J. Dairy Sci.

105:7904–7916
https://doi.org/10.3168/jds.2022-22253
© 2022, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Use of micellar casein concentrate and milk protein concentrate treated with
transglutaminase in imitation cheese products—Melt and stretch properties
P. Salunke,1* C. Marella,1† J. K. Amamcharla,2 K. Muthukumarappan,3 and L. E. Metzger1‡
1
Department of Food and Dairy Science, Midwest Dairy Foods Research Center, South Dakota State University, Brookings 57007
2
Animal Sciences and Industry, Kansas State University, Manhattan 66506
3
Department of Agricultural and Biosystems Engineering, South Dakota State University, Box 2100, Brookings 57007

ABSTRACT Comparison of IMC made using TGase-treated MCC

Melt and stretch properties in dairy-based imitation
mozzarella cheese (IMC) are affected by the amount
of intact casein provided by dairy ingredients in the
formulation. Rennet casein (RCN) is the preferred
ingredient to provide intact casein in a formulation.
Ingredients produced using membrane technology, such
as milk protein concentrate (MPC) and micellar casein
concentrate (MCC), are unable to provide the required
functionality. However, the use of transglutaminase
(TGase) has potential to modify the physical properties
of MPC or MCC and may improve their functionality
in IMC. The objective of this study was to determine
the effect of TGase-treated MPC and MCC retentates
on melt and stretch properties when they are used in
IMC and to compare them with IMC made using RCN.
The MCC and MPC retentates were produced using
3 different lots of pasteurized skim milk and treated
with 3 levels of TGase enzyme: no TGase (control),
low TGase: 0.3 units/g of protein, and high TGase:
3.0 units/g of protein. Each of the MCC and MPC
treatments was heated to 72°C for 10 min to inactivate
TGase and then spray dried. Each MCC, MPC, and
RCN powder was then used in an IMC formulation
that was standardized to 48% moisture, 21% fat, 20%
protein, and 1% salt. The IMC were manufactured in
a twin-screw cooker by blending, mixing, and heating
various ingredients (4.0 kg). Due to extensive crosslink-
ing, the IMC formulation with the highest TGase level
(MCC or MPC) did not form an emulsion. The IMC
made from MCC treatments had significantly higher
stretchability on pizza compared with their respective
MPC treatments. The IMC made from TGase-treated
MCC and MPC had significantly lower melt area and
significantly higher transition temperature (TT) and
stretchability compared with their respective controls.
Received May 1, 2022.
Accepted June 16, 2022.
*Corresponding author: Prafulla.Salunke@​sdstate​.edu
†Current address: Idaho Milk Products, Jerome, ID 83338.
‡Current address: Valley Queen Cheese, Milbank, SD 57252.
and MPC to the RCN IMC indicated no difference in
TT or texture profile analysis-stretchability; however,
the Schreiber melt test area was significantly lower.
Our results demonstrated that TGase treatment modi-
fies the melt and stretch characteristics of MCC and
MPC in IMC applications, and TGase-treated MPC
and MCC can be used to replace RCN in IMC formula-
tions.
Key words: transglutaminase, milk protein concentrate
or micellar casein concentrate, rennet casein powder,
imitation mozzarella cheese functionality, melt and
stretch properties
INTRODUCTION
Meltability and stretchability are important function-
al characteristics for process cheese products (PCP)
and imitation mozzarella cheese (IMC) applications
in pizza. Along with melting, some products require
stretchability. The ingredients used in the formulation,
especially proteins, affect meltability and stretchabil-
ity, which depend on the chemical and thermodynamic
properties of cheese (Johnson, 2000). Proteins, espe-
cially intact casein, play an important role in melt and
stretch properties (Lucey and Fox, 1993; Lucey et al.,
2003; Salunke and Metzger, 2022a,b). Intact casein
refers to unhydrolyzed casein. The presence of intact
CN results in extensive protein–protein and protein–fat
interactions and a fibrous CN network, whereas the
presence of hydrolyzed CN results in weaker protein–
protein and protein–fat interactions and a nonfibrous
CN network (Garimella Purna et al., 2006). Melting is
primarily determined by the number and strength of
the CN–CN interactions (Park et al., 1984). Addition-
ally, in stretchability along with intact casein interac-
tions (Lucey et al., 2003), the levels of intact casein,
calcium, and phosphates play an important role (Lucey
and Fox, 1993). Hydrolysis (because of proteolysis) will
break CN bonds, increasing melt but decreasing stretch
(Johnson, 2000). Increasing whey protein in process
cheese (PC) decreases meltability (Savello et al., 1989;

