International Dairy Journal 133 (2022) 105408

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International Dairy Journal

journal homepage: www.elsevier.com/locate/idairyj

Influence of chymosin type and brine concentration on chemical

composition, texture, microstructural and colour properties of Turkish
white cheeses
Dilek Türkmen, Zehra Güler*
€kmen Campus, 31034, Hatay, Turkey
University of Hatay Mustafa Kemal, Faculty of Agriculture, Department of Food Engineering, Tayfur So

a r t i c l e i n f o a b s t r a c t

Article history: The effects of chymosin type and brine concentration on chemical composition, texture, microstructure
Received 3 December 2021 and colour of white cheese were studied during ripening. The cheeses were manufactured from cows’
Received in revised form milk using microbial rennet (Rhizomucor miehei), commercial calf rennet or recombinant chymosin
25 April 2022
(Kluyveromyces lactis), and ripened in whey brines containing 10% or 12% NaCl. Increasing the brine
Accepted 30 April 2022
concentration increased the ratio of salt to moisture, hardness, gumminess, chewiness and whiteness
Available online 11 May 2022
values of cheeses and decreased the moisture, the ratio of moisture to protein and extent of proteolysis.
The similar trends were observed in cheeses with microbial rennet compared with those with calf rennet
or recombinant chymosin. pH values of all the cheeses unchanged during ripening but their brine pHs
increased. A marked effect of rennet type on microstructure of the cheeses was visualised. In microbial
rennet cheese brine salt content can be reducible to obtain satisfactory results in terms of quality
parameters.
© 2022 Elsevier Ltd. All rights reserved.

1. Introduction pepsin proteolytic enzymes. The main roles of rennet are to

be responsible for curd formation and improving proteolysis
White brined cheese (WBC) is the most popular and economi- (Akkerman et al., 2017; Bansal, Fox, & McSweeney, 2009). However,
cally the most important variety of cheese in Turkey. On an in- increased demand for cheese, a lack of calf rennet availability on the
dustrial scale, WBC is produced from pasteurised milk using a world market and its high price and also religious beliefs have
starter culture (Güler & Uraz, 2004; Hayaloglu, Guven, Fox, & encouraged the search for alternative milk coagulants. The most
McSweeney, 2005). But, in most rural areas of Turkey no starter important chymosins that meet the requirements of cheese making
culture is used for white cheese-making and the curd blocks are are microbial and recombinant chymosins as well as calf rennet
scalded into its own whey (Güler, Türkmen, & Dursun, 2021). The (Andre n, 2002). As microbial coagulants are easy to produce by
cheese is ripened in brine prepared from whey, as in feta cheese fermentation, they are easily available and have a low price (Jacob,
(McMahon, Motawee, & McManus, 2009). WBC produced both Jaros, & Rohm, 2011). The most important disadvantage of the first
industrially and traditionally is characterised by its acidity and high generation of microbial-derived coagulants has been considerably
salt content (Güler & Uraz, 2004). According to the Turkish Regu- more thermo-stable than chymosin or pepsin (Smith, Billings, &
lation (Turkish Food Codex, 2015), to improve the sensory charac- Yada, 1991), but the third generation products have thermal sta-
teristics of white-brined cheese, it should be ripened for at least 3 bility similar to that of chymosin. Even though Rhizomucor miehei
months before being consumed. protease hydrolysed casein in a manner similar to that of chymosin,
In WBC manufacturing, the coagulation of milk is traditionally is showed relatively non-specific activity on caseins (Jacob et al.,
made by calf rennet obtained from the abomasum of ruminants 2011). Cheese making experiments at laboratory scale resulted in
(Hayaloglu, Guven, & Fox, 2002). Rennet, an aspartic protease, is significantly lower cheese yield and increased protein content in
mainly composed of chymosin (rennin) and, to a lesser proportion, whey, but a high dry matter and crude protein content when
R. miehei protease were used in comparison with calf rennet (Jacob
et al., 2011). On the other hand, recombinant rennet containing
* Corresponding author. 100% calf chymosin produced by recombinant DNA technology
E-mail address: [email protected] (Z. Güler).

