BREWING INDUSTRY (AMYLASE) In beer manufacture, barley grain is malted (germinated under

controlled conditions) and kilned. This malt is extracted to give wort.

Brewery
- the place at which beer is commercially made, where distinct sets of
brewing equipment are called plant.
HISTORY
In 1831, Erhard Friedrich Leuchs (18001837) described the
hydrolysis of starch by saliva, due to the presence of an enzyme in saliva,
"ptyalin", an amylase. The modern history of enzymes began in 1833, when
French chemists AnselmePayen and Jean-Franois Persoz isolated an amylase
complex from germinating barley and named it "diastase". In 1862, Alexander
JakulowitschDanilewsky (18381923) separated pancreatic amylase fromtrypsin.
An amylase is an enzyme that catalyses the hydrolysis of starch into sugars.
Amylase is present in the saliva of humans and some other mammals, where it
begins the chemical process of digestion. Foods that contain large amounts of
starch but little sugar, such as rice and potatoes, may acquire a slightly sweet taste
as they are chewed because amylase degrades some of their starch into sugar.
The pancreas and salivary gland make amylase (alpha amylase) to hydrolyse
dietary starch into disaccharides and trisaccharides which are converted by other
enzymes to glucose to supply the body with energy. Plants and some bacteria also
produce amylase. As diastase, amylase was the first enzyme to be discovered and
isolated (by AnselmePayen in 1833). Specific amylase proteins are designated by
different Greek letters. All amylases are glycoside hydrolases and act on -1,4-
glycosidic bonds.
CLASSIFICATIONS
1. Alpha-amylase
The -amylases (alternative names: 1,4--D-glucanglucanohydrolase;
glycogenase) are calcium metalloenzymes, completely unable to function in the
absence of calcium. By acting at random locations along the starch chain, -
amylase breaks down long-chaincarbohydrates, ultimately yielding maltotriose
and maltose from amylose, or maltose, glucose and "limit dextrin" from
amylopectin. Because it can act anywhere on the substrate, -amylase tends to be
faster-acting than -amylase. In animals, it is a major digestive enzyme, and its
optimum pH is 6.77.0. In human physiology, both the salivary and pancreatic
amylases are -amylases.
The -amylases form is also found in plants, fungi (ascomycetes and
basidiomycetes) and bacteria (Bacillus).
2. Beta- amylase
Another form of amylase, -amylase (alternative names: 1,4--D-
glucanmaltohydrolase; glycogenase; saccharogen amylase) is also synthesized
by bacteria, fungi, and plants. Working from the non-reducing end, -amylase
catalyzes the hydrolysis of the second -1,4glycosidic bond, cleaving off two
glucose units (maltose) at a time. During the ripening of fruit, -amylase breaks
starch into maltose, resulting in the sweet flavor of ripe fruit.
Both -amylase and -amylase are present in seeds; -amylase is
present in an inactive form prior to germination, whereas -amylase and proteases
appear once germination has begun. Many microbes also produce amylase to
degrade extracellular starches. Animal tissues do not contain -amylase, although
it may be present in microorganisms contained within the digestive tract. The
optimum pH for -amylase is 4.05.0.
3. Gamma- amylase
-Amylase (alternative names: Glucan 1,4--glucosidase;
amyloglucosidase; Exo-1,4--glucosidase; glucoamylase; lysosomal -
glucosidase; 1,4--D-glucanglucohydrolase) will cleave (16) glycosidic
linkages, as well as the last (14)glycosidic linkages at the nonreducing end of
amylose and amylopectin, yielding glucose. The -amylase has most acidic
optimum pH of all amylases because it is most active around pH 3.
The wort is cooked and treated with hops, fermented with yeast,
filtered and stabilised.
The aim of the malting process is to achieve maximum production of
enzymes (particularly -amylase and -glucanase) and maximum
depolymerisation of starch and -glucan, with minimal respiration
losses.
Enzymes such as -amylase, exo-peptidase and carboxy-peptidase are
present in the starchy endosperm of the barley, and are activated during
malting. Other enzymes, such as -glucanase, endo-proteases, a-
amylase and pentosanases are formed in the aleurone layer of the
barley during malting.
Glucanases
These are enzymes that break down glucan, which is a polysaccharide
made of several glucose sub units that is mostly present in plants like
barley.
Used in enological practices during the aging process of wine,
particularly when aged on lees with microxygenation.
The enzyme aids in autolysis of yeast cells to release polysaccharides
and mannoproteins, which is believed to aid in the color and texture of
the wine.
It may be an -glucanase or a -glucanase
It can be produced by Neocallimastigomycota, a phylum of anaerobic
fungi found in the digestive tracts of herbivores
It is therefore essential to get as much -glucan broken down during
malting as possible, since it will cause problems later in the process by
increasing viscosity and wort run-off times and by causing hazes.
Other Applications
In production of wine, it is used in the clarification and in th
enhancement of filterability.
Feed additive in poultries, for it enhances digestability of plant based
animal feeds by glucan breakdowns.
Starch production, as well as baked goods and diet products.
May also serve as a bleaching agent in textile industries
Proteases
Are also called peptidase or proteinase that performs proteolysis or
the protein catabolism by hydrolysis of the peptide bonds that link
amino acids together in a polypeptide chain.
They have evolved multiple times, and different classes of protease can
perform the same reaction by completely different catalytic
mechanisms.
It can be found in animals, plants, bacteria, archaea and viruses.

