Enzyme in Food &Agricultural Products

Future Applications of Food Enzymes

(1)Tailoring Enzyme Properties & FunctionsTo increase the efficiency of the processultimately lower the cost of operations

Example : two key enzymes involved in corn syrup production* Gelatinization & Complete liquiefaction by α-amylase &

Ca2+ for heat stabilization at 105oCmust be cooledto 60oC & pH ~4.5

* Saccharifying enzyme by glucoamylaseLow 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 orGlucoamylase 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 30-40oC)

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 cheese-ripening 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 & processinge.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 viscosity2. Loss in ability to give a blue color with iodine3. An appearance of reducing groups4. An increase in maltose, glucose & dextrin

•Depolymeration Process•Change : Degree of Polymerization (DP)

Dextrose Equivalent (DE)

Enzyme pH Temperature (oC)

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)

70-7290

β amylase 5.0 (wheat, malt & sweet potato)6.0 (soybean & pea)

Glucoamylase 4.0-4.4 40-65

Optimum pH & Temperature

Criteria α-amylase β-amylase GlucoamylaseReducing group formation

Fixed as equal Fixed as equal Fixed as equal

Loss in viscosity Fast Slow SlowLoss in iodinecolour

Fast Slow Slow

MaltoseProduction

Slow Fast None

Glucose Production

None None Fast

Relative Rate of Amylolytic Activity

Applications of Amylase in Processing

(1)Starch Processingas processing aids to convert starch to starch derivatives& saccharification products.

Glucose syrup Production : acid & enzyme catalystThe advantages of enzyme usage :- Improved the yield- Favourable economics

* milder reaction condition (low T & neutral pH)reducing unwanted side reactionoff-flavor & off-color of HMF & salts

* low energy requirements* eliminate neutralization steps.

(2) Gelatinization & liquefaction of starch(3) Saccharification of starch

Maltooligosakarida Siklodekstrin

Hidrogenasi

Maltitol

Maltosa

Coupling sugar

GlukosaStarch Syrup Solid

Fruktosa Sorbitol

Hidrogenasi

Manitol

Enzim :(1) α-amilase(2) β-amilase(3) Glukoamilase(4) CGT-ase

(cyclodextringlukanotransferase)

(5) Glukosa-Fruktosa

Isomerase

(4)Asam/ (1)

Amilase Khusus

Starch Syrup

(2) (4)

(1) & (3)

Hidrogenasi

PATI

(5)

Penyiapan beberapa gula dari patisecara enzimatis (Kainuma, 1995)

Pengenceran

Dekstrinisasi

Purifikasi

Sakarifikasi

Isomerisasi

Pemurnian

Likuifikasi

Sirup maltosa

Sirup glukosa

Sirup campuran

Sirup fruktosa

Maltodekstrin

Larutan 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-minumankaleng, soft drink, produk susu

Sirup Campuran 42-63 Soft drink, bahan baku industripangan

Sumber : Kennedy et al. (1995)

Aplikasi Produk Hidrolisis Pati

(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

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 exo-cellulases (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.

Five general types of cellulases based on the type of reaction catalyzed

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 (beta-

glucosidase).

Mechanisms of enzymatically hydrolysis of cellulose

C4-4 C5-1

C11-1 Mixed Culture

Microscopic structure of cellulose fraction after 164 hours hydrolysis by several bacteria isolates (Light microscope, 400 x magnificence)

Microscopic structures of cellulose before and after hydrolysis

Light polarized microscope (magnificence 400x)

Light microscope (magnificence 400x)

Classification of Pectic Substances1. Pectic acid pectates2. Pectinic acids pectinates3. Pectins

Pectin Enzymes1. 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 unitvia α-elimination – endo PEL & exo-PELsubstrate : 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 mixture2. The rate of formation of reducing sugars3. The decrease in optical rotation or4. The decrease in precipitability by calcium ions or non polar solvents

Reduction in ~50% of viscosityEndo-PG : 3-5% of glycosidic bondsExo-PG : 10-15% of glycosidic bondsEffects of MineralsThe role of NaCl to prevent product inhibition of enzymeCa2+ 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 Ca2+ 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 : Ca2+

Activity of trans-eliminases can be measured as PGsSplitting of the glycosidic bond by observing the increaseat 235 nm due to formation of the double bonds

Application in Food Processing

(1)Fruit Juice Clarificationlarger use of pectinase mainly to deciduous juice & grape juice

Effects : lower viscositycauses cloud partics to aggregate to larger units

sediments are removed easily by centrifugation orultrafiltration

(2) Treatment of pulp for Juice Extractionrelease of anthocyanins of red fruits into juicee.g. Blackcurrant juice

Red wine

( (3) LiquefactionLiquified juices are almost : - clear (papaya, cucumber)- cloudy (apples, peaches)- pulpy (carrots)

(4) Maceration