https://doi.org/10.1016/j.idairyj.2022.105408
0958-6946/© 2022 Elsevier Ltd. All rights reserved.
D. Türkmen and Z. Güler International Dairy Journal 133 (2022) 105408

involving Aspergillus niger, Kluyveromyces lactis or Escherichia coli is rennet (M) at a level of 8.89 mL 100 L
1 milk, calf rennet (C1) and
mostly used today, as it is very pure and have high milk-clotting recombinant chymosin (C2) at a level of 11.11 mL 100 L
1 cheese
activity (Andren, 2002). milk, respectively. A coagulation period of 90 min was allowed for
Salt in cheese reduces water activity and affects enzymatic ac- each batch. Pre-brining process was performed at 22  C for 3e4 h
tivity, microbial growth, protein hydration, flavour and texture in brine from whey containing 14% (w/v) NaCl with 0.8% (w/v)
(Guinee, 2004), but nowadays there is an increasing focus on the CaCl2 or 12% (w/v) NaCl with 0.8% (w/v) CaCl2, followed by
development of cheese with reduced salt content since a high salt 14e16 h at 18  C. Finally, the cheese blocks were taken to ripening
in processed foods is directly relationship with various diseases, brine consisting of 12% (w/v) NaCl with 0.8% (w/v) CaCl2 or 10%
especially hypertension (Appel et al., 2011). Due to the different (w/v) NaCl with 0.8% (w/v) CaCl2 (Fig. 1). The first group cheeses
brine salt concentrations used for Turkish WBC manufacturing in were coded as 12M, 12C1 and 12C2, the latter as 10M, 10C1 and
different dairies, salt-in-dry matter of cheese are varied from 5.74% 10C2. The cheese samples were analysed on 2, 30, 60 and 90 days
to 12.88% (Güler & Uraz, 2004). Increasing the salt concentration of of ripening.
brine is given lower final cheese moisture (Guven, Yerlikaya, &
Hayaloglu, 2006). Salt concentration of 14e16% is usually used in 2.3. Chemical analysis and pH
both pre-brine and storage-brine of Turkish WBC (Hayaloglu et al.,
2002). In WBC production, as in Teleme cheese, pre-brine is applied Moisture, total nitrogen (TN), salt, water-soluble nitrogen (WSN)
at relatively high temperature and salt concentration compared and “amino acid Leu” were analysed according to the methods
with storage-brine. This practice increases salt uptake by cheese described in FIL-IDF (1982), AOAC (1990), FIL-IDF (1988), Bütikofer,
and also interrupts some of the biochemical activities of cheese, Riiegg, and Ardo€ (1993) and Tekin and Güler (2019), respectively.
especially the production of lactic acid (Hayaloglu et al., 2002; Ripening extension index (REI) is calculated as WSN percentage of
Mallatou, Pappa, & Massouras, 2003). However, brine salt concen- TN (McSweeney & Fox, 1997). S/M indicates salt in moisture. pH
tration has recently been reduced to 10% or 12% due to the negative was measured in slurry prepared from 10 g of cheese homogenised
effects of salt on health and legal regulation (Güler et al., 2021; with 10 mL deionised water and in cheese brines using a digital pH-
€
Sahingil, Hayaloglu, Simsek, & Ozer, 2014). In addition, increasing meter (Orion, Thermo, Austin, TX, USA).
the salt concentration in the brine from 12% to 18% reduced the
overall acceptability of the cheese (Guven et al., 2006). In this study 2.4. Texture profile analysis
we used pre-brine and storage-brine containing 14e12% NaCl and
12e10% NaCl, respectively. The textural parameters consisting of hardness (at first
In previous studies, the performance and proteolytic ability of compression, N), springiness (mm), adhesiveness (N mm), cohe-
microbial rennet, recombinant chymosin or commercial calf rennet siveness, gumminess (N) and chewiness (N mm) were measured
were compared (Çepog lu & Güler-Akın, 2013) and also the effect of using a Volscan profiler 32 Software in TA-XT Plus Texture Analyser
rennet and the salt concentration in brine on physico-chemical (Stable Micro Systems, Surrey, UK) fitted with a 5 kg load cell, ac-
properties of feta cheese (Prasad & Alvarez, 1999) and effect of cording to procedure described by Güler et al. (2021). Two
brine concentration on chemical composition of white cheese consecutive bites were taken from each sample at each sampling
(Guven et al., 2006). But, a combined effect of rennet type and brine time. All the measurements were replicated 6 times.
salt concentration on chemical, physical and microstructural
properties of WBC were not investigated. Therefore, we aimed to 2.5. Microstructure
determine the effect of rennet type (microbial, calf or recombinant)
and brine salt concentration (10% or 12% NaCl) on chemical, Microstructure of cheese samples were analysed using a scan-
microstructural, textural and colour parameters of White cheese ning electron microscope (SEM) according to the method of Soltani,
during ripening. Boran, and Hayaloglu (2016). The samples were cut into
1  5  10 mm dimensions from the centre of each block with a
2. Materials and methods sharp razor and then dried in a lyophiliser (Freeze Dryer, Teknosem,
Turkey). The cheese samples were covered with gold in a Sputter
2.1. Materials Coater (Polaron Thermo VG Scientific SC 7620) and then examined
in a SEM (JSM-5500LV, JEOL, Tokyo, Japan) at the Science and
Raw cows’ milk (protein, 3.2 g 100 g
1; fat, 4.4 g 100 g
1; lactose, Research Centre of Hatay Mustafa Kemal University. The micro-
4.25 g 100 g
1; non-fat solid, 8.28 g 100 g
1) used for cheese pro- graphs were represented at a magnification of 2500  .
duction was supplied by a farmer in Yayladag region in Hatay
province of Turkey. Three commercially available chymosins 2.6. Colorimetric analysis
(Mayasan Inc., Istanbul, Turkey) were used: microbial rennet
(1:20,000 strength) from R. miehei (M), animal rennet (1:16,000 The colour parameters L* (lightness), a* (þ/
: red/green) and b*
strength) from calf (C1), and recombinant chymosin (1:16,000 (þ/
: blue/yellow) were measured at a Hunter colorimeter (Col-
strength) from K. lactis (C2). Coarse salt (Oz Onur, Hatay, Turkey) orFlex-EZ, HunterLab, Virginia, USA) equipped with illuminant D65
and CaCl2 (Amik Dairy Facilities, Hatay, Turkey) were used for and observation angle 10 . The measurements were made from the
preparing brine. In the packaging of the cheeses, 1 kg poly- inner surface of cheese moulds after calibrating with black and
propylene plastic packages were supplied by ENKA milk (Konya, white ceramic calibration plates. The whiteness index (WI) was
Turkey). calculated according to Güler et al. (2021).

