Content uploaded by Ali Adnan Hayaloglu
Author content
All content in this area was uploaded by Ali Adnan Hayaloglu on Jul 19, 2022
Content may be subject to copyright.
ORIGINAL ARTICLE
Effect of blends of camel chymosin and microbial rennet
(Rhizomucor miehei) on chemical composition, proteolysis
and residual coagulant activity in Iranian Ultrafiltered White
cheese
Mostafa Soltani
1,2
•Didem Sahingil
3
•Yasemin Gokce
3
•Ali A. Hayaloglu
3
Revised: 21 March 2017 / Accepted: 21 May 2018 / Published online: 1 January 2019
ÓAssociation of Food Scientists & Technologists (India) 2019
Abstract Iranian Ultrafiltered White cheese was produced
by using different blends of coagulants (100:0, 75:25,
50:50, 25:75 and 0:100; Rhizomucor miehei and camel
chymosin, respectively) and ripened for 90 days. The
effect of different combinations of these coagulants on
chemical composition, proteolysis and residual coagulant
activity of the cheeses were studied. The results showed
that pH, fat-in-dry matter, salt-in-dry matter and protein
contents of the cheeses were significantly influenced by
type and concentration of the coagulants. The difference
between proteolytic activities of the two coagulants resul-
ted in different levels of proteolysis in the cheeses. A direct
relationship was determined between using higher con-
centrations of R. miehei and increasing the hydrolysis of
a
s1
-casein in the cheeses, during ripening. The residual
coagulant activity was influenced by the type and con-
centration of the coagulant as well. In conclusion, R.
miehei provided a higher level of proteolysis and residual
coagulant activity compared with camel chymosin.
Keywords Ultrafiltered cheese Rhizomucor miehei
Camel chymosin Residual coagulant activity Proteolysis
Abbreviations
UF
cheese
Ultrafiltered cheese
CC Camel chymosin
MR Microbial rennet
C0 Cheese produced using 100% of microbial
rennet ?0% of camel chymosin
C25 Cheese produced using 75% of microbial
rennet ?25% of camel chymosin
C50 Cheese produced using 50% of microbial
rennet ?50% of camel chymosin
C75 Cheese produced using 25% of microbial
rennet ?75% of camel chemosin
C100 Cheese produced using 0% of microbial
rennet ?100% of camel chmosin
d Day
Introduction
In many cheese varieties, proteolytic enzymes are the main
agents that play an important role during cheese manu-
facture and ripening. The activity of residual coagulant and
indigenous proteinases (i.e. plasmin and cathepsin) are
responsible for primary proteolysis and changes casein
fractions including a
s1
- and b-caseins and derived peptides.
On the other hand, secondary proteolysis is occurred by
proteolytic and peptidolytic activity of starter and non-
starter microorganisms (Fox et al. 1993; Hesari et al. 2006).
Proteolytic activity of residual chymosin can change the
rheological behavior of cheese, i.e., increase flowability
and decrease the stretch ability. However, activity of pro-
teolytic coagulant during ripening may lead to decrease of
shelf-life of high moisture cheeses, e.g., soft or UF cheeses.
So, some methods such as reducing the amount of
&Ali A. Hayaloglu
adnan.hayaloglu@inonu.edu.tr
1
Department of Food Sciences and Technology, Faculty of
Pharmacy, Tehran Medical Sciences, Islamic Azad
University, Tehran, Iran
2
Nutrition and Food Sciences Research Center, Tehran
Medical Sciences,, Islamic Azad University, Tehran, Iran
3
Department of Food Engineering, Inonu University,
44280 Malatya, Turkey
123
J Food Sci Technol (February 2019) 56(2):589–598
https://doi.org/10.1007/s13197-018-3513-3
coagulant used and storage temperature are implemented in
order to limit the activity of residual chymosin during
ripening (Sheehan et al. 2004; Moynihan et al. 2014).
Retention of milk clotting-enzymes in the curd is a
significant factor for cheese quality and influences the
proteolysis of cheese during ripening. While a small part of
rennet remains in the curd after manufacturing of tradi-
tional cheeses, almost all the rennet is retained in the curd
of UF cheeses because of addition of it to the retentate
(Broome and Limsowtin 1998; Karami et al. 2009). On the
other hand, due to whey removal occurred during the
manufacturing, the concentrations of major constituents are
increased in the conventional cheeses. However, preven-
tion of whey removal during UF cheese production lead to
presence of whey proteins at high concentration in the curd
and may inhibit the activities of chymosin and microbial
rennet. This results to slower proteolysis and production of
amino acids and alters flavor development during ripening
of UF cheeses comparing with the conventional ones
(Benfeldt 2006; Karami et al. 2009).
Calf rennet is traditionally used as a proteolytic enzyme
in conventional cheese-making; however, because of high
cost of calf rennet, it was substituted by enzymes from
other animals or microorganisms and use of these enzymes
has increasingly been expanded (Hayaloglu et al. 2014).
