Industrial processes and

products in the Agri-food

Sector
Microbial Enzymes
• The microbial enzymes are gradually replacing the traditional
chemical catalysts in many industrial processes. Enzymes in
processes have many advantages over chemical catalysts;
including the ability to work at relatively "mild" conditions of
temperature, pH, and pressure: this results in a lower
consumption of energy and the possibility of working with less
expensive vessel because less resistant to corrosion
• the enzymes are highly specific, often stereoselective and do not
produce by-products; consequently the refining and purification
processes of the target product are less needed.
• Compared to chemical processes, those involving the use of
enzymes are "environmentally friendly" because the enzymes
are biodegradable and don’t pose problems for disposal.
• Enzyme classification established by the Commission on Enzymes
of the International Union of Biochemistry (1979) states there are
six major classes of enzymes according to the type of reaction
catalyzed:

• oxidoreductase (class 1): catalyses reactions of oxidation /

reduction, and the transfer of H, 0 or electrons;
• transferase (class 2) catalyzes the transfer of groups from one
molecule to another;
• hydrolases (class 3) catalyzes the hydrolysis, the breaking of
bonds by adding water molecules;
• lyase (class 4) catalyze the breaking of bonds with other routes
which are not hydrolysis or oxidation;
• isomerase (class 5) catalyze the structural rearrangement of
molecules;
• ligase or synthetase (class 6) catalyzes the formation of new
bonds i and C-N, C-O, C-S with AT ruptureP.
Suspended or immobilized microbial cells and spores may be
also employed as catalysts in some industrial bioconversion.

In many cases, however, there may be limitations because:

 The cells loose energy in growth and / or maintenance:
 Side reactions can lead to reduction in the yield potential of the
product;
 The conditions for microbial growth, if required, may be different
from those needed for the formation of the product
 You may find difficulty in isolation and purification of the product
from the cells or from the spent fermentation medium
• To overcome these problems, you can use partially purified
enzymes "bulk" in many industrial processes

• Several thousand tons of commercial enzymes are currently

produced each year for a value of over US $ 5800 million.
• some enzymes of animals and plants are used, but the most
have microbial origin: many of them are extracellular and in
much cases derived from species of Bacillus. In particular
Bacillus protease and amylase and other various
thermostable enzymes are very common.
Application in food sector
Cleaning products BURNUS -1959, mixture of sodium hydroxyde and trypsine
• There is also growing demand for purified enzymes to be
used in molecular biology, such as endonucleases, DNA
polymerases from Thermus aquaticus (Taq DNA
polymerase) or Pyrococcus furiosus (Pfu DNA
polymerase) used for amplifications of DNA.
• Many of these enzymes are intracellular and require
purification steps. They are therefore very expensive !!!
The worldwide market for enzymes now exceeds US $ 1.5 billion in
revenue and operated by approximately 400 companies: 14 of these
are considered the major manufacturers. The 60% of the marketed
enzymes are produced in Europe, 15% in the USA and the
remaining 15% in Japan.
…..
75% of industrial enzymes are used in processes of hydrolysis
and depolymerization of complex natural molecules with a
dominance of proteases used in detergents and in the dairy
industry (transgenic rennet).

The food sector, moreover, has the greater use of enzymes, with
applications ranging from the transformation of starch into glucose
and fructose, making cheese, wine, beer and fruit juices, baking and
food flavor production.
 Immobilized enzymes
In general terms, the enzyme may be employed either in the cell in
which it is contained, or in a purified molecular form and that is deprived
of its natural around.
In both cases, however, it is possible to use the biocatalyst is in free
form, or immobilized, ie associated reversibly or irreversibly to a solid
support.
The immobilization technique is known from several decades, since it
was found that certain enzymes are able to retain its activity after being
adsorbed on solid supports such as the natural clays.
 ENZYME

