Question 1: What is fermentation? What is food fermentation?

Clarify the advantages/

disadvantages/ benefits of fermented products.
- Fermentation is a metabolic process in which an organism converts a carbohydrates such as
starch and sugar into alcohol and/or acid. It is a process of growing microorganisms (microbes)
to produce various chemicals or pharmaceutical compounds.
- Food fermentation is the oxidation-reduction process in which hydrogen and electrons
transfer from substances from foods (foods components like carbohydrates, proteins, lipids,…)
to coenzymes (NAD+, FAD+) by the help of enzymes and microorganisms under the conditions
of anaerobes (almost) and aerobes to produce alcohols (ethanol) and acids (lactic acid,
gluconic acid,…) go along with CO2.
- There are 2 types of fermentation: submerged fermentation and solid-state fermentation.
- Advantages and benefits:
 Improves digestion
 Suppresses H. pylori – an important risk factor for gastrointestinal diseases
 Enhance the immune systems
 Bio-enrichment with essential amino acids and vitamins.
 Has Anticancer effects
 Enhances bioavailability of nutrients
 Helps treat hepatic diseases
 Reduce symptoms of lactose intolerance
 Longer shelf-life with help of alcohols, lactic acids, alkaline, and acetic acid.
 Increase food flavor, reduce cooking time, and produce variety of foods.
- Disadvantages:
 Increase the risk of gastric cancer
 Risk of Botulinum contamination
 Store-bought items lose beneficial bacteria
 Slow production process

Question 2: What do you know the barley? Malt, malting, milling and mashing in beer fermentation
- Barley is a major cereal grain grown in temperate climates globally. There are two main types
of barley: Winter barley, which is sown in Autumn; Spring barley, which is sown in Spring
- Barley is the best for beer fermentation because:
 Seeds of barley are all nearly the same size.
 Seeds are tough and have thick husk  easy for transfer, preservation, and no insects
affect.
 It contains 60-65% starch and also contains the enzyme can breakdown the starch.
 It contains vitamin, mineral and low level of fat, oil  very desirable for yeast.
- Barley in beer fermentation must be choose base on:
 Rate of water uptake  it will affect malting time.
 Ease of modification.
 Extract potential.
 Enzyme producing potential.
- Further check with:
 Uniform size, color.
 Few sreening.
 Free from smell and mold or insects affect.
 - Malting: the controlled germination of the grain, to stimulate endogenous enzyme production.
- The malting process mimics the natural germination of barleycorns which would otherwise
happen in the field but with two significant differences growing conditions are manipulated to
encourage enzyme production by the aleurone, while minimizing yield losses due to
respiration and growth of the embryo and the germinated corn is gently kilned to preserve
enzymatic activities where possible.
- The purpose of malting is to activate the endogenous phytohormones and enzymes of the
barley to make it more amenable to starch/carbohydrate extraction, which is further
facilitated by milling.
- There are three main step in malting:
 Milling: On receipt of malt or adjunct into the brewery, the first step in its processing is milling.
Milling is the process where grain is broken down into small fragments. This provides a greater
surface-to-volume ratio, therefore ensuring good hydration during mashing and good access of
the malt enzymes to their substrates.
 The quality and size distribution of the milled grain fragments affect:
+ The mashing process and the saccharification time; the brewhouse yield; fermentation;
beer filterability, beer colour, taste and the overall character.
+ The overall aim of milling is to break the starchy endosperm into fine particles while
retaining the outer coat husk layer of the barleycorn with little or no starchy endosperm
adhering. This husk fragment is used as a filtration aid during the mash separation process of
lautering. In cases where mash filters are used, the whole grain is hammer milled to produce
a fine powder that can be easily extracted.
 Mashing:
 The aim of mashing, which follows the milling process, is to yield as much extract and as
good an extract as possible from the grain. Most of the extract is produced by the action
of enzymes, which are allowed to act at the optimal temperature specifics required. The
brewer will optimize liquor to grist ratios, mashing in temperature, water chemistry (pH
and salt contents) and mashing time and temperature stands to take advantage of the
optimal temperature specifics of both endogenous malt enzymes and exogenous
commercial enzymes, when added.
 The four main enzymatic reactions occurring during mashing are:
1) The hydrolysis of proteins into peptides and free amino acids.
2) The degradation of β-glucan chains.
3) The hydrolysis of pentosans (into arabinose and xylose).
4) The breakdown of gelatinized starch into fermentable carbohydrates (glucose,
fructose, sucrose, maltose and maltotriose).
These conversions happen due to the action of proteolytic, glucanolytic, pentosanolytic and amylolytic
enzyme classes, respectively.
To cover the optimal activity temperature range of each enzyme group, mashing is usually operated
by successive rests at increasing temperatures.
A simple representation of this is; a first rest at 50◦C for hydrolysis of protein, β-glucans and
pentosans, a second rest at 63◦C for the action of β-amylase, a rest at 70◦C for optimal activity of α-
amylase, followed by heating at 78◦C to promote enzyme inactivation.
Question 3: Can you list out the fermented products that you know?
- Kefir.
- Sauerkraut.
- Tempeh.
- Natto.
- Cheese.
- Kombucha.
- Miso.
- Kimchi.
- Pickle
- Yogurt
- Fish sauce/ soy sauce

Question 4: Roles of enzyme in food processing

Enzymes can modify and improve the functional, nutritional and sensoric properties of
ingredients and products, and therefore enzymes have found widespread applications in
processing and production of all kinds of food products.
In food production, enzymes have a number of advantages:
 Firstly, enzymes are used as alternatives to traditional chemical-based technology.
Enzymes can thus replace synthetic chemicals in a wide range of processes. This allows
advantages in environmental performance of processes by lowering energy
consumption levels and biodegradability of products.
 Furthermore, since enzymes are more specific in their action than chemical reactants,
enzyme-catalyzed processes have fewer side reactions and byproducts (waste
products). The result is higher quality products and less pollution. Enzymes can catalyze
reactions under very mild conditions, allowing mild processing conditions which do not
destroy valuable attributes of foods and food components.
 Finally, enzymes allow processes to be carried out which would be otherwise
impossible. The first commercial food product produced by biotechnology was an
enzyme used in cheese making. Prior to biotech techniques, this enzyme had to be
extracted from the stomach of calves, lambs and baby goats, but it is now produced by
microorganisms that were given the gene for this enzyme.
The food industry uses more than 55 different enzyme products in food processing. This
number will increase as we discover how to capitalize on the extraordinary diversity of the
microbial world and obtain new enzymes that will prove important in food processing.