2.2. Cheesemaking 2.7. Statistical analysis

Cheese production was carried out according to Güler et al. Data on physicochemical properties and textural parameters
(2021) with some modifications (Fig. 1). The first batch, the sec- from the three replications were analysed by ANOVA as a factorial
ond batch and the third batch were coagulated with microbial experiment arranged in a completely randomised block design

2
D. Türkmen and Z. Güler International Dairy Journal 133 (2022) 105408

Fig. 1. Flow diagram for cheese production.

[6 cheeses (3 rennets  2 brine concentration)  4 ripening according to the General Linear Model (GLM) procedure. A proba-
times  3 trials] using SPSS statistical program (Version 24, IBM, bility of <0.05 was used to establish statistical significance for fixed
USA). Means were compared using Duncan's multiple range test effects and interactions.

3
D. Türkmen and Z. Güler International Dairy Journal 133 (2022) 105408

3. Results and discussion brine or 10% brine, M-cheeses had moisture in lowest amounts
during ripening (Fig. 3a and b). This could be explained by
3.1. Changes in pH increased proteineprotein interaction or the more crosslinking to
occur between protein strands within protein network allowing
All the experimental gels and cheese curds had pH values of moisture loss, as a function of chymosin type (García-Go  mez,
5.96 ± 0.02 and 5.10 ± 0.02 at the moment of cutting and before Vazquez-Ode riz, Mun~ oz-Ferreiro, Romero-Rodríguez, & V azquez,
brining, respectively. It appears that there is a greater drop in pH 2019; Gumus & Hayaloglu, 2019; Jacob et al., 2011; McMahon
after cutting, due to probably variable numbers of lactic acid et al., 2009). In agreement with literature (Cuffia, Candioti, &
bacteria coming from milk. In agreement with the findings of Bergamini, 2015; Guven et al., 2006; Madadlou, Khosroshahi, &
Khosrowshahi, Madadlou, Ebrahim zadeh Mousavi, and Emam- Mousavi, 2007; McMahon et al., 2009), increased brine salt con-
Djomeh (2006), McMahon et al. (2009) and Miloradovic, centration resulted in a significant decrease in moisture (Fig. 3a
Miocinovic, Kljajevic, Tomasevic, and Pudja (2018), the pH and b), due to a dynamic mutual diffusion process between NaCl
values of the cheeses, ranging from 4.92 to 5.07, remained stable in brine and cheese moisture since the quantity of water lost is
during ripening, and a limited increase in brine salt concentration about twice the quantity of salt gained. However, despite the in-
did not have an effect on pH of the cheeses. This could be attrib- crease in salt uptake by cheeses from day 2 to day 30 (Fig. 3c and
uted to compounds contributing to the pH buffering capacity of d), the moisture increased in all samples except for cheese-12M, in
cheese such as proteins, peptides, weak acids, bases, and their which increased at the end of ripening. A similar trend has been
complexes with metal cations (Salaün, Mietton, & Gaucheron, observed in other studies (Guinee, 2004; McMahon et al., 2009;
2005). In addition, Upreti, Bühlmann, and Metzger (2006) re- Prasad & Alvarez, 1999) where moisture uptake from brine during
ported a pH buffering capacity between 4.5 and 5.5 attributed to ripening is typical for brined-cheeses due to swelling of caseins at
the solubilisation of colloidal calcium phosphate and the pro- cold storage or formation of peptides with high water absorbing
tonation equilibrium involving the side chains of protein-bound capacity in low brine pH (approximately 4.6). But, moisture un-
