International Dairy Journal 15 (2005) 929939

Application of encapsulated enzymes to accelerate cheese ripening

K. Kailasapathy

, S.H. Lam
Centre for Advanced Food Research, University of Western Sydney, Locked Bag 1797, SPDC 1797, NSW 1797, Australia
Received 30 April 2004; accepted 5 November 2004
Abstract
The suitability of gellan, k-carrageenan and a high-melting-fat-fraction of milk fat (HMFF) to encapsulate protease enzymes
(Flavourzyme) and impact in accelerating Cheddar cheese ripening were studied. The rates of enzyme entrapment were 48.2%,
55.6%, and 38.9% for gellan, k-carrageenan and HMFF, respectively. The enzyme capsules were incorporated into milk during
cheese manufacture. The moisture content of cheeses with added gum capsules was higher than control cheeses. Casein (b)
degradation was monitored by High-Performance Capillary Electrophoresis. All cheeses treated with encapsulated enzyme showed
higher rates of proteolysis than the control cheese throughout the ripening period. The rate of proteolysis was greater with cheeses
made incorporating k-carrageenan capsules containing protease. Cheese texture and sensory quality were not signicantly inuenced
by the type of encapsulating material (gum or milk fat). Differences in textural and sensory quality between treated and control
cheeses were consistent with release of protease enzymes from capsules.
r 2005 Elsevier Ltd. All rights reserved.
Keywords: Cheddar cheese; Accelerated cheese ripening; Enzyme encapsulation; Gellan; k-carrageenan
1. Introduction
Cheese maturation may take 6 months to 2 years
depending on the cheese variety. It is important for the
development of unique avour, aroma and texture of
cheese (Gripon, Monnet, Lambert, & Desmazeaud,
1991). Long maturation periods of cheeses, however,
represent a signicant cost in handling and capital (Fox,
1993; Law, 1987).
Several attempts have been made to reduce the
ripening period by addition of enzymes (Law, 1987)
some of which have been reported to halve the normal
maturation period of cheese (Law & Wigmore, 1983).
Direct addition of enzyme to the cheese milk was not
successful due to loss of enzymes in the whey, poor
enzyme distribution, reduced yield and poor-quality
cheese. Incorporation of encapsulated enzyme elimi-
nated the problems associated with direct enzyme
addition. Enzyme microcapsules physically separate
the enzyme from the substrate in the curd and the
enzyme is only released into the curd upon capsule
breakdown during ripening (Karel, 1990).
Enzyme encapsulation in milk fat (Magee, Olson, &
Lindsay, 1981; Braun & Olson, 1986a, b) and in
liposomes (Law & King, 1985; Kirby, Brooker, &
Law, 1987) for application during small-scale cheese
production trials has been reported. Milk fat, however,
is unstable at curd cooking temperatures due to its low
melting point (33 1C) and hence is unsuitable for
application in Cheddar cheese types. An alternative
would be to use higher melting fractions of milk fat. The
use of liposomes or articial lipid membrane vesicles as
enzyme encapsulating material also has drawbacks.
These drawbacks include expensive ingredients, use of
materials for liposome production that are not generally
regarded as safe and edible, lack of suitable method for
large-scale production and low encapsulation efciency
of liposomes.
One group of materials that exhibit excellent encapsu-
lating abilities are food gums or hydrophilic hydrocolloids
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doi:10.1016/j.idairyj.2004.11.006

Corresponding author. Tel.: +61 2 45 701 653;

fax: +61 2 45 701 954.
E-mail address: k.kailasapathy@uws.edu.au (K. Kailasapathy).
(Dziezak, 1988). Very little, however, has been documen-
ted on their application to encapsulate enzymes for
accelerating cheese ripening. Gums have been extensively
used for immobilisation of living cells (Willaert & Baron,
1996) and, to a lesser extent, enzymes (Roig, Rashid, &
Kennedy, 1995; Chang, Joo, & Ghim, 1984). Gum
capsules are easy to prepare and gums are relatively
widely available, cheap, and food and biologically
compatible.
Therefore, we have investigated food gums as an
alternate to liposomes and milk fat for enzyme
encapsulation for accelerating cheese ripening. Two
gums (gellan and k-carrageenan), together with higher-
melting-fat-fraction of milk fat (HMFF) were used to
encapsulate enzymes for application to cheese milk. The
cheeses produced were tested for the effect of the added
enzyme capsules on rates of maturation and quality.
2. Materials and methods
2.1. Gums, enzymes and chemicals
k-Carrageenan (Caragem 107) and Gellan (Kelcogel)
gums were supplied by Germantown Company (Sydney,
Australia). The enzyme, Flavourzyme (activity 250
LAPUg
1
), was from NOVO-Nordisk A/S (Sydney,
Australia). Emulsier, distilled monoglycerol (Myverol)
was supplied by Swift and Co., Ltd (Sydney, Australia).
Direct Set Frozen lactic acid starter cultures
(DS5CW30.1 units) were obtained from Mauri
Laboratories (Sydney, Australia). Rennet (Fromase)
and Annato (colorant) were from Home Cheese Making
Supplies (Victoria, Australia). HMFF (melting point
43 1C) was obtained from Australia Food Industry
Science Centre, (Melbourne, Australia). Casein stan-
dards, electrophoresis grade buffers, urea and D,L-
dithiothreitol (DTT) were obtained from Sigma Chemi-
cals (Sydney, Australia). All other reagents used were of
analytical grade.