Camel chymosin (CC) is a camel origin coagulant and can
be used as proteolytic enzyme in cheese manufacturing.
Although the clotting activity of CC is strong, the overall
proteolytic activity of this enzyme is reduced (Moynihan
et al. 2014).
Iranian UF white cheese is produced from UF-treated
and pasteurized bovine milk with mesophilic starter culture
and commercial recombinant chymosin in dairy plants
(Karami et al. 2009). Rhizomucor miehei as a microbial
rennet (MR) is mainly used for production of this type of
cheese. The MR has higher proteolytic activity than calf
rennet and hydrolyses both a
s1
-casein and b-casein at a
similar rate (Awad et al. 1999). Contribution of bovine
rennet to proteolysis in Iranian UF white cheese has been
previously studied (Hesari et al. 2006,2007). Nevertheless,
the effect of CC and MR on proteolysis in UF white cheese
has not been investigated in detail. The present study was
carried out to investigate the influence of CC and MR in
five different combinations on chemical composition,
proteolysis and residual coagulant activity of Iranian UF
white cheese during 90 days of ripening.
Materials and methods
Materials
Raw cows’ milk and cheese production equipment were
provided by Damaneh Sahand Co. (Tabriz, Iran). Meso-
philic homofermentative starter culture with Lactococcus
lactis spp. lactis and Lactococcus lactis spp. cremoris
(DM-230) were obtained from Danisco Deutschland
GmbH (Niebu
¨ll, Germany). Recombinant chymosin [Fro-
mase
Ò
2200 TL Granualte (C2200 International Milk
Clotting Unit (IMCU) g
-1
)] as microbial rennet from R.
miehei was obtained from DSM Food Specialties (Seclin,
Cedex, France). Camel Chymosine [CHY-MAX
Ò
M, 1000
International Milk Clotting Unit (IMCU) mL
-1
] was pro-
vided from Chr. Hansen (Hørsholm, Denmark).
Methods
Cheese manufacture
Manufacture of UF white cheese was carried out in two
separate trials in consecutive weeks in Damaneh Sahand
dairy plant (Tabriz, Iran) as introduced by Tetra-pack
incorporation (Bylund 1995) and adapted by Hesari et al.
(2006). The cheese making details are given in previous
work (Soltani et al. 2016). The experimental design was
implemented according to milk clotting activity of the
enzymes, as follows:
C0: 100% of MR ?0% of CC (32 g of MR per 1000 kg
of retentate)
C25: 75% of MR ?25% of CC (24 g of MR ?12 g of
CC per 1000 kg of retentate)
C50: 50% of MR ?50% of CC (16 g of MR ?24 g of
CC per 1000 kg of retentate)
C75: 25% of MR ?75% of CC (8 g of MR ?36 g of
CC per 1000 kg of retentate)
C100: 0% of MR ?100% of CC (48 g of CC per
1000 kg of retentate).
After production of the cheeses as described by Soltani
et al. (2016), sampling of cheeses produced for analysis
was implemented in duplicate after 1, 30, 60 and 90 days
of ripening.
Gross chemical analysis
A cheese of each trial was analyzed in duplicate for total
solids by the oven drying method at 102 ±1°C (IDF
1982), fat by the Van Gulik method (Ardo and Polychro-
niadou 1999), and total nitrogen by the micro-Kjeldahl
method (IDF 1993). The pH was monitored by mixing 10 g
590 J Food Sci Technol (February 2019) 56(2):589–598
123
of grated cheese with 10 ml of distilled water and mea-
suring the pH value of the resultant slurry using a digital
pH meter (model SevenCompact S220K, Mettler-Toledo,
Greifensee, Switzerland).
Proteolysis
Water–soluble nitrogen (WSN) and 12% trichloroacetic
acid soluble nitrogen (TCA–SN) fractions of the cheeses
were analyzed by the method described by Hayaloglu et al.
(2005) and presented as % of total nitrogen. The levels of
free amino acid (FAA) in the WSN fraction of the cheeses
were determined by the methods described by Hayaloglu
(2007).
Urea-PAGE of caseins and densitometry
After freeze-drying the water-insoluble fractions of the
cheeses, a Protean II XI vertical slab gel unit (Bio-Rad
Laboratories Ltd., Watford, UK) was used for urea poly-
acrylamide gel electrophoresis (urea-PAGE) of samples
according to the method of Andrews (1983). The gels then
were stained directly by the method of Blakesley and Boezi
(1977) with Coomassie Brilliant Blue G-250. Next, the gels
were destained by pure water and gel slabs were digitized
using a scanner (HP ScanJet software, ScanJet G4010,
Hewlett Packard, Palo Alto, CA). Scans of the elec-
trophoretograms were used to quantify bands using den-
sitometric software (Image Master TotalLab Phoretix 1D
Pro software, Keel House, Newcastle upon Tyne, UK). The
caseins were determined quantitatively by integration of
peak volumes and areas using the densitometer.
