Accepted Manuscript

High heat treatment of goat cheese milk. The effect on yield, composition,
proteolysis, texture and sensory quality of cheese during ripening

Zorana Miloradovic, Nemanja Kljajevic, Jelena Miocinovic, Nikola Tomic,

Jasmina Smiljanic, Ognjen Macej

PII: S0958-6946(16)30359-4
DOI: 10.1016/j.idairyj.2016.12.004
Reference: INDA 4121

To appear in: International Dairy Journal

Received Date: 14 June 2016

Revised Date: 9 December 2016
Accepted Date: 9 December 2016

Please cite this article as: Miloradovic, Z., Kljajevic, N., Miocinovic, J., Tomic, N., Smiljanic, J., Macej,
O., High heat treatment of goat cheese milk. The effect on yield, composition, proteolysis, texture
and sensory quality of cheese during ripening, International Dairy Journal (2017), doi: 10.1016/
j.idairyj.2016.12.004.

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 ACCEPTED MANUSCRIPT

1 High heat treatment of goat cheese milk. The effect on yield, composition, proteolysis,
2 texture and sensory quality of cheese during ripening
3
4
5
6
7
8 Zorana Miloradovic a *, Nemanja Kljajevica, Jelena Miocinovica, Nikola Tomicb, Jasmina
9 Smiljanic a, Ognjen Macej a
10
11
12
13
14
15 aUniversity of Belgrade, Faculty of Agriculture, Animal Source Food Technology Department,
16 Nemanjina 6, 11081 Belgrade, Serbia
17 bUniversity of Belgrade, Faculty of Agriculture, Department of Food Safety and Quality
18 Management, Nemanjina 6, 11081 Belgrade, Serbia
Corresponding author. Tel.: +381 11 4413368
19 E-mail address: zorana@agrif.bg.ac.rs (Z. Miloradovic)