Support

Polymeric matrix

Semi-permeable membrane

Methods of immobilization: a) adsorption; b) formation of covalent

bonds; c) trapping; d) confinement in membranes.
The main advantages of immobilized enzymes are:

a) the possibility of using the enzyme in more cycles, both in batch and in
continuous flow, recovering the catalyst at the end of this reaction being
in a different physical form from substrates and reaction products;
b) a better control of the reaction, because the enzyme can be quickly
separated from the solution;
c) the ability to develop different application technologies for continuous
reactions, either in column or in batch.
d) the possibility to carry out consecutively, more reactions co-
immobilizing two or more enzymes on the same matrix, or by
transferring the product of a reaction in a second reactor for the
following enzymatic transformation;
Immobilized Enzymes
Reduction of the bitter taste of juices from oranges, grapefruits etc
(immobilized enzyme from Pennicillum)
 Alginate Immobilized yeast

Sparkling wines
– The small balls of alginate may be easily removed avoiding the long
practice of remouage

Sweet wines
The fermentation is stopped at the desired sugar content simply
removing the alginate balls from wine without filtration
‘ Application in food sector
 The commercial production of enzymes

• Most of the enzymes are typically produced in submerged

fermentation, although they are also produced in solid-
substrate fermentations in particular for the production of
extracellular fungal enzymes

• The first preparation of commercial enzymes was produced

in solid state and it was a "Takadiastase", a fungal amylase
from Aspergillus oryzae grown on wet rice or wheat bran.
The process was first developed by Dr Jokichi Takamine
and patented in the USA in 1884.
• Most of the processes have, however, been developed in
the second half of the twentieth century after the
development of "submerged“ fermentations.
• Many enzymes are now obtained by batch fermentation and
some in continuous fermentations. The fermenters for bulk
fermentations exceed 100m3; more expensive enzymes can
also be produced in fermenters with capacities of some
hundreds liters.
• Most of the fermentors are "stirred tank" and operate under
aseptic conditions using complex low cost culture media with
an indefinite composition.
• The development of each fermentation process begins with
the search for a good microbe. The use of GRAS microbes is
critical for enzymes to be used for food or clinical applications.
• You can start a screening program to determine the ability to
secrete the enzyme and its properties such as pH optimun,
heat resistance, etc.
• The enzymes of thermophilic mo typically give advantages
such as the thermostability, the ability to operate at high
temperatures compared to those of mesophilic; operating at
high T, increases the rate of diffusion and the solubility of the
substrate and lowers viscosity and risk of microbial
contamination
• the conditions of fermentation using less expensive raw
sources of carbon-nitrogen, will be determined

• the stability of the enzyme product which may affect the

production and downstream processes, must be
considered

• The level of applied purification considerably varies in

relation to its use

• the process of strain improvement with traditional or

molecular approach, can be used
• The molecular approach is today very appreciated: you
transfer the structural gene coding for the enzyme into a
easily cultivable host together with the control of gene
expression systems. This makes possible to rapidly
develop processes providing for the fermentation of large
quantities
• The expression in GRAS or QPS microbes is preferred:
candidates are Bacillus, Aspergillus, and
Saccharomyces whose biology is well known, are easy
and safe to handle and give high yields for enzymes ,.
Moreover the fermentation media and the growth
conditions are known, in this way they minimize the
costs.
• Enzymes for starch and related
carbohydrates processing

The enzymes that are used for starch processing, have high
economic value.
Starch is a polysaccharide composed of amylose (linear
polymer: glucose units linked by α-1-4 bond) and amylopectin
(branched polymer with α 1-4 and α 1-6 bonds).

• α-amylase (1,4-α-D-glucan glucanohydrolase) is one of the

most important industrial enzymes. This endo-enzyme acts
randomly on α-1-4 bonds and generates glucose, maltose
and maltotriose

• It can also catalyze the breakage of α-1-4 internal bonds in

glycogen and oligosaccharides
Amilose: Linear polymer of glucose units linked by α-1-4 bond)

Both molecules, amylose and amylopectin, have a reducing and a

non-reducing end.