glutamate in cheese. changed in cheeses 10C1, 12C1 and 10C2 after day 30, decreased in
From the factors applied, only rennet type had a significant ef- cheese-10M and increased in cheeses 12M and 12C2 at the end of
fect on pH (Table 1). As the brine was prepared from the own whey ripening (Fig. 3a and b). This could be attributed to re-
of each cheese curd, both M-cheese and its brine had a low pH arrangements in cheese microstructure in relation to the perme-
(Fig. 2a and b). This could be attributed to the release of peptides ability with stretching and breaking the strands that allow diffu-
having free C-terminal ends from caseins during cheese production sion of moisture from the brine (Fig. 6).
or ripening (Risborg & Witt, 2013), because, unlike chymosin, it has Regarding salt, an increase in brine salt concentration from
been shown that the specificity of microbial rennet from R. miehei 10% to 12% increased the salt absorbed by cheeses from 44% to
on caseins is different (Jacob et al., 2011). In addition, the small 55% at the first 30 days of ripening, respectively. In this time,
peptides in acidic character or amino acids such as aspartate and the salt uptake by cheese was almost completed. After day 30
glutamate that are almost as acidic as lactic acid migrates more no significant changes were observed in salt contents of
easily into brine (Michaelidou, Alichanidis, Polychroniadou, & cheeses, except for cheese-10C1 (Fig. 3c and d), in consistent
Zerfiridis, 2005; Salaün et al., 2005; Soodam, Ong, Powell, with finding of Kırmacı, Hayalog €
lu, Ozer, and Türkog lu (2014)
Kentish, & Gras, 2015; Upreti et al., 2006). With increase in mois- for brined Urfa cheese. The NaCl uptake rate was higher
ture content of the cheeses on day 30 of ripening (Fig. 3a and b) or (P < 0.05) in cheese-C1, probably related to the protein struc-
diffusion of brine into the cheese body, the difference in pH of M- ture and the relatively high initial moisture content (Guinee,
cheese from other cheeses was revealed (Fig. 2a and b). 2004). The higher S/M contents (P < 0.01) in cheeses with
The pH values of brines showed a tendency increasing during 12% brine than 10% ones could be explained by lower moisture
the ripening of all the cheeses (Fig. 2a and b), due to probably in- (Fig. 3e and f).
crease of less acidic water-soluble nitrogen compounds or the
growth of yeasts in brine, although no microbial analysis has been 3.2. Total nitrogen, water soluble nitrogen and amino acid Leu
performed, since yeasts utilize the lactate and produces NH3 from
deamination of amino acids, causing the pH to increase (Seiler & The total nitrogen (TN) contents and ratio of moisture to protein
Busse, 1990). The present pH values of cheeses were within the (M/P) of cheeses were significantly influenced by rennet type, brine
ranges (3.65e5.52) reported in literature for White cheeses (Güler salt concentration and ripening (Table 1). TN showed a tendency
& Uraz, 2004; Salum, Go €vce, Kendirci, Baş, & Erbay, 2018). decreasing towards the end of ripening (Fig. 4a and b), which can be
Moisture contents of the cheeses were in accordance with explained by the hydrolysis of proteins to water-soluble nitrogen
previously reported results for brined-type cheeses (Güler et al., (WSN) compounds and their partial diffusion into brine (Abd El-
2021; McMahon et al., 2009). The applied factors significantly Salam, Alichanidis, & Zerfiridis, 1993). As shown in Fig. 4aed,
affected the moisture (Table 1). Among cheeses with either 12% M-cheeses with low moisture had the highest TN and the lowest