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20
21 ABSTRACT
22
23 White brined goat cheeses were produced from milk heated to 80 °C/5 min and 90 °C/5 min
24 using an identical cheese making procedure, and they were compared with the control cheese
25 made from milk subjected to common pasteurisation (65 °C/30 min). High heat treatment of goat
26 cheese milk resulted in significant increase of cheese yield. It also significantly affected cheese
27 composition by incorporating higher levels of moisture, fat and whey proteins. The pH values of
28 experimental cheeses decreased during ripening. Degradation of αS-casein was inhibited due to
29 the high heat treatment of milk, while β-casein degradation and secondary proteolysis were not
30 significantly affected. The effect on textural properties was significant, but the same for both
31 experimental cheeses. Contrary to expectations, the cheese obtained from milk heated to 90 °C/5
32 min received the best scores for sensory characteristics (flavour and texture) at all ripening
33 stages.
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35 1. Introduction
36
37 In Serbia, the most widely produced and consumed cheeses are white brined cheeses.
38 They come mainly from cows’ milk, while cheeses made from goats’ milk are produced in
39 relatively small quantities (Radulović et al., 2011). According to the official statistics, 1.55
40 billion litres of milk were produced in the country in 2014, but only 2.45% of it was goats’ milk.
41 However, a continuous increase in goats’ milk production has been registered ever since 2010,
42 when goats’ milk was only 1.77% of the yearly total (Anonymous, 2016). In recent years, the
43 dairy market has been showing great interest in goats’ milk products (Miloradovic, Kljajevic,
44 Jovanovic, Vucic, & Macej, 2015).
45 The supply of goats’ milk is seasonal and it is necessary to enhance both yield and quality
46 of goat cheeses if they are to become competitive with cows’ milk cheeses (Park & Guo, 2006).
47 The chemical composition of goats’ milk is highly seasonal, with very low levels of both fat and
48 protein contents during the mid-lactation period (June, July and August) (Guo, Park, Dixon,
49 Gilmore, & Kindstedt, 2004; Mayer & Fiechter, 2012), both of which directly influence cheese
50 yield (Jaeggi, Wendorff, Romero, Berger, & Johnson, 2005). As a result, cheese makers suffer an
51 unacceptable drop in goat cheese production profitability during the mid-lactation period
52 (Fenelon & Guinee, 1999).
53 High heat treatment of cheese milk has been suggested as a method for enhancement of
54 cheese yield (Sandra & Dalgleish, 2007), however, the influence of high heat treatment on milk
55 components, especially proteins, is quite different with goats’ milk as compared with cows’ milk
56 (Miloradovic et al., 2015; Pesic et al., 2012). Low pasteurisation temperature of cheese milk (65
57 °C/30 min), is a common practice in industrial cheese production (Moatsou & Govaris, 2011),
58 while the use of high heat milk pasteurisation for rennet coagulated cheeses is avoided due to the
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59 negative effect it has on cows’ milk coagulation properties. However, renneting properties of
60 goats’ milk are much less impaired by higher heat treatment than in the case of cows’ milk, due
61 to the difference in casein micelle structure of these milk types, particularly the composition, size,
62 hydration and mineralization (Raynal & Remeuf, 1998).
63 Heating of cows’ milk to 75 °C/25 min, 80 °C/20 min and 85 °C/15 min significantly
64 inhibits rennet coagulation (Montilla, Balcones, Olano, & Calvo, 1995); heating to temperatures
65 higher than 90 °C completely inhibits coagulation (Raynal & Remeuf, 1998; Sandra & Dalgleish,
66 2007). As a result, numerous modifications of cows’ milk cheese making processes have been
67 done to overcome the negative effects of high heat treatment (Ghosh, Steffi, Hinrichs, & Kessler,
68 1999; Singh & Waungana, 2001).
69 This study compared white brined cheeses produced from goats’ milk heated with either
70 80 °C/5 min or 90 °C/5 min with control cheese made from commonly pasteurised milk (65
71 °C/30min). One objective was to investigate the potential yield enhancement of cheeses produced
72 by high heat treatment compared with the standard heating method. Since milk heating may
73 interfere with cheese ripening (Faccia, Gambacorta, Caponio, Pati, & Di Luccia, 2007), the
74 second objective was to evaluate the effects of heat treatments on gross composition, protein
75 composition, proteolysis, texture properties and sensory quality of cheeses during ripening.
76 Prior to the present study, as the part of the same project, another paper was published, reviewing
77 whey from high heat treated goats’ milk (Miloradovic et al., 2016). It was found that high heat
78 treatments of milk (80 °C/5 min and 90 °C/5 min) had a significant influence on both basic
79 composition and protein composition of cheese whey. Therefore we find it reasonable to expect
80 that heat treatment would also have a significant effect on cheese composition and all the
81 mentioned properties.
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2. Material and methods
84
85 Cheese milk was taken from a commercial flock of 250 Saanen goats, during the summer
86 season (June-July), in seven day intervals. The gross composition of milk was as follows: dry
87 matter, 10.30±0.04%; protein content, 2.57±0.10%; fat content, 2.75±0.15%; pH value,
88 6.63±0.04.
89
90 2. 1. Cheese m anuf act ur e
91
92 Cheese making trials were carried out on three occasions at the Pilot Plant of the Faculty
93 of Agriculture, University of Belgrade, Serbia. For each trial, 60 L of milk was divided into three
94 20 L lots. The lots were subjected to different heat treatments: 65 °C/30 min (control cheese C),
95 80 °C/5 min (E1) and 90 °C/5 min (E2), and three cheese variants were made according to the
96 identical procedure. Starter inoculation was carried out at 31 °C with 0.005% (w/v) of mesophilic
97 starter culture - Lyofast MWO 030 (Clerici-Sacco Group, Cadorago, Italy), consisting of
98 Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris. The milk was allowed to ripen until
99 the pH value reached 6.5. Then, at 31 °C , 0.02% (w/v) CaCl2 and calf rennet Caglificio Clerici
100 (Clerici-Sacco Group, Cadorago, Italy) consisting of 94% chymosin and 6% pepsin were added.
101 The amount of added rennet was 0.2 g per 10 L of milk, which is approximately 50% less than
102 the recommended dose for cows’ milk cheese.
103 After 45 min of rennet coagulation, the curd was cut into cubes (5 cm sides), left to rest
104 for 15 min, and carefully transferred to rectangular moulds (30 × 40 cm) with cotton cloth, and
105 allowed to drain for 10 min. The curd was pressed for 1 h with 2 kg of weight per kg of curd, and
106 then, for another hour, with 4 kg kg-1 of curd, at room temperature. After pressing (2 h in total),

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107 curd was cut into blocks (15 × 7 × 2 cm). The curd blocks were dry salted with 3% (by the weight
108 of curd blocks) fine grain iodized NaCl (So Produkt, Stara Pazova, Serbia), placed into individual
109 plastic containers, and stored at 23–25 °C for 24 h. After 24 h the weight of cheese was
measured
110 and cheese yield was calculated as the weight of cheese divided by the weight of the milk used
111 (Madadlou, Mousavi, Khosrowshahi asl, Emam-Djome, & Zargaran, 2007). The plastic
112 containers were filled with 6% (w/w) NaCl brine, previously adjusted to pH 5.0 with lactic acid
113 and pasteurized at 80 °C/10 min and then cooled to room temperature. The ratio of brine volume
114 to cheese weight was 1:2. The containers were sealed and the cheeses were ripened at 13–15
°C
115 for 40 days.
116 Samples were taken from all cheeses at 1, 10, 20, 30 and 40 days of ripening to analyse
117 gross composition, textural properties and proteolysis indices. Sensory evaluations were carried
118 out at the early (3 days), mid (10 days) and late (40 days) stages of cheese ripening. Cheeses
were
119 again weighed and yield was calculated at mid and late stages of ripening (after 10 and 40 days).
120
121 2.2. Compositional analysis of cheeses
122
123 Prior to compositional analysis, cheese samples (half of each block) were ground to
124 achieve uniformity. Cheese samples were analysed according to the following methods: dry
125 matter by standard drying method at 102±2 °C (FIL-IDF, 1982); fat content according to the
126 method of Van-Gulik (FIL-IDF, 1986); nitrogen content by Kjeldahl method (AOAC, 1990);
127 protein content was calculated as nitrogen content multiplied by 6.38; NaCl content was
analysed
128 by the Volhard method (FIL-IDF, 1988). Based on these characteristics, the following parameters
129 were calculated: fat in dry matter (FDM), moisture in non-fat solids (MNFS), salt in moisture
130 (S/M) and protein-to-fat ratio (P/F). The pH was measured in cheese slurry using a digital pH-