Enzyme Source Action in Application in food technology

food
α-Amylase Aspergillus spp. Wheat Amylase dough softening,
Bacillus spp. starch increased bread volume, aid in
Microbacterium hydrolysis the production of sugars for
imperiale yeast fermentation
α-Acetolactate Bacillus subtilis Converts Reduction of wine maturation
Bacillus subtilis acetolactae time by circumventing need of
Converts to decarboxylase for secondary
acetolactate to acetoin fermentation of diacetyl to
acetoin acetoin
Amyloglucosidase Aspergillus niger Hydrolyzes One stage of high fructose corn
 Rhizopus spp starch syrup production; production of
dextrins to ‘lite’ beers
glucose
(saccharifica
tion)
Cellulase Aspergillus niger Hydrolyzes Fruit liquefaction in juice
Trichoderma spp cellulose production
Chymosin Aspergillus Hydrolyzes Coagulation of milk for cheese
awamori κ-casein making
Kluyveromyces
lactis
β-Galactosidase Aspergillus spp. Hydrolyzes Sweetening milk and whey;
(lactase) Kluyveromyces spp. milk lactose products for lactose-intolerant
to individuals; reduction of
glucose and crystallization in ice cream
galactose containing whey; improving
functionality of whey protein
concentrates; manufacture of
lactulose
Glucose isomerase Actinoplanes Converts Production of high fructose corn
missouriensis glucose to syrup (beverage sweetener
Bacillus coagulans fructose
Streptomyces
lividans
Streptomyces
rubiginosus
Glucose oxidase Aspergillus niger Oxidizes Oxygen removal from food
Penicillium glucose to packaging; removal of glucose
chrysogenum gluconic from egg white to prevent
acid browning
Lipase and esterase Aspergillus spp. Hydrolyzes Hydrolyzes triglycerides to fatty
Candida spp. triglycerides acids and glycerol; hydrolyzes
Rhizomucor miehei to alkyl esters to fatty acids and
Penicillium fatty acids alcohol
roqueforti and
Rhizopus spp. glycerol;
Bacillus subtilis hydrolyzes
alkyl esters
to
fatty acids
and alcohol
Protease Aspergillus spp. Hydrolysis Milk coagulation for cheese
(proteinase) Rhizomucor miehei of κ-casein; making; hydrolysate production
Cryphonectria hydrolysis for soups and savoury foods;
parasitica of animal bread dough improvement
Penicillium citrinum and
Rhizopus niveus vegetable
Bacillus spp. food
proteins;
hydrolysis
of wheat
glutens
 Pectinase Aspergillus spp. Hydrolyzes Clarification of fruit juices by
(polygalacturonase) Penicillium pectin depectinization
funiculosum

Question 5: What factors that we should focus on when running fermentation.

1. Temperature
- Temperature affects the growth, metabolic activity of microorganisms and the quality of
fermented foods. Every microorganism has the optimal temperature for their growth
- Usually, the fermentation at low temperatures will last. In contrast, fermentation at too high a
temperature will irreversibly reduce the metabolic activity of microorganisms. If food is
fermented at high temperatures, the loss of aromatic components in the product will increase.
2. Oxygen supply
- The oxygen exchange needs of an organism depend on the characteristics of the cell and the
culture environment. The oxygen demand of most cells is satisfied if the oxygen concentration
is about 1mg/L. If the oxygen level is lower, the anaerobic fermentation occurs and as a result
the growth rate also decreases.
- The supply of oxygen for aerobic fermentation is mandatory because it has a decisive influence
on the yield and quality of the product to be acquired. In the case of anaerobic fermentation,
the presence of oxygen can be toxic and inhibit seed microorganisms.
3. pH
- During fermentation, the pH changes due to the transport of substances through the cell
membrane, thereby changing the concentration of H + ions in the culture, some
microorganisms synthesize organic acids and release them outside the cell.
- In addition, the pH value will affect the metabolic activity of microorganisms. During
fermentation, the pH of the culture is always changing. Optimal pH for the growth or
bacteriocin production is markedly dependent on the characteristics of microbial strains used.
Optimal pH for some bacteriocins production ranged from pH 5.5 to 6.0 while for others being
less than pH 5. If the pH value is high, the losses in fermentation will increase rapidly.
4. Amount of seedlings transplanted
- If the number of seedlings is too small, then the fermentation conditions will be prolonged. In
addition, in the early stages of the fermentation process, microorganisms infected in the
environment are susceptible to growth and can adversely affect productivity and product
quality.
- Conversely, if the number of transplants is too much, it will increase the cost of propagation. In
food fermentation technology, the excessive number of seedlings will change the rate of by-
products formed in the fermentation environment, from which the sensory value of fermented
foods will be changed. Producers need to determine the appropriate number of seedlings by
experimental methods
5. Fermentation time
- Depending on the microorganism variety, the type of product to collect and many other
factors. When receiving metabolic products such as amino acids, organic acids, organic
solvents, enzymes ... fermentation time usually lasts from 1 to 3 days.
- In contrast, for fermented foods, fermentation and storage times can last for weeks, months
and even years. Manufacturers will determine the time of fermentation and storage by
experiment.

Question 6: Production of pectinases and utilization in food processing.