2.2. Preparation of gum capsules
k-Carrageenan enzyme capsules were prepared by a
modied method of Audet and Lacroix (1989). Gum
powder (1.5 g) was suspended in three lots of 50 mL
deionised water, heated to 80 1C, stirred and kept at that
temperature for 20 min to completely dissolve the
polymer. The solutions were cooled to 40 1C, and each
mixed with 5.0, 12.7 and 16.2 mL of 7.5% solution of
Flavourzyme to produce three batches of capsules. The
mix was rapidly poured into 150 mL soybean oil
containing 0.2% emulsier in a beaker kept in a water
bath at 40 1C while stirring (2000 rpm) with a marine
impeller. The water-in-oil emulsions were cooled to
25 1C to allow the gum droplets to gel. The oil phase was
decanted and the resulting gel capsules harvested by
centrifuging (100 g, 2 min). The gel beads were washed
twice with distilled water and capsules were separated
from the supernatant by sieving. The formed beads were
hardened soaking in 0.07% calcium chloride solution
for 2 h.
Gellan capsules were prepared by dispersing 0.3 g of
gellan powder in 50 mL of deionised water. The
dispersion was heated to 90 1C with magnetic stirring
for 10 min. The solutions were cooled to 45 1C and each
mixed with 5.0, 12.7 and 16.2 mL of a 7.5% solution of
Flavourzyme to produce three batches of capsules. The
rest of the preparation procedure was as described for
k-carrageenan. The size of the capsules was determined
by the method of Arnaud and Lacroix (1991).
2.3. Preparation of HMFF capsules
HMFF capsules were prepared according to a
modied method described by Magee and Olson
(1981). HMFF (150 g) melted in a steam bath was
heated further to 62 1C. Emulsier (0.25%) was added
and stirred with a magnetic stirrer. The melted HMFF
and emulsier blend was cooled to 38 1C. Flavourzyme
solution (7.5%) was added while stirring at one of the
three levels 5.0, 12.7, or 16.2 mL. The HMFFenzyme
mix was then added while stirring (1200 rpm) to 250 mL
cold distilled water (10 1C) in a beaker to form the
HMFF capsules. The capsules were left in the cold water
to stabilise for 30 min. The capsules were separated by
centrifuging (100 g, 3 min), washed twice in 250 mL of
cold distilled water (10 1C), kept for a further 1 h in cold
water and sieved using a stainless steel strainer.
2.4. Rates of enzyme entrapment
The efciency of enzyme encapsulation in three types
of capsules was measured by determining the proteolytic
activity of the enzyme using casein as a substrate
(Sarath, De La Motte, & Wagner, 1989). Proteolytic
activity of the enzyme was determined as trichloroacetic
acid (TCA) soluble peptides and amino acids following
the precipitation of intact casein with TCA. Capsules
prepared by addition of 5.0 mL of a 7.5% solution of
Flavourzyme to the encapsulant solutions were used for
this purpose. The total enzyme activity was determined
in a bulk solution of capsules (before separation of
capsules from the un-encapsulated material). The bulk
solutions (10 mL) containing k-carrageenan and gellan
capsules were separately dispersed in 50 mL of 0.4% tri-
sodium citrate solutions and stirred for 30 min at room
temperature (2324 1C) until completely dissolved.
Separated gum capsules were treated similarly in tri-
sodium citrate solution. The bulk solutions (10 mL)
containing HMFF capsules in a beaker were placed in a
water bath at 43 1C to melt the fat. The dissolved gum
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K. Kailasapathy, S.H. Lam / International Dairy Journal 15 (2005) 929939 930
capsules and melted HMFF capsule solutions (5 mL)
were added to 20 mL of 2% casein solution in buffer
(pH 7.6) and mixed thoroughly. The mixture was
incubated at room temperature for 20 min. Ten milli-
litres of 5% TCA was then added to terminate the
reaction. A blank was prepared by combining the TCA
and the enzyme capsule dispersions prior to adding the
substrate. The assay mixture and the blank were allowed
to stand for 30 min before ltering through Whatman
# 42 lter paper. Absorbances were read at 280 nm with
reference to the blank on a spectrophotometer (Ultros-
pect-LKB Biochrom 000122, Sydney).
One unit of specic enzyme activity was dened as the
increase in absorbance at 280 nm across a 1 cm path
length caused by a unit amount (1 mg) of enzyme
(expressed as total nitrogen) under the conditions of the
assay. The rate of enzyme entrapment or encapsulation
efciency was the percentage of enzyme encapsulated
(expressed as units enzyme activity) in capsules divided
by the total units of enzyme in bulk solution multiplied
by 100.
2.5. Cheese making
Control cheese was manufactured according to the
method described by the Australian Society of Dairy
Technology (1977) using pasteurised milk (72 1C; 15 s)
with 2% mixed starter culture (Lactococcus lactis and
L. cremoris) and 0.25% (v/v) calf rennet. The pas-
teurised milk was standardised to a casein:fat ratio of
0.70 using skim milk. Annato and calcium chloride
solutions were added at a rate of 0.25% each. Cheese
manufacture was carried out in a 10 L water-jacketed
vat tted with a variable speed agitator blade (Armeld
FT20-A, Ringwood, England). After milling the curd,
salt (NaCl) was applied at a rate of 2.5% (w/w) to the
curd. The curd was pressed under an 8 kg weight in a
cheese press overnight, packaged in Cryovac lm and
kept in a cheese room (910 1C) to ripen. Three batches
of the control cheeses were produced in similar ways.