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131 meter (Consort, Turnhout, Belgium) (Ardö & Polychroniadou, 1999). All analyses were carried
132 out in duplicate.
133
134 2.3. Proteolysis
135
136 The assessment of primary proteolysis was carried out by sodium dodecyl sulphate
137 polyacrylamide gel electrophoresis (SDS-PAGE). Resolving buffer and sample buffer were
138 prepared as described by (Miloradovic et al., 2015). Cheese samples (0.4 g) were mixed with
139 resolving buffer (5 mL) in polypropylene sealed tubes and placed in a boiling water bath until
140 they were completely resolved. After being cooled to room temperature, the resolved cheese
141 samples were mixed with sample buffer in a mass ratio of 1:3. A raw goats’ milk sample was
142 prepared by diluting raw goats’ milk with sample buffer in a mass ratio of 1:9.
143 SDS-PAGE was carried out according to the method of Laemmli, (1970) as described in
144 detail by Miloradovic et al. (2015). Raw goat s’milk sample and Roti-Mark 10-150 (Carl Roth,
145 Karlsruhe, Germany) protein standards were used to identify proteins detected on the gel. The
146 gels obtained from electrophoresis were scanned using a HP Scanjet G2710 (Hewlett Packard,
147 Palo Alto, CA, USA). Densitometric analysis of scanned gels was carried out by LabImage 1D
148 L340 software (Kapelan Bio-Imaging GmbH, Leipzig, Germany) and relative quantities of
149 individual proteins (αS- and β-CN) were calculated.
150 Secondary proteolysis was followed by determination of the non-protein nitrogen
151 fractions soluble in 12% of trichloroacetic acid (TCA-SN), which was fractionated from the
152 cheese water extract by the method of O'Keeffe, Fox, and Daly (1978). Results were expressed as
153 the percentage of 12% TCA soluble fraction of total nitrogen content (TCA-SN/TN).
154

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155 2.4. Cheese texture analysis
156
157 Prior to texture analysis, half of each cheese block (C, E1 and E2) was taken out of the
158 brine, placed into airtight plastic containers and put in a temperature chamber for 1 h to
159 equilibrate to 20 °C.
160 Textural properties of cheeses were measured by Texture Analyzer TA.XT Plus (Stable
161 Micro System, Surrey, UK), using a compression cell with a capacity of 5 kg. Two different tests
162 were used: a spherical probe test, that replicates squeezing cheese in hand, and fracture
wedges
163 test, that mimics the first bite from the action of molar teeth (Vincent, Jeronimidis, Khan, &
164 Luyten, 1991).
165 A spherical probe (P/1s, Stable Micro System, Surrey, UK) was used for measurement of
166 surface textural cheese properties. Cheese samples (3 × 2 × 2 cm) were compressed by 25% of
167 the initial sample height and the speed of the probe was 2 mm s-1, with a trigger force of 2.5 kg.
168 The compressive force measured at the maximum compression force (Fmax, N) was recorded as
169 an indicator of ‘Surface hardness’.
170 Fracture wedges (A/WEG) were used to determine the cheese fracture properties. Cheese
171 samples (3 × 2 × 2 cm) were placed between opposing wedges (cutting angle of 30° and 30 mm
172 width). The depth of probe penetration was 10 mm, using a speed of 2 mm s -1 with a trigger force
173 of 5 kg. The maximum measured force (Fmax, N) was expressed as a ‘Fracture hardness’. Six
174 measurements were carried out for each probe and each cheese.
175 Selection of testing parameters and data analysis were carried out by Exponent Software
176 (Stable Micro System, Surrey, UK).
177
178 2.5. Sensory evaluation