Table 1
Effects of ripening time, rennet and different brine concentrations on the composition of the cheeses.a

Source d.f. pH Moisture Salt S/M TN REI M/P Leu

Ripening 3 NS *** *** *** *** *** *** ***

Rennet 2 ** *** *** *** *** *** *** ***
Salt 1 NS *** *** *** *** *** *** ***
Ripening  Rennet 6 NS *** *** *** NS *** *** ***
Ripening  Salt 3 NS *** NS *** NS *** *** ***
Rennet  Salt 2 NS ** NS NS NS *** *** NS
Ripening  Rennet  Salt 6 NS ** NS ** NS *** *** *
a
Abbreviations are: d.f., degree of freedom; S/M, salt-in-moisture ¼ salt/(salt þ moisture)  100; TN, total nitrogen (%); REI, ripening extension index (%); M/P, ratio of
moisture to total protein; Leu, leucine amino acid (mg 100 g
1 dry matter). Asterisks indicate level of significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant,
P > 0.05.

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D. Türkmen and Z. Güler International Dairy Journal 133 (2022) 105408

Fig. 2. pHs of the cheeses and 10e12% brines (aeb) during ripening. 10Me12M, 10C1e12C1 and 10C2e12C2 indicate cheeses with microbial rennet, calf rennet and recombinant
chymosin in 10% and 12% brines, respectively. Error bars represent standard deviation (n ¼ 3). a,b,c indicate significant differences of brine pH according to the ripening time of
cheeses; X,Y indicate significant differences of brine pH according rennet type (P < 0.05).

Fig. 3. Moisture (a,b), salt (c,d) and S/M (salt-in-moisture) ¼ salt/(salt þ moisture)  100 (e,f) contents of the cheeses during ripening. 10Me12M, 10C1e12C1 and 10C2e12C2
indicate cheeses with microbial rennet, calf rennet and recombinant chymosin in 10% and 12% brines, respectively. Error bars represent standard deviation (n ¼ 6). a,b,c indicate
significant differences among cheeses according to the ripening time; x,y,z, X,Y,Z indicate significant differences among cheeses according to rennet type (P < 0.05).

M/P and REI, consistent with the finding of Jacob et al. (2011). As The most common way to assess the proteolysis in cheeses is
mentioned-above, at equally given renneting time more hydro- determined by ripening extension index (REI; McSweeney & Fox,
phobic interactions between caseins depending on the rennet type 1997). The type of rennet, brine concentration and ripening time,
may be resulted in probably more moisture loss from casein and also their interaction significantly affected REI values (Table 1).
network and an increase in TN of M-cheeses (Guinee, 2004; Jacob This could be attributed to the amount of residual rennet retained
et al., 2011). This was confirmed by the high hardness of M- in curd during cheese production depending on the type of rennet
cheeses (Fig. 5a). The TN contents of cheeses were similar to the in relation with pH at cutting and whey drainage. Because a low pH
findings of Ozer, Atasoy, and Akin (2002) and Ozer, Uraz, Beyzi- (<6.2) at cutting and whey drainage favours the retention in curd of
Yilmaz, and Atasoy (2004). chymosin but not of microbial rennet (Bansal et al., 2009). In
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D. Türkmen and Z. Güler International Dairy Journal 133 (2022) 105408

Fig. 4. Total nitrogen (TN) (a,b), ratio of moisture to protein (M/P; c,d), ripening extension index (REI) (e,f) and amino acid leucine (g,h) in the cheeses during ripening. REI ¼ (WSN/
TN)  100, Protein ¼ 6.38  TN, DM ¼ dry matter. 10Me12M, 10C1e12C1 and 10C2e12C2 indicate cheeses with microbial rennet, calf rennet and recombinant chymosin in 10% and
12% brines, respectively. Error bars represent standard deviation (n ¼ 3). a,b,c indicate significant differences among cheeses according to the ripening time; x,y,z, X,Y,Z indicate
significant differences among cheeses according to rennet type (P < 0.05).