USES
Fermentation
Alpha and beta amylases are important in brewing beer and liquor
made from sugars derived from starch. In fermentation, yeast ingest sugars and
excrete alcohol. In beer and some liquors, the sugars present at the beginning of
fermentation have been produced by "mashing" grains or other starch sources
(such as potatoes). In traditional beer brewing, malted barley is mixed with hot
water to create a "mash," which is held at a given temperature to allow the
amylases in the malted grain to convert the barley's starch into sugars. Different
temperatures optimize the activity of alpha or beta amylase, resulting in different
mixtures of fermentable and unfermentable sugars. In selecting mash temperature
and grain-to-water ratio, a brewer can change the alcohol content, mouthfeel,
aroma, and flavor of the finished beer.
In some historic methods of producing alcoholic beverages, the
conversion of starch to sugar starts with the brewer chewing grain to mix it with
saliva. This practice is no longer widely in use.

BREWING INDUSTRY (GLUCANASES & PROTEASES)

What is BREWING?
-is the production of beer by steeping a starch source (commonly cereal
grains) in water and fermenting the resulting sweet liquid with yeast

BARLEY
-a hardy cereal that has coarse bristles extending from the ears. It is
widely cultivated, chiefly for use in brewing and stockfeed
MALT
Malt is germinated cereal grains that have been dried in a process
known as "malting". The grains are made to germinate by soaking in water, and
are then halted from germinating further by drying with hot air.
BREWING INDUSTRY (BETA GLUCANASES)
Beta Glucanase - Enzyme that breaks down Beta Glucan
Beta Glucan
Long chains similar to Amylopectin found in the barley kernel. It is
much more massive and has many more 1,6 branches (and additional
1,3 branches).
sugars that create up to 60% of the cell walls of bacteria, fungi, yeasts,
algae, lichens, and plants, such as oats and barley.
 a polysaccharide made of glucose molecules linked together into long residual extract
chains that humans cannot readily digest calories
As a kind of indigestible fiber, they may become viscous in the Brewing process Extract yield Breaking down more starch more
intestinal tract and slow peristalsis (intestinal contractions). consistently
Make starch and proteins less available for enzymatic hydrolysis
The production of highly attenuated beers is typically based on:
May also prevent optimal extraction
Increasing the release of fermentable sugars from starch (amylose and
Leads to high wort and beer viscosity
amylopectin)
Make wort and beer filtration processes much more difficult
Diluting the excess of alcohol formed during fermentation
Beta-Glucanases
with water As a result, highly attenuated beers will typically
Beta-Glucanase represents a group of carbohydrate enzymes which
have lower alcohol, residual extract, and calories compared
break down glycosidic bonds within beta-glucan. It helps break down beta-linked
to their regular beer versions
glucose polymers. This digestive action is broadly classified as a gumase activity.
Beta-Glucanase works best at 104 - 113F and is denatured around 144F.
Beta-glucanase enzyme treatment will improve, via hydrolysis of beta-
glucan, the filterability of wines made from grapes. As it degrades beta glucans,
this rest will liberate free ferrulic acid. Free ferrulic acid is important in wheat
beers because it is decarboxylized into 4-vinyl guaiacol during fermentation,
which contributes to the characteristic phenolic flavor.
Beta-glucanase is particularly useful in industrial and agricultural
processing applications due to its high degree of stability. This enzymes
resistance to denaturation by higher than moderate temperature and pH extremes
makes it particularly durable. It hydrolizes beta D-glucan components, by passing
branch points which often serve as blocking points to other exo-acting enzymes.
- ensures a fast, effective breakdown of the starch molecules into
fermentable sugars
BREWING INDUSTRY (AMYLOGLUCOSIDASE AND PULLULANASE) FACTS ON ATTENUATION CONTROL
Worts produced under normal brewing conditions generate attenuation
Amyloglucosidase up to 75% Traditional brewing methods can not normally compensate
Amyloglucosidase is derived from a selected strain of Aspergillus for the lack of enzymes necessary to attenuate wort over 75%.
niger. Approximately 25 % of the carbohydrate extract is present in the beer
Potential applications include production of highly attenuated low carbohydrate as non-fermentable short chain dextrin
beers. Difficult to achieve predictable and targeted attenuation levels
Increased attenuation consistently due to variations in quality of raw materials
Improved fermentability of worts
An alternative to priming sugar
Amyloglucosidase hydrolyzes terminal a1,4- and a1,6-glucosdic bonds
(glucoseglucose bonds) in polysaccharides (e.g., starch, dextrins,
glycogen) ;
removing glucose units sequentially from the non-reducing end of the
molecule.
The enzyme will also cleave maltose and maltosides (maltotriose,
maltotetraose, etc.)

