Sie sind auf Seite 1von 36

Accepted Manuscript

Composition, microstructure and chemical interactions during the production stages of


mozzarella cheese

Mônica Correia Gonçalves, Haíssa Roberta Cardarelli

PII: S0958-6946(18)30171-7
DOI: 10.1016/j.idairyj.2018.07.005
Reference: INDA 4359

To appear in: International Dairy Journal

Received Date: 19 February 2018


Revised Date: 8 July 2018
Accepted Date: 9 July 2018

Please cite this article as: Gonçalves, M.C., Cardarelli, H.R., Composition, microstructure and chemical
interactions during the production stages of mozzarella cheese, International Dairy Journal (2018), doi:
10.1016/j.idairyj.2018.07.005.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
1 Composition, microstructure and chemical interactions during the production stages of

2 mozzarella cheese

PT
6

RI
7

8 Mônica Correia Gonçalves a, Haíssa Roberta Cardarelli b*

SC
9

10

11
U
AN
12
M

13
a
14 Postgraduate Program in Food Science and Technology, Department of Food
D

15 Engineering, Centre of Technology, Federal University of Paraíba, Campus I, Av.


TE

16 Castelo Branco s/n, João Pessoa, Paraíba, 58051-900, Brazil.


b
17 Department of Food Technology, Centre of Technology and Regional Development,
EP

18 Federal University of Paraíba, Avenida dos Escoteiros, s/ n, Mangabeira VII, Distrito

de Mangabeira, João Pessoa, Paraíba, 58055-000, Brazil.


C

19
AC

20

21

22

23

24 *Corresponding author. Tel.: 055 83 32167947

25 E-mail address: hrcarda@gmail.com (H. R. Cardarelli)

1
ACCEPTED MANUSCRIPT
26 ___________________________________________________________________________

27 ABSTRACT

28

29 Changes in chemical composition, microstructure and chemical interactions before and after

30 the stretching stage of mozzarella cheese processing were investigated. The increased acidity

PT
31 and the decreased pH resulted in the solubilisation of total calcium. The protein matrix

RI
32 became more compact and the size of the fat globules decreased with the incorporation of

33 small individual fat globules, aggregates and fat globules of irregular form into the matrix.

SC
34 The predominant bonds in the curd before the stretching stage were hydrophobic interactions,

35 whereas the number of calcium bonds was minimal. After the stretching stage, the primary

36
U
bonds responsible for maintaining the cheese structure were calcium bridges, electrostatic
AN
37 interactions and hydrogen bridges. These results clarify important aspects of the bonds
M

38 involved in the production of this type of cheese.

39 ___________________________________________________________________________
D

40
TE
C EP
AC

2
ACCEPTED MANUSCRIPT
41 1. Introduction

42

43 Worldwide production of mozzarella cheese has greatly increased in the last decade,

44 and this is presently the most produced type of cheese, primarily because of its use as an

45 ingredient in pizza (Jana & Mandal, 2011). The fast growth of the market for mozzarella

PT
46 cheese and competitive pressure has resulted in an impressive increase in industrial

RI
47 production capacities. However, large-scale manufacturing requires a precise control of the

48 production process.

SC
49 Mozzarella is a cheese of the pasta-filata type. In the stretching process, the cheese

50 curd is subjected to mechanical work in hot water to turn the amorphous structure into an

51
U
organised, elastic and compact structure (Banville, Chabot, Power, Pouliot, & Britten, 2016;
AN
52 Kindstedt, Carić, & Milanović, 2004). For the stretching process to occur, the calcium content
M

53 associated with casein (insoluble calcium) must be ideal, and this is usually achieved through

54 calcium solubilisation by acidification (Guinee, 2002). Casein hydration increases when the
D

55 amount of calcium associated with casein decreases, and this contributes to the stretching
TE

56 process (Mizuno, Matsuda, Lucey, & Ichihashi, 2009). There are two parameters that

57 determine the amount of calcium associated with the casein micelle during the stretching
EP

58 process: the total calcium content in the curd and the total calcium distribution between the

soluble and insoluble forms (Kindstedt et al., 2004). Therefore, acidity development during
C

59
AC

60 mozzarella cheese manufacturing must be controlled to maintain the desired combination of

61 total calcium content, pH and moisture (Kindstedt, 2007).

