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Enzymes in food
Enzymes
in food technology I
Dr. Otmar Höglinger
g g
1
Einsatz von Enzymen in Backwaren
Einsatz von Enzymen in Backwaren
2
Einsatz von Enzymen bei Käse und Milchprodukten
3
Eiprodukten Mayonnaisen Nudeln und Teigwaren
Eiprodukten, Mayonnaisen, Nudeln und Teigwaren
4
Fleisch Wurst Fisch
Fleisch, Wurst, Fisch
5
Süßwaren Stärkeverzuckerung
Süßwaren, Stärkeverzuckerung
6
Obst und Gemüse
Obst und Gemüse
7
Obst Gemüse und Wein
Obst, Gemüse und Wein
8
Bier
9
Bier und Spirituosen
Bier und Spirituosen
10
11
12
Amylose and Amylopectin structure
13
Starch granules
14
Retrogradation
• Gelatinization is the process in which starch becomes soluble, binds water and forms a gel. This process makes the starch more easily digestible The use of starch as a thickening agent
more easily digestible. The use of starch as a thickening agent is based on this process. • Starch is swelling up by heating and continues to swell up absorbing water and showing more viscosity and clarity g
g
y
y
along with increase of temperature. At a certain point the maximum viscosity has been reached. By further heating the starch molecules will move further apart and the viscosity is decreasing. 15
16
Retrogradation
• Viscosity is gradually increasing again when the solution is cooled down and continuous cooling makes the solution cloudy Leaving to stand the solution it will form a gel The
cloudy. Leaving to stand the solution it will form a gel. The strength of the gel is determined by the type and p
concentration of starch in the product. • When a starch gel is left to stand for some time, the amylose g
,
y
molecules will lose water and bind together. A similar process occurs when starch rich products, such as potatoes, will be stored for a long time. This process of recrystallisation of starch is called retrogradation. 17
Retrogradation
18
Effect of the action of starch degrading
enzymes
19
Typcal benifits of using enzymes in baking
20
Staling
•
Staling corresponds to loss of freshness in terms of flavour, texture, l
d
l
ff h
f fl
perceived moisture level and other product characteristics.
Anti‐staling agents used in bread include wheat gluten, enzymes, and glycerolipids,
mainly monoglycerides and diglycerides.
21
Staling
22
Staling
23
Baking process
24
Baking process
25
ENZYMES FOR BREAD, PASTA AND NOODLE PRODUCTS
• C
Consumers have certain quality criteria for bread, including h
t i
lit it i f b d i l di
appearance, freshness, taste, flavour, variety, and a consistent quality.
• Challenge:
• 1) Flour
1) Fl
• ‐ Varies due to wheat variety, weather during the growing season, and milling technology.
season, and milling technology. • ‐ Although millers attempt to blend wheat from different sources to produce flour with a good and consistent baking quality, it often proves difficult to satisfy both high‐quality lit it ft
diffi lt t
ti f b th hi h
lit
and low‐cost standards at the same time.
26
ENZYMES FOR BREAD, PASTA AND NOODLE PRODUCTS
• 2) bread preferences differ, the baking industry uses ingredients with different qualities and employs different baking procedures
baking procedures. • For instance, English sandwich bread, with its fine crumb structure and very soft texture is not popular with the
structure and very soft texture, is not popular with the French, who want baguettes with crispy crust, large holes, and good crumb chewiness.
• 3) consumer, preferences are shifting towards healthier products.
27
ENZYMES FOR BREAD, PASTA AND NOODLE PRODUCTS
• Therefore, both millers and bakers need ingredients or process aids such as chemical oxidants, emulsifiers and enzymes to standardise
to standardise the quality of the products and the quality of the products and
diversify the product range.
• For decades enzymes such as malt and microbial alpha‐
amylases have been used
y
for bread making.
g
28
Fungal alpha‐amylases
alpha amylases
• Wheat and thus wheat flour contains endogenous and indigenous enzymes, mainly amylases. However, the level of amylase activity varies from one type of wheat to another
amylase activity varies from one type of wheat to another. • The
The amount of alpha‐amylases in most sound, ungerminated
amount of alpha amylases in most sound ungerminated
wheat or rye flours is negligible. • Therefore, most bread flours must be supplemented with alpha‐amylases,
alpha
amylases, added in the form of malt flour or fungal added in the form of malt flour or fungal
enzymes
29
Fungal alpha‐amylases
alpha amylases
•
Fungal alpha‐amylases act on the damaged starch content, which can vary depending on wheat variety and milling conditions
depending on wheat variety and milling conditions. •
Generally, flour made from hard wheat contains more damaged starch than the soft wheat. •
The alpha amylases widely used in the baking industry can hydrolyse amylose and amylopectin to release soluble intermediate‐size dextrins of DP2–DP1. (DP Degree of polymerisation DP1 refers to a monosaccharide, DP2 refers to (DP=Degree of polymerisation,
DP1 refers to a monosaccharide DP2 refers to
disaccharides and so on)
•
The alpha
The
alpha‐amylases
amylases provide fermentable sugar, which results in an increased provide fermentable sugar, which results in an increased
volume, better crust colour, and improved flavour. •
There are six wheat classifications: 1) hard red winter, 2) hard red spring, 3) soft red winter, 4) durum (hard), 5) Hard white, 6) soft white wheat. The hard wheats have the most amount of gluten and are used for making bread, rolls and all‐purpose flour. The soft wheats are used for making flat bread, cakes, pastries, crackers, muffins, and biscuits. A high percentage of wheat production in the EU is used as animal feed, often surplus to human requirements or low‐quality wheat.
