting in the
formation of purer products with more consistent properties;
➢ They are more environmentally friendly and produce less
residuals (or processing waste that must be disposed of at
high costs) compared to traditional chemical catalysts.
 Advantages, Cont.
➢ Work under mild conditions of temperature, pressure
and pH. It helps to preserve the integrity of heat-labile
essential nutrients.

➢ Control of enzyme activity and reaction rate.

➢ Most of them are quite heat labile and therefore can be

readily inactivated by mild heat treatments after they
have been used to achieve the desired transformation
in foods;

➢ They are natural and relatively innocuous components

of agricultural materials that are considered “safe” for
food and other nonfood uses;

Some disadvantages: High cost, Low stability.

Undesirable effects:

➢ Autolytic changes in food products - excessive proteolysis may

produce bitterness in cheeses and protein hydrolysates, or excessive
texture softening in meats and fish products (e.g., canned tuna).

➢ Enzymes like proteases, lipases, and carbohydrases break down

biological molecules (proteins, fats, and carbohydrates, respectively)
which, if not controlled, may adversely impact flavor, texture, overall
product quality.

➢ Decarboxylases and deaminases degrade biomolecules (e.g., free

amino acids, peptides, and proteins) to form undesirable and/or toxic
components, e.g., biogenic amines in foods.

➢ Polyphenol oxidases (PPO) and lipoxygenases (LOX) promote

oxidations and undesirable discolorations and/or color loss in fresh
vegetables and fruits.

➢ Ascorbic acid oxidase cause destruction of essential components (vit.

C) in foods.
 Sources of food enzymes (plant, animal, microbial,
and recombinant).

Enzymes have been used inadvertently or deliberately in food

processing since ancient times to make a variety of food
products, such as:

➢ breads,
➢ fermented alcoholic beverages,
➢ fish sauces,
➢ cheeses.

Enzymes have been traditionally produced by

➢ extraction and fermentation processes from plant and animal

sources,
➢ from a few cultivatable microorganisms.
 Sources of food enzymes (plant, animal, microbial, and
recombinant). Cont.

Industrial enzymes have traditionally been derived from:

Plants: α-amylase, β-amylase, bromelain, β-glucanase, ficin,

papain, chymopapain, and lipoxygenase

Animals: trypsins, pepsins, chymotrypsins, catalase,

pancreatic amylase, pancreatic lipase, and rennin (chymosin)

Microorganisms: α-amylase, β-amylase, glucose isomerase,

pullulanase, cellulase, catalase, lactase, pectinases, pectin
lyase, invertase, raffinose, microbial lipases, and proteases.
 Enzymes used in food industry have mainly microbial origin.

Advantages of microorganisms as a source for enzyme

production:

➢ Easy and fast grow.

➢ Take small space to cultivate.
➢ Relatively cheep culture compounds.
➢ Their use as enzyme source is not affected by seasonal
changes and climatic conditions and are thus more
consistent.
➢ Possibility for tight control of culture conditions.
➢ Their use as sources of enzymes is not affected by
various political and agricultural policies or decisions that
regulate the slaughter of animals or felling of trees or
plants.
Microbial source of Enzymes; Even though all classes of
enzymes are expected to occur in all or most microorganisms,
in practice, the great majority of industrial microbial enzymes
are derived from only a very few GRAS (generally recognized
as safe) species.

The predominant microorganisms used for industrial production

of enzymes for food purposes are:

➢ Aspergillus species,
➢ Bacillus species,
➢ Kluyveromyces species.

Limited use is due to: co-production of harmful toxins.

There is a need for stringent evaluation for safety at high cost
before they can be put to use for food production.
 Use of enzymes in baked goods manufacturing

Baked goods are prepared from flours such as wheat flour, which
has starch as its main constituent.

Amylolytic enzymes break down flour starch into small dextrin pieces that
become better substrates for yeast to act upon in the bread-making
process.