62 Curd heating during the stretching stage causes the calcium dissolved in the aqueous

63 phase to bond again to the casein, resulting in protein interactions through calcium bridges

64 that favour whey removal. In addition, this thermomechanical treatment causes physical-

3
ACCEPTED MANUSCRIPT
65 chemical changes that strongly influence the functional properties and the proteolysis of the

66 cheese during its refrigerated storage (Banville et al., 2016).

67 The impact of different thermomechanical treatments on the characteristics of

68 mozzarella cheese was assessed by Banville et al. (2016), and their results show that the

69 amount of mechanical energy supplied was proportional to the fat loss of the cheese and that

PT
70 the amount of free whey was associated with the intensity of the thermal treatment. The

RI
71 authors concluded that the thermomechanical systems affected the cheese composition, solids

72 loss and microstructure.

SC
73 Although there have been many studies on mozzarella cheese (Bähler & Hinrichs,

74 2013; Ma, James, Zhang, & Emanuelsson-Patterson, 2013; Renda, Barbano, Yun, Kindstedt,

75
U
& Mulvaney, 1997; Rowney, Roupas, Hickey, & Everett, 2003), the interactions involved in
AN
76 the stretching stage need more studies to be elucidated. To produce pasta-filata cheeses such
M

77 as mozzarella with specific functional properties, it is necessary to better understand the effect

78 of the thermomechanical processes on the characteristics of the cheese (Banville et al., 2016).
D

79 Therefore, the objective of this work is to assess the changes in the composition,
TE

80 microstructure and chemical interactions before and after the stretching stage of mozzarella

81 cheese production.
EP

82

2. Materials and methods


C

83
AC

84

85 2.1. Processing of mozzarella cheese

86

87 To produce the cheese, 130 L of raw milk was used in each batch. The cheese

88 processing was repeated twice on different days to ensure that similar results would be

89 obtained from different milk batches of similar composition. A sample of the raw milk was

4
ACCEPTED MANUSCRIPT
90 taken from each batch to measure the fat content using the Gerber method (AOAC, 2006a)

91 and the casein content using the formaldehyde method (Lourenço & Wolfschoon-Pombo,

92 1982) for the standardisation calculations. The milk was skimmed and standardised to a

93 casein and fat ratio of 1.00. Then, the standardisation was checked by measurement of the

94 casein and fat contents using the Kjeldahl (AOAC, 2006b) and Mojonnier (AOAC, 2006c)

PT
95 methods, respectively, and a value of 0.93 ± 0.05 was obtained. The standardised milk was

RI
96 subjected to a slow pasteurisation in a 110 L electrical, automatic pasteurisation tank made of

97 stainless steel, with control of agitation and heating and cooling temperatures (Inadal,

SC
98 Mixmatic 110, Osasco, Brazil). After pasteurisation, the milk was cooled to 7 °C, placed in 50

99 L cans and stored in a cold chamber below 5 °C for one night. On the following day, the milk

100
U
was transferred to an automatic cheese fabrication tank of 210 L capacity with a cutting lyre
AN
101 and adjustable agitation (Biasinox, Lambari, Brazil). A total of 1% lactic thermophilic
M

102 bacteria, consisting of Streptococcus thermophilus and Lactobacillus delbrueckii ssp.

