Note: Descriptions are shown in the official language in which they were submitted.
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~
Milk product and method for its preparation
Field of the Invention
The present invention relates to milk products with improved struc-
ture. More specifically it relates to a method of using a particular enzyme
for
modifying the structure of milk products. The invention especially relates to
preparing acidified milk products such as yogurt. The invention enables the
use of raw or pasteurized milk instead of more severely heat-treated milk.
Background of the Invention
Milk products constitute an essential nutritional source in the human
diet. Milk is an excellent protein source, and in addition it usually contains
min-
erals, such as calcium, and vitamins. Acidified milk products that are conven-
tionally prepared by lactic acid bacterial fermentation may further comprise
health-promoting lactic acid bacteria. Milk products also contain animal fats,
which have an important contribution to the mouthfeel of the products.
In addition to the chemical and biological ingredients of a milk prod-
uct, also the physical properties such as viscosity, thickness, firmness and
wa-
ter-holding capacity have a great influence on the quality, and especially the
texture of the product. Texture is not only related to sensory perception but
also to water holding capacity, gelling, emulsifying properties and stability.
Separation of water from a product is a phenomenon called syneresis, which
may be associated with the preparation of acidified milk products. Syneresis
is
undesirable and should be avoided or at least minimized. A number of ways for
improving the physical properties of food products have been described.
Milk gels are formed during acidification due to casein gelation.
Heat treatment before acidification has been considered an important step for
the texture of the acid milk product (Pereira et al., 2003). If milk is not
heated
only casein contributes to gel formation. After heat-induced denaturation whey
proteins take part in the texture formation of acid milk gels both by
associating
partly with casein micelles, and by forming whey protein aggregates dispersed
in the continuous casein network (van Vliet et al., 2004).
Another approach to improve the structure of yogurt and other milk
products is to elevate the protein content, or add thickening agents such poly-
saccharides, pectin, gelatin and other hydrocolloids, and starch. The
thickening-
agents increase the viscosity of the yogurt, but unfortunately the
palatability
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may decrease due to a gluey mouthfeel. A further disadvantage is the custom-
ers' negative attitude towards food additives in general.
A more natural way of improving the properties of milk products is to
modify them enzymatically. Enzymes can be used for modification of the tech-
nological properties of foodstuffs e.g. by creating covalent cross-links
between
amino acid residues in proteins or by enzymes oxidizing certain amino acid
residues. Oxidation of amino acid residues can in turn also result in
formation
of cross-links. Modification of proteinaceous material by cross linking is fre-
quently used in food processing.
Enzymes suggested for food applications include transglutami-
nase, lipoxygenase, polyphenol oxidase (tyrosinase), peroxidase, lysyl oxi-
dase, protein disulfide isomerase and sulfhydryl oxidase (Matheis and
Whitaker, 1987). Further proposed enzymes are laccase, bilirubin oxidase,
ascorbic acid oxidase and ceruloplasmin, which have been proposed for cross-
linking e.g. plant and animal proteins such as cereal proteins, casein, 9-
lactoglobuiin, and egg proteins (1JS2002/000970). The applicability of a par-
ticular cross-linking enzyme for a particular use depends on a number of fac-
tors, such as availability and accessibility of substrate, possible
interfering
compounds, addition of external substrates, pH, temperature, inhibitors etc.
Currently, transglutaminases (TG, glutaminylpeptide:amine y-
glutamyltransferase, EC 2.3.2.13) are the only intensively studied and com-
mercially available enzymes for cross-linking of milk proteins. The reactivity
of
transglutaminase is dependent on the availability and accessibility of the
target
amino acids glutamine and lysine in the protein substrate.
The main proteins of cow milk consist of as1-, a s2-, (3 and ,c-caseins,
and the globular whey proteins a-lactalbumin (a-La), P-lactoglobulin, (P-Lg).
Casein is considered a very good substrate for transglutaminase, whereas
globular whey proteins, i.e. a-La and R-Lg, on the other hand have been shown
to be poor substrates. Partial unfolding of globular proteins by chemical
modifi-
cation, with reducing agents or by thermal treatment has improved their sus-
ceptibility to cross-linking by transglutaminase.
