Enzymes in food processing

APPLICATION OF ENZYMES IN FOOD
PROCESSING
BIO 691 (1+0)
SUJAYASREE.O.J
ID NO : 10877
DIVISION OF POSTHARVEST TECHNOLOGY &
AGRICULTURAL ENGINEERING
ICAR-IIHR
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• Processed foods provide convenience, improved shelf-life,
increased palatability and offer variety in the diet.
• Several processing techniques – physical and chemical- are used
for obtaining the finished product.
• Chemical methods are harsh and affect the quality of the
product adversely
ENZYMES ARE THE SOLUTIONS
GREEN CATALYSTS
Easier processes
Efficient raw material utilization
Consistent product quality
Improved sustainability
IndianInstituteofHorticulturalResearch,Bengaluru
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Enzymes are proteins with powerful catalytic function.
ADVANTAGES
• High productivity and catalytic efficiency.
• High specificity – able to discriminate between structurally similar molecules.
• They are more environmentally friendly and produce less residuals compared to
traditional chemical catalysts.
• Work under mild conditions of temperature, pressure and pH.
• Control of enzyme activity and reaction rate.
• Most of them are quite heat labile
• They are natural and relatively innocuous components of agricultural materials
that are considered “safe” for food and other nonfood uses.
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Approaches for enzyme production
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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.
SOURCES OF FOOD ENZYMES
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Conventional
• Differential solubility–pH.
• Salting out
• Organic solvents
• Chromatography – ion
exchange
• Gel filtration
• Affinity and hydrophobic
chromatography
• Electrophoresis
• Two phase aqueous
extraction and
• Affinity precipitation
NEW APPROACH
• Genetic engineering
• Higher yields and purity
• Cost effective
• Reusable, stable enzyme
preparations.
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Filamentous fungi
Aspergillus oryzae
Aspergillus niger
Fusarium venenatum
Trichoderma reesei
Yeast
Saccharomyces cerevisiae
Klyveromyces lactis
Bacteria
Bacillus licheniformis
Bacillus subtilis
Escherichia coli
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APPLICATIONS IN FOOD INDUSTRY
❖ Improving nutritional quality & bioavailability
❖ Recovery of new ingredients
❖ Food modification
❖ Improving sensory quality –flavor development
❖ Partially replacing some of the chemical additives
❖ Increasing yields
❖ Beverage clarification
❖ Bakery aids
❖ Meat tenderization
❖ Milk coagulating
❖ Protein hydrolysate preparation
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JUICE EXTRACTION & CLARIFICATION
To improve the yield of juice
To liquefy the entire fruit
Maximum utilization of raw material
To improve the colour and aroma
To clarify the juice
To break down all insoluble CHO (such as pectins,
hemicelluloses and starch)
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There are mainly two groups of enzyme which are used in fruit
juice industry : a) Pectinases b) Amylases
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Selection of fruits
Sorting and washing & crushing
Enzyme treatment
Juice extraction
Juice
Deaeration
Straining or filtration
Enzyme clarification
Flow chart of juice processing
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Filtration
Pasteurization (85° C for 5 min)
Filling and or storage (sterile tank)
Collector tank
Bottle filling
Pasteurization (85° C for 20 min)
Cooling
Labelling and
storage Srivastava, 2002
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STARCH INDUSTRY
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SACCHARIFICATION PRODUCTS AND THEIR APPLICATION
Saccharification
products
Maltose content
(%)
Glucose Content
(%)
Application Products and
Effects
High maltose
syrup by fungamyl
50-55 2-5 Confectionery Moisture and
Control in soft
confectionery
High maltose
syrup produced at
65 °C
50-55 8-10 Baking
Frozen desserts
Moisture
retention and
colour control in
final products
High maltose
syrup special
produced at 60 C
to 65 °C
55-65 8-12 Brewing Control of
fermentation via
balanced
fermentable sugar
spectrum
High percentage
Very high maltose
syrup
82-88 5-9 Brewing Control of
fermentation via
balanced
fermentable sugar
spectrum
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BAKING INDUSTRY
❖Enzymes have gained real importance in bread making, where
they improve the dough and bread quality, leading to improved
dough flexibility, stability, loaf volume and crumb structure
❖Xylanase (hemicellulases) are of great value in baking as they
improve the bread volume, crumb structure and reduce
stickiness by decreases the water absorption, and thus reduces
the amount of added water needed in baking.
❖Glucose oxidase has been used to replace the chemical oxidants
and lipases to strengthen gluten, which leads to more stable
dough and better bread quality
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Applications of starch modifying enzymes in baking
Enzyme
(classification)
Substrate in
Foods
Reaction Applications in baked
products
Amylolytic
enzymes
Starch Hydrolysis of
linkages
α-Amylases
(EC 3.2.1.1)
or α-(1,4)-
Glucanhydrolases
Amylose
and
amylopectin
α(1→4)-D-
glycosidic
[endo],
liberating α-
dextrins
Generation of fermentable compounds;
Increase in bread volume;
Reduction in fermentation time;
Improvement in dough viscosity, rheology
and bread softness;
Improvement in bread texture;