Pullulanase
Pullulanases (also known as isoamylases) cleave large molecules
of polysaccharides, such as starches, at specific locations within the molecules.

Pullulanase is used predominantly in:

conjunction with other enzymes that break down starch (glucoamylase)
in the saccharification of starch: it makes the conversion of starch
into glucose and glukose syrup more efficient
occasionally as a baking enzyme Main benefits of using enzymes
in BEER BREWING Possibility to make super-attenuated beers
Cost-effective way to make light beers
ENZYMES IN BREWING INDUSTRY Cost-effective way to keep consistent fermentabilty with varying malt
quality.

BREWING INDUSTRY (- ACETOLACTATE DECARBOXYLASE)

Adoption of Enzyme Technology in the brewing industry has been used:
In order to provide a higher degree of freedom in choice of starting
materials used in beer production
to allow simplified equipment
to reduce proper time
& to make possible a higher control over product characteristics

Beer Maturation
All steps in the beer production process which take place after the main
Improved Attenuation Control fermentation
from the point of cessation of yeast growth to the final filtration of the
What is Attenuation in brewing? beer
A measure of the degree to which sugar in wort has been fermented comprises storage of the beer for extended periods of time at low
into alcohol in beer temperatures
It is typically measured as ADF or RDF

ADF :Apparent Degree of Fermentation

The original gravity (OG) of the wort prior to fermentation
- The apparent extract (AE) of the beer after fermentation is
complete AE is the final specific gravity of beer converted to
degrees Plato. This measurement does not take into account the
lower density of alcohol compared to water
RDF :Real Degree of Fermentation The original gravity (OG) of the wort
prior to fermentation
-The real extract (RE) of the beer after fermentation is complete RE is
The final gravity of the beer, converted to degrees Plato and corrected to account
for the lower density of alcohol compared to water.

Why attenuation control is important?

Degree of starch conversion has a direct impact on several brewing
parameters Beer specifications alcohol content