RP-HPLCs of water-soluble fractions
The WSN fractions of the cheeses were freeze-dried and
analyzed by reverse-phase high performance liquid chro-
matography (RP-HPLC) using a Shimadzu LC 20 AD
Prominence HPLC system (Shimadzu Corporation, Kyoto,
Japan) according to method described by Hayaloglu et al.
(2011).
Residual coagulant activity (RCA)
The RCA was determined during ripening by RP-HPLC
according to the method described by Hurley et al. (1999),
which measures the rate of cleavage of a synthetic hep-
tapeptide substrate (H-Pro-Thr-Clu-Phe-[p-nitro-Phe]-Arg-
Leu-OH; code H-1002, Bachem, Bubendorf, Switzerland).
As described in Hurley et al. (1999), prepared sample
mixture was injected into a Shimadzu LC-20AD Promi-
nence HPLC system (Shimadzu Corp., Kyoto, Japan)
consisted of diode array detector model SPD-M20A
equipped with a pump system with an auto sampler model
SIL-20A HT, CTO-20A column heater and DGU-20A5
degasser units. A gradient solvent system consisting of
0.1% (v/v) trifuoroacetic acid (TFA) in HPLC-grade water
(solvent A) and 0.1% TFA (v/v) in acetonitrile (Merck,
Darmstadt, Germany) as solvent B was used for separation
at 300 nm using a C8 RP column with size of
250 94.6 mm, 300 A
˚pore size, 5 lm particle (Inertsil,
WP 300, GL Science, Tokyo, Japan). The column was
equilibrated initially for 5 min with 15% B. A gradient was
then generated by increasing the concentration of B as
follows: 15–45% B over 20 min, 45–95% B over 3 min,
maintaining at 95% B for 2 min, and finally returning to
equilibration conditions over 3 min. The RCA of the
cheeses were expressed as following formula.
RCAð%Þ¼ Peak area of product
Peak area of substrate 100
Statistical analysis
The data obtained from two trials were analysed statisti-
cally using the analysis of variance (ANOVA) of SPSS
program (SPSS package program, version 16.0, SPSS Inc.,
USA). Different groups were statistically defined by Dun-
can’s multiple range tests. Analysis was performed for 1,
30, 60 and 90 days of ripening. The obtained results were
considered significant at a= 0.05.
Results and discussion
Chemical composition and pH
Changes in chemical composition and pH value of Iranian
UF white cheeses during ripening are shown in Table 1.
pH, fat-in-dry matter (FDM), salt-in-dry matter (SDM) and
total protein content of the cheeses changed significantly
(P\0.05) with a change in both blends of enzymes used
and ripening period. However, no significant differences
were observed in total solids content of the cheeses
(P[0.05). A few technological changes during cheese
production particularly in the salt and rennet addition steps
may cause to differences in pH value and SDM contents of
the cheeses (Moynihan et al. 2014). The pH of the cheeses
ranged from 4.48 to 4.67 at day 1 of ripening. These values
are in accordance with previous studies for Iranian UF
White cheese (Hesari et al. 2006; Karami et al. 2009). On
the other hand, cheese pH increased towards the end of
ripening due to utilization of lactic acid, formation of non-
acidic decomposition products and liberation of alkaline
products (e.g., NH
3
) during hydrolysis of protein
(McSweeney and Fox 1993; Awad 2006).
J Food Sci Technol (February 2019) 56(2):589–598 591
123
The type and concentration of coagulants did not
influence significantly (P[0.05) the total solids content of
the cheeses. The values for total solid content of the
cheeses were similar to the values reported by Hesari et al.
(2006) and slightly higher than the values reported by
Karami et al. (2009) for Iranian UF White cheese. Several
researchers have reported that the type and concentration of
coagulant did not affect the dry matter content in various
types of cheeses (Kandarakis et al. 1999; Guven et al.
2008; Yasar and Guzeler 2011). Significant differences
(P\0.05) were determined in terms of FDM between the
cheeses at 1, 30 and 90 d of ripening. Changes in FDM of
the cheeses exhibited a parallel change for DM during
ripening. Similar results have reported by Al-Otaibi and
Wilbey (2004,2005) for UF White cheese. Diffusion
between salt and water molecules may cause DM changes
(P[0.05) and consequently led to changes in the FDM
contents of cheeses analyzed (Guinee and Fox 2004).
Significant (P\0.05), but not substantial changes were
determined in SDM contents of chesses analyzed during
ripening that were in line with changes in total solid con-
tents of the cheeses.
Total protein contents of the cheeses were significantly
(PB0.05) affected by the coagulants. The type, concen-
tration and blend rate of coagulants influence the gel
strength (Hayaloglu et al. 2014). This situation may causes
more or less differences in total protein contents between
the cheeses. On the other hand, fluctuations were observed
in total protein contents of the cheeses during ripening.
These changes may affected by proteolysis, diffusion of
water-soluble nitrogen into brine (Al-Otaibi and Wilbey
2004; Guven et al. 2006) and water holding characteristics
of some compounds formed during ripening (Hayaloglu
et al. 2002,2005).