103 bulgaricus (TCC 20; Chr. Hansen Ind. e Com. Ltda, Valinhos, Brazil) and 0.25 mL L-1 50%
D

104 calcium chloride (ECIBRA®; Ind. e Com. de produtos químicos Ltda, Santo Amaro, Brazil)
TE

105 were added to the milk at 37 °C. Pure chymosin obtained by fermentation (CHY-MAX®

106 100% Chymosin; Chr. Hansen Ind. e Com. Ltda) was used to coagulate the milk, and it was
EP

107 added at a sufficient amount to coagulate 130 L of milk in 35 min. After coagulation, the curd

was cut in cubes with 1.5 cm edges and then slowly agitated for 25 min. The curd was treated
C

108
AC

109 by indirect heating until reaching 42 °C. One third of the whey amount was removed and the

110 curd rested until the stretching point. Once the stretching pH was reached, the curd was salted

111 in a 2% (w/w) proportion (Refinaria Nacional de Sal S/A, Cisne®; Cabo Frio, Brazil) and

112 then cooled in an ice bath. The stretching process was adapted from Costa et al. (2017). After

113 cooling, the curd was chopped into cubes of the same size and then underwent a manual

114 stretching stage with water at 85 °C (62.0 ± 0.00 °C in the centre of the curd). The volume

5
ACCEPTED MANUSCRIPT
115 water and time spent in each cheese were controlled to standardise the stretching conditions.

116 The volume of water used in the stretching stage of the cheese was approximately 3.25 ± 0.24

117 L, and the time spent for each piece of cheese was 2.73 ± 0.57 min. After stretching stage, the

118 cheeses were shaped, cooled in an ice bath for 1 h, followed by drying at 12 ° C for 24 h and

119 vacuum packed in polyethylene bags.

PT
120

RI
121 2.2. Sampling during the processing

122

SC
123 Samples of pasteurised milk (PM), curd at the cutting point (CC), curd after partial

124 whey removal (CR), whey from the partial removal (WR), the curds during the acidification

125
U
stage (CpH5.8, CpH5.6, CpH5.4, CpH5.2, CpH5.1), whey at the stretching pH (WpH5.2) and stretching
AN
126 water (SW) were taken for analyses. Several studies indicate that the stretching stage of
M

127 mozzarella cheese occurs at a pH between 5.2 and 5.4 (Jana & Mandal, 2011; Lucey, Johnson

128 & Horne, 2003). The curd was also analysed at a pH of 5.1 to understand what happens to its
D

129 composition at a pH below 5.2. The stages and the samplings performed during the processing
TE

130 are shown in Fig. 1.

131 All samples were placed in a 100-mL flask and immediately cooled in an ice bath.
EP

132 After cooling, the PM, WR, WpH5.2 and SW samples were stored at 15 °C for analysis. The

samples of CC, CR and the curds during acidification (CpH5.8, CpH5.6, CpH5.4, CpH5.2, CpH5.1)
C

133
AC

134 were dispersed using a hand disperser (Ultra-Turrax, Polytron PT 1200E; Kinematica AG,

135 Luzern, Switzerland) and stored at 15 °C for analysis.

136

137 2.3. Chemical composition

138

6
ACCEPTED MANUSCRIPT
139 All samples were analysed for their casein content (AOAC, 2006b); fat content using

140 the Mojonnier method (AOAC, 2006c); acidity by titration according to the official procedure

141 (AOAC, 2006d); pH using the potentiometric method (Digimed DM20 potentiometer;

142 Digicrom Analítica Ltd, Santo Amaro, SP, Brazil); total calcium (TC) using the dry digestion

143 method (AOAC, 2006e) followed by titration with ethylenediaminetetraacetic acid (EDTA) in

PT
144 the presence of murexide (Taras, 1995); insoluble calcium (NC) calculated from the

RI
145 difference between the TC and soluble calcium contents, as described by Metzger, Barbano,

146 Rudan, and Kindstedt (2000); and moisture (AOAC, 2006f). The salt (NaCl) content of the

SC
147 cheese was also analysed using the Volhard method (Richardson, 1985). All characterisation

148 analyses were performed in triplicate.