During maturation a number of important objectives are achieved.
saturated with carbon dioxide
insoluble material is removed
colloidal stability of is improved
flavor of the beer is allowed to reach its final level
Flavor Maturation
can be considered the rate limiting factor in beer maturation
Flavor of beer and related fermentable products are due to volatile
Esters
Alcohols
Ketones
Aldehydes
Sulphur compouds
Diketone, diacetyl plays a major role
The precise adjustment of the concentration of this compound in the
beer is one of the most important objectives of the beer maturation
process since diacetyl in small concentrations contributes to the desired
flavor of the beer but conveys an objectionable character to the beer if
present in too large concentration.
"When exactly is a beer mature?"
When the diacetyl level drops below a certain limit (about 0.07 ppm)
*Diacetyl gives beer an OFF FLAVOR LIKE BUTTERMILK and one
of the MAIN REASONS for MATURING a beer is to allow the
diacetyl to drop to a level where it can't be taste
Diacetyl is FORMED by the non-enzymatic oxidative decarboxylation
of -acetolactate, which is produced by the yeast during primary
fermentation.
The YEAST removes the diacetyl again during the beer maturation
stage by conversion to acetoin, which has a much higher flavour
threshold value. In fact, acetoin is almost tasteless compared with
diacetyl.
-Acetolactate Decarboxylase
An enzyme
Family of lyases (carboxy-lyases)
Known to occur in a variety of bacteria
Strains of Enterobacter, Aeromonas, Streptococcus and etc.
Depends on this enzyme to produce acetoin and 2,3-butanediol
By adding the enzyme -acetolactate decarboxylase (ALDC) (e.g.
Novozymes' Maturex) at the BEGINNING of the primary fermentation
process, it is possible to bypass the diacetyl step (Figure 7) and convert
-acetolactate directly into acetoin.
Most of the -acetolactate is degraded before it has a chance to oxidise
and less diacetyl is therefore formed.
This makes it possible to shorten or completely eliminate the
maturation period (3) and (4). The brewery enjoys greater fermentation
and maturation capacity without investing in new equipment.
CULINARY (PAPAIN)
PAPAIN
is an endolytic plant cysteine protease enzyme which is isolated from
papaya (Carica papaya L.) latex
obtained by cutting the skin of the unripe papaya and then collecting
and drying the latex which flows from the cut
greener the fruit, more active is the papain
enzyme belongs to the papain superfamily, as a proteolytic enzyme,
papain is of crucial importance in many vital biological processes in all
living organisms
shows extensive proteolytic activity towards proteins, short chain
peptides, amino acid esters and amide links and is applied extensively
in the fields of food and medicine
STRUCTURE
single peptide chain of 211 residues folded into two parts that form a
cleft
cysteine hydrolase that is stable and active under a wide range of
conditions
very stable even at elevated temperatures (60 70degC)
Unusually defiant to high concentrations of denaturing agents, such as,
8M urea or organic solvent like 70% EtOH
PRODUCTION
usually produced as a crude, dried material by collecting the latex from
the fruit of the papaya tree
latex is collected after scoring the neck of the fruit, where it may either
dry on the fruit or drip into a container
This latex is then further dried and is classified as a dried, crude
material
A purification step is necessary to remove contaminating substances.
This purification consists of the solubilization and extraction of the
active papain enzyme
PAPAIN AS MEAT TENDERIZER
Able to break down organic molecules made of amino acids, know as
polypeptides and thus plays a crucial role in diverse biological
processes in physiological and pathological states, drug designs,
industrial uses such as meat tenderizers and pharmaceutical
preparations.
papain has been extensively used as a common ingredient in the
brewery and in the meat and meat processing (Khanna and Panda,
2007)
can act as a clarifying agent in many food industry processes
As a protein digestant, papain is used in combating dyspepsia and other
digestive disorders and disturbances of the gastrointestinal tract
(Huetet al.,2006)
OTHER USES
Medical use
Pharmaceutical preparation
MEDICAL USES
debris-removing agent with no harmful effect on sound tissues because
of the enzymes specificity, acting only on the tissues (Flindt, 1979).
Papain-based gel has also been reported as a potential useful in
biochemical excavation procedures for dentin
has advantages for being used for chemomechanical dental caries
removal since it does not interfere in the bond strength of restorative
materials to dentin (Lopes et al., 2007).
has a long history of being used to treat sports injuries, other causes of
trauma and allergies (Dietrich, 1965)
Fortunately papain has a proven track record in managing all of these
conditions with clinical evidence of significant benefits for use of
papain protease enzyme in cases of sports injury
Traditionally, as a topical agent for the treatment of cuts, rashes, stings,
burns, bedsores and wounds
Papain ointment is commonly made from fermented papaya flesh, and
is applied as a gel-like paste
Resistance to fungus
has powerful resistance to redness and may help reduce joint and
prostate irritation
Papain holds compounds that may aid in protecting the body from
cellular damage caused by free radicals
PHARMACEUTCAL AND INDUSTRIAL USES
Papain has for quite a long time been used in pharmaceutical
preparations of diverse food manufacturing applications as the
production of high quality kunafa and other popular local sweets and
pastries
has been reported to improve meltability and stretchability of Nabulsi
cheese with outstanding fibrous structure enhancing superiority in the
application in kunafa, pizza and pastries(Abu-Alruzet al., 2009)
pharmaceutical products in gel based a proteolyticcisteine enzyme,
papain presents antifungal, antibacterial and anti-inflammatory
properties (Chukwuemeka and Anthoni, 2010).
added to some toothpastes and mint sweets as a tooth whitener
DAIRY (RENNIN)
Dairy products
 - milk and any of the foods made from milk, including butter, cheese, ice
cream, yogurt, and condensed and dried milk.
The Coagulation of Milk
Rennin
- also called Chymosin
- a protease (protein-digesting enzyme) that curdles milk by
transforming caseinogen into insoluble casein
- it is found only in the fourth stomach of cud-chewing animals, such as
cows.
- its action extends the period in which milk is retained in the stomach of
the young animal. In animals that lack rennin, milk is coagulated by the
action of pepsin, as is the case in humans.
- Chymosin efficiently converts liquid milk to a semisolid like cottage
cheese, allowing it to be retained for longer periods in the stomach.
- Chymosin secretion is maximal during the first few days after birth,
and declines thereafter, replaced in effect by secretion of pepsin as the
major gastric protease.
- Chymosin is secreted as an inactive proenzyme called prochymosin
that, like pepsin, is activated on exposure to acid. Chymosin is also
similar to pepsin in being most active in acidic environments, which