 - Pectinases are the heterogeneous group of enzymes that are classified into polygalcturonase
(PG), pectinesterase (PE), and pectin lyase (PL) based on their mode of action on the substrate.
- These enzymes break the pectin polymer into smaller fragments by hydrolysis, trans-
elimination and de-esterification reactions, respectively.
- Enzyme Production
 Pectinases are produced by plants as well as by bacteria, yeast and fungus. Many
microorganisms are reported to produce pectinase enzyme such as Saccharomyces,
Bacillus, Erwinia, Aspergillus, Penicillium, Rhizopus and Fusarium etc.
 Pectinases are produced from a pure culture of one A. niger strain, selected on the
basis of its productivity.
 Fungus is preferred for production of enzymes due to high yield and ability to
withstand slight changes in the environment such as temperature, pH and pressure.
- Uses of pectinase enzyme in fruit juice industries:
 Pectin holds the juice within the mash, reduces pressability, decreases juice yield, slows
down juice clarification and makes water evaporation for concentration impossible.
 Pectinase enzyme acts on the pectin and breaks the glycosidic bonds present between
the galacturonic acid monomers thereby decreases the water holding capacity of
pectin. Hence high yield of juice is obtained.
 Apples, berries, grapes juice need an intense pectinase treatment along with cellulases for
complete clearance of the haze, turbidity and viscosity.
 Fruit juice like orange and pineapple etc need only mild pectinase treatment for the extraction
of juice and to get high yield of juice but no intense clarification as these juices are consumed
by the people, having characteristic pulpy clouds in it. Pectin along with starch complex is
responsible for the characteristic viscosity and turbidity of the juice
- Pectinases constitute a heterogeneous group of enzymes that break down complex
polysaccharides of plant tissues into simpler molecules like galacturonic acids.
 Applying in the clarification of wines; and the extraction of oils, flavors, and pigments
from plants
 These enzymes include protopectinases, polygalacturonases, lyases, and
pectinesterases on the basis of their mode of action.
 Protopectinases catalyze the solubilization of protopectin.
 Polygalacturonases hydrolyze the polygalacturonic acid chain by addition of water and
are the most abundant among all the pectinolytic enzymes.
 Lyases catalyze the trans-eliminative cleavage of the galacturonic acid polymer.
Pectinesterases liberate pectins and methanol by de-esterifying the methyl ester
linkages of the pectin backbone

Question 7: Enzymes involved in starch degradation.

- Starch is composed of amylose (a virtually linear glucose polymer in which the glucose residues
are linked via α-1,4 glycosidic linkages) and amylopectin (in which the majority of the glucose
residues are linked via α-1,4 glycosidic linkages with up to 5% α-1,6 linked side chains).
- A large variety of enzymes active towards amylose or amylopectin have evolved in nature.
- Starch-active enzymes are divided into two major groups depending on their mode of action:
(1) exo- and endo-acting hydrolases that hydrolyze the α-1,4 and/or α-1,6 glycosidic linkage
using water
(2) glucanotransferases that break an α,1-4 glycosidic linkage and form a new α-1,4 or α-1,6
glycosidic linkage
 - The first enzyme to be used on an industrial scale was the hydrolytic glucoamylase
- Glucose isomerase was introduced for the production of high fructose syrups. It converts
glucose into fructose, thus creating a product with a higher sweetness.
- Later, thermostable α-amylase and pullulanase were introduced for a faster and better
hydrolysis of starch.
- Most enzymatic starch conversion processes start with heating a water-starch slurry to disrupt
the granular structure and bring the two glucose polymers amylose and amylopectin into
solution.
- Starch processing is divided into three steps: gelatinization, liquefaction and saccharification.
 In the gelatinization step, calcium and thermostable α-amylase such as that of the
bacterium Bacillus licheniformis or Bacillus amyloliquefaciens is added.
 In the liquefaction step, the thermo stable amylase hydrolyzes the α,1-4 linkages in
both the amylose and the amylopectin to produce dextrins.
 In the saccharification step, pullulanase, glucoamylase, β-amylase or an α-amylase are
added to further degrade the liquefied starch into maltodextrins, maltose or
glucosesyrups.
- Glucoamylases (amyloglucosidase or saccharifying amylase): mainly hydrolyze α-1,4 linkages
 A pullulanase in combination with a glucoamylase: increase the glucose yield, reduce
saccharification time, produce high glucose syrup
 Combine β-amylase with glucoamylase: increase the maltose yield, produce maltose
syrup.
- Fungal acid α-amylase and thermo stable cyclodextrin glycosyltransferase: obtain higher
concentrated maltose syrups.
- Pullulanases: hydrolyze pullulan
 Type I: produce branched dextrins
 Type II: used in the saccharification process
- Isoamylases: specifically hydrolyze the α-1,6 glycosidic linkage in amylopectin or glycogen but
they do not show any activity towards pullulan.
- Glucose isomerase: a xylose isomerase converting xylose into xylulose but it also has activity
towards glucose, convert Glucose syrups into glucose–fructose mixture.
- Maltose can be converted into isomaltooligosaccharides (IMO) using specific α-glucosidases.
- Glucanotransferases: hydrolyticenzymes perform a transglycosylation reaction, break down O-
glycosidic linkage.
 The 4-α-glucanotransferases also known as amylomaltases that form an α-1,4 linkage
 The glycogen/starch branching enzymes that form an α-1,6 linkage (or branch point).

Question 8: Enzyme in bread making

- Amylase: Increase bread volume and produces better flavor, decreases the chance of starch
recrystallization
- Protease: Ensure dough uniformity, reduce dough consistency maintain gluten strength in
bread, improve flavor and texture in bread.
- Lipase: Reduce the initial firmness and increase the specific volume of breads, improve the
texture and softness by catalyzation, extend the shelf-life ; enhance flavor content by
esterification; used in the hydrolysis of butter fat used as technological additive.
- Xylanase: Gives the desired effects in terms of dough-handling properties; stability and oven
spring and volume
 - Oxidoreductase: The main action of these agents is to help rebuild the gluten network and the
GMP, in order to improve texture, volume, freshness and also dough machinability and
stability. Dough conditioners are specifically meant for gluten strengthening.
- Transglutaminate: improves the resistance of dough, particularly yeast dough from wheat
flour, in a manner comparable to potassium bromate
- Endoglycosidases
- Cellulases: Added to whole meal bread formulations to improve dough and bread
characteristics. The addition of cellulase enhances crumb structure, resulting in a more regular
and finer crumb and, consequently, improved whiteness. The positive effect is due to break
down of cellulose fibrils enabling better gluten development and improved proofing stability,
increased dough tolerance and enhanced volume, texture and appeal in wholegrain bread
types
- Mannanases: Retards staling and improves crumb structure.