7904
 Salunke et al.: MICELLAR CASEIN CONCENTRATE AND MELT PROPERTIES
Salunke and Metzger, 2022a). Various methods, includ-
ing the Schreiber melt test, tube melt test, dynamic
stress rheology (DSR), or rapid visco analyzer melt
test, have been used to study the meltability of PC
or IMC. Stretchability is the least studied technique
mainly because it is highly unstandardized and sub-
jective. Various tests have been developed, including
the pizza bake and fork test, texture profile analysis
(TPA)-stretchability, stretchability in oil, among oth-
ers; however, the most common and popular test has
been stretchability on pizza. Tests using instruments
that have repeatability and reproducibility under stan-
dard controlled conditions have been developed (Fife et
al., 2002; Ma et al., 2012).
Protein-rich powders are used in PC or IMC formu-
lation to give the desired compositional specification,
texture, structure, meltability, and stability (Guinee,
2011). Rennet casein (RCN) is the preferred choice
in PCP or IMC applications because of its superior
functional properties. The protein portion of RCN is
100% intact CN, and it provides the maximum viscos-
ity per gram of protein. Caseinates and whey protein
concentrate (WPC) added at 5 to 7% will affect sta-
bility, flavor, and structure (Caric and Kalab, 1993;
Guinee et al., 2004). Ingredients such as NDM, milk
protein concentrate (MPC), WPC, and whey powders
contribute to increased levels of whey protein in the
formulations and can lead to textural defects in the PC
and IMC. The use of membrane filtration techniques
for the manufacture of protein-enriched retentates and
powders has yielded new ingredients for IMC formula-
tions. Milk protein concentrate contributes to higher
levels of serum proteins (SP) but has low lactose and
mineral contents. More textural defects and a decrease
in PC functionality are observed because SP can
crosslink among themselves as well as with caseins at
high temperatures. The actual amount of intact CN in
PC can be balanced using casein-rich concentrate or
powders such as micellar casein concentrate (MCC).
These are very suitable proteins for use in products
where thermal stability is required because they do not
gel upon heating. Moreover, MCC has reduced levels
of whey protein (WP), lactose, and minerals. Conse-
quently, the use of MCC in PCP formulations may ad-
dress defects in PC that can be caused by using MPC.
However, CN in the MCC is soluble and is in the native
state, with glycomacropeptide (GMP) still attached to
κ-CN. This charged casein micelle surface of the MCC
may inhibit dispersion of the native CN by emulsifying
salts, causing defects in PC. This makes it difficult to
match the functionality of MCC with that of RCN in
PC, PCP, and IMC formulations.
Journal of Dairy Science Vol. 105 No. 10, 2022
7905
The use of a crosslinking enzyme, such as transglu-
taminase (TGase, EC 2.3.2.13), has the potential to
modify the protein properties of MPC or MCC and
may improve the functionality and structural proper-
ties of milk proteins. Transglutaminase catalyzes an
acyl transfer reaction between the protein-bound gluta-
minyl residues and the primary amines and gives rise to
the formation of a framework of additional isopeptide
bonds that affect the functional properties of the food.
We theorize that it is possible to create a casein-based
protein network from native casein that has functional-
ity similar to that of the rennet casein-based aggre-
gated gel network formed in IMC. Transglutaminase
has GRAS (generally recognized as safe) status in the
United States, and the USDA has authorized its use
in some dairy products, including cheese and yogurt.
However, to our knowledge, no studies have evaluated
the effect of treating MPC or MCC with TGase and
subsequently using this modified ingredient in IMC.
The objective of this study was to determine the effect
of TGase treatment of MPC and MCC retentates on
the melted (melt and stretch) characteristics of MPC
and MCC when they are used in IMC and to compare
it with IMC manufactured using RCN.
MATERIALS AND METHODS
Experimental Design
Three replicates of MCC and MPC were produced
using microfiltration (MF) and UF, respectively, from
3 different lots of milk and treated with TGase enzyme.
The TGase treatments included control (C; no TGase),
low TGase (L; 0.3 U/g protein), and high TGase (H;
3.0 U/g protein). After spray drying, the ingredients
were used as ingredients in IMC manufactured in a
twin-screw cooker. The experimental design is shown in
Figure 1. A detailed description of the process is given
in our companion paper (Salunke et al., 2022).
A 2 × 3 factorial design experiment was set up with
2 product types (MPC and MCC) and 3 TGase enzyme
levels (C, L, and H). The treatments were coded as
follows: MCC-C = IMC made with MCC powder,
control; MCC-L = IMC made with MCC powder,
low TGase level; MCC-H = IMC made with MCC
powder, high TGase level; MPC-C = IMC made with
MPC powder, control; MPC-L = IMC made with
MPC powder, low TGase level; MPC-H = IMC made
with MPC powder, high TGase level; RCN = IMC
made with rennet casein. The IMC was used for the
characterization of various melted (melt and stretch
properties) functional properties.
 Salunke et al.: MICELLAR CASEIN CONCENTRATE AND MELT PROPERTIES 7906

Manufacture of TGase-Treated MPC
and MCC Powders
The process details of manufacturing MPC and MCC
retentates using UF and MF respectively, TGase treat-
ment, and spray drying are described in our companion
paper (Salunke et al., 2022). Skim milk was produced
using a cream separator (model 392, Separators Inc.)
from 600 kg of raw milk (at 4°C). The skim milk was
batch pasteurized (63°C for 30 min) and cooled to 4°C.
The pasteurized skim milk was divided into 2 equal
portions. One portion was processed into MCC using
MF (0.5-µm polyvinylidene fluoride spiral-wound mem-
brane, Dominick Hunter Filtration Division – N.A.,
Parker Hannifin Corp.; 3,830 element format with 1.1-
mm feed spacer, area of 4.3 m2, transmembrane pres-
sure of 86.2 kPa, and 23.3°C), and the other portion
was processed into MPC using conventional UF (10-
kDa polyether sulfone spiral-wound membrane; Domi-
nick Hunter Filtration Division – N.A., Parker Hannifin
Corp.; 3,838 element format with 1.1-mm feed spacer
area of 5.7 m2, transmembrane pressure of 276 kPa, and
23.3°C). The volume reduction ratio was maintained
at 5. In the MF process, 100% diafiltration water was
added at 6 intervals, whereas in the UF process, 40%
diafiltration water in 4 intervals was used. The techni-
cal process details and compositional parameters of the
retentate and permeate are reported in our previous
paper (Salunke et al., 2021). The retentate obtained
was frozen at −18°C until further use. Each MCC and
MPC retentate lot was divided into 3 equal parts, and
TGase (Activa TI, Ajinomoto Food Ingredients LLC;
100 units/g activity) was added at 50°C by mixing in
100 mL of distilled water. After thorough mixing and
incubating for 25 min, the retentates were heated (72°C
for 10 min) to inactivate the enzyme and then cooled
to 4°C. The retentates were then spray dried (ASO
412E, Niro Inc.) with an inlet air temperature of 205°C
and outlet temperature was maintained at 90°C. The
powders were air-cooled and packaged in plastic bags
(Associated Bag Co.) and stored at room temperature
until further analysis was completed.
IMC Formulation and Manufacture
The ingredients were balanced in Techwizard (Owl
Software) to give 48.0% moisture, 21.0% fat, 1.0% salt,
and 20.0% protein content in IMC. In each formulation,
the MPC, MCC, or RCN contributed all the protein.
The ingredients used were vegetable shortening [Crisco,
all-vegetable shortening (soybean oil and hydrogenated
palm oil), The J. M. Smucker Co.] as the fat source,
sodium aluminum phosphate basic powder (Kasal, In-
nophos) as emulsifying salt, deproteinized whey powder