makes sense considering its mission.
- Aside from its physiologic role, chymosin is also a very important
industrial enzyme because it is widely used in cheesemaking. In days
gone by, chymosin was extracted from dried calf stomachs for this
purpose, but the cheesemaking industry has expanded beyond the
supply of available calf stomachs (remember that these have to be from
young calves). It turns out that many proteases are able to coagulate
milk by converting casein to paracasein and alternatives to chymosin
are readily available. "Rennet" is the name given to any enzymatic
preparation that clots milk.
Rennet vs Rennin
Rennin
Rennin is an enzyme that is found in rennet, which in turn is found in
the gastric juice of a mammal's stomach. The function of rennin is to curdle the
milk the animals digest, thereby making it easier to absorb; for this reason it is a
key element in making cheese from cow's milk. While rennet contains many
different enzymes, rennin is the only one that serves to coagulate milk. For the
home cheesemaker, rennin can be bought in tablet or powdered form from a
supermarket.
Rennet
Rennet is combinations of different enzymes that help mammals digest
milk. Each animal produces a different type of rennet, therefore specific types are
used for certain cheeses, such as kid goat rennet to make goat's cheese and lamb
rennet for sheep's cheese. It is usually only extracted from the stomachs of young
animals, as rennet in older animals contains little or no rennin.
Process
Rennet is extracted from the stomach by adding acid, which separates
the different enzymes. The rennet extract is filtered several times until a
concentrated substance is achieved. Manufacturers make 1 kilogram of cheese that
usually contains around 0.0003 grams of rennet. If you are buying rennet to make
your own cheese, you simply dissolve or stir it into water before adding it to the
milk.
Production and use of microbial enzymes for dairy processing
1. Bovine Chymosin
- Animal rennet (bovine chymosin) is conventionally used as
a milk-clotting agent in dairy industry for the manufacture
of quality cheeses with good flavour and texture.
- Rennin acts on the milk protein in two stages resulting in
coagulation of milk
by enzymatic action - the resultant milk
becomes a gel due to the influence of calcium
ions and the temperature used in the process
by nonenzymatic action
2. Recombinant Chymosin
- Due to shortage of calf stomachs and the economic value of
cheese rennet, gene for calf chymosin was one of the first
genes for mammalian enzymes that was cloned and
expressed in microorganisms
- With the development of genetic engineering, it became
possible to extract rennet-producing genes from animal
stomach and insert them into certain bacteria, fungi or
yeasts to make them produce chymosin during
fermentation.
- The genetically modified microorganism is killed after
fermentation and chymosin is isolated from the
fermentation broth, so that the fermentation-produced
chymosin (FPC) used by cheese producers does not contain
any GM component or ingredient.
- FPC contains the identical chymosin as the animal source,
but produced in a more efficient way. FPC products have
been on the market since 1990 and have been considered in
the last 20 years the ideal milk-clotting enzyme.
- Aspergillus niger
- Kluyveromyces lactis
20% of the proteins found in milk are categorized as whey (way)
proteins. The water that is expelled in cheese making is called whey
because it contains these proteins.
The chemical reaction of cheese making:
Binds proteins
Encapsulates fat cells
Expels whey
Results in a yummy treat
This is what milk looks like under a microscope at 1000x
magnification.
There are three elements we introduced to the milk:
Heat to help break down proteins
Food grade citric acid to lower the pH and remove a negative
charge.
Rennet to break down a hairy exterior layer on the casein
micelles.
The casein micelles carry a negative charge and a pH of 6.5
Introduction of heat and food grade acetic acid lowers the pH to 4.6
Rennet (chymosin) is introduced and eliminates the hairy outside layer
of the casein micelles.
Casein micelles bind together, encapsulate the fat cells and expel the
whey.
Finally, the kneading and stretching elongate protein chain to achieve
mozzarellas stringiness.
DAIRY (LIPASE)
Lipase is a naturally occurring enzyme found in the stomach and
pancreatic juice.
Lipases are one of the important groups of biocatalysts used in
biotechnological applications.
Their function is to digests fats and lipids, helping to maintain correct
gall bladder function.
It controls the amount of fat being synthesized and that which is burned
in the body, reducing adipose tissue.
Lipases are used for synthesis of fine chemicals, agrochemicals and
new polymeric materials
Research on microbial lipases, has increased due to their great
commercial potential.
Lipases have been used in various industries such as dairy, food,
detergents, textile, pharmaceutical, cosmetic, and biodiesel industries.
Applications of Lipases
Microbial lipases constitute an important group of biotechnological
valuable enzymes, mainly due to versatility of their applied properties
and ease of mass production.
Microbial lipases are widely diversified in their enzymatic properties
and substrate specificity, which make them very attractive for industrial
applications.
 They are welcomed as alternatives to traditional chemical-based
Dairy Industry technology, and can replace synthetic chemicals in many processes.
Lipases are extensively used in the dairy industry for hydrolysis of They are more specific in their action than synthetic chemicals.
milk fat. The dairy industry uses lipases to modify the fatty acid chain They allow some processes to be carried out which would otherwise be
lengths, to enhance the flavours of various cheese. impossible.
Current applications also include the acceleration of cheese ripening
and the lipolysis of butter, fat and cream. Applications
The traditional sources of lipases for cheese flavour enhancement are
animal tissues, especially pancreatic glands (bovine and porcine) and
pre-gastric tissues of young ruminants (kid, lamb and calf).
Enzyme modified cheese is used when cheese is incubated in the
presence of enzymes at elevated temperature in order to produce a
concentrated flavour.
The concentration of fat is 10 times higher in enzyme modified cheese
to that of normal cheese.
In 1976, Unilever filed a patent describing a mixed hydrolysis and
synthesis reaction to produce a cocoa butter substitute using an
immobilized lipase.
Gastric lipases have been used to accelerate ripening and flavour
development of many cheese types, including cheddar, provolone and
ras cheeses.
Lipase addition enhances the rate of fatty acid liberation, which
accelerates flavour development relative to control.
When a cock-tail of fungal protease and lipase were used, cheddar
cheese developed a highly soluble proteins and free fatty acids and
displayed better flavour within three months of ripening. But take note
high levels of enzyme during ripening may result in excessive
enzymatic reactions that impart undesired characeristics and reduce the
yield.