30
Dextrins
Dextrins are a group of low‐molecular‐
weight carbohydrates produced by the hydrolysis of starch or glycogen Dextrins
hydrolysis of starch or glycogen. Dextrins
are mixtures of polymers of D‐glucose units linked by α‐(1→4) or α‐(1→6) glycosidic bonds.
bonds
31
Fungal alpha‐amylases
alpha amylases
•
•
Due to hydrolysis of the damaged starch, a suitable dosage of alpha‐
h d l
f h d
d
h
bl d
f l h
amylases results in a desirable dough softening. However, extensive degradation of the damaged starch due to an ,
g
g
overdose of alpha‐amylases commonly leads to sticky dough.
32
33
34
Fungal alpha‐amylases
alpha amylases
Figure 1 illustrates the effect of a fungal amylase on bread f f
l
l
b d
quality in terms of bread volume, crumb structure and dough characteristics. The g
volume and crumb structure improve with increasing dosage of fungal alpha‐
amylases.
amylases
Although a high dosage can provide a larger volume increase, the dough would be g
too sticky to work with. The optimum dosage is thus defined as the dosage with maximum reachable volume
maximum reachable volume without a sticky dough. For the examples in figure 1, the optimum dosage for both flours is around 15 FAU/kg flour
(FAU = fungal alpha‐amylase units).
units)
35
Biochemical properties
36
Biochemical properties
37
Biochemical proporties
38
Biochemical proporties
39
Quality of
y wheat flour
•
1) The level of indigenous and endogenous amylases will influence the starch retrogradation as well as the yeast action, thereby affecting properties of the final as well as the yeast action, thereby affecting properties of the final
bread quality such as volume. •
2) The level of damaged starch in the flour has an influence on the action of cereal 2)
The level of damaged starch in the flour has an influence on the action of cereal
and fungal alpha‐amylase, and therefore on the final quality of the bread.
40
α
α‐Amylase
y ase
• α‐Amylase is an enzyme that hydrolyses alpha‐bonds of large alpha‐linked polysacharides such as starch and glycogen, yielding glucose and maltose
yielding glucose and maltose. • It
It is the major form of amylase found in humans and other is the major form of amylase found in humans and other
mammals.
• It is also present in seeds containing starch as a food reserve, and is secreted by many fungi.
and is secreted by many fungi.
41
Alpha‐amylase
•
Alpha‐amylase is used in ethanol production to break starches in grains into fermentable sugars.
•
The first step in the production of high fructose corn syrup is the treatment of cornstarch with alpha‐amylase, producing shorter chains of sugars called p
y
p
g
g
oligosaccharides.
•
An alpha
An
alpha‐amylase
amylase called called "Termamyl"
Termamyl , sourced from Bacillus licheniformis, is also sourced from Bacillus licheniformis is also
used in some detergents, especially dishwashing and de‐starching detergents.
42
Amylopectin
43
β amylase/ γ‐amylase β‐amylase/
γ amylase
•
β‐amylase catalyzes the hydrolysis of the second α‐1,4 glycosidic bond, cleaving off t
two glucose units (maltose) at a time. During the ripening of fruit, β‐amylase l
it ( lt ) t ti
D i th i
i
f f it β
l
breaks starch into maltose, resulting in the sweet flavor of ripe fruit.
•
γ‐amylase will cleave α(1‐6) glycosidic linkages.
44
Effect of different alpha‐amylases on bread staling
While fungal amylases are effective in partially hydrolysing damaged starch
starch,
and
are often added to flour as supplements to develop desirable properties such
as
ovenspring and a brown colour in the crust, they have limited anti-staling effect
due to their limited thermostability.
They are, for the most part, inactivated prior to the onset of starch gelatinisation
during baking when the bulk of the starch is available for modification.
The bacterial alpha-amylase
alpha amylase from Bacillus subtilis is able to inhibit staling
by hydrolysing glycosidic linkages within the amorphous areas of gelatinised
starch. However, this thermostable bacterial alpha-amylase can easily be
overdosed.
Its pure endo-action excessively degrades the starch during baking, causing
collapsing of the bread immediately after removal from the oven.
45
Effect of different alpha‐amylases on bread staling
Due tto the
D
th hi
high
hd
degree off thermostability,
th
t bilit the
th enzymes can persist
i t throughout
th
h t
baking and cooling and produce an excessive level of soluble dextrins. As a
result the final product is often unacceptable, with gummy crumb texture or
e en sticky
even
stick crumb
cr mb causing
ca sing problems d
during
ring slicing and retail storage
storage.
Maltogenic alpha-amylase has thermostability between that of fungal alpha
amylase and thermostable bacterial alpha-amylase. Therefore, it is able to
hydrolyse the glycosidic linkages of the gelatinised starch during the baking
process, but it does not excessively degrade the starch because it is
inactivated during the later stage of baking.
A major
j advantage
g with maltogenic
g
alpha-amylase
p
y
is its tolerance to
overdosing during the bread making process in the bakery.
46
Effect of different alpha‐amylases on bread staling
47
Effect of different alpha‐amylases on bread staling
48
Xylanases/pentosanases/hemicellulases
•
There are 3–4% (w/w) pentosans
There
are 3 4% (w/w) pentosans in normal wheat flour, partially soluble in normal wheat flour partially soluble
and partially insoluble. •
Xylanase or pentosanases, often commonly called hemicellulases, have or pentosanases often commonly called hemicellulases have
long been used as dough‐conditioning enzymes, especially in European‐
type bread, because they have demonstrated desirable effects as dough g
y
conditioning enzymes. •
At the optimum dosage they can improve dough machinability, dough y,
p g, g
p
stability, ovenspring, larger loaf volume and improved crumb structure. Because of the beneficial effects they have on loaf volume and crumb structure, the addition of hemicellulases results in a softer crumb. •
With the presence of fungal alpha‐amylase in the product, this effect on softness is even more pronounced.