149
U
AN
150 2.4. Assessment of the fat particle size
M

151

152 The sample was prepared according to Lopez, Carmier and Gassi (2007). One
D

153 millilitre was removed from the milk sample (PM), 10 mL of a solution of 1% (w/w) sodium
TE

154 dodecylsulphate (SDS; Synth, Diadema, SP, Brazil) and 35 mmol L-1 of EDTA (Vetec

155 Química Fina Ltda, Duque de Caxias, RJ, Brazil) were added to it, and the pH was then
EP

156 adjusted to 7.0. To determine the fat particle size in the curds (CC, CR, CpH5.8, CpH5.2) and

cheese, 1 g of the sample was dissociated with 5 mL of dissociation buffer: 6 mol L-1 urea
C

157

(Synth), 100 mmol L-1 EDTA (Vetec Química Fina Ltda), 20 mmol L-1 imidazole buffer
AC

158

159 (Sigma), pH 6.6. Next, the samples were slowly stirred at 8 rpm (mixer model AP 22;

160 Phoenix, Araraquara, Brazil) for 30 min, and the particle size was measured immediately

161 afterwards using laser diffraction in a Mastersizer instrument (Malvern Instruments Ltd.

162 Worcester, United Kingdom). The average diameter based on volume d [4,3] was calculated

163 using Eq. 1, where ηi is the number of particles of diameter equal to di. The value of d [4,3] is

7
ACCEPTED MANUSCRIPT
164 highly influenced by large fat and protein particles (Lopez-Sanchez, Svelander, Bialek,

165 Schumm & Langton, 2011).

166

167 4,3 = ∑ ⁄∑ (Eq. 1)

168

PT
169 2.5. Apparent zeta (ζ) potential

RI
170

171 The samples of milk (PM), the curds (CC; CR; CpH5.8; CpH5.2) and cheese (C) were

SC
172 prepared according to Michalski, Michel, Sainmont and Briard (2001). The zeta potentials of

173 the samples taken during processing, and of the cheese, were measured in a ZetaSizer

174
U
(Malvern Instruments Ltd). The samples (1 mL of PM and 1 g of CC, CR, CpH5.8, CpH5.2 and
AN
175 C) were diluted in a 1:25 proportion in a buffer solution (pH adjusted to 7.0) of 20 mM
M

176 imidazole (Vetec Química Fina Ltda), 50 mM NaCl and 5 mM CaCl2 (both from Synth). The

177 buffer solution was previously filtered with 0.2 µm cellulose filter paper. The samples with
D

178 the buffer were agitated and then taken to the Zetasizer at 25 °C.
TE

179

180 2.6. Types of bonds


EP

181
C

182 Specific tests were performed of the different types of bonds responsible for the
AC

183 structure of the curd before the stretching stage and of the cheese after the stretching stage.

184 The method consisted of extractions from the samples of curd at pH 5.2 and cheese with

185 different buffer solutions, to obtain a semi-quantitative estimate of the covalent and non-

186 covalent bonds that stabilise the protein structure.

187 The analysis of the bond types was performed according to the procedure described by

188 Keim, Kulozik and Hinrichs (2006) with some changes. The samples (2 g) of curd before the

8
ACCEPTED MANUSCRIPT
189 stretching stage and of cheese were transferred to a 50-mL plastic tube with 20 mL of the

190 different buffer solutions (Supplementary material Table S1) with the following reagents:

191 trisodium citrate (Vetec Química Fina Ltda); DTT and SDS (both from Sigma Aldrich);

192 EDTA (Vetec Química Fina Ltda); and monosodium phosphate, NaCl and Tris (all from

193 Synth). Hydrochloric acid (Synth) was added to adjust the pH of buffer solutions A and B.

PT
194 The buffer solutions were added and homogenised at room temperature for 5 min. Then, the

RI
195 samples were agitated in an orbital agitation table (model MA 140CFT; Marconi, Piracicaba,

196 Brazil) for 30 min and centrifuged (AllegraTM 64R Centrifuge; Beckman Coulter, São Paulo,

SC
197 Brazil) at 15,000 × g for 20 min at 20 °C. The supernatant was then filtered with 0.2 µm

198 paper, and the total nitrogen content was determined using the Kjeldahl method (AOAC,

199
U
2006g). The nitrogen contents of the buffer solutions (Nbuffer, S) and of the samples (Ncurd
AN
200 and Ncheese) were also determined. The nitrogen content of each buffer solution was
M

201 calculated using Eq. 2.

