Fermentation Technology

Module 7 (L3)

Commercial Enzymes: Proteases

Proteases act on protein and hydrolyze into peptides & di-peptides and are extracellular.
Proteases may be classified as acid proteases, neutral proteases and alkaline proteases on the
basis of pH optima. On the basis of their origin proteases may be classified as (i) Plant proteases:
papain, malt protease; (ii) Animal protease: rennin, trypsin; (iii) Microbial: bacterial, yeast,
mold.
Raw materials: Starch, dextrin, glycogen, glucose, galactose, mannose etc. Nitrogen source
includes: yeast extract, meat extract, peptone etc.
Microorganisms: Bacillus amyloliquefaciens. B. subtilis, B. licheniformis
Aspergillus flavus, A. oryzae
Process conditions: temperature: 30-45°C (mesophilles); 60°C (thermophilles), Incubation time:
2-3 days for bacterial and 5-7 days for mold.
Recovery: involves separation of the cell free liquor by filtration or centrifugation followed by
precipitation by ammonium sulfate.
Application of proteases:
(1) Bread making
(2) Cheese making & ripening
(3) Meat tenderization
(4) Protein hydrolysate (flavor)
(5) Detergent and cleaning
(6) In pharmaceuticals
Effect of proteolytic enzymes on protein quality:
(1) to increase extractability of proteins in foods.
(2) to improve the digestibility of the food's protein.
(3) to solubilize protein denatured in the course of processing of the food.
(4) to manufacture soymilk.
(5) to prepare hydrolysates parenteral nutrition.
(6) to improve the nutritional quality of the animal feeds.
(7) to use protein hydrolysate as a synergist with phenolic antioxidants.

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 Fermentation Technology

(8) to maintain protein solubility in acid media so that the proteins can be incorporated in to a
carbonated beverages.

Lipases
Lipases hydrolyze triglycerides to free fatty acid, partial glycerides and glycerol. Their natural
substrates are triglycerides of long chain fatty acids which are insoluble in water.

CH2OCOR Lipase CH2OH RCOOH

CHOCOR' + 3H2O CHOH + R'COOH

CH2OCOR" CH2OH R"COOH

Mixed triglyceride Glycerol Fatty acids

Lipases can be divided into two groups based on their positional specificity:
Non specific lipases: release fatty acids from all three positions of the glycerol molecule.
1,3 specific lipase: release fatty acids only from 1,3 positions. They convert triglycerides to 1,2 -
(2,3-) diglycerides, which in turn are chemically unstable and undergo acylmigration to give 1,3-
diglycerde & 1-monoglyceride respectively.
Substrates: wheat bran, rice bran, mustured oil cake, soybean oil cake.
Microorganisms: Aspergillus niger, A. japonicus, Candida cylendracea.
Process conditions: temperature: 30-35°C, pH: neutral; Incubation time: 5-7 days.
Recovery: involves separation of the cell free liquor by filtration or centrifugation followed by
precipitation by ammonium sulfate. For purification, gel filtration and ion exchange
chromatography are used. Recently affinity chromatographic techniques have been used to purify
lipases.
Applications:
(1) In detergent industries.
(2) As food ingredients e. g. flavour development for dairy products like cheese, butter,
margarine, alcoholic beverages, milk chocolate and sweets.
(3) For removal of pitch from pulp from pulp produced in the paper industry.

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 Fermentation Technology

Glucose oxidase
Glucose oxidase or β-D-glucopyranose aerodehydrogenase, a typical aerobic dehydrogenase
enzyme produced by Aspergillus niger, A. oryzae, Penicillium notatum, oxidises glucose to
gluconic acid in the presence of molecular oxygen.
Glucose oxidase
Glucose + O2 + H2O Glucono - δ - lactone + H2O2
H2O

Gluconic acid
The H2O2 formed is decomposed by the enzyme catalase, which is either produced by the same
organism or is added to commercial glucose oxdase preparation.
Catalase
2H2O2 2H2O + O2
The enzyme is specific for glucose which it oxidizes 400 times faster than other sugars. Its
antibacterial property is attributed to the H2O2 formation. In the presence of catalase, owing to
decomposition of the H2O2, there is no antibacterial action.
At room temperature the optimum pH is 5.5, with an active range of pH 3-8.5. The enzyme is
destroyed (90%) with in 2 min at 80°C, 3 min at 70°C & 10 min at 65°C.
The prosthetic group of the enzyme is alloxazine-adenine dinucleotide or flavin-adenine
dinucleotide. It is a dehydrogenase (uses oxygen as hydrogen acceptor) rather than an oxidase
enzyme.
The practical application of the glucose oxidase preparations is based on the removal of oxygen
from the beverages or from the air space in a closed food container, or on the removal of glucose
from a food ingredient or food product.
The presence of oxygen may change the flavour and colour of a product and may hasten the
corrosion of cans containing carbonated beverages etc. The presence of glucose may cause
darkening in some foods when drying. Glucose oxidase is used when the removal of either
oxygen or glucose is desirable e. g.
(1) in the preparation of dried egg powder, a small amount of glucose reacts with the egg protein
causing Maillard darkening of the product and some loss in flavour. Oxidation of glucose with
glucose oxide before drying the egg preserves the flavour and prevents darkening of the product.
Glucose oxidase / catalase, together with some H2O2, is added to the egg. Oxygen is supplied to
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 Fermentation Technology

the reaction as liberated from H2O2, by the catalase. The use of 25 ml glucose oxdase preparation
per 500 kg egg white (or 750 ml per 500 kg whole egg) at 30°C, with the frequent addition of
calculated amounts of 35% H2O2 is recommended.
(2) Glucose oxidase with low cellulase content, added to orange soft drinks, preserves the
freshness of flavour. The oxygen s removed by the enzyme from the head space as well as from
the liquid in bottled drinks.
(3) Added to canned beverages, glucose oxidase preparations impede fading of sensitive colours
and retard iron pick up.
(4) Oxidative deterioration of dehydrated foods such as milk powder, cake mixes, encapsulated
flavours etc., is prevented and the shelf life of the products is extended by the use of glucose
oxidase - catalase.
(5) Wrappers for cheese are coated on the inside with glucose oxidase and glucose to prevent
growth of aerobic organisms on the surface of cheese by total removal of oxygen. Process cheese
darkening by oxygen is also prevented.
(6) Glucose oxidase is used as an analytical tool in the clinical diagnosis in determining the sugar
content in blood and in urine.

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