CONCLUSION
The tremendous potential of lipases in food and allied technology
applications shows the need to develop novel cost-effective
technologies for increased production, scaling up and purification of
this versatile enzyme.
The large number of hydrolytic applications, like flavour development
in dairy products (cheese, butter, margarine), alcoholic beverages, milk
chocolate, etc., is a promising field of lipase enzyme.
Production of diet control stuff, meat technology and the processing of
sausages are some areas in food industry with commercial potential.
The properties of lipases are being improved by protein engineering
and genetic engineering to widen their applications in extreme
conditions.
Other than food industry, lipases have been applied in the synthesis of
fine chemicals, biodiesel production, the production of biopolymeric
materials, the detergent industry, organic synthesis, the paper and pulp
industry, the synthesis of imgredients for personal care products, the
synthesis of surfactants and of structural triglycerides, the
oleochemical industry, agrochemicals production, the pesticide
industry, and in environmental management.
The rapid boom in the future prospects of lipase technology is evident
from the large number of patents, publications and research reports in
the recent years and indications are that this growth will be sustained
for many years.

ENZYMES IN THE FOOD INDUSTRY

Background
Extracted from edible plants and the tissues of food animals
Produced by microorganisms (bacteria, yeasts, and fungi)
Rennet
-a natural enzyme mixture from the stomach of calves or other
domestic animals
-used in cheese making for centuries

History FOOD PROCESSING (PROTEASES)