For the experimental cheeses, enzyme capsules made
with gellan, and k-carrageenan gums and HMFF were
introduced into the cheese milk at 32 1C just before the
addition of rennet. Both gums and HMFF had capsules
prepared with three levels of enzyme; 0.34, 1.10, and
2.10 mg enzyme per kg of cheese. Stirring was continued
after enzyme capsule addition up to the point of rennet
addition. From this point, the same procedure as used in
control cheese manufacture was followed.
2.6. Estimation of enzyme capsules in cheese curd
Capsule retention in cheese curd was measured
indirectly by determining the quantity of capsules lost
in the cheese whey. The entire volume of cheese whey
was collected during the manufacturing and strained
using a 120 mm stainless steel sieve. The capsules were
collected on the sieve and the volume measured in a
50 mL measuring cylinder. Retention of enzyme cap-
sules was then expressed as a percentage of the total
volume of capsules applied in the cheese milk.
2.7. Cheese composition analysis
The fat content of cheese was determined by the
Babcock test (Bartels, Johnson, & Olson, 1987). The
moisture was determined by the oven drying method
(AOAC, 1990). Total protein was determined by the
semi-micro-Kjeldahl method. Salt (NaCl) was deter-
mined by the Volhard method as described by Bartels
et al. (1987).
2.8. Determination of proteolysis
Cheese protein (casein) degradation during ripening
was evaluated after 1 day and 2, 4, 8, 12, and 16 weeks
using a High-Performance Capillary Electrophoresis
(HPCE) method. Cheese was dispersed in a buffer
solution by the method of Gripon, Desmazeaud, Le
Bars, and Bergere (1975) and whole casein extracted
from the cheese dispersion by the method described by
McKenzie (1971). Sample solutions and running buffers
were prepared according to a modied method of
Cattaneo, Nigro, Toppino, and Denti (1996). Sample
buffer was prepared by dissolving urea and DTT in
100 mL of 0.1 M phosphate buffer (pH 7.0) to give
concentrations of 9 M and 30 mM for urea and DTT,
respectively. DTT was used as an internal marker. The
run buffer was made by dissolving urea in 100 mL of
0.1 M phosphate buffer (pH 7.0) to give a concentration
of 6 M. Sample solutions were prepared by dissolving
80 mg of the extracted casein powder or 10 mg pure
casein (standard) in 10 mL of the sample buffer. The
solution was centrifuged at 2000 rpm for 10 min and
then ltered (0.45 mm lter) before use.
Capillary electrophoresis separations were performed
according to a modied method of Kristiansen, Otte,
Zakora, and Qvist (1994) using a Hewlett Packard
3
D
CE System G1600AX (Waldbronn, Germany) com-
prising a capillary electrophoresis unit with a built-in
Diode array detector. HP Chemstation software was
used for system control, data collection and analysis. An
untreated silica capillary column of 64.5 cm total length,
56.0 cm effective length, and 50.0 mm internal diameter
was used. Each standard and sample solution was
injected hydrostatically for 15 s and separated at a
constant voltage of 10 kV and current of 112 mA at 36 1C
for 25 min. UV detection was performed at 214 nm.
After each separation, the column was sequentially
ushed for 3 s with 1.0 M sodium hydroxide and the run
buffer. Standard and sample solutions were run through
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K. Kailasapathy, S.H. Lam / International Dairy Journal 15 (2005) 929939 931
the HPCE system in duplicate and results were
expressed as concentration of b-casein in mg mL
1
.
2.9. Determination of Free Amino Groups
Proteolysis in the ripening cheese was also monitored
by measuring free amino groups by the trinitrobenze-
nesulphonic (TNBS) acid method (Polychroniadou,
1988). One gram of cheese was dispersed in 20 mL of
0.1 M sodium borate buffer (pH 9.5), warmed at 45 1C
for 15 min with stirring and centrifuged at 4300 rpm for
20 min. A 6 mL portion of the supernatant was diluted
to 100 mL with distilled water. Five dilutions of glycine
(used as standard amino acid) solution were prepared in
0.1 M HCl to give solutions of 0.01, 0.1, 1.0, 2.5, and
4.5 mg mL
1
glycine for the standard curve. A portion
of the cheese extract and the standard solution (0.5 mL)
was added to 0.5 mL borate buffer and TNBS reagent
(1 mL, 1 mg mL
1
) was added. After mixing, the
solution was incubated at 37 1C for 60 min. Blanks were
prepared with 0.5 mL of water instead of cheese extract.
The reaction was stopped by adding 2 mL of 0.1 M
sodium phosphate containing 1.5 mM sodium sulphate
and the absorbance was read at 420 nm on a spectro-
photometer (Ultrospec-LKB Biochrom 000122, Syd-
ney). Results were expressed as mg mL
1
of amino acid
with the assumption that the concentration of glycine
was equivalent to the concentration of free amino
groups
2.10. Cheese Texture Prole
Texture Prole Analysis (TPA) was conducted on
cheese curds according to the modied method of
Raphaelides, Antoniou, and Petridis (1995). Four cubic
samples (1.3 1.3 1.3 cm) were obtained from each
cheese block at different depths to minimise the effects
of surface drying (Jack, Paterson, & Piggott, 1993). The
samples were held at room temperature (2124 1C) for
1 h before testing. Each sample was compressed axially
(using a TPA, TA, XT2, Version 5.15) to 50% of their
original height in two consecutive compression cycles by
a 30 mm diameter at crosshead (crosshead speed:
1.0 mms
1
and contact speed: 5.0 g).