Figure 1. Experimental design of the study. MF = microfiltration; DF = diafiltration; MCC = micellar casein concentrate; MPC = milk
protein concentrate; TGase = transglutaminase; MCC-C = micellar casein concentrate powder–control (no transglutaminase, TGase); MCC-L
= micellar casein concentrate powder, low TGase level; MCC-H = micellar casein concentrate powder, high TGase level; MPC-C = milk protein
concentrate powder, control; MPC-L = milk protein concentrate powder, low TGase level; MPC-H = milk protein concentrate powder, high
TGase level.

Journal of Dairy Science Vol. 105 No. 10, 2022

 Salunke et al.: MICELLAR CASEIN CONCENTRATE AND MELT PROPERTIES
(Davisco Foods International Inc.) as a lactose source,
citric acid (KIC Chemical Inc.), lactic acid (85% wt/
wt; Fisher Scientific), and iodized salt (Great Value,
Wal-Mart Stores Inc.). For the RCN treatment, 3
lots of commercially available rennet casein (2 lots of
Alaren 771 edible RCN, New Zealand Dairy Board; 1
lot from Dynamix Dairy Industries) were used. The
ingredient blend and formulation used to manufacture
the IMC included lactic acid (0.8%), salt (1.0%), de-
proteinized whey (0.6–5.3%), citric acid (0.5%), Kasal
(3.0%), shortening (20.0–21.0%), treatment powder
(23.6–27.1%), and water (44.9–46.7%). The detailed
formulation and composition targets of the IMC are
described in detail in the companion paper (Salunke et
al., 2022).
A preblend (4.0 kg) of all ingredients (except lactic
acid) was prepared in a Blentech twin-screw cooker
(model CC10, Blentech Corp.) by mixing at 50 rpm
for 20 min at room temperature (20°C). The contents
were cooked at 74°C over 5 min using indirect heating
and held for an additional 4 min at an auger speed of
120 rpm. Lactic acid was added and mixed for 30 s
in the final stage of manufacture. The hot IMC was
immediately poured into plastic containers with lids
(Pro-kal-1602 Squat Polypropylene deli containers with
lids; Fabri-Kal), and immediately transferred to a cold
room (4°C). The IMC were stored at 4°C until further
analysis was completed.
IMC Functionality
Functional analysis of the IMC included melt and
stretch properties using different methods: melt area,
DSR melt temperature, pizza bake and fork test, and
stretchability test using a texture analyzer.
Functional Analysis: Melt Properties
DSR. The DSR test was carried out as described
by Biswas et al. (2008). Cylindrical IMC samples 28.3
mm in diameter were taken from the cup using a cork
borer and prepared by cutting a thin slice (2.0 mm)
using a food slicer (model 1042W, The Rival Co.) and
wire cutter. The samples were tempered to 20°C for 10
min before analysis. A stress sweep experiment for IMC
was carried out using a rheometer (Viscoanlyser, ATS
Rheosystems, Rheologica Instruments Inc.) with par-
allel plate geometry. To prevent sample slippage, fine
sandpaper (400 grit, wet or dry; 3M) was glued to the
upper plate of the rheometer plate. The stress sweep was
performed at a frequency of 1.5 Hz, with stress ranging
from 1 to 1,000 Pa at 20°C. Before starting the actual
test, pre-shear of the samples was performed at 11/s
Journal of Dairy Science Vol. 105 No. 10, 2022
7907
for 20 s, followed by a 25-s equilibrium time. The stress
sweep experiment determined that the maximum stress
limit for the linear viscoelastic region was 400 Pa. A
temperature sweep was then performed using the same
rheometer (parallel plate geometry) at a frequency of
1.5 Hz and constant stress of 400 Pa (linear viscoelastic
region). To minimize drying during measurement, the
exposed cheese surface was coated with vegetable oil
(Crisco pure vegetable oil, The J M Smucker Co.). To
measure the rheological properties, time sweep mode
was used, along with a temperature ramp from 20°C
to 90°C at a rate of 1°C/min. Before starting the test,
a pre-shear of the samples was performed at 11/s for
20 s to equilibrate the sample homogeneously. All mea-
surements were performed in triplicate for each sample,
including the elastic modulus (G′), viscous modulus
(G″), transition temperature (TT), and tangent angle
(tan δ). The TT was defined as the lowest temperature
during the temperature ramp experiment, where tan δ
equaled 1 (G′ = G″).
Modified Schreiber Melt Test. The meltability
of each IMC sample was measured using a modified
Schreiber test as described by Muthukumarappan et al.
(1999) and Kapoor et al. (2007). Each IMC sample was
cut into a cylinder 28.5 mm in diameter and 7 mm high
and tempered to 20°C for 10 min before analysis. The
4 samples were placed on four 0.95-mm-thick alumi-
num plates (100 × 100 mm), which were immediately
transferred to a forced draft oven (Fisher Scientific) at
90°C. After 7 min, the plates with the melted cheese
were cooled to room temperature. The diameter of the
melted cheese was measured using a vernier caliper at 4
places for each sample, and the average was calculated.
The meltability of IMC was reported as the change in
the area of the melted cheese (in mm2) compared with
the original area.
Functional Analysis: Stretch Properties
Stretchability is an important and essential textural
characteristic for IMC performance in baked applica-
tions such as pizza. Stretchability was measured using
the pizza bake and fork test and a cheese stretchability
rig that uses a texture analyzer.
Pizza Bake and Fork Test. In the pizza bake and
fork test, the IMC was shredded mechanically (Kitchen
Aid). The fork test was performed as described by Gu-
nasekaran and Ak (2003). In the test, shredded IMC
(150 g) was used as a topping on a pizza crust (18-