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179
180 Sensory evaluation of cheeses was carried out according to the method of Koca and Metin
181 (2004), with minor modifications. It was carried out by a sensory panel of eight members of the
182 Animal Source Food Technology Department, University of Belgrade, Faculty of Agriculture,
183 and all were experienced in dairy product quality judging. Panel consisted of 3 women and 5 men
184 age 40 to 55. Over a period of two weeks, four 2-hour training sessions were organised using
185 various brands of commercial white brined goat cheeses. Sensory quality was assessed by
186 evaluating five selected characteristics: appearance, cross-section appearance, odour
(orthonasal
187 olfaction), flavour and oral texture. The selected sensory characteristics were rated using a
188 category scale with the scores ranging from minimum (0) to maximum (5), divided by 0.5 and
189 resulting in scale with 10 alternative responses.
190 Prior to sensory analysis, one block of each cheese (C, E1 and E2) was taken out of the
191 brine, placed into airtight plastic containers and put in a temperature chamber for 1 h to
192 equilibrate to 20 °C. Evaluations of cheeses were performed in sensory booths in the Sensory
193 testing laboratory at the University of Belgrade, Serbia. The samples were labelled with random
194 3-digit codes and presented to the panellists in random order. Low-sodium bottled water and
195 bread were used for palate cleansing. Panellists were requested to describe defects that caused
196 reduced scoring.
197
198 2.6. Statistical analysis
199
200 Cheese C, E1 and E2 were manufactured in triplicate. Two-way analysis of variance
201 (ANOVA) was used to determine whether the factors (heat treatment and ripening time) and their
202 interactions had a significant effect on the measured parameters. Differences between means

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203 were determined with the LSD – test, and were considered significant at P<0.05. Error bars on
204 the graphs indicate standard deviations. Statistical analysis was conducted using the Statistica
205 10.0 software (Stat Sof. Inc., Tulsa, USA).
206
207 3. Results and discussion
208
209 3.1. Cheese yield and gross composition
210
211 The yield of young cheese C (1 day after salting) was low (9.5±1.72%), as it was expected
212 due to low fat and protein contents of the milk. According to statistical analysis, there were
213 significant effects of heat treatment on cheese yield. The yields of cheeses from high heat treated
214 milks (1 day after salting) were significantly higher than the yield of control cheese: 12.8±1.25%
215 and 13.8±1.38% for E1 and E2, respectively. The yields of experimental cheeses were also
216 significantly different from one another (P<0.05). However, the effect of ripening time on cheese
217 yield was insignificant, meaning that no significant weight loss was observed after 10 and 40
218 days of ripening in brine. Therefore, heat treatment could be considered as an effective method
219 for yield enhancement of both young and ripened white brined goat cheese.
220 Previous research (Miloradovic, 2015; Miloradovic et al., 2015) showed that heating of
221 goats’ milk at 90 °C/5 min results in significant covalent bonding of milk proteins, while, in case
222 of heating at 80 °C/5 min, the majority of protein-protein interactions are non-covalent. From this
223 work we can assume that the yield enhancement found in this study was not only a consequence
224 of formation of covalently-bonded protein aggregates, but was also due to the occurrence of both
225 hydrophobic and electrostatic interactions between proteins.

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226 The gross composition of cheeses during ripening is given in Table 1. According to
227 statistical analysis, dry matter and total protein content of C cheese were significantly higher
than
228 of E1 and E2 cheeses on the first day of ripening. This may be explained by the inferior
syneresis
229 properties of cheese made from high heat treated milk and by the increased water binding
230 capacity of the casein-whey protein complex (Singh & Waungana, 2001). The lower protein
231 content of experimental cheeses is, therefore, due to concomitant decrease in dry matter content.
232 Similar findings were reported in studies dealing with cows’ milk cheeses (Hougaard, Ardö, &
233 Ipsen, 2010; Rynne, Beresford, Kelly, & Guinee, 2004), but fat content was not found to be
234 significantly lower in experimental cheeses. The more porous matrices of curds obtained from
235 high heat treated milk (Singh & Waungana, 2001), with decreased permeability (Raynal &
236 Remeuf, 1998), likely impede the outward flux of fat from the curd, and cause higher fat
237 retent ion.
238 The differences in dry matter and protein content, observed among C, E1 and E2, were
239 equalised during the ripening because of the dynamic equilibrium between cheese and brine. It is
240 known that during ripening of white brined cheeses a significant decrease in protein content can
241 occur as a result of diffusion of small peptides, generated by proteolysis and transferred from
242 cheese to brine (Prasad & Alvarez, 1999). Such a decrease, however, was not found in our study,
243 probably because of a limited secondary proteolysis which will be discussed later.
244 According to the results of fat in dry matter content (FDM) and moisture in non-fat solids
245 content (MNFS), and also, the regulative norms (CAC, 2013), all three cheeses C, E1 and E2
246 belonged to the group of soft and full fat cheeses.
247 The study found that from the 10th day of ripening, when equilibrium between cheese and
248 brine was established, cheeses C, E1 and E2 did not differ significantly in terms of FDM and
249 MNFS content. Protein-to-fat ratio (P/F) was found to be significantly lower for E2 cheese