2000 BC
Protein is one of the three major food groups needed for proper nutrition.
-The Egyptians and Sumerians developed fermentation for use in
PROTEASES are also called as:
brewing, bread-baking and cheese-making.
Proteolytic enzyme
800 BC
-Calves' stomachs and the enzyme, chymosin, were used for cheese- Peptidase
making. Proteinase
1878 Protease
-The components of yeast cells which cause fermentation were - is the digestive enzyme needed to digest protein.
identified and the term "enzyme" was first used, derived from the Greek term - constitute the largest group of enzymes in bio-industry accounting for
meaning "in yeast". about 60% of the total enzyme market.
1926 - they play an invincible role in industrial biotechnology, especially in
detergent, FOOD and pharmaceutical area.
-Enzymes were first shown to be proteins.
1980s
Best Proteolytic Enzymes Food Sources
-Enzyme preparations were developed to improve the digestibility and -pineapple, papaya, ginger, sauerkraut, kefir, kiwi, yogurt, miso soup
nutrient-availability of certain animal feeds.
1982
-The first food application of a product of gene technology, alpha-
amylase, took place.
1988
-Recombinant chymosin was approved and introduced in Switzerland,
marking an early approval of a product of gene technology for a food use.
1990
-Two food processing aids obtained using gene technology: an enzyme THE 3 MOST COMMON PROTEOLYTIC ENZYMES
for use in cheese-making in the US, and a yeast used in baking in the UK. 1. PEPSIN
- Is naturally occurring in the gut and essential for the digestion and breaking
Advantages down of proteins.
2. BROMELAIN
- Is a protease enzyme found in the juice and stem of pineapple.
3. PAPAIN
- Another protease enzyme and seemingly the most popular, is derived from the
latex of the papaya and is also similar to pepsin.
FUNCTIONS OF PROTEASES ENZYMES
1. Improved digestibility
2. Improved solubility
3. Foaming properties, whipping properties, gel strength
Food processing
-utilizes several enzymes for production of quality and nutritional food products.
Proteases have important roles mainly in:
Baking, production of soy sauce, preservation of juices, cheesemaking, meat
tenderizing.
APPLICATION IN BAKER INDUSTRY
Used on a large commercial scale in the production of bread, baked
goods, crackers and waffles.
To reduce mixing time,
To decrease dough consistency,
To dough uniformity,
To regulate gluten strength in bread,
To control bread texture and to improve flour.
APPLICATION IN DAIRY INDUSTRY
The major applications of proteases in the dairy industry is the
manufacture of cheese.
In cheese making, the primary function of proteases is to hydrolyze the
specific peptide bond to generate para-k-casein and macro peptides.
Trypsin
It is a serine protease from the PA clan superfamily, found in
the digestive system of many vertebrates, where it hydrolyses proteins.
It is formed in the small intestine when its proenzyme form,
the trypsinogen produced by the pancreas, is activated.
Trypsin cleaves peptide chains mainly at the carboxyl side of the amino
acids lysine or arginine, except when either is followed by proline. It is
used for numerous biotechnological processes. The process is
commonly referred to as trypsin proteolysis or trypsinisation, and
proteins that have been digested/treated with trypsin are said to have
been trypsinized.
It catalyzes the hydrolysis of peptide bonds, breaking down proteins
into smaller peptides. The peptide products are then further hydrolyzed
into amino acids via other proteases, rendering them available for
absorption into the blood stream.
Trypsin is produced as the inactive zymogen trypsinogen in
the pancreas. When the pancreas is stimulated by cholecystokinin, it is then
secreted into the first part of the small intestine (the duodenum) via the pancreatic
duct. Once in the small intestine, the enzyme enteropeptidase activates
trypsinogen into trypsin by proteolytic cleavage. Auto catalysis can happen with
trypsin using trypsinogen as the substrate. This activation mechanism is common
for most serine proteases, and serves to prevent autodegradation of the pancreas.
Tryptic digestion is a necessary step in protein absorption as proteins
are generally too large to be absorbed through the lining of the small
intestine.
Human trypsin has an optimal operating temperature of about 37C.
As a protein trypsin has various molecular weights depending on the source. For
example, a molecular weight of 23.3 kDa is reported for trypsin from bovine and
porcine sources.
Trypsin is available in high quantity in pancreases, and can be purified
rather easily. Hence it has been used widely in various biotechnological
processes.
In a tissue culture lab, trypsin is used to re-suspend cells adherent to
the cell culture dish wall during the process of harvesting cells. Some
cell types have a tendency to "stick" - or adhere - to the sides and