Amilopectin: Branched polymer with α 1-4 and α 1-6 bonds

Enzymatic attack on part of an
amylopectin molecule. Glucose
molecules are indicated as circles
and the reducing ends are marked
by a line through the circle.
• Since the '50 fungal amylases have been used in the manufacture of
sugar syrups, containing specific blends of sugars, not obtainable by the
conventional acid hydrolysis of starch.
• In addition, the α-amylases are used in many industries including brewery
and bakery where they are needed to obtain starch liquefaction and
hydrolysis of dextrin.

• These enzymes are secreted by many microorganisms, bacteria and

yeasts. The thermostable α-amylase of Bacillus licheniformis are
particularly useful
• Other important amylases are the amyloglucosidase (glucoamylase)
of Aspergillus niger and Rhizopus, available from the '60. This
enzyme is able to completely hydrolize starch and dextrins into glucose.
After its introduction into the market, practically all the products obtained
by acid hydrolysis have been replaced by products obtained by enzymatic,
hydrolysis for both the higher yield and for the highest level of purification
and better crystallization of the products. This process has been further
improved by the introduction of highly heat stable bacterial amylase for the
pre-treatment of starch (liquefaction).
• A huge success was obtained by the glucose isomerase. This enzyme,
obtained by many bacteria including Bacillus and Streptomyces, is
generally used in the immobilized form for the glucose to fructose
conversion
• After the conversion, the final product has about the same calories of
glucose, but double sweetening power.
• The glucose used as a substrate usually results from the hydrolysis of corn
starch, then it is converted into a mixture of glucose and fructose by the
glucose isomerase. I
• The conversion yield is around 40-50%; the end product is called high
fructose corn syrup "or 'high fructose corn syrup' (HFCS) or"
isosyrup ". The concentration of fructose can be increased to 55% by the
chromatographic enrichment
• Alternatively, the fructose can be separated from glucose-fructose mixture
and the remaining fraction of glucose isomerized to produce a final product
containing over 80% fructose

• These derived starch syrups are less expensive than sucrose derived from
sugar cane or beet but have the same sweetening power. As a result they
have become the main ingredients of foods in particular in North America
and Europe.

• The annual production of HFCS now exceeds 8 x 109 kg

 Glucose-isomerase

Glucose-6-phosphate Fructose-6-phosphate
Immobilized Enzymes
 Corn starch

Fungal amyloglucosidase
and/or bacterial amylases

Glucose Isomerase

Glucose and Fructose

(HFCS)

Fructose

Fructose Glucose
Isomerase
Layout of a corn wet milling plan

Corn-derived starch is first converted into D-

glucose by the steps of liquefaction and
saccharification through the action of
thermostable α-amylases and glucoamylase,
respectively. The saccharified liquor is then
clarified by filtration and refined by passage
through carbon and ion-exchange filters.
Evaporation results in concentration to
glucose syrup with a dry solids content of 45
to 55% (w/w). Magnesium is added to a level
of 30 to 50 ppm in order to maintain the
activity of the immobilized glucose isomerase,
in addition to sodium bisulfite (100 mM) which
acts as a preservative. The pH is adjusted to
between 7.8 and 8.2 prior to a final filtration
step. The glucose liquor feed is fed in a down-
flow manner into a series of fixed bed reactors
arranged in parallel and held at around 60 °C.
The flow rate through a given column is
controlled so as to achieve the desired degree
of isomerization, which is a function of the
catalytic activity of the IGI bed and the flow
rate. Suppliers of immobilized GI typically
provide recommended operating parameters
and kinetic rate equations that allow their
customers to calculate the required process
conditions for optimal use of their products.53
Effluent with a fructose content of 42% w/w is
typical output.
• INVERTASE
• Sucrose is the most common sugar; after hydrolysis with
dilute acids or by action of the enzyme invertase, it form
equal amount of D (+) glucose and D (-) fructose;
• Invertase is an enzyme that catalyzes the hydrolysis of specific
glycosidic linkages, is located in yeast and the leaves of several
plants; bees provide invertase “inverting” sugar contained
in honey.
• The hydrolysis leads to a change of the direction of rotation of
the sugar, from positive to negative (hence the name inversion
of sugar). The mixture of sugars obtained takes the name of
inverted sugar.