Question 9: Enzyme in non-bread wheat-base foods:

- In cake and muffin production:
 Cake or muffin is produced from: wheat flour, sugar, eggs.. It’s batter may be considered as an
oil-in-water emulsion which can be stabilized by emulsifiers.
 Eggs (contain lecithin) have surface-active properties and may act as an emulsifier to stabilize
the emulsion.
 Enzymes used and their activities: Phospholipase A1 or A2.
 Egg lecithin are phospholipids consisting of phosphatidylcholine and
phosphatidylethanolamine. These lipids have emulsifying properties which can be improved
when part is hydrolyzed into the lyso-form.
 This hydrolysis is catalyzed by the action of phospholipase A1 or A2.
- Addition of this commercial lipase:
 Reduce the surface tension and surface viscosity at the air/water interface of batter.
 Increasing in cake specific volume and maintaining a fine crumb structure.
 Eating quality and perceived freshness over 14 days of chilled storage were improved.
 Improve cake quality and/or to reduce costs because of reduction in egg quantity in cake
recipes.
- Amylase:
 Starch-degrading enzymes have been applied in cake production to prevent cake staling.
 Specialized amylase, bacterial amylase improve the quality of cake in general and more
specifically the softness of the crumb and the shelf life of the product (ex: Maltogenic α-
amylase).
 Amylomaltase has been applied to branch potato starch into a product which can be used as a
fat replacer and mouthfeel improver in cake recipes.
- Proteases:
 Proteases have been used to:
 Lower the viscosity of cereal flour suspensions.
 Retard staling of the cake crumb.
 Prolong shelf life (alkaline proteases such as keratinase and thermitase ).
 Improve the flavour of cakes.
- Pasta and noodle production:
 Enzyme in pasta:
o Transglutaminase: Cross-linking agent => high steady state viscosities of strong durum
dough.
 o Glucose oxidase, peroxidase, dehydrogenases, lactonohydrolases: Improve gluten strength in
pasta dough and overall quality of the end product.
o Endoxylanases: Reduce the pasta dough viscosity, head pressures and/or significantly higher
throughputs in commercial pasta extrusion systems.
o Lipases: Extends shelf life, keep the characteristic yellow color over time.
 Enzyme in noodle:
o Problems: Darkening of raw noodles or noodle dough sheets during production or shelf life
and speckiness. It is cause by polyphenol oxidase
→ Fungal lipase helps to brighten noodle sheets or raw noodles during shelf life and also reduce
the increase of the number of dark spots over storage time.
o Lipase or the combination of lipoxygenase and lipase make boiled noodles firmer, smoother
and less sticky → easier disentanglement and improved eating quality.
Explain: Monoglycerides by the action of triacylglycerol lipase, form stable complexes with
amylose.
o Phospho- or galactolipase activity may be applied to obtain similar effects.
o Transglutaminase and Glucose oxidase make internal cross-links between gluten and other
protein fragments within the dough → More elastic and glutinous noodles having an
improved texture, resilience and mouthfeel.
o Application of fungal α-amylase on the surface of pre-gelatinized noodles prevents clumping
of the cooked noodles and improves its flavour and texture even after a long period.
 Biscuit, cookie and cracker production:
o By using a protease that may be only active at the beginning of the dough preparation,
shrinking of the dough may be reduced and more regular sizes of baked products, such as
biscuits, can be obtained.
o α-Amylases is able to produce dextrins from damaged starch -> play a role in the enzymatic
browning during baking, resulting in darker biscuits. They also prevent checking as well as
creating a leavening effect and improved flavour development.
o Papain is an interesting protease to use since it has a strong hydrolytic action on glutenins
but can be spontaneously stopped by natural oxidation of the dough.
 Waffer production:
o Proteases, especially bacterial proteases, added to the wafer batter to prevent gluten
development and liquefy the gluten, resulting in a uniform mixture with optimum flow
properties.
o The addition of endoxylanase (preferably originating from Trichoderma sp.) is able to
hydrolyze the backbone of the arabinoxylan -> moderate release of water due to the
decrease in water binding capacity of the hemicellulose.
o The use of a thermalstable α-amylase can manipulate textural attributes of flour-based food
products like wafers, without resulting in an increased batter viscosity.

Question 10: Brewing with enzymes

- Brewing process required a great knowledge of enzymology as each enzyme has its own
temperature point.
 - Beer brewing is based on the action of enzymes activated during malting and fermentation.
The action of activated enzyme in time of malting and fermentation is one of the vital step in
beer brewing
- The first step of brewing beer is the mash, or mashing. The mash is the process of activating
enzymes in the grain to change starches into sugar, ultimately providing the necessary “food”
for the yeast. It will also create the base for the color, body and overall flavor of your beer.
- The next step is lautering, which is the process of separating the wort from the grain. The
purpose is to remove sugars that may be trapped within the grain following the mash.
- The boil can sometimes be confused with the mash, but they are actually two very different
processes.
- The reason for the difference between the brewing methods is that the minerals in the water
can affect the starch conversion of the mash, but once the sugars have been produced, the
affect of water chemistry on the flavor of the beer is greatly reduced. When brewing with malt
extract, if the water tastes good to begin with, the beer should taste good.
- The four most common enzymes used in brewing are beta glucanase (cellulase), protease,
alpha-amylase, and beta-amylase