2.11. Sensory evaluation
Samples of mature experimental cheeses (enzyme-
treated) were assessed by a sensory panel when the level
of casein degradation (HPCE method) was equivalent to
that observed in a 6 month old untreated cheese
(according to the procedure of Muir, Hunter, Banks,
and Horne, (1995). Panel members were asked to rate
the sensory attributes of each cheese on a 12.5 cm
undifferentiated scale with anchor points at each end.
3. Results and discussion
3.1. Encapsulation efciency
The encapsulation efciencies for the three encapsu-
lants were signicantly different (po0.05) (Table 1)
possibly due to the enzyme release method. While the
enzyme from the gum capsules was released by
redissolving them in a buffer solution, the enzyme from
HMFF was released by melting the capsules. Also, the
ionic strength of the capsule hardening solution (calcium
chloride) may have had an effect on the activity of
enzyme (Kilara & Shahani, 1985).
The observed encapsulation efciencies for the three
encapsulants in this study were, however, higher than
those for liposomes as reported by Kirby et al. (1987).
3.2. Retention of Capsules in Cheese Curd
All encapsulants showed very high entrapment rates
(Table 2). The retention rate for the two gum capsules,
k-carrageenan (90.0%) and gellan (91.5%) was not
signicantly different (p40.05) but was signicantly
higher (po0.05) than that of milk fat (73.5%) capsules.
The retention rates of the two gums are comparable to
that of dehydrated-rehydrated (DRV) liposomes (90%),
the highest reported for liposomes (Kirby et al., 1987).
The retention rates for both gums and milk fat in this
study were higher than those observed by Alkhalaf
et al.(1988) for free Neutrase in cheeses (20%).
Unlike smaller-sized liposomes that were poorly
retained in the cheese curd, compared with larger sized
ARTICLE IN PRESS
Table 1
Encapsulation efciency of Flavourzyme in different capsules
Capsule type Encapsulation efciency (%)
a
Gellan 48.273.4
b
k-Carrageenan 55.672.1
HMFF
c
38.971.5
a
Mean of triplicate determinations.
b
Results are given as mean7standard deviation.
c
High melting milk fat fraction.
Table 2
Retention of enzyme capsules in cheese curd
Capsule type Capsule size (mm)
a
Capsule retention (%)
b
k-Carrageenan 473.6 90.0 (2.1)
c
Gellan 389.1 91.2 (2.3)
Milk Fat 658.2 73.5 (7.4)
a
Mean of duplicate determinations.
b
Mean of the three levels of capsules applications; each determined
in duplicate.
c
SEM in brackets.
K. Kailasapathy, S.H. Lam / International Dairy Journal 15 (2005) 929939 932
liposomes as described by Kirby et al. (1987), gum
capsules in this study were smaller in size than milk fat
capsules, but exhibited higher retention (Table 2). The
difference in the retention rate between the two gum
capsules and HMFF capsules observed could be due to
the fact that while the gum capsules remained in the
cheese milk during cheese making and were entrapped in
the curd matrix when the milk set, a proportion of the
milk fat capsules tended to oat, hence reducing their
chance of being trapped in the curd matrix. Both gellan
and k-carrageenan gums are known to interact with
milk proteins (Sanderson & Clark, 1983) and this could
be another reason for their higher retention. The
retention rate of capsules in the cheese curd is important
because it inuences the amount of enzyme activity
available for casein degradation. The rate of capsule
entrapment in the cheese curd is also another factor that
signicantly inuences the cost of using enzyme capsules
in cheese ripening. A high level of capsule retention
would therefore not only guarantee increased proteoly-
tic activity but also reduces the cost of the process of
enzyme encapsulation.
3.3. Effect of treating cheese with enzyme capsules on
cheese composition
The compositions of cheeses are shown in Table 3.
Moisture content ranged from 37.4% to 40.3%, fat
ranged from 31.5% to 33.8% and the salt content
ranged from 1.67% to 1.89%. The moisture content of
all gum capsules-treated cheeses was signicantly higher
(po0.01) than that of control cheese. The moisture
content of HMFF capsules-treated cheeses were, how-
ever, not signicantly different from that of control
cheeses. The moisture content of gum capsules treated-
cheeses was also above the maximum allowable moist-
ure content (39%) in Cheddar cheese as stipulated by
the Australian Standards (Anonymous, 1994). This
nding is similar to that of Manning, Witt, and Ames
(1986) and Kanombirira and Kailasapathy (1995) who
reported high moisture content in cheeses where gum
was incorporated. The high moisture content of gum
capsules-treated cheese curds was due to the hydrophilic
nature of both carrageenan and gellan gums retaining
moisture in cheese.
The salt and fat contents of the experimental cheeses
were comparable with those of the control. The fat
content of the milk fat capsules-treated cheeses was,
however, higher; but not signicantly higher (p40.05)
than the other cheeses (both experimental and control).
This may be due to the extra fat added to the cheese, as
the capsules were made in an oil emulsication process.
Thus, it may be concluded that application of enzyme
capsules had minimal effects on the fat and salt contents
of the cheeses. Application of gum capsules however
increased the moisture content of cheeses.