202
D

+
, = . , − . , Eq. 2!
TE

203
EP

204 where Nsup is the nitrogen content of the supernatant in the curd or in the cheese, mS is the

205 mass of each buffer solution, and ms is the mass of the samples (curd or cheese).
C

206 The interpretation of the data on the bond types was based on the solubility of the
AC

207 samples of the different buffer solutions, with the set of equations (Eq. 3 to 10) below:

208

, "
= # $%! + # &'! + # &%! + # ! Eq. 3!

, %
= # $%! + # &'! + # &%! + # ! + # ! Eq. 4!

9
ACCEPTED MANUSCRIPT
, (
= # $%! + # &%! + # ! Eq. 5!

, *
= # $%! + # &%! + # (+%! + # ! Eq. 6!

, % , "
# != − Eq. 7!

PT
, " , (
# &'! = − Eq. 8!

, (

RI
# $%! + # &%! + # #/! = Eq. 9!

SC
, * , (
# (+%! = − Eq. 10!

209

210
U
where Nbond,A, Nbond,B, Nbond,C, Nbond,D are the nitrogen contents of the supernatant
AN
211 obtained from the treatment of the samples with buffer solutions A, B, C and D (g g-1); Ns is

the nitrogen content in the curd (g g-1) or in the cheese (g g-1); and P (j) is the amount of
M

212

213 protein (%) stabilised by j interactions, where j corresponds to electrostatic interactions (EB),
D

214 hydrophobic interactions (Hy), hydrogen bridges (HB), disulphide bridges (SS), calcium
TE

215 bridges (CaB) and free proteins.

216
EP

217 2.7. Assessment of the microstructures of the curds (CC; CR; CpH5.8 and CpH5.2) and the

cheese (C)
C

218
AC

219

220 The curds and cheese samples were prepared according to the methodology described

221 by Lopez et al. (2007). Initially, the samples were cut in cubes with 5 mm edges with a steel

222 blade and placed in the centre of a glass slide. The samples were stained with the addition of

223 0.5 mL of acridine orange fluorescent dye and Nile red dye (both from Sigma Aldrich),

224 previously dispersed in anhydrous ethylic alcohol 99.8% (Synth). The solvent was evaporated

10
ACCEPTED MANUSCRIPT
225 in the dark. Next, a slide with the sample was covered with a coverslip and stored in the dark

226 for 30 min at 4 °C to evaporate the solvent and then viewed under a microscope (AGZeiss

227 LSM780-NLO; Carl Zeiss, Berlin, Germany). The wavelengths used to excite the Nile red

228 and the acridine orange fluorescent dyes were 568 and 488 nm, respectively. Images of slices

229 of 100 to 350 µm with a 1024 × 1024 pixel resolution were obtained.

PT
230

RI
231 2.8. Experimental design

232

SC
233 All experiments were performed in duplicate. The results of composition and apparent

234 zeta (ζ) potential of samples CC, CR, CpH5.8, CpH5.6, CpH5.4, CpH5.2 and CpH5.1 and of particle

235
U
size of samples CC, CR, CpH5.8, CpH5.2 and cheese taken during the manufacturing stages of
AN
236 mozzarella cheese were analysed using analysis of variance (ANOVA). The Tukey test was
M

237 applied to verify differences among the means. Differences were considered significant at a

238 95% probability level (P ≤ 0.05). The standard deviations are shown as error bars in the
D

239 figures. The data were analysed using STATISTICA 7.0 software (StatSoft Inc., Tulsa, OK,
TE

240 USA).

241
EP

242 3. Results and discussion


C

243
AC

244 3.1. Composition of the standardised and pasteurised milk, whey after partial removal,