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250 comparing with C and E1 until day 30. This suggests that the protein matrix of E2 was more
251 porous/less permeable than the matrices of E1cheese, and therefore able to keep more fat
droplets
252 inside the cheese. In the last stage of ripening (30th and 40th day), the P/F ratios of C, E1 and
E2
253 did not differ significantly.
254 Besides its preservation role, salt might have a great influence on the ripening process, in
255 terms of texture, flavour and overall quality of cheeses (Guinee, 2004). This study showed that
256 heat treatment of cheese milk did not cause significant effect on salt in moisture content of the
257 cheeses (S/M). Therefore, the influence of salt content on cheese characteristics during ripening
258 was eliminated. As expected, a rapid increase of S/M was found only during the first 10 days of
259 ripening.
260
261 3.2. Changes in pH
262
263 At the very beginning of ripening, all cheeses had a similar pH. Later on, the pH of E1
264 and E2 cheeses was lower than the pH of the control cheese. This demonstrates the significant
265 influence that the heat treatment of milk has on the pH (Fig. 1). The lower pH of experimental
266 cheeses was a consequence of their higher initial moisture level, which was related to the higher
267 initial lactose level, higher microbial activity, and thus, higher lactic acid level (Hougaard et
al.,
268 2010; Rynne et al., 2004).
269 After 20 days of ripening, the pH of cheeses E1 and E2 remained unchanged until the end
270 of ripening (Fig. 1). The pH value of control cheese C did not change significantly throughout the
271 whole ripening period.
272
273 3.3. Protein composition of cheeses
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274
275 It could be seen from the electrophoretogram shown on Fig. 2 that the high heat treatment
276 of goats’ milk affected the protein composition of cheeses. Cheeses E1 and E2 showed higher
277 amounts of both the major whey proteins [α-lactalbumin (α-la), f3-lactoglobulin (f3-lg)] and of the
278 whey proteins with high molecular mass [lactoferrin (LF), serum albumin (SA), immunoglobulin-
279 heavy chain (Ig-HC)] compared with cheese C. This result is in agreement with our previous
280 study (Miloradovic et al., 2016) that showed that these proteins were detected at lower levels in
281 whey samples obtained from cheeses produced from high heat treated goats’ milk.
282 Although at the pH of E1 and E2, whey proteins are soluble and could be extracted by
283 brine (Michaelidou, Alichanidis, Polychroniadou, & Zerfiridis, 2005), the electrophoretogram
284 (Fig. 2) shows that they remained in experimental cheeses until the end of ripening, which
means
285 that they were bond within the cheese matrix. As mentioned before, interactions between
proteins
286 in goats’ milk heated at 80 °C/5 min are mainly non-covalent. According to the electrophoresis
287 results, it appears that non-covalent bonds were sufficiently strong to incorporate large amount of
288 whey proteins into the cheese and keep them in the cheese matrix throughout ripening. The
289 incorporation of biologically-valuable whey proteins enhanced the nutritional value of
290 experimental cheese proteins, as compared with the control cheese proteins.
291
292 3.4. Pr imary proteolysis
293
294 Primary proteolysis during ripening of C, E1 and E2 cheeses was monitored by SDS-
295 PAGE electrophoresis (Fig. 2). This technique does not allow a complete resolution of caseins,
296 but it is considered the most effective way to evaluate proteolysis in dairy product in which whey
297 proteins are also present (Faccia et al., 2007). The extent of primary proteolysis was monitored

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298 by the relative amount of residual αS-casein (αS-CN) and f3-casein (f3-CN) obtained by
299 densitometric analysis of the gel.
300 Statistical analysis showed that the heat treatment of milk, the ripening time and the
301 interaction between these two factors have significant effect (P<0.05) on the amount of residual
302 αS-CN.
303 Fig. 3 showed that a more pronounced proteolysis of αS-CN was found during ripening of
304 cheese C compared with cheeses E1 and E2. Proteolysis of experimental cheeses was intensive
305 during the first 10 days, but was limited after 10 days. This is in agreement with previous
306 findings that during the ripening of white brined cheeses, the most intensive biochemical
changes
307 occur within the first two weeks (Barac et al., 2016; Moatsou & Govaris, 2011). In case of E1
308 and E2, the significant drop of pH value after ten days of ripening (Fig. 1) certainly contributed to
309 the inhibition of further degradation of αS-CN. Also it is likely that a matrix of E1 and E2, with
310 lower permeability, as compared with C, reduced the accessibility of chymosin to αS-CN.
311 Fig. 3 also shows the level of f3-CN degradation. Due to the significant incorporation of
312 whey proteins in E1 and E2, there was a noticeable difference in the relative amount of f3-CN in
313 C versus E1 and E2 during the entire ripening period. However, according to ANOVA results, no
314 statistically significant effect of ripening time or of heat treatment was found, meaning that the
315 extent of f3-CN degradation in all the cheeses was limited. This is explainable by the fact
that this
316 casein fraction undergoes proteolysis by plasmin activity, which is inhibited by low pH, one of
317 the generally recognised characteristics of white brined cheeses (Kelly & McSweeney, 2003;
318 Upadhyay, McSweeney, Magboul, & Fox, 2004).
319 It is known that in goat cheeses, proteolytic changes of αS-CN are more pronounced than
320 of f3-CN (Asteri, Kittaki, & Tsakalidou, 2010; Bontinis, Mallatou, Pappa, Massouras, &