Formation of reducing sugars and
subsequent Maillard reaction products,
Intensifying bread flavor and color;
Decrease of bread crumb firming rate;
Anti-staling effects.
β-Amylases
(EC 3.2.1.2)
Amylose
and
amylopectin
α(1→4)-D-
glycosidic
[exo], liberating
β-
dextrins and β-
maltose
Glucoamylase
(EC 3.2.1.3) or
Amyloglucosidase
Amylose
and
amylopectin
α(1→4)- and
α(1→6)-
Dglucosidic,
liberating β-
glucose
Pullulanase
(EC 3.2.1.41)
Amylopectin α(1→6)-D-
glycosidic
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BREWING INDUSTRY
• Employs the fermentation process to produce the wine, beer,
sake and soy sauce
• Many glycosidic enzymes such as cellulase, xylanase,
amylases, amyloglucosidase, glucanase, acetolactase, and also
other enzymes such as decarboxylase, lipase, pentosanase,
proteinase, etc. are used in this industry
• In the wine industry, enzymes such as amylase,
amyloglucosidase, cellulase, glucanases, hemicellulases,
pectinases, proteases, glucose oxidase and catalase are needed
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STARCH TRANSFORMATION
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Main enzymes from leaven and their characteristics
Enzyme
pH
Optimal
temperature
, °C
Inactivation
temperature, °C
Hydrolyzed bond
Hydrolyzed
products
α-amylase 5.6-5.8 70-75 80
α -1,4 from any place
inside amylose and
amylopectine
linear dextrines
from amylose
branched dextrines
from amylopectine
maltose and
glucose
β- amylase 5.4-5.6 60-65 70
α -1,4 from the non-
reducing end of the
catena not only at
amylose but also at
amylopectine
branched dextrines
maltose
Dextrynase 5.1 55-60 65
α 1,6 from amylose
and amylopectine
linear dextrines
with small
molecular mass
Maltase 6 35-40 40 Maltose 2 Glucose
Invertase 5.5 50 55 Saccharose Glucose
Fructose
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• Enzymes from microbial sources such as
cellulases, hemicellulases, galactomanna- nase,
pectinases are used from Leuconostoc
mesenteroides, Saccharomyces marscianus,
Flavobacterium spp., Fusarium spp.
Coffee
Industry
• Requires cellulases, glucanases, pectinases and
tannaseTea processing
• Fermentation of Aspergillus oryzae and later
inoculated with a bacterium, Peiococcus soyae,
and yeasts such as Saccharomyces rouxii and
Torulopsis sp., which ferment the mixture for
approximately six months
Asian food products
(soy sauce, koji,
moromi, etc.)
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DAIRY INDUSTRY
• Milk is the chief dairy product and is processed and converted
in to yoghurt, buttermilk, cheeses, butter, cream, soured cream,
etc. through fermentation.
• The enzymes proteases, lactases and lipases are chiefly used in
the dairy industry to develop the flavor compounds.
• Rennin acts on the milk protein in two stages, by enzymatic and
by nonenzymatic action, resulting in coagulation of milk.
• The principal components of milk are lactose (ca. 4.8%), lipids
(ca. 3.6%) and proteins (ca. 3.5%). Thus, enzymes modifying
these substrates are mainly used in dairy technology: β-
galactosidases, lipases and peptidases.
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ENZYMES FUNCTIONS
Chymosin Milk coagulation
Proteases Flavour improvement,
decrease ripening time of
cheeses
Lipases Flavour improvement,
decrease ripening time of
cheeses
Sulphydryl oxidase Remove cooked flavour
β-Galactosidase Lactose removal
Proteases Soyabean milk coagulation
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Crosslinking Enzymes
✓Using crosslinking enzymes to improve textural and
other properties of food
✓Better rheological properties
✓Enzymatic crosslinking of food biopolymers is an attractive
option owing to the specificity of enzymes and mild reaction
conditions.
✓Both food proteins and carbohydrates can be crosslinked by
enzymes.
✓via aromatic groups present in proteins or carbohydrates or
through certain amino acid moieties present in proteins
✓It is possible to improve the strength of weak flours,
containing low quality protein or fibre, improve dough
handling properties and baking quality
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• Making low fat fermented milk products with sensory properties
identical to those of products with normal fat content
• Meat industry: strengthening the structure of fermented sausages
and ham and restructuring of fresh meat with the aid of enzymes
thus increasing the value of the products.
• Transglutaminase- based crosslinking technology is well patented
and the markets are dominated by the Japanese company
Ajinomoto
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Potential crosslinking enzymes for food structure engineering
Crosslinking Enzyme Reaction Acting on protein
(target amino acid)
Direct Transglutaminase Formation of
isopeptide Linkage
Glutamine
Lysine
Peroxidase Oxidation of aromatic
groups to radicals
Tyrosine
Laccase Oxidation of aromatic
groups
Tyrosine
Cysteine
Tyrosinase Oxidation of tyrosine
to dopaquinone
Tyrosine
Glutathione oxidase * Cysteine
Sulphydryl oxidase * Cysteine
Indirect Lipoxygenase Production of hydro-
peroxide
radical from
unsaturated fatty acids
Glucose oxidase Production of H2O2 in
conjunction with
glucose oxidation
Hexose oxidase Production of H2O2 in
conjunction with
hexose oxidation
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Enzymatically modified whey protein and
other protein-based fat replacers
• Protein-based fat replacers belong to fat mimetics that imitate
one or more of the organoleptic and physical functions of fat
in food, but do not replace fat on a one to-one basis