addition, only ~10% of chymosin or 2e3% of microbial rennet added making technique and type of milk. Microbial rennet exhibited a
to cheese milk is retained in the curd (McSweeney & Fox, 1997). In high REI value in 12M-cheese only on day 2 of ripening compared
the present study, to standardise to equal clotting time the con- with the other cheeses. Decreased the zeta-potential with an in-
centration of microbial rennet added to milk was low. In this crease of 2% in brine salt concentration might have increased the
context, the more residual calf rennet or recombinant chymosin in interactions between the casein micelles and Mucor miehei prote-
curd in relating with the high moisture might have been caused the ase that had a broader specificity than that of chymosin and pepsin
rapid production of water-soluble peptides at the primary phase of (Bansal et al., 2009). After day 2, cheese-12M had the lowest REI
ripening, as a result of enhanced chymosin activity on aS1-casein values, possibly related to low residual enzyme content and
(Madadlou, Khosroshahi, Mousavi, & Farmani, 2007). This resulted moisture content. In addition, more compact interactions between
in high REI values in C1- and C2-cheeses compared with M-cheeses, proteins (Fig. 6) may cause the enzyme susceptible bonds to
contrary to the finding of García et al. (2012), who found no sig- become inaccessible, resulting in the lower degree of protein hy-
nificant differences. This may be related to the different cheese- drolysis, and higher TN contents of M-cheeses (Madadlou,

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D. Türkmen and Z. Güler International Dairy Journal 133 (2022) 105408