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321 Alichanidis, 2012; Hayaloglu, Tolu, & Yasar, 2013). However, it is not yet clear why the high
322 heat treatment of goats’ milk inhibits αS-CN degradation during cheese ripening.
323
324 3.5. Secondary proteolysis
325
326 The amount of nitrogen compounds soluble in 12% trichloroacetic acid, expressed as a
327 percentage of total nitrogen compounds (TCA-SN/TN), is considered as an index of “ripening
328 depth” (Tejada, Abellán, Cayuela, Martínez-Cacha, & Fernández-Salguero, 2008). The TCA-
329 SN/TN was influenced by neither heat treatment nor ripening time (Fig. 4), therefore, secondary
330 proteolysis of both the control cheese and experimental cheeses was very limited. The results of
331 TCA-SN/TN were in agreement with the constant level of total proteins found throughout the
332 ripening period (Table 1).
333
334 3.6. Textural properties
335
336 The textural properties of cheeses changed within the ripening period, mainly as a result
337 of changes of moisture content (Watkinson, Crawford, & Dodds, 2002), proteolysis, NaCl
338 diffusion, calcium equilibrium changes (Buňka et al., 2013), and pH value (Watkinson et al.,
339 2001). Surface and fracture hardness were followed throughout the ripening of C, E1 and E2
340 cheeses (Fig. 5).
341 Statistical analysis demonstrated that heat treatment of milk had a significant effect on
342 both surface and fracture hardness of cheeses C, E1 and E2. However, there was no statistical
343 difference (P<0.05) between both parameters of the two experimental cheeses, E1 and E2.

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344 Significant differences between surface hardness of all cheeses were recorded on the first
345 day of ripening. Cheese C exhibited higher surface hardness compared with E1 and E2, mainly
as
346 a result of higher dry matter content. It can be observed from Fig. 5 that there was a significant
347 increase of surface hardness of cheeses E1 and E2 during the first ten days of ripening, which
348 was caused by a rapid drop of pH and by a significant intake of salt from brine to the cheese
349 during that period (Table 1). It has already been reported that, during the first days of
ripening,
350 the influence of NaCl appears to be predominant and leads to an increase in rigidity of the
351 material, after which the influence of NaCl gradually decreases (Buňka et al., 2013). Beginning
352 on day 10 until the end of ripening (40 th day), the surface hardness of all three cheese variants
353 remained relatively constant, and did not differ significantly between the cheeses (Fig. 5).
354 The fracture hardness of cheeses E1 and E2 was lower than that of cheese C on the first
355 day of ripening. Values for fracture hardness for E1 and E2 remained mostly unchanged for 40
356 days. The fracture hardness of the control cheese followed a very different pattern: from a high
357 value at the 1st day, it then decreased significantly until day 20, and then increased back to the
358 level measured on the first day. Thus, in the early and late stages of ripening, the force required
359 for the fracture to occur was higher in C than in E1 and E2. It has been stated in the literature
that
360 the protein matrices of cheeses made from high heat treated milk (E1 and E2) are generally
361 coarser, with a body that is less homogeneous and contains numerous small holes and cracks
362 (Singh & Waungana, 2001). The decrease of fracture hardness of cheese C after 20 days of
363 ripening could be attributed to the more brittle structure caused by the pH drop in that period,
364 while the increase at the end of the ripening is explainable by the αS-casein degradation.
365 The way food fractures at the first bite is a very important component of perceived texture
366 (Vincent et al., 1991). Heat treatment of goats’ milk has great influence on this particular
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367 property, and more research is required, possibly in the area of cheese microstructure, to
368 understand the mechanism of this influence.
369
370 3.7. Sensory evaluation
371
372 It is known from the literature that the development of both body and flavour during
373 ripening of products from cheese milk heated at elevated temperatures differs from traditionally
374 produced cheeses (Benfeldt & Sørensen, 2001). The instrumental analysis of texture in this study
375 confirms that finding.
376 Some authors reported off-flavour development in cow cheeses as a result of high heat
377 treatment of milk (Singh & Waungana, 2001; Vasbinder, 2002). To our best knowledge, no study
378 has yet dealt with the effect of high heat treatments of goats’ milk on the sensory quality of white
379 brined cheeses.
380 According to our sensory panel, high heat treatment of goats’ milk significantly affected
381 both cheese flavour and oral texture (P<0.05), while the appearance, the cross-section
appearance
382 and the odour remained unaffected, and, for all cheeses, most received excellent scores (Table
2).
383 In the early ripening stage (3 days), E1 received a significantly higher score for texture than
the
384 control cheese, while E2 was significantly better graded for both texture and flavour. The
385 panellists had the impression that, at the beginning of ripening, E1 and E2 cheeses seemed as if
386 they were already ripened, while the control cheese was described as rubbery and empty-
387 flavoured. After 10 days of ripening, E1 and E2 received lower scores for both flavour and
388 texture than in the early stage (3 days). The deficiencies that caused the decrease of scores
were
389 the same for both experimental cheeses: undesirable crumbliness, dry texture and dominant salty
390 flavour. At day 3, the cross-section appearance was also graded with lower scores for all three