Nonaka et al., 1992 have studied the reactivity of transglutaminase
to casein in skim milk powder, and compared it to its reactivity to caseinate.
They found that the reactivity of transglutaminase to casein in the milk was
in-
ferior to that to caseinate, which indicates that casein in milk is not
susceptible
to enzymatic cross-linking in the same way as caseinate.
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It has been shown that heating the milk prior to cross-linking with
transglutaminase dramatically increases the reactivity of the milk proteins.
Preheating at 35 C for 15 minutes has been found to enhance the susceptibil-
ity of milk proteins to cross-linking (Sharma et al. 2001). It has further
been
found that cow milk heat treated at low temperature (63 C, 30 min.) had a
lower susceptibility to transglutaminase than cow milk sterilized at 130 C (2-
3
sec). When the former was additionally heat-treated to 90 C, the reactivity to
transglutaminase significantly improved. Preheating raw milk to 95 C for 2
seconds before reaction with transglutaminase has also been reported to be
effective in yoghurt production (1JS2002/0061353).
However, too severe heat-treatment before acidification and/or en-
zymatic treatment is also associated with disadvantages. First the heat-
treatment is an additional step that complicates the production of the milk
product, and of course the time and energy needed increases the production
costs. Further the heat-treatment denaturates the milk proteins, which leads
to
a loss of flavor, bitter taste, and an adverse effect on the mouthfeel.
It has been reported that heat-treatment may be avoided in connec-
tion with transglutaminase by adding a reducing agent such as a thiol com-
pound to the milk. The reducing agent enables the use of transglutaminase on
raw milk without the need of preheating. Suitable reducing agents are e.g. glu-
tathione, cysteine, y-glutamylcysteine, sulfurous acid, ascorbic acid and
erythorbic acid. Milk treated with transglutaminase in the presence of a reduc-
ing agent has been suggested for use in the production of yogurt, cheese and
powdered milk to improve texture and mouthfeel (US2002/0061358). However,
the addition of a reducing agent may also be problematic, because food addi-
tives should be avoided whenever possible. Further, the reducing agent may
influence other properties of the final product, such as taste or flavor.
The present invention relates to a new method of producing struc-
ture modified milk products without the need of detrimental preheating, or add-
ing reducing agents, which are associated with transglutaminase.
Tyrosinase is another cross-linking enzyme that has been sug-
gested for food applications. Tyrosinase has been reported to affect for exam-
ple wheat proteins (Takasaki and Kawakishi, 1997; Takasaki et al., 2001).
Lantto et al. (in press) have studied the effect of transglutaminase,
tyrosinase
and freeze-dried apple pomace powder on gel forming and structure of ho-
mogenized pork meat. Tyrosinase was not able to affect gel forming in the ex-
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periments conducted, but it improved gel hardness of an unheated meat ho-
mogenate to a certain extent.
Tyrosinase has further been reported to cross link the whey proteins
a-La and (3 Lg in the presence of caffeic acid. In contrast to (3-Lg a-La was
even capable of direct cross-linking by tyrosinase without addition of caffeic
acid (Thalmann and Loetzbeyer, 2002). Further the effect of tyrosinase on ca-
sein in the presence or absence of L-tyrosine and L-DOPA as cross-linkers
has been studied. The presence of cross-linkers was found to be indispensa-
ble for the cross-linking of casein (Halaouli et al., 2005).
None of the above references disclose the use of tyrosinase in
cross-linking the proteins in milk. Applicants tried to do this, but with poor
re-
sult. As could be seen with transglutaminase isolated milk proteins do not
react
in the same way as they do in milk, Tyrosinase did not show significant cross-
linking activity in whole cow milk. Preheating the milk did not significantly
en-
hance cross-linking. Surprisingly, however, it was found that reduction of the
fat content of the milk resulted in desired modification of the texture of the
product when using tyrosinase. This was especially surprising, because fat re-
duction generally has an adverse effect on the texture and water holding of
the
product.