3.4. Effect of treating cheese with enzyme capsules on
casein degradation during ripening as determined by the
HPCE method
Most of the a
s1
-casein in milk was broken down
during the cheese making process (Fig. 1). Therefore, b-
casein remained as the major component of milk protein
in the cheese curd. This observation is in agreement with
that of McSweeney, Olson, Fox, Healy, and Hojrup
(1993), who reported that chymosin substantially breaks
down a
s1
-casein during the early stage of cheese
manufacture. In our study, proteolysis during cheese
ripening was assessed based on the degradation of b-
casein, as determined by the HPCE method. The
observed degradation of b-casein was used as a basis
for determining the extent and rate of acceleration of
cheese ripening.
Cheeses were sampled at various time intervals during
ripening and the protein separated by HPCE. There was
a marked reduction in b-casein content in all cheese
treatments after the 5 months storage period (Table 4).
All k-carrageenan capsules-treated cheeses, plus cheeses
treated with gellan and HMFF capsules with a high level
of enzyme content showed signicant differences
(po0.05) in b-casein content compared to control
cheeses after 5 months. Treatments incorporating either
gellan or milk fat capsules at both low- or medium-level
enzyme content were not signicantly different. In
ARTICLE IN PRESS
Table 3
Chemical composition of experimental cheeses
a
Cheese Enzyme
b
Salt Moisture Fat MNFS
c
FDM
d
S/M
e
(%) (%) (%)
Control C 1.89 37.6 32.6 55.8 50.8 5.0
k-Carrageenan Kl 1.88 38.4
*
31.8 56.3 51.6 4.9
Km 1.86 38.7
*
32.1 57.0 52.4 4.8
Kh 1.90 38.2
*
31.7 56.0 51.3 4.9
Gellan Gl 1.82 39.7
*
32.0 58.4 53.1 4.6
Gm 1.87 40.3
*
31.5 58.8 52.8 4.6
Gh 1.91 39.9
*
31.8 58.5 52.9 4.8
Milk fat Fl 1.80 37.8 33.0 56.4 53.1 4.8
Fm 1.89 37.5 33.4 56.3 53.4 5.0
Fh 1.87 37.6 33.8 56.8 54.2 5.0
a
Results are the mean of six determinations;
*
Indicates signicant
difference (po0.01), compared with control cheese.
b
C, control (no enzyme); K, k-carrageenan; G, gellan; F, milk fat.
l, m and h are low, medium and high levels (respectively) of enzyme
applications where low, medium, and high refers to capsules prepared
by adding 5.0, 12.7 and 16.2 mL of a 7.5% solution of Flavourzyme,
respectively, to 50 mL of the encapsulant solutions.
c
MNFS
moisture %
100fat %
100:
d
FDM
fat %
100moisture %
100:
e
S=M
salt %
moisture %
100:
K. Kailasapathy, S.H. Lam / International Dairy Journal 15 (2005) 929939 933
general, cheeses treated with capsules containing larger
concentrations of enzyme contained progressively lesser
b-casein from 1 day to 5-month period of ripening. The
observed levels of b-casein for all treatments after 5
months of ripening were, however, below that of a
6-month-old untreated cheese (1.39 mg mL
1
) prepared
in the conventional way.
The percentage of b-casein remaining (expressed as
percentage of the amount present in control cheese
immediately after pressing) for all treatments showed a
similarly decreasing trend as ripening progressed.
Control cheese had 60.68% b-casein remaining after 5
months while the treated cheeses had 24.6558.26% of
b-casein remaining. This nding is consistent with that
of Kirby et al. (1987) who reported that 68% of the b-
casein remained intact in the untreated cheese after 3
months of ripening. Law and Wigmore (1982) also
noted a progressive decrease in b-casein levels in free
bacterial proteinase enzyme treated cheeses after 2
months ripening and a reduction in the amount of b-
casein of approximately 60% in treated cheeses after
4 months.
The higher rate of b-casein hydrolysis in the k-
carrageenan capsules-treated cheeses was probably due
to low stability of k-carrageenan gels in solutions with
acidic pH similar to that encountered in ripening cheese
(Lam, 1997). Thus lactate in the cheese may have played
a role in the release of enzyme from k-carrageenan
capsules by chelating calcium and disrupting the
capsules (Roy, Goulet, & Le Duy, 1987). Interaction
that normally occurs between k-carrageenan and casein
(Snoeren, Payens, Jeunink, & Both, 1975) may have also
inuenced the rate of b-casein breakdown in cheese.
This interaction, especially that between b-casein and
carrageenan with Ca
2+
as a cross-linker (Dalgleish &
Morris, 1988), might bring the substrate (casein) into
closer proximity to the enzyme entrapped in the
carrageenan gel. This may enhance the chance for the
encapsulated enzyme to interact with casein and hence
the observed higher rate of b-casein degradation. It may
be said that the interaction between casein and
carrageenan has both minimal effect on casein con-
formation or poses no steric hindrance on casein
ARTICLE IN PRESS
(a)
80
70
60
50
40
30
20
10
0
5 10 15 20 25
D
T
T

-
c
a
s
e
i
n

-
c
a
s
e
i
n

-
c
a
s
e
i
n
(b)
80
70
60
50
40
30
20
10
0
5 10 15 20 25
DTT
-casein
DTT
-casein
(c)
80
70
60
50
40
30
20
10
0
5 10 15 20 25
(d)
80
70
60
50
40
30
20
10
0
5 10 15 20 25
DTT
Time (min)
A
b
s
o
r
b
a
n
c
e

(
m
A
U
)
-casein
Fig. 1. Electrophoretograms of: (a) a mixture of pure caseins (a, b, and
k-caseins) and protein from cheese treated with encapsulated enzyme
(k-carrageenan capsules-low level of enzyme after (b) 1, (c) 30, and
(d) 90 days of ripening. (DTT: D,L- dithiothreitol, internal marker).