• SPECIFIC OPTICAL ROTATION D sucrose + 66.5 ° + 52.7 ° D

D glucose fructose -92.4°
• The invertase (β-fructofuranosidase) was the first enzyme
to be immobilized on an industrial scale; It was developed in
the 40s in the UK for the production of syrups. The conversion
of sucrose into glucose and fructose by immobilized invertase
replaced the conventional acid hydrolysis. The enzyme was
originally prepared from S. cerevisiae autolysate and
stabilized at pH 4.7, clarified by filtration through calcium
sulfate.

• Today invertase is also prepared from other yeasts or

filamentous fungi such as A. niger and A. oryzae.

• In addition to the production of syrups, it is used in the

production of chocolates and candy; for example chocolate
with fondant filling: they are prepared from a solid filling of
sucrose and invertase and covered with chocolate. Within two
weeks, the center becomes soft and contains a fructose-
glucose syrup.
• Lactase (β-galactosidase) is used in various industrial processes
which require hydrolysis of lactose from milk, where the disaccharide
is present at concentrations of 4.7% (w / v). The hydrolysis of lactose
is performed in milk and milk products for infants who are lactose
intolerant and in some regions of the world where a large percentage
of the adult population is lactose intolerant, for example in certain
regions of Africa and South East Asia.
• The lactose hydrolysis is also useful in the production of ice cream
because the low solubility of this disaccharide leads to the formation of
crystals that can give a "sandy texture" unpleasant to the product.
Moreover, the conversion to glucose and galactose increases the
relative sweetness of about 4 times.
• Commercial lactase is obtained from Kluyveromyces marxianus
(formerly K. fragilis), A. niger or A. oryzae.
• The optimum pH of yeast enzyme is 6.0-7.0; pH for fungal enzyme is
3.0-4.0. These enzymes can be used either free or immobilized;
• the fungal enzyme is particularly used in whey processing.
• The yeast enzymes have been immobilized in cellulose acetate fibers,
those fungal porous silica of 0.5mm diameter for use in packaged
reactors (packed bell reactor).
Parzialmente scremato
Valori nutrizionali medi (100 ml)
ENERGIA (Kcal/Kj) 46/195
PROTEINE (g) 3,1
CARBOIDRATI (g) 4,9
di cui
GLUCOSIO (g) 2,2
GALATTOSIO (g) 2,2
LATTOSIO (g) 0,5
GRASSI (g) 1,6
CALCIO (mg) 120
 Enzymes for cheese production

• Rennin from the stomach of calves lambs etc has been used in the
production of cheese for centuries. These rennins contain chymosin
(an aspartate protrease) that causes the proteolysis of milk casein to
form the curd, the starting point in the manufacture of cheese. Even the
fig leaf juice that contains the proteolytic enzyme ficin, has been used
for the same purpose.

• The animal rennins, especially from veal, dominated the market until
the 60s when an increase in cheese production caused the shortage of
animal enzyme. At that time specific fungal protease were developed
with properties very similar to those of calf chymosin from Rhizomucor
miehei and R. pusillus. In this way the shortage of enzyme was
overcome and the production of "vegetarian“ cheese made easier.
• Recently the gene of calf chymosin was
introduced into various microorganisms
including E. coli, K. lactis, Aspergillus nidulans
and A. niger var. awamori

• These genetically modified microorganisms

are able to secrete the enzyme. The
recombinant chymosin has been approved
in more than 20 countries.