Proteolysis
Soluble nitrogen fractions
The effects of type or concentration of coagulant on the
level of WSN in experimental cheeses were significant
(PB0.05). The levels of WSN in the cheeses increased
during ripening (Table 2). The cheeses produced using
higher level of MR (C0 and C25) had higher levels of WSN
Table 1 The chemical composition and pH value of Iranian UF
White cheeses produced using different combinations of proteases
from MR and CC [100% of MR ?0% of CC (C0), 75% of
MR ?25% of CC (C25), 50% of MR ?50% of CC (C50), 25% of
MR ?75% of CC (C75) and 0% of MR ?100% of CC (C100)] after
1, 30, 60 and 90 days of ripening
Variables Days C0 C25 C50 C75 C100 Ptreatment Pripening
pH 1 4.49 ±0.01 4.41 ±0.05 4.67 ±0.01 4.49 ±0.08 4.53 ±0.14 * *
30 4.76 ±0.09 4.67 ±0.14 4.79 ±0.07 4.65 ±0.02 4.72 ±0.08 *
60 4.61 ±0.01 4.60 ±0.03 4.64 ±0.04 4.53 ±0.01 4.60 ±0.01 *
90 4.80 ±0.03 4.69 ±0.07 4.81 ±0.01 4.88 ±0.02 4.78 ±0.01 *
Dry matter (%) 1 38.52 ±0.13 38.59 ±1.06 38.50 ±0.31 38.57 ±0.20 38.15 ±0.32 n.s. n.s.
30 38.65 ±0.33 38.79 ±0.17 38.05 ±0.34 39.02 ±0.13 39.00 ±0.30 n.s.
60 37.47 ±0.28 37.76 ±0.11 37.91 ±1.22 37.97 ±0.79 37.71 ±0.24 n.s.
90 38.55 ±0.38 38.86 ±0.22 38.51 ±0.39 38.69 ±0.19 38.51 ±0.38 n.s.
Fat-in-dry matter (%) 1 50.62 ±0.18 50.38 ±1.43 50.49 ±0.35 51.48 ±0.33 50.41 ±0.45 * *
30 51.29 ±1.05 50.72 ±1.26 50.86 ±1.81 50.24 ±1.10 50.74 ±0.11 *
60 49.86 ±1.22 49.43 ±1.13 49.49 ±0.76 50.20 ±0.51 49.33 ±0.09 n.s.
90 50.96 ±0.80 51.25 ±0.62 50.62 ±0.13 51.28 ±0.85 50.79 ±0.14 *
Salt-in-dry matter (%) 1 5.88 ±0.11 5.85 ±0.05 5.92 ±0.03 5.91 ±0.02 5.89 ±0.07 * **
30 5.59 ±0.08 5.63 ±0.09 5.59 ±0.04 5.54 ±0.16 5.60 ±0.06 *
60 2.97 ±0.07 2.93 ±0.01 2.97 ±0.03 2.94 ±0.01 2.96 ±0.03 *
90 4.89 ±0.07 4.88 ±0.05 4.87 ±0.05 4.93 ±0.05 4.90 ±0.02 *
Total protein (%) 1 12.20 ±0.39 11.97 ±1.29 13.02 ±0.30 11.81 ±0.29 12.32 ±0.22 * *
30 14.07 ±0.67 12.77 ±0.18 13.73 ±0.04 14.27 ±0.89 14.82 ±0.26 *
60 14.69 ±0.39 12.80 ±0.59 14.97 ±0.51 14.40 ±0.17 14.49 ±0.29 *
90 13.02 ±0.07 14.15 ±0.01 15.62 ±1.03 13.99 ±0.41 13.86 ±0.45 *
Presented values are the means of two replicate trials
n.s. non-significant
*P\0.05; **P\0.01
592 J Food Sci Technol (February 2019) 56(2):589–598
123
values than the cheeses produced using higher level of CC
(C75 and C100) during ripening period (P\0.05) proba-
bly due to lower general proteolytic activity of CC com-
pared with MR (Kappeler et al. 2006; Govindasamy-Lucey
et al. 2004). A similar trend was observed in TCA–SN
values of the cheeses during ripening (Table 2). However,
because of the liberation of intermediate and lower
molecular weight peptides, the differences in the level of
TCA–SN in the cheeses were greater as ripening pro-
gressed (Hayaloglu et al. 2014). Hayaloglu et al. (2014)
and Moynihan et al. (2014) reported that different coagu-
lant enzymes caused significant differences in pH 4.6-SN,
WSN or TCA–SN in Malatya and Mozzarella cheeses.
Similar trends during ripening for pH 4.6-SN or TCA–SN
have been reported by Kandarakis et al. (1999) for Feta
cheeses produced using calf or fermentation produced
rennet. Due to the proteolytic activity of starter bacteria,
the levels of WSN and TCA–SN increased as ripening
progressed for all experimental cheeses, as reported by
other authors (Hayaloglu et al. 2005; Hayaloglu 2007).
