Enzyme in Food & Agricultural Products

Enzyme in Food &
Agricultural Products

Future Applications of Food Enzymes
(1)Tailoring Enzyme Properties & Functions
To increase the efficiency of the process
ultimately lower the cost of operations
Example : two key enzymes involved in corn syrup production
* Gelatinization & Complete liquiefaction by α-amylase &
Ca 2+ for heat stabilization at 105 o C
must be cooled
to 60 o C & pH ~4.5
* Saccharifying enzyme by glucoamylase
Low heat stability & acidic pH optimum
Highly desirable enzymes : glucoamylase that is heat stable
at liquifaction temperature
Simultant process of liquifaction & saccharification

Example : Saccharifying Enzymes
Glucoamylase : catalyze the hydrolysis of α-1,4 about 30-50
times faster than the branching (α-1,6) linkage.
The yield ~96% but takes long time (~48 hours)
It is desirable to add a debranching enzyme or
Glucoamylase must be modified to enhance its action on (α-1,6)
bonds.
Dextrozyme (mixture of glucoamylase & pullulanase)
Reduce the process time

Example : Lipase
Transesterification of inexpensive oils (e.g. palm) oil to produce
substitutes for cocoa butter
Melting properties (low and sharp melting point at 30-40 o C)
Palm oil with most of its 1,3 position occupied by palmitic acid
can be transesterified with stearic acid to produce CBS
Desirable enzyme : High stability & high catalytic effiency lipase
Example : Addition of lipase & protease to accelerate the cheeseripening
process is of economic significance
Delicate control a proper flavor profile

(2) In vivo modification of Food Quality
The potential use of modification of enzymatic pathways in plants
(1) Improving functional properties of protein & other components
more suitable for food & feed formulation & processing
e.g. high lysine corn, golden rice
(2) Tailoring the chemical composition and physical properties of
food components for value added products.
e.g. high starch amylose & low moisture potato french fries
absorb less oil on frying
(3) Removing or reducing toxicants or undesirable compounds in
food crops to improve their nutritional value.
e.g. Low HCN cassava

Amylolytic Enzyme
β-amylase
pullulanase
α-amylase

Amylolytic Activity Assay
During hydrolysis, there is :
1. Decrease in viscosity
2. Loss in ability to give a blue color with iodine
3. An appearance of reducing groups
4. An increase in maltose, glucose & dextrin
•Depolymeration Process
•Change : Degree of Polymerization (DP)
Dextrose Equivalent (DE)

Optimum pH & Temperature
Enzyme pH Temperature
( o C)
α-amylase 6.0-7.0 (mammalia)
4.8-5.8 (A. oryzae)
5.8-6.0 (B. subtilis)
5.5-7.0 (B. licheniformis)
β amylase 5.0 (wheat, malt & sweet potato)
6.0 (soybean & pea)
70-72
90
Glucoamylase 4.0-4.4 40-65

Criteria α-amylase β-amylase Glucoamylase
Reducing group
formation
Fixed as equal Fixed as equal Fixed as equal
Loss in viscosity Fast Slow Slow
Loss in iodine
colour
Maltose
Production
Glucose
Production
Relative Rate of Amylolytic Activity
Fast Slow Slow
Slow Fast None
None None Fast

Applications of Amylase in Processing
(1)Starch Processing
as processing aids to convert starch to starch derivatives
& saccharification products.
(2) Gelatinization & liquefaction of starch
(3) Saccharification of starch
Glucose syrup Production : acid & enzyme catalyst
The advantages of enzyme usage :
- Improved the yield
- Favourable economics
* milder reaction condition (low T & neutral pH)
reducing unwanted side reaction
off-flavor & off-color of HMF & salts
* low energy requirements
* eliminate neutralization steps.

Penyiapan beberapa gula dari pati
secara enzimatis (Kainuma, 1995)
Enzim :
(1) α-amilase
(2) β-amilase
(3) Glukoamilase
(4) CGT-ase
(cyclodextrin
glukanotransfer
ase)
(5) Glukosa-
Fruktosa
Isomerase
Maltitol
Hidrogenasi
Maltosa
Maltooligosakarida Siklodekstrin
Starch Syrup
Starch Syrup Solid
Amilase
Khusus
Asa
m/
(1)
Hidrogena
si
Manitol
PATI
(2)
Glukosa
(5)
(1) &
(3)
(4)
(4)
Hidrogena
si
Fruktosa Sorbitol
Coupling sugar

Larutan Pati
Pengenceran
Dekstrinisasi
Sakarifikasi
Purifikasi
Isomerisasi
Pemurnian
Likuifikasi
Maltodekstrin
Sirup maltosa
Sirup glukosa
Sirup campuran
Sirup fruktosa