Xylanase – preferred are those that act on

the non-water soluble arabinoxylan fraction. It
interferes with the formation of gluten
network. Removal of not-extractable with
water arabinoxylan fraction results in increase
of high molecular weight solubilized in water
arabinoxylans that in turns increase viscosity
and dough stability; provide better crumb
texture and increased loaf volume.
Broader application of enzymes in the baking industry is replacement of
chemicals that are conventionally used in bread making.

For example, an enzyme like glucose oxidase (GOX) is used in baked

goods to strengthen dough texture and enhance elasticity in place of
chemicals such as potassium bromate (flour improver - E number E924).

Potassium bromate - 2B carcinogen (possibly carcinogenic to humans).

Baneed for use in EU; Allowed for use in the USA.
Proteases:

To break down protein molecules in the dough and improve dough

handling;

Enhance flavor development;

May be used to degrade gluten and protect individuals that are gluten
intolerant.

Asparaginase breaks down asparagine in the flours to reduce its

availability for reaction with reducing sugars to form acrylamide at high
temperature.
 Horváthová et al. Biologia, Bratislava, 55/6: 605|615, 2000
Enzymes in starch modification

Biologia, Bratislava, 55/6: 605|615, 2000

Fig. 1. Action of starch-degrading enzymes. () Reducing -D-glucose residue; () non reducing -D-
glucose residue. Arrows indicate the -1,6-branching points in the starch molecule.
Major steps of starch conversion
Enzymes in starch modification: Liquefaction
Bacterial thermophilic α-amylase: an endo-amylase which hydrolyses
the α- 1,4-linkages in starch (amylose and amylopectin) almost at
random. The breakdown products formed are mainly soluble dextrin and
oligosaccharides. In a concentrated solution of starch, the hydrolysis
results in a rapid viscosity reduction. In consequence the bacterial
thermophilic α-amylase is often referred to as a 'liquefying amylase'. The
process is called liquefaction.

Starch liquefaction results in formation of maltodextrin (Dextrose

equivalent (DE) = 15-25 means partial hydrolysis.

Enzymes in food technology. 2002. Whitehurst R.J., Law B.A. (Eds.), Sheffield
Academic Press Ltd., Sheffield, UK
 Saccharification of liquefied starch

Maltodextrin is commercially available and used for its

rheological properties. They are used in the food
industry as fillers, stabilizers, thickeners, pastes
and glues.

Further degradation of maltodextrins is known as

saccharification. Depending on the degree of
hydrolysis and enzymes used variety of sweeteners
can be produced which differ by their carbohydrate
composition and rheological properties.

Dextrose equivalent (DE) = degree of hydrolysis. Expresses the

reducing power as a percentage of pure dextrose, calculated to dry
weight basis.
 Saccharification enzymes

Production of maltose syrup:

Fungal α-amylase: exo-amylase, which hydrolyses the

alpha-1,4-linkages in liquefied starch (amylose and
amylopectin); A prolonged reaction results in the
formation of large amounts of maltose.

The Fungal alpha-amylase is used for production of

high maltose syrups or high conversion syrups.

β-amylase are exo-enzymes, which attack amylose

chains resulting in maltose production. β-Amylase is
used for the production of maltose syrups.
Maltose syrups (maltose content from 50 to 80%) are
produced by saccharifying liquefied starch with maltogenic
exo-enzymes - fungal α-amylase or barley β-amylase, also
known as malt extracts.

Properties of maltose syrups:

➢ Low glucose content and a high maltose content.

➢ Because of the low glucose content, high maltose syrups
show a low tendency to crystallize.
➢ They are relatively non-hygroscopic.
Production of glucose syrup:

Glucoamylase (amyloglucosidase): (exo-amylase)

which hydrolyses alpha-1,4-linkages as well as alpha-
1,6-linkages in liquefied starch (amylose and
amylopectin).

The breakdown product formed is glucose (as β-

glucose), which has been split off from the non-reducing
end of the substrate molecule.

Eventually, almost complete conversion of starch into

glucose can be obtained.
Glucose syrups (95-97% glucose) may be produced
from most starch raw materials (corn, wheat, potatoes,
tapioca, barley and rice).
Isoamylase and pullulanase (de-branching enzymes)

Isoamylase and pullulanase hydrolyse alpha-1,6-glycosidic

bonds of starch, which has been partly hydrolyzed by
alpha-amylase.