Khosroshahi, & Mousavi, 2005). Similarly, the REI values of C1-and 12% brine. With the exception of 10C1-cheese on day 60, 10C1-
C2-cheeses also decreased with increased brine salt concentration and 12C1-cheeses had the highest amount of Leu at all times of
or S/M value (Fig. 4e and f), which is not consistent with the finding ripening. Recombinant rennet was more markedly affected by an
of Guven et al. (2006) for white cheeses. It has been shown that increase of 2% in brine salt concentration compared with calf
non-starter lactic acid bacteria counts during ripening exhibit a less rennet, in terms of the production of Leu. As a result, both REI
increase in cheese with high S/M compared with its low S/M and Leu values can be useful indices of ripening in brined-
counterpart (Gobbetti et al., 1999). This, along with a reduction in cheeses.
water activity, might have contributed to the lower levels of pro-
teolysis in cheeses with 12% brine. 3.3. Texture profile analysis
It seems that the moisture content together with the enzyme
type has a more pronounced effect on proteolysis of brined cheese All the textural parameters were significantly influenced by
under the current experimental conditions. Hesari, Ehsani, rennet type (Table 2). M-cheeses with the lowest moisture had the
Khosroshahi, and McSweeney (2006) have reported that rennet is highest values for hardness, gumminess and chewiness values,
a principal ripening agent in both primary and secondary proteol- while C1- and C2-cheeses with high moisture had the lowest values
ysis of UF white cheese. Although recombinant chymosin or calf (Fig. 5aef). With increased brine salt concentration, resulting in
rennet was added to milk at the same concentration, 10C1-cheese increase in salt of cheeses, the three textural parameters increased.
had the highest REI values on the 60th and 90th days of matura- This may be due to increased proteineprotein interaction resulting
tion, but REI values did not differ between 12C1- and 12C2-cheeses from the ionic strength of Naþ and Cl
, and also a low M/P and
on corresponding days. This may indicate that pepsin in calf rennet proteolysis degree (Akkerman et al., 2017; Madadlou et al., 2007).
compared with its corresponding chymosin is influenced by in- At the first 30 days of ripening, the three textural parameters
crease in brine salt concentration or S/M since pepsin is more increased in the cheeses except for cheese-10C2 and reached their
proteolytic in cheese than chymosin (Risborg & Witt, 2013). All highest levels. It can be said that cheeses reached the highest levels
values obtained for REI were within ranges (from 3.84 on day 1 of due to the increase in salt or S/M content (Güler et al., 2021;
ripening to 18.23 on day 90) reported for Malatya cheese, produced Miloradovic et al., 2018). From 30th day onwards, the tendency to
by microbial or calf rennet and brined at %12 NaCl (Hayaloglu, decrease in hardness, gumminess and chewiness was probably due
Karatekin, & Gurkan, 2014). to increased WSN, weakened hydrophobic interactions of protein
It has also been reported that REI is not a useful criterion for matrix and internal redistribution of moisture and also the sol-
casein hydrolysis in brined-cheeses because of somewhat ubilisation of calcium phosphate (Koca, Balasubramaniam, &
migration of WSN compounds from cheese to brine (Michaelidou Harper, 2011). 10C2-Cheese with a low S/M content showed a
et al., 2005). In this sense, irrespective of the rennet type, the tendency decreasing in the three textural parameters from day 2 to
hydrophobic amino acid Leu increase continuously in brined- day 90.
cheeses such as white, teleme and feta during ripening and In the present study, increased TN and S/M and decreased
also is one of the principal free amino acids (Balabanova, Ivanova, moisture were correlated with increased hardness values of
& Vlaseva, 2017; Hayaloglu et al., 2005; Mallatou, Pappa, & cheeses, as in the finding of Chen, Larkin, Clark, and Irwin (1979). In
Boumba, 2004; Michaelidou et al., 2005). Based on these find- addition, cheeses with a similar TN, moisture, S/M content, as in
ings we quantified amino acid Leu in the present cheeses to see if 10C1- and 10C2- or 12C1- and 12C2-cheeses, had almost identical
it could be a useful index in cheese ripening (Fig. 4g and h). As in hardness values (Fig. 5a and b). From other textural parameters,
the aforementioned studies, Leu showed a continuous increase in adhesiveness and cohesiveness were not influenced by brine con-
all the cheeses during ripening. No significant effect of brine salt centration. But, both parameters were significantly influenced by
concentration on Leu was observed until the first 30 days of the interaction of rennet, brine salt concentration and ripening
ripening. However, at days 60 and 90 of ripening, the amount of time (Table 2). Adhesiveness values, varied from
0.06
Leu was significantly higher in cheeses with 10% brine compared to
0.23 N mm, decreased steadily only in C2-cheeses during
with those with 12% brine. Presumably, after established a salt ripening (Fig. 5g and h). In the findings of Zheng, Liu, and Mo
balance between the cheese and the brine, the high salt in the (2016), adhesiveness correlated negatively with protein content
cheese mass inhibited the activity of non-starter lactic acid and positively with S/M levels. Contrary to these findings, in the
bacteria since they contribute to free amino acid production at present study, it was observed that the adhesiveness may be related
the secondary phase of ripening (Hayaloglu et al., 2005; to the number of protein bonds and their strength rather than S/M
Madadlou et al., 2007). It may also be due to a decrease in the and TN contents (Fig. 6). C1- and C2-cheeses with relatively high
supply of peptides which the bacteria act on. Regardless of brine REI and moisture had a high cohesiveness for 10% brine at day 60 of
salt concentration and ripening time, calf rennet cheese (C1) had ripening (Fig. 5i and j), which is in agreement with Pastorino,
the highest amount of Leu, followed by recombinant rennet (C2) Hansen, and McMahon (2003). No significant changes in springi-
and microbial rennet (M) cheeses, as in REI values. However, Leu ness values (0.89e0.93 mm) of the cheeses during ripening were
showed a different trend from REI between cheeses with 10% or observed (Fig. 5k and l). As a consequence, increasing salt content
Table 2
Effects of ripening time, rennet and different brine concentrations on the textural parameters of the cheeses.a

Source d.f. Hardness (N) Gumminess (N) Chewiness (N mm) Springiness (mm) Adhesiveness (N mm) Cohesiveness

Ripening 3 *** *** *** NS *** ***

Rennet 2 *** *** *** *** *** ***
Salt 1 *** *** *** NS NS NS
Ripening  Rennet 6 *** *** *** NS *** NS
Ripening  Salt 3 *** *** *** * NS NS
Rennet  Salt 2 NS NS * NS NS NS
Ripening  Rennet  Salt 6 *** ** *** NS ** *
a
Abbreviation: d.f., degree of freedom. Asterisks indicate level of significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant, P > 0.05.