49
Fungal Xylanase
50
Fungal Xylanase
51
Fungamyl
52
Fungamyl
53
Fungamyl
54
Fungamyl
55
Fungamyl
56
Fungamyl
57
Fungal amylase
58
Fungal amylase
59
Bacterial Amylase
60
Bacterial amylase
61
62
63
Synergistic effects of enzymes
•
Combining a hemicellulase
b
h
ll l
or xylanase
l
with fungal alpha‐amylase has a hf
l l h
l
h
synergistic effect.
64
Synergistic effects of enzymes
65
Synergistic effects of enzymes
66
Cell‐Wall Degrading Enzymes
•
Many microorgansms produce enzymes that degrade fruit cell walls.
•
Commercial pectinases for the fruit juice industry come from selected
strains of Aspergillus sp.
•
Enzymes are produced during fungal growth, purified and concentrated.
•
•
Hemicellulases
Hemicellulases are enzymes
y
that hydrolyze
y
y arabinogalactans, galactans, g
,g
,
xyloglucans, and xylans.
•
Amylases
l
In fruit
f
juice industry, fungal
d
f
l acid
d amylase
l
and
d amyloglucosidase
l l
d
are used to process fruits which contain starch. Aspergillus niger produces
acid amylase and amyloglucosidase.
67
Fruit cell wall compositionin g/kg fresh matter
68
Pectin
•
•
Pectin forms a familiy of complex polysaccharids that contain 1,4 linked α-D
galacturonic acid.
Three groups: homogalacturonan,
homogalacturonan rhamnogalacturonans and substituted
galacturonans.
69
Pectin structure
Pectin
P
ti is
i composed
d off two
t regions
i
: a rhamnogalacturonan
h
l t
region
i (alternate
( lt
t
Rhamnose ‐Rha‐ and Galacturonic Acid ‐GalA‐ linear chain substituted by Arabinose ‐
Ara‐ chain) and an homogalacturonan region. Soybean does not contain
homogalacturonan regions. Galacturonic
regions Galacturonic acid units might also be
also be substituted by
Methyl (Me) or Acetyl (Ac) groups. 70
Pectinstructure
71
Pectin
72
Biochemistry of fruit cell walls
•
Three major independent domains are distinguished: the xyloglucan
network, the pectin matrix, and the structural proteins.
•
The cellulose xyloglucan network is embedded in the pectin matrix.
•
Pectin
P
ti is
i the
th major
j structural
t t l polysaccharide
l
h id componentt off fruit
f it
lamellas and cell walls.
73
Pectinase action
Rhamnogalacturonase cuts the bonds between galacturonic acid and rhamnose in the rhamnogalacturonan region. Polygalacturonase cuts the linear chain of
ggalacturonic acid in the homogalacturonan
g
region. Pectin
g
esterase releases the
methyl residue linked to the galacturonic acid. Pectin acetylesterase releases the
acetyl residue linked to the galacturonic acid.
74
Pectinase
•
The first
Th
fi application
li i off enzymes in
i the
h fruit
f i juice
j i industry
i d
was the
h
use of pectinases for apple juice clarification in the 1930s.
•
Pectinase is an enzyme that breaks down pectin.
•
Commonly referred to as pectic enzymes, they include
pectolyase, pectozyme and polygalaturonase.
•
One of the most studied and widely used commercial pectinases is
polygalacturonase.
75
Polygalacturonase
76
Polygalacturonase
77
Polygalacturonase
78
Cell wall degrading Enzymes
79
Pectin degrading enzymes
80
Enzymes in wine production
Enzymes in wine
81
Enzymes in wine production
Enzymes in wine
• Th
The main application of industrial enzymes in i
li ti
fi d t i l
i
winemaking concerns the use of pectinase, hemicellulase and glucanase
g
preparations.
p
p
• Enzyme preparations can be supplied in granular, liquid or powder form. • The powder form is not recommended for use because of the allergic potential of enzyme dust. In granular form enzyme preparations do not produce dust due to
form enzyme preparations do not produce dust due to fixation of the enzyme particles. • Other advantages of granular enzyme products are the g
g
y
p
lack of preservatives and the good storage stability. • Liquid enzymes normally contain preservatives.
82
Enzymes in wine production
Enzymes in wine
• Applications of enzymes
en mes
Enzyme preparations are used throughout the whole winemaking process:
• on the grapes (weakening, maceration)
• in the must and the press wine (clarification and sedimentation)
• in the young wine at the end of the fermentation
in the young wine at the end of the fermentation
Enzymes are added to the process in different ways. In all cases solutions are made of the enzyme (granular enzyme preparations are also readily soluble)
also readily soluble).
• Actual addition proceeds via:
• spraying the enzyme solution on the grapes
• dosing the solution via a pump system directly into the inlet of the f
press
• adding the solution to the tank before clarification
83
Enzymes in wine production
Enzymes in wine
• The activity and efficiency of an enzyme can vary enormously, depending on temperature and pH.
• Must pectinases can be used in the temperature range of 10–
M t
ti
b
d i th t
t
f 10
55°C. Below 10°C the enzyme dosage should be increased. Above 55°C
Above 55
C the enzyme will be inactivated. the enzyme will be inactivated
• Enzymes are not inhibited by sulfur dioxide (SO2) at levels that are acceptable in wine. Inhibition by polyphenols may occur in p
y p yp
y
red wines. Increasing the enzyme dosage can counteract the effect. • Alcohol up to a level of 14% (w/v) has no negative influence on enzyme action. 84
Enzymes in wine production
Enzymes in wine
• EEnzyme activities involved in hydrolysing
ti iti i l d i h d l i pectic
ti
substances are pectin esterase, polygalacturonase, pectin lyase, rhamnogalacturonase, p
y ,
g
,
rhamnogalacturonan acetylesterase, arabinase and
galactanase.