• The microbial chymosin covers about 1/3 of

the market to a value of approximately USD
75 million !!!
•Microbial lipases are used in the production of fresh
products (dairy) species for cheese for the hydrolysis of
esters of fatty acids to speedthe development of aromas.

triacylglycerols

Fatty acids glycerol

 Enzymes in the production of juices

• Several enzymes are used in the manufacture of fruit juices,

but probably the most important are the pectinase. These
process adjuvants are often produced by solid-substrate
fermentation of A.niger or species of Penicillium.

• Fruits and berries contain various amounts of pectins that act

as binders between the plant cells. Pectin is a heteropolymer
of galacturonic acid and sugars.

• In the production of juices, part of the pectin is extracted

during the pressing. This causes increase in the viscosity of
the juice, and then leads to difficulty to obtain optimum juice
yields and in the clarification and filtration processes.
• These problems can be overcame by adding pectinases to the fruit
pulp before pressing. Similar enzymes are used to increase the yield
from the pulp of olives, palm and coconut.
• Commercial pectinase is not a single enzyme, but a complex
cocktail of enzymes (pectin methyl-esterase, poligacturonasi and
pectin lyase) capable of attacking a variety of different bonds in
pectin.
• Several commercial preparations contain different proportions of the
various enzymes.
• Fungal pectinases are also available: they remain active in highly
acidic juices such as citrus, where the pH is 2.2-2.8. They can be
used in the enzymatic treatment of citrus canned juice.

• Apple juice contains starch which must be degraded to obtain a

clear juice and concentrated. This result can be obtained by adding
amylase together with pectinase
Nome

LAFAZYM CL

Codice

ENZIMA 06

Descrizione

Lafazym CL è un preparato enzimatico pectolitico

altamente concentrato e purificato, privo di
attività cinnamil esterasi(FCE). Specifico per
la chiarifica e la defecazione dei mosti
bianchi e rosati.
La concentrazione elevata di pectinasi
permette un idrolisi rapida delle pectine
dell'uva.

La confezione è disponibile da 100 e 250 g

The citrus juices such as grapefruit and bitter orange
contain a bitter flavonoid called naringin. The bitterness of
the juice can be reduced using the immobilized naringinase
(α+β-glucosidase-rhamnosidase) of Penicillium decumbens
and Aspergillus. The first enzyme converts naringin in
prunine, less bitter, the second in the prunin into the not
bitter naringenin. The level of hydrolysis of naringin can be
adjusted from the juice stream through a column of
immobilized naringinase.
Hydrolysis of naringin into prunin, rhamnose, naringenin and
glucose by naringinase containing α-L-rhamnosidase and β-D-
glucosidase activities
Glucose oxidase from A. niger or Penicillium species, often coupled with catalase,
can be employed to remove molecular oxygen from wines, beer, fruit juices or soft
drinks. The aim is to prevent oxidative damage that lower the quality

Glucose oxydase + catalase

2 glucose + O2 2 gluconic acid

SALIBABA.com
Glucose Oxidase
Quick Details 1 Introduction

CAS No.: Glucose Oxidase is made from a fungal strain through cultivation and
9001-37-0 extraction technique. It is mainly used in flour and baking processing to
Other Names: hydrolyze glucose into gluconic acid and peroxide to improve dough
Habio Glucose Oxidase and bread quality.
Place of Origin:
Sichuan, China (Mainland)
Type:
Enzyme Preparations, Nutrition
Enhancers
Brand Name:
Habio
Color:
White to Yellow
Production of Aminoacids
and
Organic Acids
• Several amino acids are produced in commercial quantities by fermentation
using hyperproducers strains

• They are mostly used as human food or animal feed supplements or as

compounds that contribute to the flavour formation.