development of reduced-fat foods.
• Micro particulated protein-based fat replacers, containing
milk and/or egg or whey protein are commercially produced
by either heat-induced gelation or aggregation and
crosslinking under shear stress of whey proteins.
• Proteases
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Food category Function of fat Type of fat replacer
Meat products
Contributes to juiciness and
tenderness; carries flavour;
absorbs frying-generated
flavours; reduces sharpness
of acid compounds
Microparticulated proteins,
soybean isolate, caseinates,
protein blends
Dairy products
Imparts smooth mouthfeel;
affects meltability, viscosity
body, crystallinity, spread
ability and palatability
heat- or enzyme-denatured
whey protein concentrates,
protein blends
Baked products
Inhibits formation of tough
gluten strands; softens crumb;
imparts tenderness; delays
staling
heat- or enzyme-denatured
whey protein concentrates,
protein blends
Cooking and salad oils, salad
dressings, soups, sauces,
gravies
Emulsifies fat; stabilizes
emulsion; imparts smooth
mouthfeel and palatability;
affects meltability and
viscosity
protein blends, protein
concentrates
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Type of fat replacer Function
Egg white Fat extender
Gelatin
Texture and viscosity enhancer,
smooth mouthfeel
Soybean and whey protein
concentrates and isolates
creaminess, opacity, water/
foam/emulsion stabilizer
Microparticulated proteins
creaminess, opacity, clean
flavour base, good flavour
release
Protein-gum/starch blends
Flavour and texture enhancer,
mouthfeel, water binding
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Enzymatic production of bioactive
peptides from milk and whey proteins
Bioactive peptides contain 2–20 amino acids per molecule and have
been defined as specific protein fragments that have a positive impact
on body function or condition and may ultimately influence health
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Production of flavours, flavour enhancers and other
protein-based speciality products
• The proteins can be hydrolysed into peptides and this will
produce flavours such as those found in soy sauce or cheeses
like brie and camembert.
• The protein can be more extensively hydrolysed and then
reacted with sugars via the Maillard reaction to form strong
savoury flavours
• Amino- and carboxy-peptidases
• Monosodium glutamate
• Aspartame
• Vanilla extraction
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Applications of cold-adapted proteases in
the food industry
• Proteolytic cold-adapted ‘superactive’ enzymes from marine
sources offer advantages for the food industry.
• The high catalytic efficiency of cold-adapted serine proteases
is especially useful in the processing of fresh foods where
protein digestion at low temperatures is required
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Flavorings and other value-added
products from sucrose
• Introduction of corn syrup-based sweeteners, especially
high-fructose corn syrup
• Introduction of microbial gums to replace the more
traditional plant-based gums
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Production of structured lipids with
functional health benefits
• Structured lipids (SL) are broadly referred to as modified or
synthetic oils and fats with functional or pharmaceutical
applications.
• Triglycerides and Diglyceride oil
• Lipases as biocatalysts
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Potential Application of Phytases in Food Processing
• Recently, phytases have been found increasingly interesting
for processing of food for human consumption, particularly
because the decline in food phytate results in an enhancement
of mineral bioavailability
• Phytases degrade phytate by sequentially removing
phosphate from the myo-inositol ring in a regio- and
stereospecific manner and the majority of phytases generates
only one single myo-inositolpentakis-, tetrakis-, tris-, and
bisphosphate isomer
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Tenderizing Enzymes in Meat Technology
• Tenderizing effects of marinating meat with brine containing
proteolytic enzymes from fruit extracts, such as ficin from
fig, papain from papaya fruit and bromelain from pineapple
have been shown.
• Actinidin enzyme from kiwi acted in the raw meat by a
restricted degradation of proteins in both connective tissue
and in myofibrils for improving tenderness in processed
meat.
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Bio-protective enzymes (preservatives): 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.
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IMMOBILISED ENZYMES
When purified enzymes are used to make large
quantites of another product, down stream
processing can be difficult and expensive.
Immobilising enzymes is cheaper.
Enzyme molecules are attached to a support
matrix rather than free in solution. They still
function properly but can be kept separate from
the reactants and the products.
Immobilized enzymes are usually used in
continuous flow-through re-actors, which have
a low volume.
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• Production of trans-free oils
• Production of cocoa butter equivalents
• Production of modified triacylglycerols
• Modification of phospholipids
• Production of diacylglycerols
• Production of high-fructose corn syrup
• Synthesis of functional oligosaccharides
• Lactose hydrolysis
• Tagatose production
• Production of protein hydrolysates
• Aspartame production
APPLICATION OF IMMOBILIZED ENZYMES