• Other enzyme activities are of hemicellulase
Oth
ti iti
f h i ll l
and d
cellulase type and are normally present in varying p
p p
amounts in pectinase preparations. • The combined action of all these enzymes leads to a partial hydrolysis and solubilisation of acid and neutral polysaccharides present in the pectocellulose
l
h id
i h
ll l
wall and ll d
middle lamella of the grape cells. 85
Enzymes in wine production
Enzymes in wine
86
The increase in highest quality first press juice yield can go up to at least 10% and the pressing time can be reduced by 20‐50% as a result of the presence of enzymes.
87
Cinnamylesterase
•
•
•
For production of white and pink wines it i
it is recommended that enzyme d d th t
preparations that have been purified to remove cinnamyl esterase activities are used. Enzyme preparations used for d E
ti
df
making of white wines should have none of this activity. It is produced in nature by Aspergillus
niger and Botrytis cinerea species, and is responsible for the hydrolysis of coumaric and ferulic acids, which, after decarboxylation, lead to the formation of vinyl‐4‐phenol and vinyl‐4‐guaiacol. These compounds give a characteristic unwanted 'pharmaceutical‘ off‐flavour
in white
te wines.
es
88
Enzyme applications for must and press wines
• Clarification enzymes
en mes
• The clarification of must before alcoholic fermentation is of utmost importance.
• Proper settlement of floating particles considerably reduces the formation of aromatic C6 compounds, which lead to spicy or p
p y
savoury flavours.
• Enzyme
Enzyme preparations for clarification purposes have predominantly preparations for clarification purposes have predominantly
pectolytic activities. • Hydrolysis
Hydrolysis of pectic
of pectic substances leads to significant reduction of the substances leads to significant reduction of the
viscosity of the must and for reduction of the protecting colloidal effect of macromolecules.
89
Enzyme applications for must and press wines
• Settlement proceeds in three stages. • The first stage is depectinisation, characterised by partial b kd
breakdown of pectins
f
ti and decrease of the viscosity of the dd
f th i
it f th
must. • The second stage, the flocculation, is characterised
The second stage the flocculation is characterised by an by an
increase in turbidity and formation of insoluble complexes. • The third stage, the sedimentation, is mainly characterised
The third stage the sedimentation is mainly characterised by by
strong reduction of the turbidity and precipitation of the complex molecules. 90
Pectinase
91
Pectinase
A depside
A
depside is a type of polyphenolic compound composed of two is a type of polyphenolic compound composed of two
or more monocyclic aromatic units linked by an ester bond
92
93
Production of apple concentrate
94
Apple Juice depectinization
•
Pectin is the main cause of juice turbidity.
•
One liter of juice with 13% dry matter can contain 2‐5 g of pectin after pressing., depending on the ripeness of the fruit.
•
Pectinlyase or pectin methylesterase plus polygalacturase and arabanase
are the most important enzymes.
•
Aspergills niger is the main microorganism used for large scale
production of pectinases for the fruit industry.
industry
•
g secrete large amounts
g
of different enzymes
y
to
Wild strains of this fungus
break down the substrate on which they grow into nutrients for their own
metabolism.
95
Production of blackcurrant concentrate
96
Enzymic modification of food protein
modification of food protein
• Th
The hydrolysis of proteins with enzymes is an attractive way h d l i f
t i
ith
i
tt ti
of giving better functional and nutritional properties to food proteins of vegetable origin or from by‐products (e.g. scraps of meat from slaughterhouses). f
f
)
97
Alcalase, Subtilisin, Serin Endopeptitase
98
Subtilisin
• SSubtilisin
btili i is a non‐specific protease initially obtained from i
ifi
t
i iti ll bt i d f
Bacillus subtillis.
• Subtilisins belong to subtilases, a group of serine proteases g
, g p
p
that ‐ like all serine proteases ‐ initiate the nucleophilic attack on the peptide (amide) bond through a serine residue at the active site
residue at the active site. • Subtilisins typically have molecular weights of about 20,000 to 45,000 dalton. They can be obtained from certain types of soil bacteria, for example, Bacillus amyloliquefaciens
f il b t i f
l B ill
l li
f i
from which they are secreted in large amounts.
• The active site features a charge
The active site features a charge‐relay
relay network involving network involving
Asp‐32, His‐64, and active site Ser‐221 arranged in a catalytic triad. 99
Proteases
• P
Proteases have been used for more than 50 years to t
h
b
df
th 50
t
produce infant milk formulas from cow’s milk. • The proteases are used to convert the milk proteins into p
p
peptides and free amino acids. • The main reason is that non‐degraded cow’s milk protein can induce sensitization in infants when they are fed the
can induce sensitization in infants when they are fed the milk. • When a higher percentage of the milk protein is degraded, g
p
g
p
g
the risk of inducing sensitization or an allergic reaction is minimized. • This is very important for infants who are in a high risk This is very important for infants who are in a high risk
group for developing allergies or who are already allergic to cow’s milk. 100
101
Cow milk protein
milk protein and soy protein
102
Hydrolyzed formulas
103
Milk allergy
Milk allergy
• A
A milk allergy
ilk ll
i f d ll
is a food allergy, an adverse immune reaction d
i
ti
to one or more of the constituents of milk from any animal (most commonly alpha S1‐casein, a protein in cow's milk). This milk‐induced allergic reaction can involve anaphylaxis, a potentially life‐threatening condition.