The present invention is directed to a method of modifying milk
proteins, which is suitable for treating raw, or moderately heat-treated, fat-
reduced milk, and which has superior texture modifying and syneresis prevent-
ing effects compared to transglutaminase. The method is convenient for pre-
paring acidified milk products, and it enables the use of pasteurized milk in-
stead of using pasteurized and conventionally reheated milk, as well as the
preparation of milk products with reduced fat and protein content without the
need of other additives, such as thickening agents.
In processed milk products fat and added proteins, starches and
polysaccharides greatly affect the technological and sensory properties. The
present invention now contributes to the preparation of healthier, less
additives
containing low-energy milk products suitable for e.g. weight management.
Summary of the Invention
The present invention provides a method of preparing a milk prod
uct, said method comprising adding tyrosinase to raw or pasteurized fat-'
reduced milk and incubating it to form a milk product with modified structure.
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The invention further provides the use of tyrosinase in modifying
the structure of raw or pasteurized fat-reduced milk.
The invention still further provides a rnilk product comprising raw
or pasteurized fat-reduced milk and tyrosinase, and having a tyrosinase modi-
5 fied structure.
Specific embodiments of the invention are set forth in the depend-
ent claims.
Other objects, details and advantages of the present invention will
become apparent from the following drawings, detailed description and exam-
1o ples.
Brief Description of the Drawings
Figure 1 shows an SDS-PAGE gel of non-heated and preheated
(90 C) skim milk without tyrosinase treatment, or with 500 nkat tyrosinase/g
of
protein.
Figure 2 shows the effect of tyrosinase on the firmness of a 2.7%
Na-caseinate gel.
Figure 3 shows the effect of tyrosinase (TYR) on the firmness of an
acidified milk gel prepared from pasteurized whole milk or skim milk.
Figure 4 shows the effect of tyrosinase (TYR) on the firmness of an
2o acidified milk gel prepared from pasteurized skim milk.
Figure 5a shows the effect of tyrosinase (TYR) and transglutami-
nase (TG) on the firmness of an acidified milk gel prepared from pasteurized
skim milk.
Figure 5b shows the effect of tyrosinase (TYR) and transglutami-
nase (TG) on liquid released from an acidified milk gel prepared from pasteur-
ized skim milk.
Figure 6 shows an amino acid sequence alignment of two Tricho-
derma reesei tyrosinases TYRI and TYRII. The sequences are shown up to the
C-terminal cleavage site of TYRII. The signal sequences are on the first row.
A
putative propeptide cleavage site of TYRI is shown by an arrow. The amino
acid residues involved in coordination of the two Cu atoms in the active site
are
shaded. The thioether bond between cysteine and histidine involved in the ac-
tive site of the tyrosinases is shown by a vertical line above the sequence.
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Detailed Description of the Invention
Tyrosinase belongs to the group phenol oxidases, which use oxy-
gen as electron acceptor. Traditionally tyrosinases can be distinguished from
other phenol oxidases i.e. laccases on the basis of substrate specificity and
sensitivity to inhibitors. However, the differentiation is nowadays based on
structural features. Structurally the major difference between tyrosinases and
laccases is that tyrosinase has a binuclear copper site with two type III
coppers
in its active site, while laccase has altogether four copper atoms (type I and
II
coppers, and a pair of type I I I coppers) in the active site.
Tyrosinase oxidizes various phenolic compounds to the corre-
sponding quinones. The quinones are highly reactive and may react further
non-enzymatically. A typical substrate of tyrosinase is tyrosine (or tyrosine
residue in proteins), which is first hydroxylated into DOPA (dihydroxyphenyla-
lanine or DOPA residue in proteins), which is then further oxidized by the en-
zyme to dopaquinone (or dopaquinone residue in proteins). Dopaquinone may
react non-enzymatically with a number of chemical structures such as other
dopaquinones, thiol and amino groups. Tyrosinase thus has two enzyme activi-
ties in one and the same protein i.e. monophenol monooxyganase activity (EC
1.14.18.1) and catechol oxidase activity (EC 1.10.3.1) as shown below.