Treatment of amylopectin with pullulanase generates

linear amylose fragments.

Using heat-stable and acid-stable pullulanase in

combination with saccharification enzymes makes the
starch conversion reactions more efficient.
 Fructose syrup
High-fructose corn syrup (HFCS):
contain 42% or 90% fructose
based on dry substance.

A sweetener alternative to white

sugar (sucrose) produced from
sugar cane or beets.
Produced by the use of the enzyme glucose isomerase.

Glucose can reversibly be isomerized to fructose. The

equilibrium conversion for glucose to fructose is 50%
under industrial conditions.

The isomerization reaction can only be economically

efficient by using immobilized enzyme.
This is done by using an immobilized
isomerase in a fixed-bed reactor process in a
column through which glucose flows
continuously.

The enzyme granules must be rigid enough

to prevent compaction during the operation.

Sweetzyme IT (Novozymes A/S) is produced

by a mutant of a selected Streptomyces
murinus strain. The immobilization procedure
consists of a disruption of a cell concentrate
through with a homogenizer. The cells are
then cross-linked with glutaraldehyde. The
concentrated aggregate is extruded and
finally fluid-bed dried and sieved.

Depending on parameters such as

temperature, pH, feed purity, and so on, the
operating lifetime of this isomerase will
typically be 200–360 days.
Saccharification products and their application
 Use of enzymes in dairy products manufacturing

Proteases: To act on milk proteins to modify texture and

solubility properties of milk and other dairy products;
accelerate cheese ripening and improve flavor intensity.

Rennet is the stomach extract that contains the enzyme

chymosin in a stabilized form that is usable for cheese
making. It is a coagulant which degrade kapa-casein to
produce cheese curds.

For the manufacture of traditional rennet, calves, lambs,

or kids that are no more than about 2-weeks-old and fed
only milk are used.
Genetic engineering technology has been used for the commercial
production of a 100% pure chymosin product from microbes. This type
of chymosin is often called fermentation-produced chymosin.

The microbes used for the production of this type of rennet include
nonpathogenic microorganisms

➢ Escherichia coli K-12,

➢ Kluyveromyces marxianus var. lactis
➢ Aspergillus niger var. awamori.

Pro-chymosin genes obtained from young calves are transferred

through DNA plasmid intervention into microbial cells. Fermentation
follows to produce pro-chymosin, cell destruction, activation
of the prochymosin to chymosin (by cleavage of 42 amino acids), and
harvesting/producing large yields of pure, 100% chymosin.
Lactase

Lactose is present in milk (about 4.7% (w/v)) and remains in the

whey (supernatant) left after the coagulation stage of cheese-
making. Lactose has low solubility resulting in crystal formation at
concentrations above 11 %.

If lactase is added to milk or liquid whey (2000 U kg-1) and left for
about a day, about 50% of the lactose is hydrolyzed, giving a
sweeter product which will not crystallise if condensed or frozen.
Therefore, it can be used in the production of ice cream and
sweetened flavored and condensed milks to prevent “sandy” taste.

Hydrolyzed lactose is 4 times sweeter than non-hydrolyzed lactose.

It also improves the 'scoopability' and creaminess of the product.

Use of lactase protect individuals that are lactose intolerant.

Of the Thai, Chinese and Black American populations,

97%, 90% and 73% respectively, are reported to be
lactose intolerant.

Some individuals suffer from inborn metabolic lactose

intolerance (lactase deficiency).

Severe tissue dehydration, diarrhea and even death

may result from feeding lactose containing milk to
lactose-intolerant children and adults.
Lipases

Lipases are used to break down milk fats and give characteristic
flavors to cheeses. Stronger flavored cheeses, for example, the
Italian cheese, Romano, are prepared using exogenous lipases.

The flavor comes from the free fatty acids produced when milk fats
are hydrolyzed.

Animal lipases are obtained from kid, calf and lamb.