• Hydrolyzed formulas are available in partially hydrolyzed Hydrolyzed formulas are available in partially hydrolyzed
and extensively hydrolyzed varieties. • Partially hydrolyzed formulas are characterized by a larger proportion of long‐chain peptides and are considered more ti
fl
h i
tid
d
id d
palatable. • However, they are intended for milder cases and are not However, they are intended for milder cases and are not
considered suitable for treatment of moderate to severe milk allergy or intolerance. 104
Milk allergy
Milk allergy
•
Extensively
E
i l h
hydrolyzed
d l
d fformulas
l are composed
d off proteins
i that
h h
have
been largely broken down into free amino acids and short peptides.
•
Casein and whey are the most commonly used sources of protein in
hydrolyzed formulas because of their high nutritional quality and
their amino acid composition.
composition
105
Enzymatic hydrolysis
•
Enzymatic
E
i h
hydrolysis
d l i off proteins
i ffrequently
l results
l in
i bi
bitter taste,
which is due to the formation of low molecular mass peptides
composed of mainly hydrophobic amino acids.
•
The formation of bitter peptide is the most serious problem in the
practical use of food protein hydrolysates.
hydrolysates
•
y the release of p
peptides
p
containing
g
Bitter taste is caused by
hydrophobic amino acids, such leucine, isoleucine, valine,
phenyloalanine,tyrosine and tryptophan.
106
Enzymatic hydrolysis
•
Isolated three bitter peptides with the structure of Gly‐Pro‐Phe‐Pro‐Val‐
l d h
b
d
h h
f l
h
l
Ile, Phe‐Phe‐Val‐Ala‐Pro‐Phe‐Pro‐Glu‐Val‐Phe‐Gly‐Lys and Phe‐Ala‐Leu‐Pro‐
Gln‐Tyr‐Leu‐Lys from trypsin casein hydrolysates. •
The peptides are built mostly of amino acids containing hydrophobic
groups.
groups
107
Milk based Formular
Milk based
108
Milk based formular
Milk based
109
Meat extracts
Meat extracts
• A range of raw materials from the meat industry can be utilized and processed into valuable ingredients for the
utilized and processed into valuable ingredients for the food industry. • An example is the meat left on the bones after cutting off p
g
the lean meat parts. This valuable meat protein normally makes up a high percentage of the total weight of the bone material. material.
• In order to utilize this the meat has to be made soluble, separated from the bone and fat material, and then dried t bt i th
to obtain the extracted protein. t t d
t i
• By using proteases for the process the protein can be made soluble by a gentle hydrolysis process, and products y g
y
y p
,
p
with different properties can be produced, mainly by varying the enzyme composition and dosage. 110
Meat extract
Meat extract
• P
Protein extracted with a relatively low degree of t i
t t d ith
l ti l l d
f
hydrolysis possesses some very good functional properties making it ideal for use as a marinade
properties, making it ideal for use as a marinade for meat products like ham or bacon. • These functional extracts can be used to improve the meat products with respect to flavour,
the meat products with respect to flavour, cooking loss and sliceability. • Other important applications for meat extracts p
pp
are as flavour improvers in soups, sauces, snack food and pot‐noodles. 111
Maltodextrins
•
Maltodextrins may be manufactured either by acid or by acid–enzyme
acid enzyme
processes.
•
Maltodextrins produced by acid conversion of starch from dent corn contain
a high percentage of linear fragments, which may slowly reassociate into
insoluble compounds causing haze in certain applications.
•
Haze formation,
H
f
ti
which
hi h results
lt ffrom retrogradation,
t
d ti
can be
b overcome b
by use
of alpha-amylases.
•
Alpha-amylases preferentially cleave the alpha-1,4-D-glucosidic
alpha-1 4-D-glucosidic bonds of
amylose and amylopectin , leaving a higher proportion of branched
fragments, decreasing the ability of the fragments to reassociate.
•
Maltodextrins made from waxy corn starch also have a lower tendency to
haze, because such starch is composed almost entirely of the highly
branched molecule, amylopectin.
112
Maltodextrin process
• IIn a maltodextrin
lt d t i process using enzyme‐catalyzed i
t l d
conversion, the starch slurry (30% to 40% dry solids) is first pasted at a temperature of 80–
solids) is first pasted at a temperature of 80
90°C, and is then treated with a ‘heat‐stable’ bacterial alpha‐amylase for liquefication. p
y
q
• When stabilized with calcium ions, alpha‐
amylases from B. licheniformis or B. Stearothermophilus can withstand temperatures of 90–105°C for at least 30 minutes, allowing sufficient process time to split the 1 4 bonds and
sufficient process time to split the 1,4 bonds and form maltose and limit dextrins.
113
114
115
116
117
Acid Enzym Process
Acid‐Enzym Process
• A
As in the case of acid‐catalyzed hydrolysis, the starch i th
f id t l d h d l i th t h
molecule is hydrolyzed to the desired starting DE in a converter, but further conversion is carried out with enzymes until the final DE or carbohydrate profile is f
f
reached. • This is done by adding the appropriate enzymes to the acid
This is done by adding the appropriate enzymes to the acid‐
converted slurry and allowing them to react in a holding vessel called an ‘enzyme tank.’ Several enzymes may be used to achieve the desired carbohydrate profile.
used to achieve the desired
profile
• The alpha‐amylases (EC3.2.1.1) used are bacterial or fungal enzymes that hydrolyze alpha‐1,4 linkages in both amylose and amylopectin, eventually producing dextrose and maltose. 118
Acid Enzym Process
Acid‐Enzym Process
• Th
The beta‐amylases (EC3.2.1.2) used are enzymes of barley b t
l
(EC3 2 1 2)
d
fb l
and yeast that act on the non‐reducing ends of starch molecules and produce maltose in the beta form from the starch polymers. These enzymes are used to produce high‐
maltose syrups.