245 whey before stretching stage (pH 5.2) and stretching water

246

247 The compositions of the standardised and pasteurised milk (PM), whey after

248 partial removal (WR), whey from the stretching stage (WpH5.2) and stretching water

249 (SW) are shown in Table 1. The acidity and total solids contents of the milk meet the

11
ACCEPTED MANUSCRIPT
250 technical regulations for pasteurised milk (Brasil, 2011). The pH of samples WR, WpH5.2

251 and SW decreased during the production stages of the mozzarella cheese. In addition,

252 the pH reduction resulted in calcium solubilisation, indicated by the decrease in

253 insoluble calcium from 0.040% in sample CR to 0.020% in sample WpH5.2 and by the

254 7.3% reduction of the apparent zeta potential. The neutralisation of the charges occurs

PT
255 as the pH decreases, which causes a decrease in the electrostatic repulsion, and

RI
256 consequently, decreasing the zeta potential.

257 The total calcium concentration in the milk was 0.154%, with 58.4% in the colloidal

SC
258 form; this was slightly lower than the value reported by Walstra (2006) of 66% in the

259 colloidal form (bonded to casein) and 34% in the soluble form as Ca2+ bonded to citrates and

260 phosphates.
U
AN
261 The milk studied in this work had a fat content of 2.3%. A milk fat content of 2.5% is
M

262 considered optimum for making mozzarella cheese that is to be consumed in slices (Valle,

263 Campos, Yotsunagi, & Souza, 2004), i.e., very close to the value of this study. According to
D

264 Huppertz and Kelly (2006), fat is initially dispersed in milk as lipid droplets with a size
TE

265 distribution between 0.2 and 15 µm. The size of the fat globules of the standardised and

266 pasteurised milk was determined as d43 = 4.66 ± 0.65 µm in this study. Lopez et al. (2007)
EP

267 reported that, in the case of Emmental cheese, the size of the milk fat globules varied between

1.0 and 10 µm, with d43 = 4.04 ± 0.03 µm, slightly lower than that of this study.
C

268
AC

269 The total solids and fat in the samples WR, WpH5.2 and SW show losses during the

270 production stages of mozzarella cheese. There was a 12.6% loss of fat in whey at pH 5.2,

271 corresponding to whey after its total removal. There was practically no loss of casein.

272 However, the loss of components has a direct influence on the yield and may affect

273 functional, rheological and sensory characteristics of mozzarella cheese.

274

12
ACCEPTED MANUSCRIPT
275 3.2. Influence of the production stages on the curds’ and mozzarella cheese’s compositions

276

277 The compositions of the curds at different production stages and of the mozzarella

278 cheese are shown in Table 2. The acidification of the curd resulted in more whey release and

279 in the consequent reduction of 31.87% of the curd moisture after the cutting point (CC) when

PT
280 compared to the cheese moisture (C). The mozzarella cheese had 17.07% ± 1.07 fat, 52.63%

RI
281 ± 0.39 moisture and 1.63% ± 0.04 salt. The low fat % in the cheese is a result of the casein

282 /fat ratio of the milk and the high moisture content of the cheese. After the stretching stage

SC
283 (CpH5.2), only 66.87% of the insoluble calcium in the curd after the cutting point (CC) was still

284 bonded to the casein (insoluble calcium/casein ratio expressed in dry basis). This value

285
U
decreased to 58.20% in the cheese after the stretching stage and was 59.75% in the curd at pH
AN
286 5.1, below the ideal for stretching (Table 2). These results confirm that calcium bonded to
M

287 casein must be partially solubilised to promote the stretching of the curd.