Question 11: Enzymes in alcohol and wine production (enzymes là gì, mục đích sử dụng ez trong chế
tạo thức uống cồn, các loại ez thường dùng, các quá trình diễn ra chủ yếu)
- Enzymes are proteins which have specific functions and serve as catalysts for biochemical
reactions. Enzymes possess specifically shaped active sites for reacting with one specific
substrate -> generating pure products free from unwanted by-products
- Most of the enzymes used in wine are hydrolases, meaning they hydrolyse substrates and
liberate products; moreover, Enzymes are biodegradable -> cause less damage to the
environment.
- The main benefits of using these enzymes during wine making include better maceration,
improved color extraction, easy clarification, easy filtration, improved wine quality, and
improved stability
- Some typical enzymes used in alcohol and winemaking:
o Amylases are found in malt, moulds (as Aspergillus oryzae), bacteria (Bacillus subtilis), as well
as in human and animal saliva (phytalin), and in the pancreas (pancreatin), are utilized mainly
in breaking down the starch, maltose, and glucose molecules as a part of the malting process
o Amyloglucosidase is Produced by the controlled fermentation of non-pathogenic and non-
toxicogenic strains of Aspergillus niger in a growing medium rich pectin
o Cellulases are important in brewery, winemaking and soon being accepted as a vital
ingredients in distillery enzymes preparations group, and being synthesized by large variety
of microbes insisting of fungi and bacteria

Question 12: Enzyme in dairy product manufacture

 The best-known dairy enzyme preparation is rennet, a collective name for commercial
preparations containing acid proteases extracted from animal tissues.
 In addition to the use of milk-clotting enzymes to make cheese, the dairy industry also
makes use of enzymes such as lipases, non-coagulant proteases, aminopeptidases,
lactases, lysozyme, lactoperoxidase and transglutaminase.
  Some of these applications are traditional (lipase for flavour enhancement) whilst
others are relatively new (lactose hydrolysis, accelerated cheese ripening, control of
microbiological spoilage, modification of protein functionality)

Enzymes Application examples

Acid proteinases Milk coagulation
Neutral proteinases and Accelerated cheese ripening, debittering, enzyme-modified
peptidases cheese, production of hypoallergenic milk-based foods
Lipases Accelerated cheese ripening, enzyme-modified cheese,
flavour-modified cheese, structurally modified milk fat products
β-Galactosidase Lactose-reduced whey products
Lactoperoxidase Cold sterilization of milk, milk replacers for calves
Lysozyme Nitrate replacer for washed-curd cheeses and cheeses with eyes
(e.g. Emmental)
Lactase  Lactase also increases the sweetness of the final product
such as cheese or ice cream, avoiding the requirement
for sugars.
 Cheese manufactured from hydrolyzed milk ripens more
quickly than the cheese manufactured from normal milk.
 The lactase enzyme is used increasingly in higher yields
in dairy products during today’s society to efficiently
breakdown lactose sugars to allow these lactose-
intolerant people to consume dairy products.
Transglutaminase  Reducing syneresis in acid milk gels and has been
investigated as a method of improving the texture and
shelf life of yoghurt.
 Improve the emulsifying properties of milk proteins and
increases the viscosity and water-holding capacity of
yoghurt

Question 13: Enzyme in fruit and vegetable processing and juice extract

 Roles of enzyme in fruit and vegetable processing and juice extract

 Increase extraction of fruit from raw material
o Ex: Pectinase and cellulases enzymes were used for extraction of pineapple
juice at enzymatic concentration of 0.025% => The TSS of the final pooled juice
was around 12°Brix
o For carrot, pectinase and cellulases at concentration 2% in (3:2) ratio increase
yield of final juice TSS
 Improve quality of fruit juice (clarity, viscosity, filterability, color)
 Increase processing efficiency
 Generate a final product that is clear and visually attractive
 Enzymatic hydrolysis of the cell walls: increases the extraction yield, reducing sugars,
soluble dry matter content and galacturonic acid content and titratable acidity of the
products.
 Enzymatic degradation of the biomaterial: depends on the type of enzyme, incubation
time, incubation temperature, enzyme concentration, agitation, pH and use of different
enzyme combinations
 Enzyme in fruit processing
  Economic: When added to fruit after crushing, pectinases quickly decrease the viscosity of the
mash by pectin hydrolysis. They facilitate juice extraction from pulpy fruit, increase press
loading and increase juice yield. Residual waste, such as apple pomace, is reduced and is drier.
Enzymes increase the overall productivity of the processing plant. Once added to the juice,
pectinases and amylases hydrolyze residual pectin and starch. Their use is vital for fast juice
clarification, filtration, pasteurization and concentration. As a result, the juice remains stable,
long term, without additives or preservatives, storage volumes are reduced and shipping
weight reduced
 Quality: Fast juice processing with enzymes lowers the risk of microbial spoilage, reduces
oxidation and improves juice and concentrate shelf life. Pectin hydrolysis of fruit cell walls
weakens cells and vacuoles and thereby maximizes extraction of their components such as the
red colour from berries (anthocyanins), aromas and antioxidants of phenolic type – known for
their positive effect on human health, particularly heart disease prevention.
 Sustainability: The use of enzymes has a positive effect on sustainable production. They lower
energy consumption (electricity, steam and water), reduce waste flow by maximizing fruit use
and reduce dependency on chemicals used in equipment cleaning products.
 Enzyme in vegetable processing
Vegetable juice processing therefore requires more cellulases in addition to pectinases to
reduce viscosity sufficiently for juice extraction using a decanter
 Applications of some enzymes
 Pectic enzymes or pectolytic enzymes:
o Increase yields
o Improve liquefaction, clarification and filterability of juices
o Maceration and extraction of plant tissues
o Release flavors, enzymes, proteins, polysaccharides, starch and agar
 Cellulase
Used in:
o Extraction and clarification of fruits and vegetable juices for production of nectars
and purees, oil extraction from oil seeds, animal feed preparation
o Improvement in soaking efficiency
o Homogeneous water absorption by cereals
o The nutritive quality of fermented foods
o The re-hydrability of dried vegetables and soups
o The production of oligosaccharides as functional food ingredients and low-calorie
food substituent’s and biomass conversion
o Carotenoid extraction in the production of food coloring agents
 Hemicellulases:
o Include endo- and exo-xylanases, galactanases, xyloglucanases and mannanases
o One of the most abundant groups of polysaccharide in nature
 Amylases:
o Catalyse the breakdown of starch into sugars
o Are extensively employed in clarification of fruit juice