Microbial lipase is derived by fermentation with the fungal species
Mucor meihei.

Microbial lipases are readily available for cheese-making, but less

preferred, since they are less specific in what fats they hydrolyze.

Animal enzymes are more partial to short and medium-length fats.

Hydrolysis of the shorter fats is preferred because it results in the
desirable taste of many cheeses. Hydrolysis of the longer chain
fatty acids can result in either soapiness, or no flavor at all.
Bio-protective enzymes (preservatives)

Bio-protective enzymes offer a natural means to improve

food safety and reduce costs associated with microbial
contamination during storage.

Lysozyme: An antimicrobial enzyme that limits the growth of

Clostridia in aged cheese. These bacteria can cause swelling
of the cheese shape and/or development of unpleasant taste
and smell.

Nisin: An antimicrobial peptide effective against Gram-positive

and spore-forming bacteria in cheese. Useful in non-thermally
processed dairy products. No widespread agreement on the
maximum level application.
Use of enzymes in meat and seafood products
manufacturing

Proteases- heat stable forms preferred , e.g., papain, ficin, and

bromelain (mixture of enzymes found in pineapples)

➢ To modify texture and induce tenderness in meats and squid,

➢ To improve chewability and digestibility,
➢ To reduce bitterness and improve flavor as well as nutritive
value,
➢ Produce hydrolysates from meat scraps, underutilized fish
species and fish processing discards;
➢ Enhanced flavors in fermented herring (fish).
Transglutaminase:

➢ To improve texture in meats and seafood products,

➢ Form restructured meats from trimmings and surimi-type
products,
➢ Form “umami” flavors for use as additives to meat products (After
cross-linking treatment the content of 1000–5000 Da peptides increases, 2012,
Food Chemistry 136 (1), Pages 144–151)

Umami taste – the fifth taste of food (basic tastes: sweet, sour,
salty and bitter). It can be described as a pleasant "meaty" taste
with a long lasting, mouthwatering and coating sensation over the
tongue.

Umami taste represents the taste of the amino acid L-glutamate

and 5’-ribonucleotides such as guanosine monophosphate (GMP)
and inosine monophosphate (IMP).

GMP and IMP amplify the taste intensity of the sodium glutamate.
Use of enzymes in fruit, vegetable and cereal processing

Pectolytic enzymes (pectinase): a collective name for several

enzymes that degrade pectin;

Cellulolytic complex;

Hemicellulases.
Enzymatic mash treatment: why are exogenous enzymes needed?

Cell walls contain high-molecular weight compounds.

Protopectin is insoluble and inhibit the extraction of the juice

from the fruit and keep solid particles suspended in the juice.

In addition, polymers of xylose, galactose, and arabinose

(hemicelluloses) form a link with cellulose. The entire system
forms a gel that retains the juice in the mash.

The goal:

Enzymatic mash treatment (for example, in juice production) is

performed to: improve the pressabiliy of the mash and,
respectively juice yield.
What happens?

Pectinase pretreatment acts mainly on the cell wall, breaking the

structure and freeing the juice. Pectinases with a high proportion of
pectinesterase and liquefying polygalacturonases are suitable for
mash treatment.

➢ The hydrolysis of the protopectin that binds the cells weakens the
fruit tissue, causing the protopectin to dissolve thus increasing
the juice viscosity. More juice can be released from the mash.

➢ The high content of pectin esterase (PE) causes the formation of

de-esterified pectin fragments, which have a low water-binding
capacity and reduces the slipperiness.

➢ Greater yield and press capacity.

 Cellulases and hemicellulases
The use of cellulases and hemicellulases in fruit
processing is not allowed in the EU for legal reasons.

They are, however, allowed without any legal restrictions

for vegetable processing.

However, cellulases and hemicellulases can in fact be

detected in commercially available pectinase, amylase
and protease products as secondary activities. Their
proportion depends on the strain used for the enzyme
production.

Cellulolytic enzymes are usually used in combination

with pectolytic enzymes. These enable further viscosity
reduction and facilitate solid/liquid separation.