Characteristics of some commercially available
immobilized enzymes
Name Supplier Organism Specificity Matrix
Lipozyme TL
IM
Novozyme
A/S
Thermomyces
anuginose,
TLL-1
sn-1,3
specific
Silica granules
Lipozyme RM
IM
Novozyme Rhizomucor miehei, RML sn-1,3
specific
Macroporous ion
exchange resin
Novozyme 435 Novozyme Candida antartica
lipase B
non-specific Macroporous
acrylic resin
Lipase PS-C Amano Pseudomonas cepacia
lipase
non-specific Ceramic particles
Lipase AK-C Amano Burkholderia cepacia lipase
(formerly Pseudomonas
Fluorescens lipase PFL)
sn-1,3
specific
Ceramic particles
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Commercial enzyme Fruit juice Applications
PECKTOZYMEᵀᴹ MAX Liq Apple and pear juice High juice yield
PECKTOZYMEᵀᴹ MAX juice Apple pomace juice Enhance extraction & yield
PECKTOZYMEᵀᴹ POWER clear Apple and pear juice High clarity
PECKTOZYMEᵀᴹ POWER clear L Lemon juice Clarification
CITROZYME TM Citrus juice Peel extraction, viscosity reduction
PECKTOZYMEᵀᴹ Ultra C Citrus juice Fast viscosity reduction
Diazyme® X4NP Fruit juice Starch breakdown
RAPIDASE Intense ᵀᴹ Lemon juice Improves clarification and conc.
Commercial enzymes used in fruit juice
Maria Henriques
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Brewing with prolyl endopeptidase from Aspergillus niger : the
impact of enzymatic treatment on gluten levels, quality attributes
and sensory profile
International Journal of Food Science and Technology, 2017, 52, 1367–1374
Ghionno et al
Italy
OBJECTIVE: To evaluate the impact of treatment with PE on gluten
level, quality attributes and sensory profile of three different styles
of barley/wheat malt beer .
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MATERIALS AND METHODS
1. Beer production
2. Beer Analysis (Assessment of gluten content,
Standard quality attributes, Volatile compounds,
Sensory analysis)
Recipe of beer types
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Gluten levels throughout the brewing process of untreated American Pale Ale
(APA) trial determined by RIDASCREEN R5 gliadin competitive assay.
*Guerdrum & Bamforth (2012).
UBC = Unfiltered bottle-conditioned beer. n = 2 technological replicates;
data shown as mean standard deviation.
48