cm diameter, Mama Mary’s Traditional Pizza Crusts,
Spartan Foods of America Inc.) covered with a thin
layer (10 g) of pizza sauce (Ragu Homemade Style,
Unilever). The pizza was baked at 205°C for 12 min
 Salunke et al.: MICELLAR CASEIN CONCENTRATE AND MELT PROPERTIES
in a forced draft oven (Fisher Scientific). One minute
after baking, a 4-pronged stainless-steel fork was in-
serted into the cheese and lifted vertically; the distance
at which the cheese could be lifted before breaking was
measured. The extent of stretch was measured at 5 dif-
ferent places on the pizza, and each IMC was tested in
duplicate.
Cheese Stretchability Using a Texture Analyz-
er. A cheese stretchability rig (TA-426, from Texture
Technologies Corp.) was used for the stretchability test
with the experimental setup shown in Figure 2. The
IMC samples were shredded mechanically (Kitchen
Aid). The post was placed in the center of the base with
a ring, and 12 g of shredded IMC was packed around it;
then, the base, post, and ring with cheese were placed
in an oven at 95°C for 12 min. To minimize drying dur-
ing heating, the cheese was covered with an aluminum
dish. After the stipulated heating time, the fixture with
the melted IMC was placed on the texture analyzer
stage (TA. XTPlus, Texture Technologies Corp.) and
clamped using the accessories provided within 1 min of
removal from the oven. The post was then raised to 270
mm at a rate of 20 mm/s.
Figure 2. Stretchability test rig setup.
Journal of Dairy Science Vol. 105 No. 10, 2022
7908
Figure 3 shows various stages of the stretchability
experiment, and Figure 4 shows a typical graph ob-
tained. Each graph was analyzed for initial resistance to
stretch (initial gradient pull force; point A in Figure 4),
peak resistance to stretch (peak force; point B in Figure
4), ease of extensibility or stretch quality (downward
slope gradient; point C in Figure 4), ease of extensibil-
ity (mean force during extension), total stretchability
(distance of stretch; point E in Figure 4), and strength
(area of work during extension; point F in Figure 4).
During the analysis, a small drop of oil was placed un-
der the post to prevent suction and an increase in pull
force. However, this did not solve the problem fully, and
test results were discarded when it occurred. An expo-
nent software macro was used to calculate the various
point forces, gradients, and areas, as well as the stretch
distance. Each treatment was replicated 5 times.
Statistical Analysis
Statistical analysis of the collected data was done
using Proc GLM (SAS Institute Inc.). Because IMC
using a high TGase level (MPC-H and MCC-H) did not
 Salunke et al.: MICELLAR CASEIN CONCENTRATE AND MELT PROPERTIES
Figure 3. Stages of stretchability test: (A) start of test; (B) initial stretching; (C) final stretching; and (D) return of post.
form an emulsion, a 2 × 2 factorial analysis with a type
I error rate (α) of 0.05 was used to test for significant
differences among the treatments. Additionally, the
IMC manufactured using RCN as an ingredient was
compared with all treatment IMC using the Dunnett
test.
RESULTS AND DISCUSSION
MCC and MPC Powder
The analytical details of the MCC and MPC pow-
ders are discussed in the companion paper (Salunke
et al., 2022). The protein on DM basis (79.1–80.6%)
and calcium content (2.06–2.07%) of the MCC powders
was higher than that of the MPC powders (protein on
DM: 75.7–76.4%; calcium: 1.84–4.87%), whereas the
lactose content of the MCC powder (4.4–4.5%) was
significantly (P < 0.05) less than that of MPC powder
(9.0–9.1%). Differences in fat (2.4–2.9%) and moisture
(2.8–3.3%) were small and not significant.
IMC Manufacture and Composition
No functional analysis was performed on MCC-H
and MPC-H treatments because they did not form an
emulsion during IMC manufacture due to extensive
crosslinking following TGase treatment. This may have
been caused by the high level of crosslinking that limits
the emulsion capacity of casein (Salunke et al., 2022).
To form a proper emulsion, de-aggregation of casein is
essential, and strong covalent crosslinking of casein by
Journal of Dairy Science Vol. 105 No. 10, 2022
7909
TGase resists the heat and mechanical forces encoun-
tered during IMC manufacture.
Even though the MCC, MPC, and RCN powders
were different compositionally, all IMC treatments were
balanced for similar proximate composition. No signifi-
cant differences (P > 0.05) were observed in the pH or
fat, moisture, and protein contents of the IMC. The
IMC samples had pH (5.77–5.88), fat (20.8–21.0%),
moisture (47.91–47.98%), and protein (19.89–19.91%)
close to the targeted proximate composition (Salunke
et al., 2022).
Melted Functional Properties
The melted functional properties in IMC are due to
protein network and molecular interactions. The mean
square values and P-values for the melted functional
properties (melt area and TT) are shown in Table 1.
Enzyme level had a significant effect on melt area and
TT; mean values of melt area and TT are shown in
Table 2.
Melt Area and TT
The melt of cheese is defined as the ability of cheese
to flow and spread, along with a visual loss of integ-
rity of individual cheese shreds (Gunasekaran and Ak,
2003; Lucey et al., 2003), whereas the TT measure-
ments of the initial melt characteristics of cheese in-
dicate molecular interactions. The TT has been used
to quantify the melting characteristic of IMC; using
DSR to measure the TT, where tan δ (G″/G′) = 1,
 Salunke et al.: MICELLAR CASEIN CONCENTRATE AND MELT PROPERTIES 7910