Question 14: Enzymes in meat processing

( short )
- Protease: tenderise meat
- Lipase: remove fat and form flavor
 - Transglutaminase: increase meat hardness and texture
- Oxidative enzyme: include Tyrosinase and Laccase can alternative for Transglutaminase to
generate cross – link in protein matrices.
- Glutaminase: create flavor for meat by increasing glutamate.
( Detail )
1. Proteases
- Catalyze the hydrolytic cleavage of the peptide bond present in proteins
- Capable of digesting connective tissue and muscle proteins. Present in a variety of sources
including plants, microbes and animals.
- Common proteolytic enzymes: Papain, Bromelain, Ficin, Actinidin, Collagenase
2. Papain (the most commonly proteolytic enzymes in meat tenderization)
- Produced as a crude, dried material by collecting latex from papaya
- Highly aggressive, indiscriminate enzyme causing significant degradation to both myofibrillar
and collagen proteins.
- Optimum pH: 5.0 - 7.0
- Optimum temperature: 65°C
(+) Advantages:
- As a meat tenderizer, act even on the toughest cuts of meat and make them soft and
appetizing.
- Better penetration of spices and marinades into the meat’s interior ⇒ enhancing taste & helps
in cutting fuel cost by reducing cooking time.
(-) Disadvantages:
- When use papain, meat must be injected with marinade containing papain ⇒ some off-
flavoured for products
3. Bromelain
- Prepared from the stump or root portion of the pineapple plant after harvest of the fruit and
all supplied as powders
- Optimum pH: 5.0 - 8.0
- Optimum temperature: 55°C
- Tenderise beef, mutton, chicken meat & pork. Recognised by the United States federal
agencies as Generally Recognised As Safe (GRAS) to improve the meat tenderness
- Example: dipping beef cube in its solution before freeze drying ⇒ improved texture, marked
effect on collagen solubilization
( - ): some effect on texture and negative effect on visual appearance.
4. Ficin
- A vegetable-based enzyme that derived from figs latex
- Exhibit less activity against all types of proteins when compared to papain & bromelain
- Optimum temperature: 60 - 70°C
- Optimum pH for collagen and myofibrillar activity: 7
- Optimum pH for elastin degradation: 5.0-5.5
- Sensory evaluations tend to show only a marginal increase in tenderness when compared to
untreated samples
5. Actinidin
- Protease extracted from gooseberry or the kiwi fruit
- Commercially used in meat industry to tenderize meat and enhance the chemical processes
related to degradation of the myofibrillar proteins into peptides
- Show mild tenderizing activity even at high concentrations, preventing surface mushiness
- Optimum pH: 6.0 – 6.5 Optimum temperature: 50°C
6. Lipases
- Lipases are ubiquitous in nature, found in various organisms including : animals, plants, fungi
and bacteria.
- Esterases which are key enzymes involved in fat digestion.
- Optimum temperature : 30 - 40°C. Optimum pH : around 7
- Application
- Catalyze the formation of flavour esters.
- Used for flavour formation in sausage production.
- Facilitate the removal of fat from meat.
- Essential roles in digestion, transport and processing of dietary lipids ( e.g. triglycerides, fats,
oils )
7. Transglutaminase
- Transglutaminase is an enzyme capable of creating cross-linking with the protein of meat (fish)
and also with vegetable protein (in soy).
- It can catalyze the reaction of cross-linking within and between protein molecules, so TG can
improve the ability of food proteins.
- Optimum temperature: 40°C. Optimum pH: 5-8
- Sources
 Widely discovered in animal tissues and body fluids, fish, birds, invertebrates,
amphibians, plants and microbes.
 Transglutaminase is derived from bacterial fermentation. So, large amounts of growth
can be produced on an industrial scale.
- Enzyme production
 The extremely high costs of manufacturing transglutaminase from animal origin
 Producing enzymes synthesised by microorganisms – New Source
 Microbial transglutaminase was isolated from Streptoverticillium spp.
(+) Advantages
 Easy to use
 Mix with meat directly
 Create the natural cross linking of the protein veins of meat (fish).
 Does not affect the properties of meat.
 Stable during processing such as cutting, marinating, drying, and packaging.
 Can standardize and control components to create added value. Products can be
further processed to satisfy customers' wishes in food processing
- Application
 TG-SA has been developed for processing applications in poultry, red meat and
seafood.
 Strengthen the texture of homogenized sausages made of pork, beef or poultry meat
 Instead of high-quality meat, lower quality raw materials and additives: skimmed milk
powder, soy or wheat flour. The impact of the enzyme on the proteins of these raw
materials yields products which do not differ in appearance, texture, odour, taste and
nutritional value from analogical products made exclusively of high quality meat.
 The using of lower quality raw materials: mechanically deboned meat, collagen, blood
proteins
 Adding it with amino acids in which it is deficient (e.g. exogenous lysine).
 Higher nutritive value
8. Oxidative enzyme
- Can be an alternative for TGases to generate cross – link in protein matrices.
 - Include tyrosinases and laccases: use for creating covalent cross-links protein. Bind fresh meat
pieces together.
- Not common because of limited availability
- Main target: myofibrillar protein myosin. Affect gelation and the texture of meat gel
- Traditionally, using salt or phosphates with heat to bind meat pieces together. Now consumers
require lower salt content, fresh meat. => use enzyme
- Source: found in whole cells, tissues from mushrooms, fruits, and vegetables, fungi.
9. Tyrosinase
- In fruits and vegetables, tyrosinase controls the production of melanin, through the oxidation
of the amino acid tyrosine. The production of melanin cause enzymatic browning ( such as the
dark color of banana).
- Tyrosinases are copper - containing proteins, containing two copper atoms (𝐶𝑢3+)in their
active site to shuttle electrons from the substrate to molecular oxygen.
→ oxidation tyrosine residue to create the tyrosine-tyrosine, tyrosine-cysteine
or tyrosine - lysine cross-links
→ increase gelation of meat protein, increase firmness of meat gel
10. Laccases
- Laccases are also copper-containing enzymes, they contain four copper atoms in their active
site and use molecular oxygen as a terminal electron acceptor.
- Source: plant, fungi
- In plants, laccases are involved in cell wall formation and lignin biosynthesis. Can cross – link
protein or peptides.
- Laccases oxidize tyrosine and form tyrosine – tyrosine cross-link → Improve the firmness of
meat gel
- Excessive laccase protein fragmentation → the gel formation declined
11. Glutaminase
- Glutaminases is an enzyme that catalyzes the hydrolytic deamidation of L-glutamine to
glutamate and ammonia.
Glutamine + H2O → Glutamate + NH3
- Increased concentration of glutamate in meat improves the taste of meat
- Glutaminase appear to be involved in the increase in the muscle free glutamate level
- Fermented foods: In addition to nutritional benefit, protein associated glutaminase gives
texture and sensory properties to the foods
- Ammonia used in the fermentation industry as a source of nitrogen for microorganisms and to
adjust pH during fermentation.
- Source of glutaminase: in bacteria and eukaryotes; lack in archaea, thermophiles, plants.
- Glutaminases are mostly produced by Bacillus and Pseudomonas sp.
- For instance: from Aspergillus oryzae, Rhiobium etli, Debaryomyces.
- Optimum condition: range of 40 - 50oC and at neutral pH.
- Application
 Adding Mucor racemosus and Penicillium aurantiogriseum increases Ammonia - a
neutral acidity and Umami flavor.
 In Neam – a Thai fermented pork, they add rice into pork. Rice acts as a medium for
growing lactic acid bacteria. Glutaminase is produced from LAB to create flavor and
increase ammonia for neutralization of the acidity in fermented pork.