• Although beta
Although beta‐amylase
amylase converts linear chains completely to converts linear chains completely to
maltose, the enzyme cannot cleave branch points and the yield of maltose from amylopectin is only 55% of the molecule.
molecule
• Glucoamylases (EC3.2.1.3) are fungal enzymes which
hydrolyze maltose to produce glucose (dextrose). These enzymes catalyze hydrolysis of alpha‐1,3, alpha‐1,6 and
beta1,6 linkages. 119
Acid Enzym Process
Acid‐Enzym Process
• Pullulanase (EC3.2.1.41) and isoamylase (EC3.2.1.68) are so‐
called debranching enzymes because they catalyze the hydrolysis of the 1 6 linkages without effect on the 1 4
hydrolysis of the 1,6 linkages without effect on the 1,4 linkages. These enzymes are particularly useful in the p
production of extremely high maltose syrups with maltose y g
y p
levels of 50 to 90%.
120
High fructose Syrups
High‐fructose Syrups
• U
Using immobilized enzyme technology, it is possible to i i
bili d
t h l
it i
ibl t
produce high‐fructose syrups containing 42%, 55% or 90% fructose. • A starch solution at about 35% solids and a pH of about 6.5 is drawn into a steam jet at 82°C in the presence of a calcium‐stabilized
calcium
stabilized, thermostable alpha amylase. thermostable alpha amylase
• The slurry is maintained at this temperature through a series of loops for 3–5 minutes and then cooled to 95°C in a secondary reactor, where further alpha‐amylase additions d
t
h
f th
l h
l
dditi
occur. • A holding time of up to 120 minutes in the secondary A holding time of up to 120 minutes in the secondary
reactor produces a solution of approximately 12 DE. The pH is adjusted to about 4.3 and glucoamylase is added. 121
High fructose Syrups
High‐fructose Syrups
• Th
Then the product is pumped to saccharification
h
d i
d
h ifi i tanks k
where the enzyme reacts for 24–90 hours. The glucoamylase reaction produces liquor containing 94% reaction produces liquor containing 94%
dextrose, which is then filtered to remove residual p
protein and fats before being passed through beds of gp
g
activated carbon.
• Following carbon purification, the hydrolyzate
g
p
y
y
is demineralized through anion and cation exchange resins prior to being isomerized.
• The conversion step to high‐fructose syrup takes place in a reactor containing immobilized glucose isomerase. 122
123
Cellulasen
•
Cellulase refers to a class of enzymes produced chiefly by fungi, bacteria,
and protozoans that catalyze cellulolysis (i.e. the hydrolysis of celluloses).
•
However, there are also cellulases produced by a few other types of
organisms,
g
, such as some termites and the microbial intestinal symbionts
y
of
other termites.
•
Several different kinds of cellulases are known
known, which differ structurally and
mechanistically.
124
Cellulases
•
•
Five general types of cellulases based on the type of reaction catalyzed:
Endocellulase breaks internal bonds to disrupt the crystalline structure of cellulose and expose individual cellulose polysaccharide chains
and expose individual cellulose polysaccharide chains
•
Exocellulase cleaves two to four units from the ends of the exposed chains produced by endocellulase, resulting in the tetrasaccharides or disaccharides, such d db
d ll l
li i h
h id
di
h id
h
as cellobiose. •
Cellobiase or beta‐glucosidase hydrolyses the exocellulase product into individual monosaccharides.
•
Oxidative cellulases depolymerize cellulose by radical reactions, as for instance cellobiose dehydrogenase.
•
Cellulose phosphorylases depolymerize cellulose using phosphates instead of water.
125
Cellulases
•
In many bacteria, cellulases in-vivo are complex enzyme structures
organized in supramolecular complexes, the cellulosomes.
•
They contain roughly five different enzymatic subunits representing
namely
•
endocellulases, exocellulases,
endocellulases
exocellulases cellobiases,
cellobiases oxidative cellulases and
cellulose phosphorylases wherein only endocellulases and cellobiases
participate in the actual hydrolysis of the β(1→ 4) linkage.
126
Cellulases
127
Cellulase
•
Cellulase
C
ll l
i used
is
d ffor commercial
i l ffood
d processing
i iin coffee.
ff
It performs
f
hydrolysis of cellulose during drying of beans.
•
Furthermore, cellulases are widely used in textile industry and in laundry
detergents.
•
They have also been used in the pulp and paper industry for various
purposes, and they are even used for pharmaceutical applications.
•
Cellulase is used in the fermentation of biomass into biofuels, although this
process is relatively experimental at present.
128
Asparaginase‐an enzyme for acrylamide reduction in food products
•
IIn 2002,
2002 it was discovered
di
d that
th t relatively
l ti l high
hi h concentrations
t ti
off
acrylamide are found in common carbonhydrate rich foods prepared by
baking or frying.
•
Acrylamide is classified as probably cancerogenic to humans and ist
occurence in food products has therefore caused intensive debate
concerning the potential health risk through dietary exposure.
129
Acrylamid content of several typical food categories
130
Schematic overview of acrylamide formation from a reducing sugar and the
amino acid asparagine
•
Asparaginase
A
i
catalyze
t l
th hydrolysis
the
h d l i off the
th amide
id group off the
th side
id chain
h i
of asparagine to produce aspartic acid and ammonium.
•
Asparaginases participate in basic amino acid catabolism by shuttling
asparagine to aspartic acid which, after subsequent conversion to
oxalacetate, can enter the citric acid cycle.
•
Implementing an asparaginase treatment broadly into a range of food
products is not a simple
p
p undertaking,
g, since for each p
product the food matrix
or components may influence enzyme action and reactivity.