OH Monophend OH 0
mancmxygenase OCafechet oxidase
20 a 2 F~ #~ 010
2 ~ 2 g
2Hz0 _2N20 /
R EG 1.14.18.1 R EC 1.10.3.1 ~
The substrate specificity of tyrosinase is relatively broad, and the
enzyme is capable of oxidizing a number of polyphenoles and aromatic
amines. Contrary to laccase (EC 1.10.3.2), however, tyrosinase does not oxi-
dize syringaldazin. At least tyrosine, lysine and cysteine residues in
proteins
form covalent bonds with dopaquinones formed by tyrosinase.
Tyrosinase activity can be measured by techniques generally
known in the art. L-DOPA or L tyrosine can be used as a substrate, whereafter
dopachrome formation may be monitored spectrofotometrically, or alternatively
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substrate consumption may be monitored by following the oxygen consump-
tion.
Tyrosinases are widely distributed in nature, and they are found in
animals, plants, fungi (including mushrooms) and bacteria. The only commer-
cially available tyrosinase at present is derived from the mushroom ,4garicus
bisporus. The tyrosinase used in the present invention may originate from any
animal, plant, fungus or bacteria capable of producing tyrosinase. According
to
one embodiment of the invention the tyrosinase is of microbial origin. Tyrosi-
nases may be found e.g. in Neurospora crassa, Streptomyces, Bacillus, My-
rotheium, Mucor, Miriococcum, Aspergillus, Chaetotomastia, Ascovaginospora,
Chaetorniurn, Trametes, Serpula lacrymans, Conidiophora puteana,
Pycnoporus, Scytalium and Trichoderrna. According to a specific embodiment
of the invention the tyrosinase is derived from a filamentous fungus. Suitable
tyrosinases are described in PCT/F12006/050055. The tyrosinase may for ex-
ample be an extracellular tyrosinase obtainable from Trichoderma reesei, or a
tyrosinase variant thereof. According to one particular embodiment of the in-
vention the tyrosinase comprises an amino acid sequence having at least 70,
80, 90, 95, 98 or 99% identity to the amino acid sequence of l 1 R1 or TYR2 as
shown in Figure 6, or to a fragment thereof having tyrosinase activity.
An effective amount of tyrosinase is added to raw or pasteurized fat-
reduced milk under conditions sufficient to modify the structure of the milk
product obtained. The structure of a milk product refers both to its chemical
and physical properties. Structure influences the sensory perception of the
product i.e. the texture. The relationship between structure and water-holding
capacity is complicated and not straightforward, but "structure" as used
herein
includes both texture and water-holding properties.
The milk to be treated with tyrosinase is obtained from milking ani-
mals, preferably from cows or goats. Before enzyme treatment fats and oils are
removed to obtain fat-reduced milk. Milk may contain up to about 10 / fat,
but
normally whole milk has a fat content of 3.5-3.6 weight-%. "Fat-reduced milk"
in the present context refers to milk having a maximum fat content of 2.0% by
weight. Preferably it has a fat content of no more than 1.5, 1.0, or 0.5
weight-
%. According to a specific embodiment of the invention all fat is removed to
ob-
tain "skim milk" having a fat content of 0 / by weight. The preparation of
fat-
reduced including skim milk is general knowledge.
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"Raw" milk as used herein refers to milk that has not been heat-
treated to decrease its biological burden.
"Pasteurized" milk refers to milk that has been moderately heat-
treated to destroy pathogens, undesirable enzymes and milk spoilage bacteria.
Pasteurization is a generally used term in the dairy industry, and refers to a
minimum of heat treatment needed for obtaining a microbiologically safe prod-
uct with prolonged shelf life. The extent of microorganism and enzyme inacti-
vation depends on the combination of temperature and time. Heat-treatment at
62 to 65 C for about 30 minutes is used in some countries, whereas other use
temperatures between 70 to 75 C for much shorter times. Milk is normally pas-
teurized at 72 C for 15 seconds. Milk is deemed pasteurized if it tests
negative
for phosphatase but positive for peroxidase.