Figure 4. Example of a typical texture profile analysis (TPA) stretchability graph showing the force required to stretch imitation mozzarella
cheese. The points of interest include initial pull force (initial gradient) (A), maximum pull force (B), downward slope gradient and resistance
to extension (C), point of break-in cheese strands (D), stretch distance (E), and total work done (F).

is a convenient measure of the melting point of IMC
because it is the lowest temperature where a material
changes from primarily elastic to primarily viscous
(Sutheerawattananonda and Bastian, 1998; Prow and
Metzger, 2005). The control IMC samples (MCC-C and
MPC-C) had significantly (P < 0.05) higher melt area
and significantly (P < 0.05) lower TT than the samples
containing TGase (MCC-L and MPC-L), indicating
that the addition of enzymes restricted the meltability
of cheese (Table 2). The TT of the IMC made from
MCC-L was significantly (P < 0.05) higher than that
of MCC-C, MPC-C, and MPC-L samples.
Meltability is dependent on CN interactions in the
cheese (Lucey et al., 2003). As expected, samples that
had a low melt area had a high TT and vice versa. A
cheese that melts easily will have a low DSR melt tem-
perature and vice versa (Prow and Metzger, 2005). The
addition of TGase had a significant (P < 0.05) effect on
melt area and TT, indicating changes at the molecular
level via crosslinking. The covalent bond formation and
surface modification of casein micelle due to TGase
crosslinking (Salunke et al., 2022) may have restricted
the melt characteristics and cause an increase in TT.
The covalent bonds formed are not disrupted easily
under the prevailing conditions in IMC manufacture,
and thus will form long strands and not melt easily,
resulting in a lower melt area and higher TT. Salunke
and Metzger (2022b) reported an increase in TT in
PCP when TGase-treated MCC was used.
Lucey et al. (2003) stated that hydrophobic interac-
tions (electrostatic repulsion, hydrogen bonds, colloidal
casein phosphate crosslinks, and disulfide bonds) play
a critical role in the melt characteristics of cheese; how-
ever, the role of covalent bonds is not clear. Casein
molecules (number, strength, and type of bond) are the
basic building blocks of rheological properties (Lucey
et al., 2003), and the melting of cheese is determined
primarily by the number and strength of CN–CN in-
teractions above 50°C (Park et al., 1984). It seems that
CN–CN interactions and formation of long strands due
to new and more crosslinked covalent bonds explain the
higher TT in MCC-L compared with MPC-L.
We believe that the intact casein coupled with TGase
action created the crosslinked protein network that had
longer protein strands, resulting in low meltability. The
higher covalent crosslinking due to TGase action modi-
fies CN and SP at the molecular level, causing restricted
melt characteristics (Salunke et al., 2012; Salunke and
Metzger, 2022b). A further detailed study is required at
the molecular level to determine the effect of covalent
bonds. Salunke and Metzger (2011, 2022b) and Salunke
et al. (2012) have reported on the increase in TT and
the decrease in the melt area of PCP manufactured
using MCC and MPC treated with TGase.

Journal of Dairy Science Vol. 105 No. 10, 2022

 Salunke et al.: MICELLAR CASEIN CONCENTRATE AND MELT PROPERTIES 7911
Stretchability on Pizza and Stretchability Test Using

Table 1. Statistical analysis of melt area, transition temperature, and stretchability (on pizza and texture profile analysis rig) of the 4 imitation mozzarella cheese treatments

distance
a Texture Analyzer

Stretch

*
*

*
Stretch is the ability of the CN network or strands
to maintain their integrity and not break when a con-
Extensibility
tinuous tensile force or stress is applied (Lucey et al.,
2003); it represents the ease and extent to which the

NS
*

*
* strands are formed (Gunasekaran and Ak, 2003). On
pizza, stretch is the ability of cheese to form fibers or
strands of reasonable tension upon heating when a slice
Maximum
pull force

is lifted from the rest of the pizza (Lucey et al., 2003).

NS

NS

The statistical analysis and P-values for the melted

*

functional properties—pizza stretchability test and

IMC stretchability test using a texture analyzer (re-
sistance to extension, stretch quality, total work done
Initial
pull

NS
NS
*
*

area, initial pull force, maximum pull force, extensibil-

ity, and stretch distance) are shown in Table 1. The
product type, enzyme level, and interaction term
work done)
Area (total

(product × enzyme level) had a significant (P < 0.05)

NS

NS
NS

NS

effect on pizza stretch. There was a significant (P <

0.05) replicate effect in initial pull force, extensibility,
and stretch distance. We found a significant (P < 0.05)
Stretch
quality

effect of product type on initial pull force, maximum

NS

NS
NS

pull force, extensibility, and stretch distance, whereas

enzyme level had a significant effect on stretch quality
and stretch distance. The significant (P < 0.05) effect
Resistance to
extension

of the interaction term product type × enzyme level in

NS
NS
NS
NS

maximum pull force, extensibility, and stretch distance

indicated that these changes were not linear. We found
no difference (P > 0.05) in resistance to extension and
total work done area values; however, the work done
Stretchability

values were less for IMC treated with TGase.

on pizza
NS

The mean values of the pizza stretch of the 4 IMC

*
*
*

are shown in Table 2. The IMC containing MCC

(MCC-C and MCC-L) had significantly (P < 0.05)
Enzyme level = control, low, and high levels of transglutaminase.

higher stretchability than the corresponding samples

temperature
Transition

containing MPC (MPC-C and MPC-L). The stretch-

ability of IMC manufactured from MCC-L was highest
NS

NS
NS

(P < 0.05) and that of MPC-C was lowest (P > 0.05)

of the 4 samples.
Stretchability depends on intact CN and critical lev-
Change

els of calcium and phosphorus (Lucey and Fox, 1993;

area

NS

NS
NS

Lucey et al., 2003); it also depends on a cohesive CN

manufactured in a twin-screw cooker

network, the linking of CN molecules, and the stor-

age and dissipation of energy by CN molecules (Lucey
df

Product type = MCC or MPC.