Aplikasi Produk Hidrolisis Pati
Produk Hidrolisis Pati DE Aplikasi
Maltodekstrin 3-20 Stabilizer, thickener,
filler, lem dan pasta
Sirup Maltosa 48-63 Permen keras,
mencegah higroskopis,
bahan baku untuk fermentasi
Sirup Glukosa 96-98 Soft drink, bahan baku untuk
fermentasi
Sirup Fruktosa - Industri makanan-minuman
kaleng, soft drink, produk susu
Sirup Campuran 42-63 Soft drink, bahan baku industri
pangan
Sumber : Kennedy et al. (1995)

(4) Baking
Main reasons for supplementation of flour with amylase
• Amylase increase the level of fermentable sugar in dough.
• Amylase improve crust colour reducing sugars produced
reacts with other components in bread to give Maillard
Reaction products golden crust
• The flavor of bread is improved by simple sugar and Maillard
reaction products.
• Gas retention properties of the dough are improved by
starch modification resulting from amylase
• The crumb has improved moisture retention properties
• Heat stable amylase retard the staling of bread
Amylase as anti-staling agents

Five general types of cellulases based
on the type of reaction catalyzed
1. Endo-cellulase breaks internal bonds to disrupt the crystalline
structure of cellulose and expose individual cellulose polysaccharide
chains
2. Exo-cellulase cleaves 2-4 units from the ends of the exposed chains
produced by endocellulase, resulting in the tetrasaccharides or
disaccharide such as cellobiose. There are two main types of exocellulases
(or cellobiohydrolases, abbreviate CBH) - one type working
processively from the reducing end, and one type working
processively from the non-reducing end of cellulose.
3. Cellobiase or beta-glucosidase hydrolyses the exo-cellulase product
into individual monosaccharides.
4. Oxidative cellulases that depolymerize cellulose by radical reactions,
as for instance cellobiose dehydrogenase (acceptor).
5. Cellulose phosphorylases that depolymerize cellulose using
phosphates instead of water.

The three types of reaction catalyzed by cellulases:
1. Breakage of the non-covalent interactions present in the crystalline structure
of cellulose (endo-cellulase)
2. Hydrolysis of the individual cellulose fibers to break it into smaller sugars
(exo-cellulase)
3. Hydrolysis of disaccharides and tetrasaccharides into glucose (betaglucosidase).

Mechanisms of enzymatically hydrolysis of
cellulose

Microscopic structure of cellulose fraction after
164 hours hydrolysis by several bacteria isolates
(Light microscope, 400 x magnificence)
C4-4 C5-1
C11-1
Mixed Culture

Microscopic structures of cellulose
before and after hydrolysis
Light microscope (magnificence 400x)
Light polarized microscope (magnificence 400x)

Classification of Pectic Substances
1. Pectic acid pectates
2. Pectinic acids pectinates
3. Pectins

Pectin Enzymes
1. Polygalacturonase (PG) α-1,4 glycosidic bonds
- exo-PG : cleaves from non-reducing ends
- endo-PG : attacks the substrate randomly
2. Pectinesterase (PE)
catalyse the hydrolysis of the methyl ester group
3. Pectat Lyase (PEL)
catalyse the cleavage of non-esterified galacturonate unit
via α-elimination – endo PEL & exo-PEL
substrate : pectate and low methoxyl pectin
4. Pectin lyase (PNL)
catalyse cleavage of esterified galaturonate unit by β-elimination
(endo-enzyme)

Activity Assay of Polygalacturonase
1. The rate of decrease in viscosity of the reaction mixture
2. The rate of formation of reducing sugars
3. The decrease in optical rotation or
4. The decrease in precipitability by calcium ions or non polar solvents
Reduction in ~50% of viscosity
Endo-PG : 3-5% of glycosidic bonds
Exo-PG : 10-15% of glycosidic bonds
Effects of Minerals
The role of NaCl to prevent product inhibition of enzyme
Ca2+ performs a role in binding and or catalysis, maintaining
the conformation of the enzyme
pH optima for most PG enzymes are pH 4.5-6.0

Activity Assay of Pectinesterase (PE)
• The rate of methanol production
• The Ca 2+ precipitability of the pectic acid formed
Activity Assay of Pectate Lyase (PEL)
Acidic (pH 4-5), neutral (pH 7-8.5) & alkaline (pH 9-10)
Activator : Ca 2+
Activity of trans-eliminases can be measured as PGs
Splitting of the glycosidic bond by observing the increase
at 235 nm due to formation of the double bonds

Application in Food Processing
(1)Fruit Juice Clarification
larger use of pectinase mainly to deciduous juice & grape juice
Effects : lower viscosity
causes cloud partics to aggregate to larger units
sediments are removed easily by centrifugation or
ultrafiltration

(2) Treatment of pulp for Juice Extraction
release of anthocyanins of red fruits into juice
e.g. Blackcurrant juice
Red wine

( (3) Liquefaction
Liquified juices are almost :
- clear (papaya, cucumber)
- cloudy (apples, peaches)
-pulpy (carrots)
(4) Maceration