Question 15: Enzymes in protein modification;

( Short )
- There are 4 enzymes used in protein modification
  Protease: hydrolysed protein into smaller peptide
 Transglutaminase: cross-links proteins by forming an isopeptide bond between glutamine and lysine
 Tyrosinase: form covalent tyrosine–tyrosine, tyrosine–cystein or tyrosine–lysine cross-links by oxidative
reaction.
 Laccase: form tyrosine–tyrosine cross-links, forms disulphide bridges, oxidize thiol groups by oxidative
reaction
( Detail )
1. Proteases
- Proteases is an enzyme that performs protein catabolism by hydrolysis of peptide bonds. They are also
called proteolytic enzymes or proteinases.
- Proteases break down protein into smaller peptides. The primary goal of hydrolysis of proteins is to
disrupt the protein structure by breaking peptide bonds in amino acid chains to generate smaller
peptide fragments.
- This process reduces the molecular weight of the original protein, reduces the antigenicity and
allergenicity of the protein, and may increase digestibility.
- The protease catalyzed reaction cleaves a peptide bond in the protein:

Reaction scheme for protease catalyzed reaction

- Properties:
a) Taste: One of the most important properties for food protein hydrolyzates is taste.
 Intact proteins are tasteless in purified form. When proteins are hydrolyzed there are two main
contributions to the taste of the peptides.
 One is the release of flavour components ‘hidden’ in the protein structure, which are released
when the protein is hydrolyzed.
 But most important is the formation of small peptides with a relative high content of hydrophobic
amino acids which tend to create bitterness
b) Solubility
 Hydrolyzed proteins are used mostly as ingredients in beverages because of solubility property.
 The solubility of hydrolyzed proteins is higher, comparing with native protein
c) Viscosity
 A decrease in the viscosity of the protein solution caused by hydrolysis of protein.
 The reduction of protein viscosity is applied widely in production of protein-fortified drinks and
clinical nutrition products.
d) Emulsification
 Hydrolyzed protein generally has the inferior emulsifying property, compared to the native protein.
 By this property, hydrolyzed protein can be applied in low allergenic baby food products and other
nutritional products.
- Applications of hydrolyzed protein

Source Product Application

Hypoallergenic baby food ingredient

Peptigen
Fast absorbance peptides for dietetic and sports nutrition
Whey protein
Biozate Reduction of blood pressure

Casein Low allergenic peptides Hypoallergenic baby food ingredient

 Di-and tri-peptide peptides for sports nutrition
PeptoPro
Casein-phosphopeptides for dental care product

CE90GMM Casein-phosphopeptides for better mineral absorption

InsuVitalTM Manage blood glucose level – help type 2 diabetics

TensGuardTM Milk protein tri-peptide for blood pressure control

Immune enhancing enteral nutrition

Soya Soya protein hydrolyzate
Fully soluble peptides for protein fortification of acid drinks

Pea Pea protein hydrolyzate Protein fortification of drinks

Peptides for treating arthritis and

Gelatin Gelatin-hydrolyzate
osteoporosis

Nutripeptin
Lowers postprandial blood glucose
Fish Salmon protein
Nutritional food supplement
hydrolyzates

Egg white Benefit Protein fortification of drinks

Hydrolyzed meat protein

Meat Soluble meat protein/meat stocks for flavour and nutrition
extract

2.Transglutaminase
- Transglutaminase catalyzes the formation of isopeptide bonds between proteins in several food proteins
such as soy, milk, egg and wheat proteins. Transglutaminase has considerable potential to improve the
firmness, viscosity, elasticity and water binding capacity of food products.
- Sources
 Transglutaminases are widely distributed in animal tissues and body fluids, plants and
microorganisms
- Principle in protein modification
 Cross-links proteins by forming an isopeptide bond between glutamine and lysine and is used in
modifying the proteins in a food product but hardly ever used to produce protein ingredients
 Applications
 In commercial food processing, transglutaminase is used to bind proteins together (imitation crab
meat, fish balls). It is produced by Streptoverticillium mobaraense fermentation in commercial
quantities or extracted from animal blood.
 Transglutaminase can be used as a binding agent to improve the texture of protein-rich food such
as surimi and ham.