1
1

6
1
2

et al., 2003). The stretchability test requires close CN

molecular interactions but, at the same time, the CN
Product × enzyme level

bonds should relax and reform quickly (Lucey and Fox,

1993). In the current study, the MPC treatment contrib-
uted 2.55% whey proteins to the formulation, whereas
Product type1
Enzyme level2

MCC treatments contributed only 1.38%; thus, MCC

Replication

*P < 0.05.

samples had more intact casein than MPC samples.

Factor

Consequently, the MCC, having more intact CN (Table

Error

3), had more stretch, whereas IMC containing MPC

1

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 Salunke et al.: MICELLAR CASEIN CONCENTRATE AND MELT PROPERTIES 7912
Table 2. Mean values (n = 3) of the functional properties of the 4 imitation mozzarella cheese treatments
manufactured in a twin-screw cooker

Imitation mozzarella cheese treatment1

Parameter MCC-C MCC-L MPC-C MPC-L

2 2 a b a
Change in area, mm 846.1 ± 69.24 424.2 ± 123.0 795.6 ± 150.45 434.4b ± 123.45
Transition temperature,3 °C 70.3c ± 3.28 80.9a ± 2.96 70.9c ± 3.77 76.9b ± 3.04
Stretchability on pizza,4 mm 13.9b ± 2.39 24.9a ± 2.29 6.4d ± 1.41 12.7c ± 1.48
a–d
Means within a row not sharing a common superscript are significantly different (P < 0.05).
1
MCC-C = micellar casein concentrate powder, control (no transglutaminase, TGase); MCC-L = micellar ca-
sein concentrate powder, low TGase level; MPC-C = milk protein concentrate powder, control; MPC-L = milk
protein concentrate powder, low TGase level.
2
Change in area measured using modified Schriber melt test.
3
Transition temperature was measured using dynamic stress rheology.
4
Strtechability on pizza was measured using pizza bake and fork test.

(high in whey proteins) had reduced stretch. Also, the
calcium content in the MCC powders was higher than
that of MPC powders, as reported in the companion
paper (Salunke et al., 2022).
The strands of MPC-C samples were weak and
broke soon after lifting the fork, whereas the MCC-C
samples had longer strands, indicating that the whey
proteins had a detrimental effect on the stretchability
of the cheese. The addition of TGase significantly (P <
0.05) increased the stretchability in both MCC-L and
MPC-L samples. This may be due to the higher level of
crosslinking (Salunke et al., 2022) and modification of
surface properties due to TGase action. The strands in
TGase-treated samples were longer and did not break
immediately after lifting the fork. The strong covalent
bonds formed due to TGase may help with an increase
in the casein network, greater energy storage, and
delayed energy dissipation, resulting in higher stretch-
ability phenomenon as explained above. Process cheese
with more intact CN will have longer protein strands
(Berger et al., 1998), which will sustain the stretching
and relaxing forces.
The mean values of stretch properties obtained on a
TPA rig are shown in Table 3. Control samples (MCC-
C and MPC-C) had lower stretch quality than the
respective samples treated with TGase (MCC-L and
MPC-L). Once cheese starts to weaken, it may weaken
at a faster rate, which is measured by the downslope
gradient. This gradient in the stretch region defines
stretch quality (point C in Figure 4) or ease of stretch-
ability (extensibility). Initial pull and maximum pull
force values were higher for MCC samples than for the
corresponding MPC samples. Strong cheese will have a
higher resistance force to stretch and peak force com-
pared with weak cheese. Nonmelting cheese or very-
high-moisture cheese will form a plug and not stretch;
hence, the stretch test is considered negative for those
cheeses.
The extensibility of MPC-L was significantly (P <
0.05) higher than in other samples (Table 3) and can
be attributed to TGase treatment along with the in-
volvement of SP in the IMC network. Any resistance
to extension (point C in Figure 4) indicates the tough-
ness of the cheese. The stretch distances of MCC-L and

Table 3. Mean values (n = 3) of the functional properties obtained in the cheese stretchability rig of the 4
imitation mozzarella cheese treatments manufactured in a twin-screw cooker

Imitation mozzarella cheese treatment1

Parameter MCC-C MCC-L MPC-C MPC-L

Resistance to extension, g 349.04 ± 143.3 424.06 ± 182.7 336.15 ± 148.3 424.06 ± 172.2
Stretch quality, g/mm −3.95a ± 1.94 −7.11b ± 3.48 −4.29a ± 2.01 −7.01b ± 5.02
Total work done, g.mm 47,448 ± 12,642.5 43,396 ± 7,563.0 51,005 ± 11,547.7 47,996 ± 14,732.4
Initial pull, g/mm 2,450.3ab ± 1,022.4 2,720.1a ± 734.5 1,223.8c ± 709.9 1,357.0bc ± 722.9
Maximum pull force, g 2,273.9a ± 1,417.1 1,334.9b ± 962.1 834.4b ± 641.9 1,106.8b ± 583.5
Extensibility, mm 1.14b ± 0.59 0.70b ± 0.51 1.16b ± 0.68 2.24a ± 1.51
Stretch distance, mm 24.75c ± 3.98 47.38a ± 6.74 21.19c ± 4.62 38.08b ± 3.73
a–c
Means within the same row not sharing a common superscript are significantly different (P < 0.05).
1
MCC-C = micellar casein concentrate powder, control (no transglutaminase, TGase); MCC-L = micellar ca-
sein concentrate powder, low TGase level; MPC-C = milk protein concentrate powder, control; MPC-L = milk
protein concentrate powder, low TGase level.