Table 4
Changes in b-casein content of ripening cheeses (HPCE method)
Treatment
a
b-Casein content (mg mL
1
) Casein remaining
after 5 m (%)
1 d 1 m 3 m 5 m 6 m
C 2.34 1.83 1.64 1.42 1.39 60.68
Kl 2.32 1.69 1.55 0.79
*
34.05
Km 2.22 1.62 1.43 0.65
*
29.28
Kh 2.11 1.48 1.26 0.52
*
24.65
Gl 2.49 1.87 1.77 1.34 53.82
Gm 2.38 1.92 1.57 1.24 52.10
Gh 2.32 1.73 1.48 1.05
*
45.26
Fl 2.30 1.96 1.84 1.34 58.26
Fm 2.19 1.91 1.81 1.25 57.08
Fh 2.16 1.85 1.78 1.08
*
50.00
a
Results are the mean of duplicate determinations;
*
Indicates
signicant difference (po0.05), compared with control cheese. C,
control; K, k-carrageenan; G, gellan; F, HMFF. l, m and h are levels
low, medium and high, respectively, of enzyme applications.
K. Kailasapathy, S.H. Lam / International Dairy Journal 15 (2005) 929939 934
molecular structure. Thus enzymecasein coupling
occurs even when the casein has already interacted with
carrageenan. This is in agreement with the views of
Oakenfull, Miyoshi, Nishinari and Scott (1996) who
indicated that interaction between casein and carragee-
nan does not affect their individual molecular beha-
viour.
The observed slower rate of b-casein degradation in
cheeses treated with gellan capsules suggests that these
capsules probably release their enzyme contents very
slowly. This implies that gellan capsules remain rela-
tively stable within the cheese curd. This is consistent
with an observation that gellan gels are relatively stable
under acidic pH conditions similar to those encountered
during cheese making (Lam, 1997). Stability of the
gellan capsules could be attributed to its ability to form
gels with a wide variety of ions (Grasdalen & Smidsrod,
1987) besides Ca
2+
. Thus, even if lactic acid in cheese
chelated Ca
2+
in gellan capsules, the capsules main-
tained their integrity due to interaction with other ions
present in the complex cheese curd system. Gellan gum
interaction with casein under normal conditions of
acidity has not been conrmed (Sanderson & Clark,
1983). Hence, unlike carrageenan that interacts with
casein, there is a lower chance for casein to come in
contact with gellan-encapsulated enzyme. This may have
contributed to the lower rate of b-casein degradation as
compared to carrageenan-encapsulated enzyme.
HMFF capsules seem to have been stable because
cheese treated with these capsules showed a slow
degradation of b-casein. Magee et al. (1981) suggested
that the activity of lipase enzyme on milk fat capsules
facilitates enzyme release from these capsules. In our
study, this mechanism was probably very slow to bring
about a signicant initial reduction in b-casein content.
Heat is another means by which milk fat-encapsulated
enzyme is released when the milk fat melts. Heat was not
effective in this study because the milk fat used in the
preparation of capsules in this experiment had a higher
melting point (43 1C) than the highest temperature
(38 1C) applied during cheese production. The slower
rate of degradation of b-casein in milk fat capsules
treated cheeses could also be attributed to the relatively
higher loss (18%) of the added enzymes into cheese
whey (Lam, 1997) as compared to the gum capsules
(5.68.6%).
3.5. Effect of adding enzyme capsules on proteolysis in
cheese as determined by the TNBS method
There were signicantly (po0.05) higher levels of free
amino groups in treated cheeses as compared to the
control cheese and the difference increased as ripening
progressed (Table 5).
Cheeses treated with k-carrageenan capsules showed a
high rate of increase in free amino groups during the
rst month of ripening before dropping to a similar level
as those of gellan and HMFF capsules treatments. The
increase in free amino groups paralleled b-casein
breakdown (Table 4). This was expected because amino
groups are produced in cheese as a consequence of
protein breakdown during ripening (Fox, Law,
McSweeney, & Wallace, 1993). Thus an increase in
free amino groups in treated cheese over that of control
at any given time during ripening indicates an accelera-
tion of ripening. Using the TNBS method, Kuchroo,
Rahilly, and Fox, (1983) and Lemieux, Puchades, and
Simard (1990) also showed that the concentration of
free amino acids generally increased with cheese ripen-
ing time.
3.6. Inuence of adding gum and milk-fat enzyme
capsules on ripened cheese texture
There were signicant differences between the experi-
mental and control cheeses for all the textural para-
meters (as determined by TPA) between the
experimental and the control cheeses (Table 6). There
was reduction in the mean score of most textural
parameters when k-carrageenan capsules of higher
enzyme concentrations were used. The mean scores for
gumminess, hardness and chewiness were signicantly
ARTICLE IN PRESS
Table 5
Changes in the content of free amino group in ripening cheese by TNBS method
Age (d) Free amino group (mg mL
1
)
a
C Kl Km Kh Gl Gm Gh Fl Fm Fh
0 0.055 0.026 0.068 0.072 0.047 0.055 0.302 0.421 0.622 0.681
14 0.695 1.364 1.496 1.262 1.100 1.382 1.276 0.664 0.727 0.800
30 0.708 1.454 1.682 3.187 1.171 1.524 1.584 0.795 1.041 1.176
60 0.821 1.550 1.794 3.306 1.242 1.671 1.758 0.941 1.273 1.368
90 0.972 1.676 1.805 3.570 1.351 1.738 1.947 1.266 1.385 1.574
120 1.139 1.794 2.013 3.641 1.402 1.815 2.198 1.301 1.502 1.639
150 1.286 1.983 2.172 3.686 1.497 1.873 2.233 1.390 1.567 1.715
a
Results are the mean of triplicate determinations. C, control; K, k-carrageenan; G, gellan; F, milkfat. l, m and h are levels low, medium and high,
respectively, of enzyme applications.