288 The insoluble calcium/casein*DB ratio of the cheese (C) was 1.88, a 41.79% (P =
D

289 0.00) reduction of the insoluble calcium of the curd after cutting (CC) (Table 2). According to
TE

290 Guinee, Feeney, Auty, and Fox (2002), calcium and pH are interdependent and are the

291 primary factors that influence the microstructure of mozzarella cheese. The pH defines the
EP

292 amount of soluble calcium lost in the curd after whey removal and the soluble

calcium/insoluble calcium ratio in the final cheese. The proportion of soluble calcium
C

293
AC

294 increases with decreasing pH, which helps neutralise the protein charges. This allows the

295 association of proteins through hydrophobic interactions; thus, at a low pH, the degree of

296 solvation of the protein is thought to decrease. In turn, at a high pH, the protein matrix will

297 swell and absorb more water and the size of the whey channels will decrease. Fig. 2 shows the

298 evolution of pH and soluble calcium during the processing stages of mozzarella cheese. The

13
ACCEPTED MANUSCRIPT
299 pH from CC until CpH5.2 decreased (P < 0.0001) and the calcium bonded to casein solubilised

300 more with the pH reduction.

301

302 3.3. Microstructure of the curd, apparent (ζ) potential, particle size and predominant bond

303 types before the stretching stage and of the cheese after the stretching stage

PT
304

RI
305 The confocal microscopy technique enabled the analysis of the fat globules without

306 modifying the internal structures of the curds during production as well as that of the cheese,

SC
307 so it was possible to visualise what occurs during the different processing stages of

308 mozzarella cheese. Fig. 3 shows the microstructure of the curds during the production stages

309
U
(cutting, whey removal, acidification and stretching) and of the mozzarella cheese after the
AN
310 stretching. The images of the curd after cutting (Fig. 3A) and after partial whey removal (Fig.
M

311 3B) show fat globules dispersed in an open protein matrix with an amorphous and

312 unorganised structure. Because the pH was near 6.3 during cutting and whey removal, the
D

313 membrane of the fat globule was negatively charged, which resulted in electrostatic repulsion
TE

314 between the fat globules and their consequent separation from each other (Fig. 3A,B).

315 The curd was agitated and heated after cutting. The thermomechanical treatment of the
EP

316 curd caused syneresis, a result of the grain’s contraction that enables whey expulsion, which

caused changes in the structure with the formation of a more compact protein matrix and,
C

317
AC

318 consequently, fat globules closer to each other (Fig. 3B). This approach was favoured by the

319 reduction of the apparent (ζ) potential (Fig. 4).

320 The apparent (ζ) potential measures potential in volts at the surface of hydrodynamic

321 shear. High values of apparent (ζ) potential mean a high level of charge-charge interactions or

322 repulsion. This means that the high the apparent (ζ) potential, the greater the strength of the

323 electrostatic interactions. In turn, a lower apparent (ζ) potential decreases the repulsion among

14
ACCEPTED MANUSCRIPT
324 the particles. Fig. 4 shows the apparent (ζ) potential of the curd after cutting, curd after whey

325 removal, curd at pH 5.8, curd at stretching pH (pH 5.2) and the cheese, the apparent (ζ)

326 potential decreased with increasing acidity. In parallel, the negative charges of the casein

327 reduce with the pH decreases caused by the dissociation of the calcium ions from the

328 micelles.

PT
329 The sharp 56.04% increase in total solids from the curd after cutting to that after whey

RI
330 removal (Table 2), as a result of heating, caused the syneresis and contraction of the protein

331 matrix. The increase in density of the protein matrix during the heating reduced the size of the

SC
332 whey pores (Fig. 3B); therefore, the fat globules and/or fat globules aggregates became very

333 large and separated the protein matrix. The fat retention inside the cheese matrix is strongly

334
U
influenced by the initial fat and protein content of milk and by the curd cutting (Johnston,
AN
335 Luckman, Lilley & Smale, 1998). In addition, native fat globules with hydrophilic surfaces
M

336 that did not interact with the protein network were expelled with the whey and the stretching

337 water during the cheese processing stages (Table 1). Therefore, the external region of the curd
D

338 grains had a low concentration of fat globules (Fig. 3B).