17
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391 cheeses, because they tended to crumble while being cut. The excessive perception of salty
392 flavour was reported for all three cheeses.
393 At the end of ripening, after 40 days, the score for flavour of E2 increased again
394 significantly. It was described by panellists as being rich in flavour and with a pleasant, creamy
395 texture. However, the score was still significantly lower than at the beginning of ripening, mostly
396 due to excessive saltiness. According to the Serbian Regulation (2014), seven days is a minimum
397 time for the ripening of soft cheeses. Therefore, 3 days old soft cheese could be categorised as
398 young cheese, while 10 days old and also 40 days old is mature cheese. Thus, the panellists’
399 expectations differed depending on the category, and 3 days old E2 cheese received the excellent
400 score being mild, milky, and not excessively salty. However after 10 days it was obviously not
401 mature enough, and saltiness dominated, lowering the flavour score. Then after 40 days, with
402 developed richness of flavour, it received again the excellent score.
403 It was interesting to notice that throughout the entire ripening period and for each sensory
404 characteristic, except for the appearance, E1 was graded significantly inferior to E2, although the
405 great majority of parameters investigated in this study (composition, extent of proteolysis,
406 instrumental texture parameters) did not differ significantly between those two treatments.
407 Further studies are required to understand these differences more fully.
408
409 4. Conclusion
410
411 Taking into account all the results obtained, it can be concluded that high heat treatment
412 could be successfully used for the enhancement of young and ripened white brined goat cheese
413 yield, without any adjustment to the cheese making process.

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414 High heat treatment of cheese milk significantly affected the composition, textural
415 properties and sensory qualities of white brined goat cheeses. It caused an inhibition of αS-
casein
416 degradation, but had no effect on β-casein degradation and on secondary proteolysis. Cheese
417 fracture hardness followed very different patterns for the experimental cheeses versus control
418 cheese.
419 Contrary to expectations, E2 cheese obtained from milk heated to 90 °C/5 min received
420 the best scores for all sensory characteristics at all ripening stages (3 days, 10 days, 40
days). The
421 best score in the early ripening phase (3 days) makes it possible for this cheese variety to be
422 released for sale very soon after production, which is economically very attractive.
423 This study could serve as a platform for the development of new cheese types with
424 increased yield, increased biological value and with unique texture and flavour characteristics.
425 More work is required for detailed texture and sensory analysis, and in the assessment of
426 lipolysis, microstructure and of microbiological quality of cheeses during ripening, and with
427 optimization of cheese technology to aim for lower salt content, and to develop different ways of
428 cheese packaging.
429
430 Acknowledgements
431
432 We are grateful to the Ministry of Education, Science and Technological Development of
433 Republic of Serbia for providing Grant III 46009 for this study. We are also grateful to the dairy
434 plant Beocapra, Kukujevci, Serbia for the supply of milk.
435
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Figure legends
Fig. 1. pH value of cheeses made from goats’ milk heated to 65 °C/30 min (C: •), 80 °C/5 min (E1: •),
and 90 °C/5 min (E2:▲), after 1, 10, 20, 30 and 40 days of ripening
Fig. 2. SDS-PAGE electrophoretogram of cheeses made from goats’ milk heated to 65 °C/30 min (C),
80 °C/5 min (E1) and 90 °C/5 min (E2), after 1 day (1 d), 10 days (10 d), 20 days (20 d), 30 days (30 d)
and 40 days (40 d) of ripening. M, raw goats’ milk; S, standard molecular mass protein mixture (10–150
kDa).
Fig. 3. Residual αS-casein (a) and f3-casein (b) in cheeses made from goats’ milk heated to 65 °C/30
min (C: •), 80 °C/5 min (E1: •), and 90 °C/5 min (E2:▲), after 1, 10, 20, 30 and 40 days of ripening.
Fig. 4. Percentage of TCA-SN/TN in cheeses made from goats’ milk heated to 65 °C/30 min (C: •), 80
°C/5 min (E1: •), and 90 °C/5 min (E2:▲), after 1, 10, 20, 30 and 40 days of ripening
Fig. 5. Surface hardness (a) and fracture hardness (b) of cheeses made from goats’ milk heated to 65
°C/30 min (C: •), 80 °C/5 min (E1: •), and 90 °C/5 min (E2:▲), after 1, 10, 20, 30 and 40 days of
ripening.
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Table 1
Gross composition of cheeses made from goats' milk heated at 65 °C/30 min (C), 80 °C/5 min (E1) and
90 °C/5 min (E2) during ripening.a
Parameter Cheese Ripening time (days)
1 10 20 30 40
DM (%) C 44.27±1.62Aa 40.34±1.31b 38.76±0.43b 39.82±1.66b 38.16±3.82b
E1 38.54±1.18B 39.93±1.52 38.98±0.90 39.48±1.74 39.84±0.99
E2 38.08±0.30B 38.76±0.43 39.25±1.07 39.21±2.05 39.49±1.38
TP (%) C 18.43±0.25Aa 16.35±0.87b 16.76±2.13b 15.69±1.17b 16.03±1.32b
B
E1 14.81±1.31 16.17±0.96 16.05±0.19 16.23±1.63 15.63±0.90
E2 14.77±1.45B 14.94±0.33 15.56±0.79 15.33±1.21 15.86±0.90
Fat (%) C 18.50±0.71 17.67±1.53 18.00±1.73 18.83±1.04 17.50±2.64
E1 17.50±2.12 18.00±0.87 18.50±0.71 18.83±1.04 18.58±0.52
E2 18.25±0.35 18.17±0.28 19.17±0.29 18.50±0.71 19.00±1.73
NaCl (%) C 1.27±0.05a 2.71±0.38b 2.45±0.20b 3.20±0.82b 3.28±0.53b
E1 1.09±0.14a 2.58±0.26b 2.68±0.17b 3.12±0.48b 3.09±0.46b
a b b b
E2 1.09±0.36 2.67±0.45 2.60±0.23 3.01±0.33 3.39±0.92b
MNFS (%) C 67.52±1.29 72.46±0.67 72.22±0.77 73.62±2.35 b 74.92±2.99 b
Aa b b