bottom of a dish when cultivated in vitro. Trypsin is used to cleave
proteins bonding the cultured cells to the dish, so that the cells can be
suspended in fresh solution and transferred to fresh dishes.
Trypsin can also be used to dissociate dissected cells (for example,
prior to cell fixing and sorting).
Trypsin can also be used to dissolve blood clots in its microbial form
and treat inflammation in its pancreatic form.
In Food:
Trypsin can be used to break down casein in breast milk. If trypsin is
added to a solution of milk powder, the breakdown of casein will cause
the milk to become translucent. The rate of reaction can be measured
by using the amount of time it takes for the milk to turn translucent.
Commercial protease preparations usually consist of a mixture of various protease
enzymes that often includes trypsin. These preparations are widely utilized in food
processing:[12]
as a baking enzyme to improve the workability of dough;
in the extraction of seasonings and flavourings from vegetable or
animal proteins and in the manufacture of sauces;
to control aroma formation in cheese and milk products;
to improve the texture of fish products;
to tenderize meat;
during cold stabilization of beer;
in the production of hypoallergenic food where proteases break down
specific allergenic proteins into nonallergenic peptides. For example,
proteases are used to produce hypoallergenic baby food from cows milk
thereby diminishing the risk of babies developing milk allergies.
FOOD PROCESSING (CELLULASE and PECTINASE)
A. CELLULASE
Are enzymes that hydrolyze -1, 4 link- ages in cellulose chains. They
are produced by fungi, bacteria, protozoans, plants, and animals.
Contain non-catalytic carbohydrate-binding module s (CBM s) and/or
other functionally known or unknown modules, which may be located
at the N- or C-terminus of a catalytic module.
Are increasingly being used for a large variety of industrial purposes
in the textile industry, pulp and paper industry, and food industry, as
well as an additive in detergents and improving digestibility of animal
feeds.
Breaks down the cellulose molecule into monosaccharides ("simple
sugars") such as beta-glucose, or shorter polysaccharides and
oligosaccharides.
Have a wide range of potential applications in food biotechnology as
well. The production of fruit and vegetable juices requires improved
methods for extraction, clarification, and stabilization.
Have an important application as a part of macerating enzymes
complex (cellulases, xylanases, and pectinases) used for extraction and
clarification of fruit and vegetable juices to increase the yield of juices.
Used for commercial food processing in coffee. It performs hydrolysis
of cellulose during drying of beans.
Most animals (including humans) do not produce cellulase in their
bodies and can only partially break down cellulose through fermentation, limiting
their ability to use energy in fibrous plant material.
Mechanism of Cellulolysis
The three types of reaction catalyzed by cellulases:
1. Breakage of the noncovalent interactions present in the amorphous structure of
cellulose (endocellulase)
2. Hydrolysis of chain ends to break the polymer into smaller sugars
(exocellulase)
3. Hydrolysis of disaccharides and tetrasaccharides into glucose (beta-
glucosidase).
B. PECTINASE
An enzyme that breaks down pectin, a polysaccharide found in plant
cell walls.
Commonly referred to as pectic enzymes.
It is useful because pectin is the jelly-like matrix which helps cement
plant cells together and in which other cell wall components, such as
cellulose fibrils, are embedded.
Pectinase enzymes are commonly used in processes involving the
degradation of plant materials, such as speeding up the extraction of
fruit juice from fruit, including apples and sapota.
Pectinases have also been used in wine production since the 1960s.
Extracted from fungi such as Aspergillus niger. The fungus produces
these enzymes to break down the middle lamella in plants so that it can
extract nutrients from the plant tissues and insert fungal hyphae.
If pectinase is boiled it is denatured (unfolded) making it harder to
connect with the pectin at the active site, and produce as much juice.
Are used for extracting juice from pure. This is done when the
enzyme pectinase breaks down the substrate pectin and the juice is
extracted. The enzyme pectinase lowers the activation energy needed
for the juice to be produced and catalyzes the reaction.
 used in processes involving the degradation of plant materials, such as 2. The presence of pectin in finished wine causes a haze or slight cloudiness.
speeding up the extraction of fruit juice from fruit Pectinase is used to break this down and so clear the wine.

The function of pectinase in brewing is twofold

1. It helps break down the plant (typically fruit) material and so helps the
extraction of flavours from the mash.