131
Schematic overview of acrylamid formation from a reducing sugar and the
amino acid asparagine
132
Asparaginase
133
Asparaginase in cereal food Products
•
Asparaginase can be successfully applied for acrylamide reduction in a range
a ge o
of ce
cereal‐based
ea based recipes
ec pes without
t out cchanging
a g g tthee taste
taste and
a d appea
appearance
a ce
of the final product.
•
Asparaginase has been shown to work in both hard and short doughs, and
at temperatures between 10°C and 40°C.
•
Acrylamide reduction of 50‐90% was seen for the different recipes and
process parameters. •
For cereal food applications, enzyme performance is dependant upon processingg conditions, for
p
,
example
p temperature, pH and
p
,p
restingg time, and
,
most importantly water availability. 134
Lactase
•
Lactase is a glycoside hydrolase enzyme that cuts lactose into it's constituent sugars, galactose and glucose. sugars, galactose
and glucose.
•
Without sufficient production of lactase enzyme in the small intestine, humans become lactose intolerant resulting in discomfort (cramps gas and diarrhea) in
become lactose intolerant, resulting in discomfort (cramps, gas and diarrhea) in the digestive tract upon ingestion of milk products. •
Lactase is used commercially to prepare lactose‐free products, particularly milk, for such individuals. •
It is also used in preparation of ice cream, to make a creamier and sweeter‐tasting product. Lactase is usually prepared from Kluyveromyces sp. of yeast and Aspergillus sp. of fungi.
135
Lactase properties
•
•
The properties of the enzyme depend on its source. Temperature and pH optima differ from source to source and with the type of
Temperature and pH optima differ from source to source and with the type of particular commercial preparation.
•
Immobilization of the enzymes, method of immobilization, and type of carrier can bili i
f h
h d fi
bili i
d
f
i
also influence these optima values. •
In general, fungal lactase have pH optima in the acidic range 2.5–4.5, and yeast and bacterial lactases in the neutral region 6–7 and 6.5–7.5, respectively.
g
, p
y
The variation in pH optima of lactases makes them suitable for specific applications, for example fungal lactases are used for acid whey hydrolysis, while y
yeast and bacterial lactases are suitable for milk (pH 6.6) and sweet whey (pH 6.1) (p
)
y (p
)
hydrolysis. •
136
Using lactase
•
The enzymatic hydrolysis of lactose can be achieved either by free enzymes, h
i h d l i fl
b
hi d i h b f
usually in batch fermentation process, or by immobilized enzymes or even by immobilized whole cells producing intracellular enzyme. •
Large‐scale systems which use free enzyme process have been developed for Large‐scale
systems which use free enzyme process have been developed for
processing of UHT‐milk and processing of whey, using K. lactis lactase (Maxilact, Lactozyme). 137
Using lactase
•
Snamprogretti process of industrial‐scale milk processing technology in Italy is one such working systems. They make use of fibre‐entrapped y
yeast lactase in a batch process, and the milk used is previously sterilized p
p
y
by UHT. •
FFor pilot plants, there are three other processes designed and developed il t l t th
th
th
d i d dd l
d
to handle milk; (i) by Gist‐Brocades, Rohm GmbH (Germany), and (ii) by Sumitomo, Japan. These are continuous processes with short residence times. Processing of whey UF‐permeate is accomplished by the system developed by Corning Glass, Connecticut, Lehigh, Valio and Amerace corp. The process by Corning Glass is being applied at commercial scale in the bakers yeast production using hydrolysed whey. 138
Lactase properties
•
Product inhibition, e.g. inhibition by galactose, is another property which also depends on the source of lactase The enzyme from A niger is more strongly depends on the source of lactase. The enzyme from A. niger
is more strongly
inhibited by galactose than that from A. oryzae. This inhibition can be overcome by hydrolysing lactose at low concentrations by using immobilized enzyme systems or by recovering the enzyme using ultrafiltration after batch hydrolysis. by recovering the enzyme using ultrafiltration
after batch hydrolysis
•
Lactase from Bacillus species are superior with respect to thermostability, pH operation range, product inhibition, and sensitivity against high‐substrate ti
d t i hibiti
d
iti it
i t hi h b t t
concentration. •
Thermostable enzymes, able to retain their activity at 60°C or above for prolonged periods, have two distinct advantages viz. they give higher conversion rate or shorter residence time for a given conversion rate, and the process is less prone to microbial contamination due to higher operating temperature. 139
Catalase
•
The enzyme Catalase has found limited use in one particular area of cheese production.
•
Hydrogen peroxide is a potent oxidizer and toxic to cells. It is used instead of pasteurization, when making certain cheeses such as Swiss, in order to preserve natural milk enzymes that are beneficial to the end product and flavour
natural milk enzymes that are beneficial to the end product and flavour
development of the cheese. These enzymes would be destroyed by the high heat of pasteurization. •
•
However, residues of hydrogen peroxide in the milk will inhibit the bacterial cultures that are required for the actual cheese production, so all traces of it must be removed. •
Catalase enzymes are typically obtained from bovine livers or microbial sources, y
yp
y
,
and are added to convert the hydrogen peroxide to water and molecular oxygen.
140
Enzymatische Verfahren der Lebensmittelhaltbarmachung
Biokonservierungg
141
Glucose Oxidase
Glucose‐Oxidase
142
Glucose Oxidase
Glucose‐Oxidase
• Di
Die enzymatische Entlüftung von Apfelsaft ist nur ein Beispiel für ti h E tlüft
A f l ft i t
i B i i l fü
eine erfolgreiche Hemmung des Hefewachstums und die Antioxidation. Der Anstieg der Produkthaltbarkeit ist die Folge. Es ist außerdem möglich die die Reaktivität der L Ascorbinsäure
ist außerdem möglich die, die Reaktivität der L‐Ascorbinsäure effektiver zu nutzen oder Schwefeldioxid zu minimieren oder zu substituieren.