The type or concentration of coagulant enzymes sig-
nificantly (P\0.05) influenced the total FAAs of the
cheeses. As shown in Fig. 1, the highest and the lowest
values of FAA were observed during ripening (with
exception of day 60) in the cheeses produced by using
100% of MR (C0) and 25% of MR (C75), respectively. The
levels of total FAA declined steadily by increasing levels
of CC. According to Fig. 1, the total FAA concentration of
C100 was higher than C75 and close to C25 or C50. So, it
is noticeable that not only the type but also the level of
coagulant affects the formation of total FAA. Total FAAs
are the final product of proteolysis and liberation of them
from casein is the cause of their presence in the cheese at
any stage of ripening (Sousa et al. 2001). So, the higher
Table 2 Levels of soluble nitrogen fractions of Iranian UF White
cheeses produced using different combinations of proteases from MR
and CC [100% of MR ?0% of CC (C0), 75% of MR ?25% of CC
(C25), 50% of MR ?50% of CC (C50), 25% of MR ?75% of CC
(C75) and 0% of MR ?100% of CC (C100)] after 1, 30, 60 and
90 days of ripening
Variables
a
Days C0 C25 C50 C75 C100 Ptreatment Pripening
WSN, % of TN 1 11.71 ±0.21 11.49 ±0.07 9.78 ±0.25 9.56 ±0.56 9.13 ±0.53 * *
30 11.90 ±0.20 11.41 ±0.31 11.07 ±0.48 10.95 ±0.51 9.71 ±0.08 *
60 12.67 ±0.76 12.22 ±0.80 11.56 ±0.61 11.25 ±0.41 10.38 ±0.17 n.s.
90 13.89 ±0.63 12.42 ±0.50 12.09 ±0.20 11.95 ±0.47 12.26 ±0.62 n.s.
TCA–SN, % of TN 1 5.80 ±0.43 6.49 ±0.26 5.72 ±0.14 6.17 ±0.30 4.95 ±0.45 n.s. **
30 8.14 ±0.44 7.63 ±0.11 5.82 ±0.38 6.10 ±0.38 5.36 ±0.55 *
60 10.48 ±1.30 10.15 ±0.47 8.97 ±0.67 8.76 ±0.22 6.26 ±0.80 *
90 13.12 ±0.48 10.38 ±0.11 9.05 ±0.22 9.97 ±0.93 8.99 ±0.14 **
Presented values are the means of two replicate trials
n.s. non-significant, WSN water-soluble nitrogen, TCA–SN 12% trichloroacetic acid-soluble nitrogen, TN total nitrogen
*P\0.05; **P\0.01
0,00
0,10
0,20
0,30
0,40
0,50
0,60
1
30 60 90
Ripening time, day
Total Free Amino Acids, mg Leu/g cheese
C0 C25 C50 C75 C100
Fig. 1 Levels of free amino
acid in Iranian UF White
cheeses produced using
different combinations of
proteases from MR and CC
[100% of MR ?0% of CC
(C0), 75% of MR ?25% of CC
(C25), 50% of MR ?50% of
CC (C50), 25% of MR ?75%
of CC (C75) and 0% of
MR ?100% of CC (C100)]
after 1, 30, 60 and 90 days of
ripening
J Food Sci Technol (February 2019) 56(2):589–598 593
123
NaCN A E C B D A E C B D NaCN A E C B D A E C B D
DAY 1 DAY 30 DAY 60 DAY 90
β-Casein
αs1-Casein
αs1-Casein (f24-199)
(b)
(a)
Fig. 2 Urea-PAGE electrophoretogram (a) and dendrogram (b)of
the water-insoluble fractions of Iranian UF White cheeses produce-
dusing different combinations of proteases from MR and CC [100%
of MR [(A=C0]) ?0% of CC, 75% of MR ?25% of CC [(B=C25)],
50% of MR ?50% of CC [(C=C50)], 25% of MR ?75% of CC
[(D=C75)] and 0% of MR ?100% of CC [(E=C100)]] after 1, 30, 60
and 90 days of ripening
594 J Food Sci Technol (February 2019) 56(2):589–598
123
proteolytic activity of MR compared with CC caused a
higher hydrolysis of casein and higher levels of FAA in the
C0 compared to other cheeses (Hayaloglu et al. 2014;
Moynihan et al. 2014).