K. Kailasapathy, S.H. Lam / International Dairy Journal 15 (2005) 929939 935
(po0.01) higher for control cheese than for the
experimental cheeses. This nding was in agreement
with that of Alkhalaf et al. (1988) who reported a
reduction in hardness, gumminess and chewiness in
Saint Paulin cheeses treated with free or liposome-
encapsulated Neutrase.
The reduction in gumminess, hardness and chewiness
in experimental cheeses was most likely due to the
degradation of casein as a result of the activities of the
enzymes released from the capsules. It is known that
casein is largely responsible for forming the cheese
structure. Thus, lower scores noted for k-carrageenan
were expected since cheeses treated with enzymes
encapsulated in k-carrageenan exhibited the highest
level of b-casein degradation (Table 4).
The changes in the texture of the experimental cheeses
are also consistent with the values of MNFS and S/M of
these cheeses (Table 3). Most of the treated cheeses had
higher MNFS value and lower S/M values than the
control cheeses. High MNFS values and lower S/M
values bring about a high rate of casein breakdown in
cheeses (Lawrence, Creamer, Gilles, & Martley, 1987)
hence the low scores for texture parameters as obtained
in treated cheeses.
Although use of enzyme capsules resulted in a higher
rate of protein breakdown in the cheese and hence
accelerated ripening, the lower mean score for
textural parameters of the experimental cheeses as
compared to the control cheese (Table 6) may present
a problem with product acceptance. The lower mean
score for textural parameters for experimental cheeses
could have also been as a result of moisture retention in
gum-treated cheese. Excessive moisture retention in
cheese during manufacture is known to result in
soft and crumbly texture in nished cheese (Manning,
1985).
3.7. Inuence of adding gum and milk fat enzyme
capsules on sensory quality
The control cheese scored highest for appearance but
was not signicantly different (p40.05) from the scores
of cheeses treated with Gl and Fh capsules (Table 7).
Cheeses treated with k-carrageenan capsules (Kl, Km
and Kh) gained the lowest score for appearance. This
could be due to higher level of proteolysis in the cheese
as compared to the other experimental cheeses, which
led to a crumbly and less cohesive texture. Excessive
proteolysis in cheese is considered to lead to a low score
for the textural properties of cheese (Law & Wigmore,
1982).
There were no signicant differences in avour and
aroma characteristics (p40.05) between the experimen-
tal cheeses (5 months ripened) and the control cheese (6
months ripened). This may be because of the accelerated
ripening of the experimental cheeses. However, the
strongest avour was noted in cheeses treated with k-
carrageenan capsules. This may be due to the increased
level of amino acid produced in those cheeses (Table 5).
Free amino acids are considered to be avour precursors
in ripening cheese (Law & Wigmore, 1982).
Cheeses treated with Fm, Fl, Gh and Gm capsules
scored higher for texture but were not signicantly
different (p40.05) from the control cheese. Increased
peptidolysis as experienced in the experimental cheeses
in this study, expose more ionic groups, which would
bind the available water resulting in less solvation of the
remaining casein (Creamer & Olson, 1982). In addition,
when the elasticity of the cheese has been reduced by
proteolytic cleavage of the a
s1
-casein (mainly caused by
chymosin), which is regarded as a link in the protein
network (McSweeney et al., 1993), the cheese might be
perceived by the sensory panel as harder, more brittle
ARTICLE IN PRESS
Table 6
Changes in textural properties of cheeses ripened for 5 months
Sample Textural properties
a
Springiness Gumminess ( 10
2
) Cohesiveness Hardness ( 10
2
) Chewiness ( 10
2
)
Control
b
0.79 7.41
*
0.49 14.37
*
5.54
*
Kl 0.61 3.39 0.33 9.49 1.91
Km 0.48 0.72 0.26 2.71 0.37
Kh 0.27 2.11 0.18 1.35 0.24
Gl 0.79 2.09 0.51 4.08 1.67
Gm 0.76 4.92 0.38 13.38 3.45
Gh 0.61 1.73 0.36 4.80 1.24
Fl 0.74 2.28 0.41 9.68 3.11
Fm 0.65 1.99 0.21 8.54 1.29
Fh 0.78 2.77 0.36 5.96 2.16
a
Textural properties were determined using a TPA, values are given as mean of four measurements;
*
Indicates signicant difference (po0.01),
compared with experimental cheeses (Kl to Fh).
b
Untreated cheese ripened for 6 month.
K. Kailasapathy, S.H. Lam / International Dairy Journal 15 (2005) 929939 936
and less elastic. This was observed for cheeses treated
with Fm, Fl, Gh and Gm capsules (less proteolytic
activity as discussed earlier, Section 3.4) because they
were perceived as rmer than the control cheese. This
was however, not observed for k-carrageenan capsules
treated cheeses probably because of the very high level
of proteolysis experienced.