3. Tyrosinase
- Tyrosinase is a copper-containing enzyme that catalyzes sequential oxidation steps with various phenolic
substrates.
- Tyrosinases are mainly involved in the biosynthesis of melanin pigments from tyrosine amino acid as well
as from other polyphenolic compounds.
- Tyrosinases are important factors in wound healing and primary immune response.
- Source
  Tyrosinase can be produced and extracted through a number of organisms such as bacteria, fungi,
plants and mammals. Tyrosinases can be purified from these sources and studied for specific
functioning.
 Ex: Bacteria (Symbiobacterium thermophilum, Pseudomonas maltophilia,…)
Fungi (Agaricus bisporus, Aspergillus oryzae,…)
Plants (apple, sunflower seed, monastrell grape,…)
- Structure
 Due to multiple sources of tyrosinase its structural properties are diverse in nature along with their
distribution in tissues and cells, so no common protein is observed across all species.
 The difference is observed in primary structure, size, in post modification sites like active sites.
Common thing in all tyrosinases is their binuclear type III copper centre containing two copper atoms
each connected with six histidine molecules in their active site.
- Principle in protein modification
 An oxidative reaction can form covalent tyrosine - tyrosine, tyrosine–cystein or tyrosine–lysine cross-
links
- Applications
 Tyrosinase has been known for a long time to create intra- and intermolecular cross-links via tyrosine
residues in tropocollagen.
 The firmness of unheated pork homogenate gels have been improved by tyrosinase Sweet tasting
thaumatins (sweetener) have been stabilised by tyrosinase against proteolytic activities.
 The whey proteins β-lactoglobulin and α-lactalbumin have been polymerised by tyrosinase
4. Laccase
- Laccases are a group of enzymes belonging to the blue multicopper oxidases.
- Laccases have a broad substrate range, being able to oxidize various phenolic compounds, diamines,
aromatic amines and benzenethiols . Some laccases can also oxidize monophenols such as cresol and
tyrosine.
- Sources
 Laccases are common in nature and occur widely in fungi (Ascomycetes, Deuteromycetes and
Basidiomycetes), whereas in higher plants their occurrence is more limited. Laccases or laccase-like
proteins have also been identified in insects and bacteria.
- Function
 The physiological functions of laccases are all related to polymerisation or degradation processes.
 In higher plants laccases are involved in cell wall formation and lignin biosynthesis. In fungi, laccase
have a role in fungal morphogenesis and can protect fungal pathogens from toxic compounds in host
environments, acting as virulence factors in many fungal diseases. In insects laccases have been
reported to be involved in cuticle sclerotization. In the bacterial species Bacillus subtilis in pigment
production of endospores.
- Applications
 With substrates BSA, casein and pork, cross-linking of BSA and caseins by laccase can improve gel
strength of pork sausage.
 With substrates gelatin and fish, cross-linking of gelatin by laccase can improve elasticity of kamaboko
(surimi) gels.

Question 16: Lipases for the production of food components

Today the four main areas in which lipolytic enzymes (lipases and phospholipases) are applied are as
follows:
- Interesterification (hydrogenation, chemical and enzymatic), to modify melting properties for
fats for margarines and shortenings, without production of trans fats or by-products.
- Degumming, to render water soluble the phospholipid gums in a range of oils to allow them to
be removed without yield loss.
 - Ester synthesis within the cosmetic/oleochemical industry to produce esters and waxes with
lowered energy requirements and by-product formation.
- Speciality fats, the synthesis of speciality fats of nutritional importance such as high omega-3
fish oils.

Question 17: Cold active enzymes in food processing

- Cold-active enzymes are extremozymes produced by the psychrophiles (extremophiles) and
have attracted much attention as biocatalysts due to their capacity to resist unfavourable
reaction conditions in the industrial process.
- Cold-active enzymes possess wide applications in the food industry; these enzymes are not
only secreted by bacteria but also from yeasts and moulds.
- Although enzymes are derived from plant and animal sources, cold-active microbial enzymes
have taken advantage, due to their productivity and thermostability.
- Psychrophilic microorganisms produce a wide range of cold-active enzymes with immune
application in food processing.
- The use of ß-galactosidase for the removal of lactose from refrigerated milk, application of
pectinase for the reduction of viscosity and turbidity in chilled juice and use of amylase for
hydrolysis of polysaccharides in starch processing industries and processing of meat with the
help of cold-active proteases are the representative examples of application of cold-active
enzymes.
- In food processing industries, cold-active pectinases have been used for the removal of pectin
which is important in fruit juice and wine processing, coffee and tea processing and macerating
of plants and vegetable tissue, for degumming of plant fibres, for extracting vegetable oils and
for adding poultry feed and in the alcoholic beverages.
- The potential of cold-active enzymes provides numerous opportunities for industrial
applications. However, specific properties of cold-active enzymes may be improved and
modified through enzyme engineering.

Question 18: Production of industrial enzymes

- Improvements in protein engineering have yielded enzymes which are more efficient, stable
and have less side activities.
- Efforts have been made towards the development of plug-in platforms, that is limiting the
number of hosts in which an enzyme can be produced.
- Baseline processes, including fermentation, downstream processing and formulation,
developed for each host, are a starting point for the production of a new enzyme.
- Most fermentations for food enzyme production require the preparation of a sterile medium.
They are usually aerobic, requiring pressure, airflow, power and cooling.
- Fermentations are most often run in the fed-batch mode, although batch and continuous
processes can also be found. Since most food enzymes are secreted into the fermentation
medium, downstream processing simply requires removal of cells by filtration or
centrifugation, followed by a concentration step to make the product ready for formulation.
- Finally, a formulation step is often required, to allow for use in the application and/or to
extend shelf life.
- Most often for food applications, the enzyme is formulated in the solid state, although some
liquid formulation examples can also be found.
- Tablets are a popular form of delivery, as they can allow for homogenous blends of products
and a consistent dose delivered.
- Preservatives are used to control microbial growth in enzyme products. Customer preferences
have been evolving towards less preservative use, or at least more natural preservatives.