Urea-PAGE patterns of caseins
Urea-PAGE electrophoretogram and dendrogram of the
water-insoluble fractions of the cheeses during ripening are
presented in Fig. 2a and b, respectively. The degradation of
a
s1
- and b-caseins were considerably slow until 30 d of
ripening; however, the hydrolysis of a
s1
-CN and formation
of its degradation products accelerated after 30 d and it was
more intense after 60 d of ripening. The degradation
patterns of a
s1
-CN in the cheeses were affected by the type
or concentration of the coagulants during ripening. The
level of degradation in a
s1
-CN was lower in cheeses pro-
duced using higher levels of CC (C75 and C100) compared
to cheeses produced using higher levels of MR (C0 and
C25). While the a
s1
-CN (f 24-199) was cleaved in cheeses
produced using coagulant containing MR after 30 d of
ripening, a
s1
-CN (f 24-199) was not hydrolysed further in
cheese produced using CC (C100). The C100 was not
grouped with other cheeses at any sampling time as shown
Fig. 2b. Cheeses were distinguished by the levels of
enzyme types on the dendrogram; however, the best sep-
aration was observed by ripening time. Bansal et al. (2009)
reported that Cheddar cheese made using recombinant
Retention time, min
010 30 5040 60 7020 80 90
Day 90
Day 30
Day 60
C0
C100
C75
C50
C25
C0
C100
C75
C50
C25
C0
C100
C75
C50
C25
Absorbance unit,mAU
12
7865
4
3
Fig. 3 Reversed phase-HPLC
peptides profiles of Iranian UF
White cheeses produced using
different combinations of
proteases from MR and CC
[100% of MR ?0% of CC
(C0), 75% of MR ?25% of CC
(C25), 50% of MR ?50% of
CC (C50), 25% of MR ?75%
of CC (C75) and 0% of
MR ?100% of CC (C100)]
after 1, 30, 60 and 90 days of
ripening
J Food Sci Technol (February 2019) 56(2):589–598 595
123
camel chymosin as coagulant presented lower level
hydrolysis of a
s1
-CN compared to cheese made by calf
chymosin. On the other hand, type or concentration of the
coagulants did not affect the degradation of b-CN in the
cheeses. Hayaloglu et al. (2014) reported that the concen-
tration of coagulants did not influence the patterns of both
a
s1
-CN and b-CN in this type of cheese, while using pro-
tease from calf rennet and MR resulted in differences in
urea-PAGE patterns of Malatya cheese after 60 d of
ripening. The same urea-PAGE patterns for a
s1
-CN and b-
CN is reported by Yasar and Guzeler (2011) for Kasar
cheese produced by using proteases from calf rennet and
MR. In this context, Kubis et al. (2001) and Dave et al.
(2003), reported that no relation was found between the
concentration of rennet and the degradation of b-CN, while
the direct relationship was observed between the rennet
concentration and the degradation level of a
s1
-CN in
Cheddar and Mozzarella cheeses, respectively.
RP-HPLC peptide profiles of water-soluble fractions
RP-HPLC peptide profiles of the cheeses after 30, 60 and
90 d of ripening are shown in Fig. 3. Major differences
were observed between the chromatograms of the water-
soluble fractions of the cheeses at early elution time
(5–20 min) during ripening. Moreover, the quantitative
differences between the peak concentrations in the cheeses
were pronounced after 25 min and particularly between 40
and 80 min of elution time. With increasing CC concen-
tration in the composition of coagulant, the peak concen-
trations decreased. So, C75 and C100 which produced
using higher concentration of CC had lower peptide peak
concentration than C0, C25 and C50. These differences
reflected the lower general proteolytic activity of CC
(Bansal et al. 2009; Moynihan et al. 2014). Hydrophobic
and high molecular mass peptides along with whey pro-
teins are eluted between 70 and 100 min of retention time
(Hayaloglu et al. 2011). In our study, the peaks eluted at 79
and 85 min was belonged to a-lactalbumin and b-lac-
toglobulin, respectively that is similar to results reported by
Hesari et al. (2006). These two proteins were identified and
confirmed by injection of their analytical standards under
the same chromatographic conditions.
Residual coagulant activity
The amount of residual coagulant changes the biochemical
and physical characteristics of cheeses. Activity of residual
coagulant can also catalyze the primary hydrolysis of caseins
into peptides and lead to secondary proteolysis by enzymes
from lactic acid bacteria (Hesari et al. 2006; Hayaloglu et al.
2014). The levels of residual coagulant activity of the
experimental cheeses during ripening are shown in Fig. 4.
Significant differences were observed between the activity of
MR and CC in the cheeses during ripening and the residual
coagulant activity increased as ripening progressed. In this
context, Maniou et al. (2013) reported that the level of
residual chymosin after 60 d of ripening compared with the
first day was increased and decreased in Feta cheese made
with HP-treated starter and non-treated starter, respectively.
The level of residual MR was significantly higher than the
level of residual CC in the cheeses (P\0.05). Therefore,
C75 and C100 which were produced using higher levels of
CC had the lowest level of residual coagulant among the
cheeses analyzed. This was in agreement with the results of
proteolysis and urea-PAGE (see Table 2, Fig. 2), which
showed lower contents of WSN and TCA–SN and less
hydrolysis of a
s1
-CN in the C75 and C100 due to lower
proteolytic activity of CC compared with MR (Bansal et al.
2009; Moynihan et al. 2014).