143
Einsatz von Enzymen als Antioxidantien
144
Lactoperoxidase Reaktion
145
Lactoperoxidase und Myeloperoxidase
und Myeloperoxidase
146
Lactoperoxidase und Myeloperoxidase
und Myeloperoxidase
•
Die antimikrobiellwirkenden
k b ll k d Komponenten sind die Intermediärprodukte
dd
d
d k
der Reaktion, das Hypothiocyanat und höhere Oxidationsprodukte. •
Ihre Wirkung besteht mit essentiellen SH‐Gruppen von Enzymen, auf der Permeabilisierung von Zellen und auf der Oxidation von Reduktionsäqivalenten.
Reduktionsäqivalenten
147
Lytische Enzyme
148
Lysozym
149
Lysozym
•
•
Als Lysozym werden Enzyme mit N‐acetylmuramyl‐Hydrolase ktivität
l
d
l
l d l
k
bezeichnet, welche die Hydrolyse der glykosidischen Bindung zwischen N‐
Acetylmuraminsäure und N‐Acetylglucosamin der Peptidoglucanstränge
der Zellwände gram‐positiver und einiger gram‐negativer Bakterien katalysiert.
Zur Lyse von Schimmelpilz
Zur Lyse
von Schimmelpilz‐ und Hefezellen werden vorwiegend Chitinasen
und Hefezellen werden vorwiegend Chitinasen
und Glucanasen eingesetzt.
150
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
1) Struktur von Amylose und Amylopektin
2) E klä di B iff G l i
2) Erkläre die Begriffe Gelatierung
und Retrogradation
dR
d i
3) Wie verläuft die Viskositätskurve bei der Erwärmung von Stärke
4) Welche Vorteile bringt der Enzymeinsatz beim Brotbacken
5) Was versteht man unter Staling, welche Veränderungen gehen beim Staling vor
6) Beschreibe den Backprozess
7) Warum werden Enzyme im Backprozess eingesetzt
8) Welche Amylasen werden eingesetzt, welche Vorteile und Nachteile haben sie.
9) Was bedeutet DE wie sind Dextrine aufgebaut
9) Was bedeutet DE wie sind Dextrine aufgebaut
10) Welche biochemischen Parameter charakterisieren Enzym wie die Amylase
11) Wo findet die alpha Amylase Anwendung
12) Welche Enzyme gibt es die Polysaccharide spalten können, wie wirken sie
13) Wie wirken Amylasen beim Staling Effekt
13) Wie wirken Amylasen beim Staling
14) Welche Funktion haben Xylanasen/Pentosanasen und Hemmicellulasen
15) Wie sind Pektine aufgebaut, beschreibe die Struktur
16) Wie ist die Zellstruktur bei Pflanzenzellen aufgebaut
17) B h ib d Abb
17) Beschreibe den Abbaumechanismus von Pektin
h i
P ki
18) Wie wirkt Polygalacturonase
19) Auf welchen Stufen der Weinproduktion werden Enzyme eingesetzt. Welche? Welche Aufgaben haben sie?
20) Welche Rahmenbedingungen beeinflussen die Enzymaktivität bei der Weinherstellung. )
l h
h
b d
b
fl
d
k
b d
h
ll
Beschreibe diese.
21) Welche Bedeutung hat die Cinnamylesterase?
22) Auf welcher Stufe der Mostherstellung werden Enzyme eingesetzt? Welche Funktion haben sie.
151
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
23) Wie wird Apfelkonzentrat hergestellt. Auf welcher Stufe werden Enzyme eingesetzt?
24) Welche Enzyme werden zur Modifikation von Proteinen eingesetzt.
25) Welche Bedeutung hat Subtilisin, wie ist das aktive Zentrum ausgebildet
26)Wodurch wird Milchallergie ausgelöst, wie werden Produkte hergestellt die keine Allergien verursachen?
27) W l h V und Nachteil bringt die Hydrolyse von Proteinen, wodurch entsteht der bittere 27) Welche Vor‐
d N h il b i
di H d l
P
i
d h
h d bi
Geschmack, 28) Kann Soyamilch als Ersatz für Kuhmilchallergie eingesetzt werden?
29) Wie können Fleischextrakte hergestellt werden, welche Enzyme werden verwendet, wo werden diese Fleischextrakte verwendet
Fleischextrakte verwendet
30) Wie wird Maltodextrin hergestellt, beschreibe den Prozess
31) Beschreibe den Säure Enzym Prozess für die Hydrolyse von Stärke, welche Enzyme werden eingesetzt
32) Wie wird High Fructose Syrup hergestellt. Welche Enzyme werden verwendet.
33) Welche Enzyme können Cellulose abbauen. Beschreibe diese. Wo werden sie in der
33) Welche Enzyme können Cellulose abbauen. Beschreibe diese. Wo werden sie in der Lebensmittelindustrie eingesetzt.
34) Asparaginase hat welche Funktion, wo wird es eingesetzt, beschreibe den Reaktionsmechanismus
35)Wie wird Acrylamid in Lebensmitteln gebildet?
36) Welche Funktion hat Lactase. Wie wird Lactase zur Herstellung lactosefreier Milch eingesetzt. Welche Eigenschaften hat Lactase
37) Nach welchen Prinzipien erfolgt die Biokonservierung
38) Beschreibe das Reaktionsprinzip der Glucoseoxidase, wo wird sie eingesetzt
39) Wo werden Enzyme als Antioxidantien eingesetzt
40) Lactoperoxidase hat welche Funktion, 41)Lytische Enzyme haben welche Funktionen, welche lytrischen Enzyme gibt es
42 Wie wirkt Lysozym
152