Both the raw and the pasteurized milk may have undergone other
processing, such as centrifugation, filtration or homogenization before pas-
teurization and/or the treatment with tyrosinase. Conventional food additives
may be added, if desired.
The tyrosinase modified fat-reduced milk may be used e.g. in pre-
paring milk powder by spray drying. According to a particular embodiment of
the invention the tyrosinase modified fat-reduced milk is used in the prepara-
tion of acidified milk products, such as yogurt, clabber milk, desserts, creme
fraiche, ripened cream and the like. The preparation of these products is car-
ried out without rennet or other proteolytic enzymes. The "milk product"
formed
by the method of the invention may thus be e.g. a milk concentrate or powder,
yogurt, clabber milk, cr6me fraiche, ripened cream, fat-reduced cream, a dairy
dessert, or the like.
As set forth above, heat treatment before acidification is consid-
ered essential for the texture of acid milk products. If transglutaminase is
used,
the heat treatment is further necessary for enhancing the susceptibility of
milk
proteins to cross-linking. "Heat treatment" or "preheating" in this context is
harsher than pasteurization and leads to peroxidase inactivation. The conven-
tional preheating temperatures for different processes may vary between 85
and 140 C, and the treatment times may vary from 30 minutes down to a few
seconds, usually $5-95 C for 30-10 minutes is used. Skim milk is normally
preheated at about 85 to 95 G for about 5 to 15 minutes, the higher the tem-
perature the shorter the time.
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Milk contains naturally about 3.2% protein. Yogurt is convention-
ally prepared from milk having a protein content of at least 3-5% by weight de-
pending on the amount of other additives used, such as hydrocolloids and
starch to obtain the desired texture. If additives are not used, the protein
con-
tent is elevated up to about 5 / e.g. by evaporation or by adding external
pro-
tein. Pasteurized milk with optionally elevated protein content is heat-
treated at
about 90-95 C for about 5-10 minutes, and sugar, fruit, jam or other tasters
may be added. The mixture is then cooled and inoculated with a starter i.e.
acid producing microorganisms such as lactic acid bacteria, which contribute
both to acidification and taste. Fermentation is normally carried out at 40-45
C
for 3-6 hours, Possible enzymes are added prior to or concomitantly with the
starter. After fermentation the yogurt may optionally be heat-treated once
more
to prolong its storage tirne. However, this postheating may also be avoided in
order not to destroy the fermentation organisms of the final product, which
might have health-promoting properties. For experimental acidification
gluconic acid lactone (GDL) may be used instead of fermenting organisms.
According to the present invention acidified milk products may be
prepared with tyrosinase in the same way as described above, except that no
heat treatment exceeding 30 C is carried out before acidification. If the milk
has been pasteurized already at the farm, it may optionally be repasteurized
in
the dairy at a temperature not exceeding 30 C before the enzyme treatment.
The tyrosinase may be added before or after the addition of the starter. Con-
veniently the tyrosinase is added simultaneously with the starter and allowed
to
react during the lactic acid fermentation. The reaction mixture may be further
supplied with oxygen to enhance the effect of the enzyme. Tyrosinase enables
the use of milk with a protein content of less than 5 weight-%, and especially
less than 4 weight- / e.g. with a natural protein content of about 3.2% for
yo-
gurt preparation.
Tyrosinase is dissolved in an aqueous solution. An amount of at
least 10, 20, 40, 80, 120, 160, 320, or 640 nkat/g milk protein is usually
suffi-
cient to modify the texture and/or water-binding properties of the milk
product.
Tyrosinase is normally allowed to react at a temperature of about 4-40 C for
at
least 10 minutes up to 24 hours or more. Naturally incubation at low tempera-
tures requires longer incubation times and vice versa. An incubation time of
at
least 1 hour up to at least 18 h is convenient at 4 C, whereas reaction times
of
at least 10 minutes up to 4 hours at 40 C are efficient. The pH may range from
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3 to 8, preferably from 3 to 5 during the process. If desired, the incubation
may
be terminated with postheating, but this is not necessary.