0
10
20
30
40
50
60
1306090
Ripening time, day
Residual coagulant activity, %
C0 C25 C50 C75 C100
Fig. 4 Residual coagulant
activity (as percentage of peak
area ratio of product and
substrate) of Iranian UF White
cheeses produced using
different combinations of
proteases from MR and CC
[100% of MR ?0% of CC
(C0), 75% of MR ?25% of CC
(C25), 50% of MR ?50% of
CC (C50), 25% of MR ?75%
of CC (C75) and 0% of
MR ?100% of CC (C100)]
after 1, 30, 60 and 90 days of
ripening
596 J Food Sci Technol (February 2019) 56(2):589–598
123
Conclusion
The type and concentration of coagulants used for produc-
tion of the cheeses significantly influenced the pH, FDM,
SDM and protein contents of the experimental cheeses
during ripening. While pH was established in the lowest
level in C75 after 30, 60 and 90 d of ripening, FDM and
protein contents were generally increased during ripening in
cheeses produced using higher levels of CC (C75 and C100).
SDM content of C0 was also higher than C100 during
ripening with exception of the first day. Because of higher
proteolytic activity of MR compared with CC, cheeses
produced using higher levels of MR (C0 and C25) had higher
proteolysis values than the other cheeses during ripening.
While the degradation of a
s1
-CN was influenced by the type
or concentration of the coagulants used, the b-CN degra-
dation was not influenced by any of these factors. Using MR
or CC as the coagulating agent resulted in higher and lower
residual coagulant activity in the cheeses, respectively. To
optimize the coagulating and proteolytic action of coagulant
during ripening in UF white cheese, MR and CC can be
blended at a ratio of 75:25 and 50:50, respectively. The
results indicated that CC in combination with MR can be
used in the production of Iranian UF White cheese.
Acknowledgements The authors thank the Damaneh Sahand dairy
plant (Khosroshah, Tabriz, Iran) for providing raw materials and
production equipment for cheese making. The study was partially
supported by Inonu University (Malatya, Turkey) Scientific and
Research Projects Unit (Project No: 2013/30).
Compliance with ethical standards
Conflict of interest The authors on this manuscript declare that they
have no conflict of interest.
Human and animal rights This article does not contain any studies
with human participants or animals performed by any of the authors.
References
Al-Otaibi MM, Wilbey RA (2004) Effect of temperature and salt on
the maturation of white salted cheese. Int J Dairy Technol
57:57–63
Al-Otaibi MM, Wilbey RA (2005) Effect of chymosin and salt
reduction on the quality of ultrafiltrated white-salted cheese.
J Dairy Res 72:234–242
Andrews AT (1983) Proteinases in normal bovine milk and their
action on caseins. J Dairy Res 50:45–55
Ardo Y, Polychroniadou Y (1999) Laboratory manual for chemical
analysis of cheese. Office for Official Publications of the
European Communities, Luxembourg
Awad S (2006) Texture and flavour development in Ras cheese made
from raw and pasteurised milk. Food Chem 97:394–400
Awad S, Luthi-Peng QQ, Puhan Z (1999) Proteolytic activities of
Suparen and Rennilase on buffalo, cow, and goat whole casein
and b-casein. J Agricul Food Chem 47:3632–3639
Bansal N, Drake MA, Piraino P, Broe ML, Harboe M, Fox PF,
McSweeney PLH (2009) Suitability of recombinant camel
(Camelus dromedarius) chymosin as a coagulant for Cheddar
cheese. Int Dairy J 19:510–517
Benfeldt C (2006) Ultrafiltration of cheese milk: effect on plasmin
activity and proteolysis during cheese ripening. Int Dairy J
16:600–608
Blakesley RW, Boezi JA (1977) A new staining technique for
proteins in polyacrylamide gels using coomassie brillant blue
G250. Anal Biochem 82:580–582
Broome MC, Limsowtin GKY (1998) Milk coagulants. Austr J Dairy
Technol 53:188–190
Bylund G (1995) Dairy processing. Tetra pak processing systems AB.
Lund University Publications, Lund
Dave RI, McMahon DJ, Oberg CJ, Broadbent JR (2003) Influence of
coagulant level on proteolysis and functionality of Mozzarella
cheese made using direct acidification. J Dairy Sci 86:114–126
Fox PF, Law J, McSweeney PLH, Wallace J (1993) Biochemistry of
cheese ripening. In: Fox PF (ed) Cheese: chemistry, physics and
microbiology, vol 1, 3rd edn. Chapman & Hall, London,
pp 379–438
Govindasamy-Lucey S, Jaeggi JJ, Bostley AL, Johnson ME, Lucey
JA (2004) Standardization of milk using cold ultrafiltration
retentates for the manufacture of Parmesan cheese. J Dairy Sci
87:2789–2799
Guinee TP, Fox PF (2004) Salt in cheese: physical, chemical and
biological aspects. In: Fox PF, Mc Sweeney PLH, Cogan TM,
Guinee TP (eds) Cheese: chemistry, physics and microbiology.