E1 74.41±0.10B 73.26±1.92 75.82±1.50 74.56±1.22 73.89±0.77

E2 75.81±0.16B 74.83±0.77 75.16±1.06 76.04±0.58 74.70±0.11
FDM (%) C 41.14±0.17 Aa 43.75±2.59 ab 47.55±1.24 c 45.89±2.80 bc 45.86±0.47 bc
E1 45.25±3.55B 45.12±2.55 47.62±0.30 47.69±0.67 46.64±0.32
E2 47.99±0.42B 46.87±1.24 48.85±0.59 48.65±1.78 48.05±2.66
P/F C 1.00±0.01A 0.93±0.11A 0.93±0.07A 0.89±0.07 0.92±0.04
E1 0.90±0.12A 0.90±0.06A 0.86±0.02A 0.85±0.02 0.84±0.04
E2 0.85±0.05B 0.82±0.03B 0.81±0.03B 0.79±0.05 0.84±0.05
a
S/M (%) C 2.24±0.12 4.52±0.54b 4.33±0.49b 5.15±1.02b 5.27±0.40b
E1 1.76±0.22a 4.28±0.28b 4.37±0.22b 5.21±0.99b 5.19±0.77b
a b b b
E2 1.76±0.57 4.37±0.72 4.31±0.29 5.02±0.76 5.67±1.69b
a
Values are means of three replicated trials ± standard deviation; different superscript uppercase
letters indicate significant difference between values in terms of milk heat treatment (P<0.05), different
superscript lowercase letters indicate significant difference between values in terms of ripening time
(P<0.05), no superscript letters indicates no significant differences in the respective factors (P > 0.05).
Abbreviations are: DM, dry matter; TP, total protein; MNFS, moisture in non-fat solids; FDM, fat in dry
matter; P/F, protein-to-fat ratio; S/M, salt in moisture.
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Table 2
Sensory properties of cheeses made from goats' milk heated at 65 °C/30 min (C), 80 °C/5 min (E1) and
90 °C/5 min (E2) after 3, 10 and 40 days of ripening. a
Odour (orthonasal olfaction)
Ripeni Chee Appearanc Cross- 4.8±0.45 3.9±0.22A
A
ng time se e section 3.5±0.35
(days) appeara 4.8±0.27 4.3±0.27Aa 4.2±0.27B
3 C 5.0±0.00 nce
4.8±0.45a 5.0±0. Flavour Oral texture
E1 5.0±0.00 b
4.8±0.45a 00
b 5.0
E2 5.0±0.00 5.0±0.00
±0.00Ba
10 C 5.0±0.00 4.4±0.55 4.9±0.22C
a
E1 5.0±0.00 4.4±0.42 4.7±0.45 3.9±0.42 A B 3.9±0.22 A B
a
E2 5.0±0.00 4.7±0.27 4.8±0.23 3.7±0.27Ab 3.5±0.00A
5.0±0.00 4.9±0.22 4.3±0.45Bb 4.4±0.55B
40 C 5.0±0.00 A
b 4.6±0.65 3.9±0.65 3.8±0.83A
E1 5.0±0.00 4.9±0.22 4.6±0.42 3.6±0.22 Ab
3.7±0.27A
b
E2 5.0±0.00 5.0±0.00 4.7±0.27 4.8±0.27Bc
B
4.7±0.45
a
Different superscript uppercase letters indicate significant difference between values in terms of milk
heat treatment (P < 0.05, different superscript lowercase letters indicate significant difference between
values in terms of ripening time (P < 0.05), lack of superscript letters indicates no significant differences
in the respective factors (P > 0.05).
 ACCEPTED MANUSCRIPT

pHvalue

Ripening time (days)

Figure 1
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Figure 2
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Residual αS-casein (%)

Ripening time (days)

b

Ripening time
(days)
Residual β-casein (%)

Figure 3
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TCA-SN/TN (%)

Ripening time (days)

Figure 4
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a
Surface hardness (N)

Ripening time (days)

b
Fracture hardness (N)

Ripening time (days)