Tyrosinase especially modifies the texture and the water-holding
capacity of the milk product. The effect of tyrosinase on the proteins in fat-
5 reduced milk can be seen e.g. as increased molecular weight of milk
proteins,
increased firmness measured as maximum compression force or area, and/or
improved water holding capacity. Water holding capacity may be measured
e.g. as liquid released from the cross-linked gel.
According to a particular embodiment of the invention raw or pas-
10 teurized skim milk is inoculuted with microorganisms capable of forming
lactic
acid to obtain a fermentation mixture, and the mixture is incubated in the
pres-
ence of tyrosinase under conditions sufficient to acidify the milk product and
modify the texture and water-holding capacity thereof.
The invention is illustrated by the following non-limiting examples.
It should be understood, however, that the embodiments given in the descrip-
tion above and in the examples are for illustrative purposes only, and that
vari-
ous changes and modifications are possible within the scope of the invention.
Example 1. Tyrosinase catalysed cross-linking of heated and non-heated
milk products
A tyrosinase gene encoding a protein comprising the sequence of
TYR2 as shown in Figure 6 was amplified by PCR from genomic DNA of
Trichoderma reesei, and the isolated gene was overexpressed in T reesei un-
der a CBHI promotor, and excreted into the growth medium.
Tyrosinase activity was assayed using 15 mM L-DOPA (Sigma,
USA) as substrate at pH 7 and room temperature according to Robb (1934).
Enzyme activity is expressed in nanokatals (nkat). One nkat is defined as the
amount of enzyme activity that converts one nmol per second of substrate
used in the assay conditions. Enzyme dosage nkat/g protein means the
amount enzyme calculated as activity and dosed per one gram of milk protein.
Changes in the molecular weight and mobility of milk proteins
caused by the T. reesei tyrosinase were analysed by sodium dodecylsulphate-
polyacrylamide gel electrophoresis (SDS-PAGE). Tyrosinase was added to
raw or heated (90 C, 5 min) skim milk at a dosage of 500 nkat/g protein in
open tubes (oxygen available) and closed tubes (less oxygen available). Incu-'
bation was carried out at 30 C for 2 hours.
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The results are shown in Figure 1. The samples were as follows:
lanes 1 and 10: molecular weight standard, lane 2: unheated milk in closed
tube, lane 3: unheated milk + tyrosinase in closed tube, lane 4: unheated milk
in open tube, lane 5: unheated milk + tyrosinase in open tube, lane 6: heated
milk in closed tube, lane 7: heated milk + tyrosinase in closed tube, lane 8:
heated milk in open tube, lane 9: heated milk + tyrosinase in open tube, lane
10: molecular weight standard.
Tyrosinase caused the following detectable electrophoretic
changes: 1) Appearance of high molecular weight compounds around 198
KDa and below the well, 2) disappearance of caseins (especially in open
tubes). The results show that tyrosinase was capable of catalyzing cross-links
formation of both unheated and heated milk, the effect being higher in the
presence of oxygen.
Example 2. I prove errt of firmness of Na caseinate protein gels by ty
rosinase
Gels were prepared from IVa-caseinate dissolved in tap water over
night at room temperature. Caseinate solutions were chemically acidified with
GDL ( -gluconic acid lactone; 0.4% GDL in 2.7% (w/w) caseinate, final pH 4.6
4.7) during the enzyme treatment. T. a eesei tyrosinase (0, 10 and 50 nkat/g
caseinate) and GDL were mixed to 13 ml of caseinate solution and 2.35 ml ali-
quots of the solution were pipetted in cylindrical aluminium vessels (cross
sec-
tion 16 mm). The samples were covered with a glass Petri dish and they were
incubated at 30 C for 4 hours. Firmness of the 1 day old gels was measured at
room temperature (Texture Analyzer TA-FiDi, Stable Micro Systems) by meas-
uring the maximum compression force needed to compress the gels 5 mm (0.5
mm/s, 4 replicate samples). For comparison the firmness of a 4.5% caseinate
gel without enzyme treatment was measured. The results are shown in Figure
2.