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D. Türkmen and Z. Güler International Dairy Journal 133 (2022) 105408

Fig. 5. Hardness (a,b), gumminess (c,d), chewiness (e,f), adhesiveness (g,h), cohesiveness (i,j) and springiness (k,l) textural parameters of the cheeses during ripening. 10Me12M,
10C1e12C1 and 10C2e12C2 indicate cheeses with microbial rennet, calf rennet and recombinant chymosin in 10% and 12% brines, respectively. Error bars represent standard
deviation (n ¼ 3). a,b,c indicate significant differences among cheeses according to the ripening time; x,y,z, X,Y,Z indicate significant differences among cheeses according to rennet type
(P < 0.05).

and use of microbial rennet compared with the other rennet types in cheeses with microbial rennet or recombinant chymosin. The
resulted in the cheese matrix being more hard and chewy. first appeared to have an even denser protein network, also inter-
spersed with spaces of varying sizes. No cross-linked was observed
3.4. Microstructure in cheese with calf chymosin (C1), even displaying a smooth and
thin appearance with a few small spaces.
Microstructure images of the cheeses are shown in Fig. 6. At the From 2nd day onwards, the re-arrangements in the micro-
beginning of ripening casein micelles formed a continuous network structures of all the cheeses and increases in the number and size of
8
D. Türkmen and Z. Güler International Dairy Journal 133 (2022) 105408

voids were observed. Protein strands became visible in cheese with structure was still visible. The elongated protein strands together
calf chymosin (C1). Cheeses with 12% brine compared with those with spaces were evident in cheese-10C2, in comparison with
with 10% brine appeared denser, this can be due to the lower M/P 10C1.This resulted in a relatively lower hardness in the cheese. In
values and the high chloride anion, a kosmotropic ion, promoting cheese-12C2 the micelles were compressed into a uniform protein
hydrophobic interactions and resulting in a more homogenous matrix and considerable curd knitting occurred. The microstruc-
matrix (Madadlou et al., 2007). At day 60, a relatively thin tures of cheeses 12C1and 12C2 were similar to findings of Soltani
appearance with the opens in protein strands of all the cheeses et al. (2016).
except for 12M displayed. At the end of ripening, a more layered
and compact appearance together with breakages in protein 3.5. Colour parameters
strands and occasionally irregular deep large voids was observed in
12M-cheese, probably resulting in the higher hardness values. The Colour indices may served to categorising the cheese on the
globular spaces were evident in cheese-10M exhibiting an scale of white (WI) better than only L* (Ramo ~ rez-Navas & De
appearance like honeycomb, as reported by Hayaloglu et al. (2014). Stouvenel, 2012). The results of the whiteness index (WI) are pre-
A thinner protein strand was observed in cheeses C1 and C2, in sented in Fig. 7. At the beginning of ripening, the high moisture in
which micelles were fused into chains, but the micelle-based cheeses with 10% brine resulted in higher WI value, as reported by

Fig. 6. Microstructures of the 2, 30, 60 and 90-day old cheeses. 10Me12M, 10C1e12C1 and 10C2e12C2 indicate cheeses with microbial rennet, calf rennet or recombinant chymosin
in 10% and 12% brines, respectively.

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D. Türkmen and Z. Güler International Dairy Journal 133 (2022) 105408

Fig. 7. Whiteness index (WI) values of the cheeses during ripening. 10Me12M, 10C1e12C1 and 10C2e12C2 indicate cheeses with microbial rennet, calf rennet or recombinant
chymosin in 10% and 12% brines, respectively. Error bars represent standard deviation (n ¼ 3). a,b,c indicate significant differences among cheeses according to the ripening time; x,y,z,
X,Y,Z
indicate significant differences among cheeses according to rennet type (P < 0.05).

Kaya (2002). On day 30, WI values increased only in cheeses in 12% Acknowledgements
brine, due to possibly even salt distribution in the cheese structure.
From day 60 to day 90, WI values decreased in the cheeses, in This work was supported by the Scientific Research Projects
especially cheeses with 10% brine, due to probably increased pro- (BAP) Commission of Hatay Mustafa Kemal University, Turkey
tein hydration resulting in a decrease in the number of free mois- (Project No: 18.YL.055).
ture droplets which reflects a reduced degree of light scattering
(Sheehan et al., 2005). At the end of ripening, 10C1- and 10C2-
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