Elsevier Academic Press, London, pp 207–259
Guven M, Yerlikaya S, Hayaloglu AA (2006) Influence of salt
concentration on the characteristics of Beyaz cheese, a Turkish
white-brined cheese. Lait 86:73–81
Guven M, Cadun C, Karaca OB, Hayaloglu AA (2008) Influence of
rennet concentration on ripening characteristics of Halloumi
cheese. J Food Biochem 32:615–627
Hayaloglu AA (2007) Comparisons of different single strain starter
cultures for their effects on ripening and grading of Beyaz
cheese. Int J Food Sci Technol 42:930–938
Hayaloglu AA, Guven M, Fox PF (2002) Microbiological, biochem-
ical and technological properties of Turkish White cheese
‘Beyaz Peynir’. Int Dairy J 12:635–648
Hayaloglu AA, Guven M, Fox PF, McSweeney PLH (2005) Influence
of starters on chemical, biochemical, and sensory changes in
Turkish White-brined cheese during ripening. J Dairy Sci
88:3460–3474
Hayaloglu AA, Topcu A, Koca N (2011) Peynir analizleri [Cheese
analysis]. In: Hayaloglu AA, Ozer B (eds) Peynir Biliminin
Temelleri [Principles of Cheese Science]. Sidas, Izmir,
pp 489–562
Hayaloglu AA, Karatekin B, Gurkan H (2014) Thermal stability of
chymosin or microbial coagulant in the manufacture of Malatya,
a Halloumi type cheese: proteolysis, microstructure and func-
tional properties. Int Dairy J 38:136–144
Hesari J, Ehsani MR, Khosroshahi A, McSweeney PLH (2006)
Contribution of rennet and starter to proteolysis in Iranian UF
White cheese. Le Lait 86:291–302
Hesari J, Ehsani MR, Mosavi MAE, McSweeney PLH (2007)
Proteolysis in ultra-filtered and conventional Iranian white
cheese during ripening. Int J Dairy Technol 60:211–220
Hurley MJ, O’Driscoll BM, Kelly AL, McSweeney PLH (1999)
Novel assay for the determination of residual coagulant activity
in cheese. Int Dairy J 9:553–558
IDF (1982) Determination of the total solid content (cheese and
processed cheese). IDF Standard 4A, International Dairy Fed-
eration, Brussels, Belgium
J Food Sci Technol (February 2019) 56(2):589–598 597
123
IDF (1993) Determination of the nitrogen (Kjeldahl method) and
calculation of the crude protein content. IDF Standard 2B,
International Dairy Federation, Brussels, Belgium
Kandarakis I, Moschopoulou E, Anifantakis E (1999) Use of
fermentation produced chymosin from E. coli in the manufacture
of Feta cheese. Milchwissenschaft 54:24–26
Kappeler SR, van den Brink HM, Rahbek-Neilsen H, Farah Z, Puhan
Z, Hansen EB, Johansen E (2006) Characterization of recom-
binant camel chymosin reveals superior properties for the
coagulation of bovine and camel milk. Biochem Biophys Res
Commun 342:647–654
Karami M, Ehsani MR, Mousavi SM, Rezaei K, Safari M (2009)
Changes in the rheological properties of Iranian UF-Feta cheese
during ripening. Food Chem 112:539–544
Kubis I, Sousa MJ, Walsh-O’Grady D, Kelly AL, McSweeney PLH
(2001) Proteolysis in Cheddar-type cheese made from goat’s
milk. Milchwissenschaft 56:557–560
Maniou D, Tsala A, Moschopoulou E, Giannoglou M, Taoukis P,
Moatsou G (2013) Effect of high-pressure-treated starter on
ripening of Feta cheese. Dairy Sci Technol 93:11–20
McSweeney PLH, Fox PF (1993) Methods of chemical analysis. In:
Fox PF (ed) Cheese, chemistry, physics and microbiology.
Chapman & Hall, New York, pp 389–438
Moynihan AC, Govindasamy-Lucey S, Jaeggi JJ, Johnson ME, Lucey
JA, McSweeney PLH (2014) Effect of camel chymosin on the
texture, functionality, and sensory properties of low-moisture,
part-skim Mozzarella cheese. J Dairy Sci 97:85–96
Sheehan JJ, O’Sullivan K, Guinee TP (2004) Effect of coagulant type
and storage temperature on the functionality of reduced-fat
Mozzarella cheese. Lait 84:551–566
Soltani M, Boran OS, Hayaloglu AA (2016) Effect of various blends
of camel chymosin and microbial rennet (Rhizomucor miehei)on
microstructure and rheological properties of Iranian UF White
cheese. LWT-Food Sci Technol 68:724–728
Sousa MJ, Ardo Y, McSweeney PLH (2001) Advances in the study of
proteolysis during cheese ripening. Int Dairy J 11:327–345
Yasar K, Guzeler N (2011) Effects of coagulant type on the
physicochemical and organoleptic properties of Kashar cheese.
Int J Dairy Technol 64:372–379
598 J Food Sci Technol (February 2019) 56(2):589–598
123