Gluten content, standard quality attributes and foam stability in control beer (CB) and in
treated sample with prolyl endopeptidase from Aspergillus niger (PE) of American Pale Ale
(APA), American Stout (AS) and Weizen (W) bottle-conditioned beers
49

CONCLUSION
• PE addition during fermentation was very effective in gluten content
reduction, without negatively affecting the quality attributes and the
sensory characteristics of beer.
• A combination of enzyme addition and maximisation of those brewing
steps where relevant proteins are removed (i.e. vigorous wort boiling,
lowest temperature and prolonged time of maturation phase, beer
filtration) could be even more effective in gluten equivalent reduction.
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CASE STUDY 2
OBJECTIVE: To establish the optimal enzymatic treatment conditions to
clarify pomegranate juice using pectinolytic and proteolytic enzymatic
clarification.
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MATERIALS AND METHODS
• Fruits :Fresh pomegranate (Punica granatum L., var Wonderfull)
• Enzymes
• Native plant cysteine protease, papain from papaya latex (A)
• Klerzyme 150 pectinase preparation (B) from Aspergillus niger
• Orthogonal test design to optimize protease:pectinase ratio mixture
• Optimization of pomegranate clarification conditions
• Turbidity measurement and heat stability test
• Clarity
• Assessment of haze-active proteins and phenols
52

3D response surfaces for chill haze (aeb), turbidity (ced), heat turbidity (eef), clarity (geh) of pomegranate
juice as a function of complex enzyme amount and incubation time at 37.5 C (left-side) and of complex
enzyme amount and temperature at 75 min (right-side).
53

Haze active proteins (a) and phenols (b) developed by adding tannic acid or
gelatin respectively, turbidity (c) and potential turbidity (d) of treated juice with
0.1 0.25 0.4 g of enzyme complex per 100 g of juice after 1, 7 and 21 d of storage
at 4 C.
The values are mean ± SD of triplicate analysis of juice samples. For each time of
storage (1, 7 and 21 d) data with different superscript letters indicate
significantly different (P < 0.05).
54

The optimum region by overlaying contour plots of the four responses (chill haze - CH, turbidity - T, heat
turbidity - HT, clarity - Cl) evaluated as a function of incubation time and temperature (a), incubation time
and complex enzyme amount (b), incubation temperature and complex enzyme amount (c).
55

CONCLUSION
• Incubation time (30-120 min), temperature (25e50 C) and complex
enzyme amount (0.1e0.4 g/100 g of juice). All these variables markedly
affect the chill haze, turbidity, potential turbidity and clarity of the
pomegranate juice.
• Using the contour plots, the optimum set of the operating conditions
were graphically obtained: temperature 25-30 C for 100e110 min using
0.22e0.25 g of protease-pectinase complex enzyme amount (the ratio of
protease:pectinase was 1:2) per 100 g of juice.
• This kind of clarification treatment caused a substantial decrease of
protein and phenol haze forming activity, thus reducing the potential
juice turbidity.
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CONCLUSIONS & FUTURE THRUST
• The integration of enzymes in food and feed processes is a well-
established approach; however there are clear evidences that
dedicated research efforts are consistently being made to make the
applications of biological agents more effective as well as diversified.
• Various techniques have been employed such as rDNA technology
and protein engineering (site-directed mutagenesis and random
mutation) for the design of new/improved biocatalysts
• Advances in molecular biology, evolution- ary protein engineering
expertise, the (bio) computational tools, and the implementation of
high-throughput meth- odologies enabling the efficient and timely
screening/ characterization of the biocatalysts.
• There needs to be continue efforts in the direction to have more
diverse, versatile and robust enzymes to be applied in food
technology
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Enzymes in food processing

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