Tyrosinase increased the gel firmness at both dosages. Further
the firmness of the tyrosinase-treated 2.7% caseinate gel was almost the same
as that of a 4.5% caseinate gel without tyrosinase; max compression force for
both samples was around 100 g, indicating that the protein content of the gel
can be decreased by using tyrosinase.
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Example 3. Tyrosinase catalyzed cross-linking of whole milk and skim
milk
Tyrosinase from T. reesei was added to pasteurized whole milk
(3.5 / fat) and skim milk (0 / fat) at a dosage or 50 nkat tyrosinase/g
protein at
a milk temperature of 40 C, and the milk was incubated at 40 C for 1 h. During
incubation, the samples were continuously supplied with oxygen. Also a control
solution without enzyme was prepared. Then the milk sample was acidified
with 1.2% GDL at 40 C for 4 h (final pH 4.6). After over night storage at 4 C,
the maximum compression force was measured with a Texture Analyzer (l"A-
HDi, Stable Micro Systems). The results are shown in Figure 3. Tyrosinase
greatly increased the firmness of the acidified milk gel. The effect was
signifi-
cantly greater with skim milk than with whole milk.
Example 4. Tyrosinase-induced cross-linking of pasteurized skim milk
Mushroom tyrosinase (Fluka, Switzerland) was added (50 nkat ty-
rosinase/g protein) to warm (40 C) pasteurized skim milk and the milk was in-
cubated at 40 C for 1 h. During incubation, the sample was continuously sup-
plied with oxygen. Also a control solution without enzyme was prepared. Then
the milk sample was acidified with 1.2% GDL at 40 C for 4 h (final pH 4.6). Af-
ter over night storage at 4 C, the maximum compression force was measured
with a Texture Analyzer (TA-HDi, Stable Micro Systems). The results are
shown in Figure 4. Mushroom tyrosinase increased the hardness of the acidi-
fied skim milk gel.
Example 5. Comparison of tyrosinase with transglutaminase
Tyrosinase from T. reesei, or transglutaminase (Activa MP, Ajino-
moto) was added to warm (40 C) pasteurized skim milk both at a dosage of 50
nkat enzyme/g protein and the milk was incubated at 40 C for 1 h. Tyrosinase
activity was determined as described in Example 1, and transglutaminase ac-
tivity was determined using 0.2 M N-carbobenzoxy (CBZ)-L-glutaminylglysine
(Sigma, USA) as the substrate at pH 6 (Folk, 1970)). One nkat is defined as
the amount of enzyme activity that converts one nmol per second of substrate
used in the assay conditions. Enzyme dosage nkat/g protein means the
amount enzyme calculated as activity and dosed per one gram of milk protein.
During incubation, the sample containing tyrosinase was continu-'
ously supplied with oxygen. Also a control solution without enzyme was pre-
pared. Then the milk sample was acidified with 1.2% GDL at 40 C for 4 h (final
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13
pH 4.6). After over night storage at 4 C, the liquid released from the gel was
gently poured out from the sample cup and weighed. After that the maximum
compression force was measured with a Texture Analyzer (TA-H i, Stable Mi
cro Systems).
Although the enzyme activities of tyrosinase and transglutaminase
are not directly comparable due to differences in reaction mechanisms and
model substrates, it could be seen that tyrosinase was superior to transgluta-
minase.
The results of the compression test are shown in Figure 5a. The
firmness of the acid milk gel was greatly increased by the use of tyrosinase,
while the gel containing transglutaminase was only s9ightly firmer than the
control gel without enzyme.
The amount of liquid released from the gels during over night stor-
age at 4 C is shown in Figure 5b. The water binding of the acid milk gel was
reduced to zero by the use of tyrosinase. Transglutaminase did not prevent the
liquid released compared to the no enzyme control.
CA 02654186 2008-12-03
WO 2007/141385 PCT/F12007/050324
14
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