Except for the cheese treated with k-carrageenan
(high level) capsules (Table 7) the difference in texture as
assessed by the sensory panel was not signicant
(p40.05) between the experimental cheeses and control
cheese. This implies that although hydrocolloids (San-
derson & Clark, 1983) and fat (Stampanoni & Noble,
1991) are known to modify food texture, the levels at
which they were used in this study had little or no
inuence on cheese curd texture. The results of our
study showed that textural attributes as determined by
TPA do not necessarily agree with the scores from a
human sensory panel.
Except for cheeses treated with Gl capsules, all
experimental cheeses scored higher than control cheese
for bitterness, but only cheeses treated with k-carragee-
nan and Gh capsules were signicantly (po0.05) more
bitter than control cheese. This could be attributable to
the relatively high proteolytic activities in cheeses
treated with k-carrageenan and Gh capsules leading to
bitter avour. This is consistent with earlier reports from
studies with slurries and whole cheese (Kosikowski &
Iwasaki, 1975; Sood & Kosikowski, 1979) where
excessive proteolysis brought about bitter taste in
cheese. The bitter defect is due to the production of
peptides that characteristically contain a high propor-
tion of aromatic and bulky hydrophobic amino acid
residues (Richardson & Creamer, 1973). Bitterness was
however not expected in this study as Flavourzyme was
supposed to bring about increased proteolysis without
generating bitterness according to its manufacturer
(Novo-Nordisk). The bitterness experienced in cheeses
treated with Kl and Gh capsules therefore shows that
beyond a certain level of proteolysis, irrespective of the
type of enzymes involved, there can be bitterness in
cheese.
There were no signicant differences in overall
acceptance (p40.001) for the experimental cheeses and
the control cheese except for cheeses treated with Gh
and k-carrageenan capsules. This was due to both their
high levels of bitterness and poor textural appearances
observed in this study. Cheeses treated with Gl capsules
were the most acceptable, even more so than the control
cheese. This is consistent with earlier studies where it
was reported that gellan gum-treated cheeses have
superior avour to those treated by carrageenan
(Kanombirira & Kailasapathy, 1995). This is because
gellan gum has excellent avour release properties
(Sanderson & Clark, 1983).
4. Conclusions
This study has demonstrated that encapsulated
enzyme in gum capsules can be effectively incorporated
into cheese matrix. k-Carrageenan and gellan capsules
showed higher retention (90.0% and 91.5%) than milk
fat capsules (73.5%). Enzyme losses from gum gel
capsules were also lower (5.62% and 8.66% for gellan
and k-carrageenan gums, respectively, compared with
17.93% for HMFF capsules).
The study has shown that the encapsulation of
enzyme in food gum gels for accelerated cheese ripening
is feasible. This study also reveals that the impact of
encapsulated enzymes in cheese ripening is inuenced
greatly by the nature of the gum itself. However, the
particular combination of gum capsules and added
enzyme used in this study did not signicantly improve
the sensory or textural properties of the experimental
cheeses. This is despite cheeses treated with enzyme
ARTICLE IN PRESS
Table 7
Sensory scores of cheeses ripened for 5 months
a
Treatment Appearance Flavour Aroma Texture Bitter aftertaste Overall acceptance
Control
**
8.48 6.18 6.19 5.06
b
5.11 7.05
Kl 3.74 7.13 5.51 4.83
b
9.55 3.26
Km 3.29 6.98 5.23 4.30
b
9.49 3.04
Kh 2.51 7.54 3.47 2.79
a,*
9.87 1.18
Gl 7.09 6.67 6.59 4.11
b
4.92 7.34
Gm 5.70 6.46 6.84 5.25
b
5.81 6.57
Gh 4.94 5.63 5.37 5.33
b
7.32 4.43
Fl 4.55 6.14 6.36 5.91
b
6.20 6.08
Fm 5.28 6.44 6.32 6.10
b
5.46 6.00
Fh 7.22 6.45 6.85 4.75
b
5.80 6.57
a
Sensory scores were assessed by a sensory panel. Values with different superscript letters are statistically different from each other within a
column;
*
Texture score for treatment Kh is signicantly (po0.05) different from all other texture scores.
**
Untreated cheese ripened for 6 months.
K. Kailasapathy, S.H. Lam / International Dairy Journal 15 (2005) 929939 937
capsules showing increased rates of proteolysis as
compared to the untreated cheese.
Cheeses treated with k-carrageenan capsules showed
the highest rate of proteolysis as compared to those
treated with gellan and HMFF capsules. Conditions in
cheese such as the presence of ions and lactic acid
appeared to inuence the stability of gum capsules in
cheese. Interaction between milk protein and k-carra-
geenan gum capsules most likely enhanced the inter-
action between the encapsulated enzyme and milk
proteins.
Gum gels disrupted under cheese manufacturing
conditions appeared to be more suitable than HMFF
capsules for encapsulating enzyme to be used for
accelerating cheese ripening. However, the use of
capsules from a gum easily disrupted under cheese
manufacturing conditions may lead to rapid release of
enzyme and excessive proteolysis early during ripening.
This is consistent with what was observed in this study
for k-carrageenan. Cheeses that showed high levels of
proteolysis scored poorly for texture and sensory
properties. This was one major drawback observed
from this study. It is therefore important in future
studies to consider combining such unstable gums with
gums that produce more stable gels.
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