Journal of Dairy Science Vol. 105 No. 10, 2022

 Salunke et al.: MICELLAR CASEIN CONCENTRATE AND MELT PROPERTIES 7913

MPC-L were significantly (P < 0.05) greater than the
control samples. The control samples had the lowest
stretch distance, and differences between MPC- and
MCC-C were not significant (P > 0.05). When cheese
elongates to its maximum point and the strands are no
longer holding together, it breaks, causing a sudden dip
in the force in the graph (point D in Figure 4). This
stretch distance can be measured (point E in Figure 4)
and is termed the “net stretch” of the cheese. The total
work done (point F in Figure 4) was calculated from
the area under the curve (the area from initial pull to
breaking point, D); no significant (P > 0.05) differences
were found among samples.
Crosslinking of proteins by TGase had a significant
effect on stretch quality and distance. The MCC-L
and MPC-L treatments had higher crosslinking than
the control samples, causing modifications to the
surface properties of the CN micelle, which is evident
from the capillary gel electrophoresis graph (Salunke
and Metzger, 2022b; Salunke et al., 2022). As the CN
matrix strengthens, stretchability increases, as was
the case with MCC treated with TGase. Higher cal-
cium content in cheese has been shown to influence
stretchability of cheese (Fife et al., 2002; Joshi et al.,
2004; McMahon et al., 2005; Ma et al., 2012); in the
current study, MCC had higher calcium content than
MPC samples (Salunke et al., 2022). The whey protein
content in MPC influences stretchability; however, an
increase in stretch was found after TGase treatment.
The stretchability obtained in the texture analyzer
(Table 3) was higher than that obtained by the pizza
bake and fork test (Table 2). These differences may
be due to different heating and stretching conditions
because heating cheese on a pizza would change the
heat transfer rate. However, in MPC-L, stretchability
differed between the pizza bake and fork test and the
TPA-stretchability test. The problem with the pizza
bake and fork test is that it is subjective. Although
temperature, time, and addition of ingredients are
controlled, fork insertion, tine orientation, direction
of stretch, the amount of tine covered by cheese, and
speed of lifting the cheese are variable (Fife et al., 2002;
Hicsasmaz et al., 2004; Ma et al., 2012), and some
variation is related to the pizza base. The meltability
of MCC-L and MPC-L samples was lower than that
of MCC-C and MPC-C samples but stretchability was
higher, implying that covalent bonding at the molecu-
lar level of casein plays a significant role in the melting
and stretching qualities of cheese.
Comparison with RCN
The IMC manufactured using RCN as an ingredient
was compared with IMC manufactured using MCC and
MPC powders using the Dunnett test. The mean values
and the Dunnett test comparison of treatment powders
for all functional properties are shown in Table 4. We
found significant (P < 0.05) differences in melt area of
IMC made using RCN and MCC and MPC powders.
Differences in melt area could be attributed to TGase
crosslinking and the presence of SP in MCC and MPC
samples. Rennet casein powder does not contain SP
and the casein present is 100% intact. No significant (P
< 0.05) differences were observed in RCN and treat-
ment powders in DSR melt temperature. However, in
IMC, the DSR melt temperature of RCN IMC was
significantly (P < 0.10) lower and (P < 0.10) higher
than that made from MPC-C and MCC-L, respectively.

Table 4. Statistical comparison of mean values (n = 3) of the functional properties of the rennet casein
imitation mozzarella cheese and imitation mozzarella cheese treatments manufactured in a twin-screw cooker

Imitation mozzarella cheese treatment1

Parameter RCN MCC-C MCC-L MPC-C MPC-L

2
Change in area, mm 1,108.19 ± 222.4 * * * *
TT,2 °C P < 0.05 73.7 ± 2.96 NS NS NS NS
TT, °C P < 0.10 73.7 ± 2.96 NS * * NS
Stretchability on pizza, mm 25.6 ± 3.50 * NS * *
Resistance to extension, g 461.26 ± 180.8 NS NS NS NS
Stretch quality, g/mm −6.75 ± 2.35 NS NS NS NS
Total work done, g.mm 46,579.98 ± 13,476.5 NS NS NS NS
Initial pull, g/mm 1,737.53 ± 442.8 NS NS NS NS
Maximum pull force, g 2,001.65 ± 967.9 NS NS NS NS
Extensibility, mm 2.44 ± 1.13 NS * NS NS
Stretch distance, mm 48.39 ± 5.58 * NS * NS
1
RCN = imitation mozzarella cheese treatment- rennet casein; MCC-C = micellar casein concentrate powder,
control (no transglutaminase, TGase); MCC-L = micellar casein concentrate powder, low TGase level; MPC-C
= milk protein concentrate powder, control; MPC-L = milk protein concentrate powder, low TGase level.
2
Transition temperature.
*P < 0.05.