• Amino acids also are used in pharmaceutical products and in cosmetics as

well as in the chemical industry for the production of polymers
L-Glutamic acid

• Among all the processes of production of amino acids, probably that of

L-glutamic acid is the most important in terms of quantity. It is mainly
used as a flavor enhancer in the form of monosodium L-glutamate
(MSG) that increases and intensifies the flavors of food without
significantly adding aroma.
• MSG is naturally present in some foods and it was discovered to be the
"active" component in giving the traditional flavor containing the Far
East algae. Firstly it was isolated in the alga Laminaria japonica in
1908.
• In 1959 the US Food and Drug Administration (FDA) classified MSG as
'Generally Regarded As Safe' (GRAS) based on its history and the Joint
Food and Agriculture Organization (FAO) / World Health Organization
(WHO) Expert Committee gave in 1970 the daily acceptable doses of 0-
120 mg / kg body weight
Glutamic acid
• Since the 60's classic production processes from algae, have been
replaced by fermentation processes which are now responsible for
producing more than 400,000 tonnes a year of MSG.
• The price of the MSG is about 1.2 USD / kg, and, besides the traditional
use in the East, it is added to a wide range of food products especially
soups, sauces, etc snaks

• Microbial species that produce glutamic acid are all species closely
related to the genus Arthrobacter, Brevibacterium, Corynebacterium,
Microbacterium and Micrococcus. They are Gram-positive bacteria,
which require biotin, and all have an intense activity of glutamate
dehydrogenase

Oxoglutaric acid + NH4+ + NAD(P)H + H+ Glutamate dehydrogenase

Acido L-Glutammico + NAD(P)+ + H2O

• Brevibacterium and Corynebacterium are the most used species in

industrial fermentations
• In the wild type strain of
Corynebacterium glutamicum the
feedback inhibition occurs when the
concentration of glutamic acid reaches
5% of the dry weight. The strains used
in production, subjected to intense
selection and development programs
through mutagenesis, are auxotrophic
mutants and regulated. They
accumulate up to 30% of glutamic
acid in the cytoplasm and give yields
of 1 mole of glutamate every 1.4
moles of glucose. They are also
resistant to the phage.
• Recently, molecular biology techniques
have been used to increase the specific
activity of biosynthetic enzymes for
transformation with a multicopy plasmid
which carries the structural genes for
the enzymes.
The strategy to get the hyper-producing strains provided that:

• the control of anabolic enzymes increased

• the feedback control system of enzymes was removed
• metabolic pathways that were to lead to undesirable by-products
were blocked
• During growth these mutants produce intermediates of the TCA from
isocitrate via glyoxylate.
• Furthermore, since these bacteria normally do not secrete
glutamate, the cells are made more permeable to the amino acid
limiting biotin, decreasing the synthesis of phospholipids by adding
to the medium during the growth C16-C18 saturated fatty acids,
surfactants (Tween 40) and penicillin
 The industrial production of L-glutamic acid

The industrial fermenters are made of stainless steel up to

450 m3. The batch processes operate aerobically at 30-37 °
C depending on the utilized microbe.

In addition to the sources of carbon and nitrogen, the

culture media normally contain inorganic manganese salts,
phosphates and potassium and limited levels of biotin.
Corynebacterium are troublesome from a nutritional point
of view and needs vitamins, amino acids, purine-
pyrimidine. The preferred carbon sources are
carbohydrates especially sucrose and glucose. Molasses
from sugar beet or cane can be used, but the medium
requires modifications as the level of biotin is too hight.
Moreover saturated fatty acids, penicillin or surfactants that
promote the secretion are added
• The source of nitrogen (ammonium salts, urea or ammonia) is
given slowly to prevent the inhibition of the production of L-
glutamate. The pH is maintained at 7-8 by adding alkali otherwise
it quickly falls down for the secretion of the amino acid.

• The amino acid accumulation becomes more or less evident at

mid-fermentation which lasts about 35-40 hours and reaches
levels in the broth of 80 g / L

• The recovery of the product involves the separation of the cells

from the broth.

• The l-glutamic acid is crystallized from the broth by lowering the

pH to the isoelectric point of 3.2 using hydrochloric acid

• The crystals are filtered and washed

• MSG is prepared by adding crystalline amino soda and

subsequent re-crystallization
ADDITIVES AND SUPPLEMENTS