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 Salunke et al.: MICELLAR CASEIN CONCENTRATE AND MELT PROPERTIES
In the pizza stretch test, results for RCN IMC were
similar to those of MCC-L; however, RCN IMC showed
significant (P < 0.05) differences for other properties
(P < 0.05). Extensibility was significantly (P < 0.05)
higher for RCN IMC compared with MCC-L IMC
but similar to that of other samples. The RCN IMC
had a significantly (P < 0.05) higher stretch distance
compared with MCC-C and MPC-C; however, stretch
distance was similar to that of MCC-L and MPC-L.
These differences in the IMC were due to differences in
CN availability, SP content, and calcium level.
The native casein obtained through membrane fil-
tration (either UF or MF) is different from the intact
casein found in the natural cheese or RCN powder
(Salunke and Metzger, 2022b; Salunke et al., 2022).
All casein micelle models state that κ-CN covers the
casein micelle surface, and the protruding flexible hair
has hydrophilic properties that provide steric stability
to casein micelles (de Kruif et al., 2012). This steric
stability of the casein micelles is due to the κ-CN on
the micelle surface having a negative charge because of
GMP, which provides strong repulsive steric stability,
preventing casein aggregation and collapse of micelles.
During manufacture of RCN powder or natural cheese,
due to the action of chymosin, the GMP portion is
cleaved off (Fox and McSweeney, 1998), changing the
charge profile and causing collapse of the casein micelles,
leading to aggregation of para-casein. This para-casein
complex found in RCN powder and natural cheese is
highly insoluble, whereas the casein obtained from the
membrane process still has intact GMP and is soluble.
The RCN powder has high ash content, especially
colloidal casein phosphate, Ca, and P, along with
higher pH (Mulvihill, 1992; Augustin et al., 2011). The
use of RCN provides the desirable strong IMC network
because when insoluble RCN is used, the small, aggre-
gated form of insoluble para-casein remains even after
the emulsifying salts are added. After heating, mixing,
and cooling, it forms a strong IMC network that gives
firmer texture and long strands during melting. The
intact casein obtained through MF and UF is soluble
and, depending on the amount of DF used, may have
reduced levels of SP and soluble constituents (calcium,
other minerals, and lactose) but MF and UF concen-
trates the casein micelles, maintaining their integrity
and soluble nature. Because GMP is still present in in-
tact form, the casein micelle surface is highly negatively
charged, and this charged surface of the membrane
products (MPC and MCC) inhibits full dispersion of
the native casein by emulsifying salts. The action of
TGase changes the surface properties by crosslinking
(Salunke and Metzger, 2022b; Salunke et al., 2022)
and reduces negative charges on casein micelles so that
these products can function similarly to RCN.
Journal of Dairy Science Vol. 105 No. 10, 2022
7914
The main IMC requirements of meltability and
stretchability improved in MCC-L after TGase action.
The IMC made from MCC-L was higher in TT, similar
in stretchability (as measured by both methods), but
lower in meltability than RCN, whereas IMC made from
MPC-L was lower in meltability and stretchability but
had higher TT compared with RCN. The IMC manu-
factured from MPC treatments could not provide bet-
ter functionality partly because of the presence of SP.
The SP content in the formulation provided by MPC,
MCC, and RCN was 2.55, 1.38, and 0%, respectively.
These results indicate that SP had a clear detrimental
effect on the functional properties of IMC.
The TGase treatment of MPC did modify some
functional properties; however, they could not match
those provided by MCC or RCN. However, the TGase
treatment on MCC had a positive effect and improved
certain functional properties. An increase in TT in PCP
manufactured using TGase-treated MCC was reported
by Salunke and Metzger (2022b). Abou El Nour et al.
(1998) found that replacement of RCN in a spread-type
cheese analog with MPC85 (MPC with ~85% protein)
increased firmness but decreased meltability of the
product. Model PC samples prepared with RCN and
sodium aluminum phosphate as emulsifiers were not
emulsified properly but had good meltability (Savello et
al., 1989). In the model of PC samples made with RCN,
as SP content increased, meltability decreased (Savello
et al., 1989; Mleko and Foegeding, 2000, 2001; Solowiej
et al., 2010). Overall, our experiment showed that at a
higher TGase level, there was no emulsion formation,
and the product formulation, processing conditions,
and emulsifying salts could not overcome the strong
covalent bonds formed by TGase. The low TGase treat-
ment restricted melt and increased stretchability of the
resulting IMC. Salunke and Metzger (2022b) reported
higher cooked viscosity, increased hardness, and re-
stricted melt in PCP made with TGase-treated MCC
(TGase used at 7 U/g of protein). Figure 5 depicts and
summarizes the effect of TGase treatment on MPC and
MCC and the effect of crosslinking on melt and stretch
functional characteristics.
CONCLUSIONS
The TGase treatment significantly affected the func-
tionality of the IMC by changing proteins at a molecu-
lar level through covalent crosslinking. The IMC made
from MCC (with or without TGase) had higher stretch-
ability on pizza compared with their respective MPC
treatments. The IMC made from TGase-treated MCC
and MPC had decreased Schreiber melt test area and
higher TT, stretchability on pizza, and TPA-stretch-
ability values compared with the respective controls.
 Salunke et al.: MICELLAR CASEIN CONCENTRATE AND MELT PROPERTIES
Figure 5. Effect of transglutaminase (TGase) crosslinking on melt and stretch qualities of imitation mozzarella cheese (IMC). MCC-H =
micellar casein concentrate powder with high TGase level; MPC-H = milk protein concentrate powder with high TGase level.
Covalent bonds appear to be involved in the stretching
characteristics of IMC, and this role of covalent bonds
needs to be explored further. A comparison of IMC
made using TGase-treated MCC and MPC to that of
IMC made using RCN showed no difference in TT or
TPA-stretchability; however, the Schreiber melt test
area was significantly lower, and TT was significantly
higher. The study demonstrates that TGase treatment
modifies the melt and stretch characteristics by modi-
fying the casein surface of MCC and MPC through
covalent bonding and can be used to replace RCN in
IMC formulations.
ACKNOWLEDGMENTS
We thank Dairy Research Institute (Rosemont, IL)
and Midwest Dairy Foods Research Center (St. Paul,
MN) for funding this project. The authors have not
stated any conflicts of interest.
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ORCIDS
P. Salunke https:​/​/​orcid​.org/​0000​-0002​-1168​-4765
C. Marella https:​/​/​orcid​.org/​0000​-0002​-3903​-596X
J. K. Amamcharla https:​/​/​orcid​.org/​0000​-0001​-6620​-0089
L. E. Metzger https:​/​/​orcid​.org/​0000​-0003​-3929​-4539