TE

339 Fig. 3C shows the curd at pH 5.8 (CpH5.8), with a protein matrix even more compact,

340 with 24% of whey lost compared with the curd after cutting, and with few voids compared to
EP

341 the protein matrix after cutting (Fig. 3A) and after whey removal (Fig. 3B). The average fat

globules sizes were 7.58 ± 0.82 µm and 5.73 ± 0.35 in the curd after cutting and in the curd at
C

342
AC

343 pH 5.8 (CpH5.8), respectively (Fig. 3A,C). After the stretching stage, the protein matrix was

344 more organised and porous, interwoven with fat globules of different sizes (Fig. 3D). At the

345 end of the mozzarella cheese production, the protein matrix became more compact, with more

346 agglomeration of fat particles and with the incorporation of small individual fat globules (of

347 approximately 2.79 ± 0.83 µm, Table 2) (Fig. 3E). The mozzarella cheese had a lower fat

348 content because of the standardisation of the casein and fat ratio (Table 1); therefore, the

15
ACCEPTED MANUSCRIPT
349 higher volume fraction of the casein matrix formed thicker para-casein fibres with reduced fat

350 and whey channel inclusions, as observed in the micrographs.

351 The average particle sizes varied from 7.58 ± 0.82 µm in the curd after cutting to 3.74

352 ± 0.01 µm in the curd at the stretching pH (Table 2). The membranes of the fat globules are

353 negatively charged at the cutting point (pH 6.37), which increases the electrostatic repulsions

PT
354 among the fat globules. The average size of the fat particles decreased to 2.79 ± 0.83 µm after

RI
355 the stretching stage due to the thermomechanical treatment during this process.

356 The changes in the zeta potential (Fig. 4) suggest protein adsorption on the surface of

SC
357 the fat globules, which may induce the formation of aggregates of fat globules. However,

358 some globules seem to be trapped in the protein matrix (Fig. 3A).

359
U
The rupture of the fat globules entrapped with the protein matrix were also observed in
AN
360 the confocal microscopy (Fig. 3E). The heating during the stretching stage induced some
M

361 changes in the microstructure of the fat globules, such as coalescence and rupture. The

362 stretching process stretched the fat globules that filled the voids between the protein fibres.
D

363 The density increase in the protein matrix reduced the space occupied by the whey, and
TE

364 therefore, the fat globules and/or the fat globule aggregates became very large and separated

365 the protein matrix. In addition, fat globules with hydrophilic surfaces that had not interacted
EP

366 with the protein network may have been expelled with the whey during the contraction of the

globules. The concentrations of the fat expelled from the heated curd grains were 0.36 ±
C

367
AC

368 0.03% and 0.28 ± 0.07% in the whey partially removed (WR) and in the whey at pH 5.2

369 (SpH5.2; Supplementary material Table S1), respectively.

370 The high temperature applied to the curd during the stretching stage favoured the

371 hydrophobic interactions (protein-protein), which caused the aggregation and contraction of

372 the protein matrix. The contraction initiates the partial separation between the protein phase

373 and the water inside the curd structure. The application of shear forces on the heated curd

16
ACCEPTED MANUSCRIPT
374 during the stretching stage aligned the aggregated protein matrix in dense elastic fibres

375 separated by free fat and whey channels (Fig. 3E).

376 The change induced by the temperature resulted in the increase in the insoluble

377 calcium content, which presumably has the effect of reinforcing the protein interactions and

378 strengthening the dense para-casein fibres through bonds with calcium (Calcium bridges

PT
379 (CaB), Fig. 5B). According to Joshi, Muthukumarappan, and Dave (2003), non-covalent

RI
380 bonds, such as calcium bridges, are responsible for stabilising the gel structure obtained by

381 enzymatic coagulation. During enzymatic coagulation, the soluble calcium, in its ionic form,

SC
382 helps form the curd and favours cross bonds among the casein micelles. In cheese, the

383 decrease in the insoluble calcium content reduces the electrostatic interactions among the

384
U
casein molecules and improves the softness, while making the casein molecules more
AN
385 susceptible to proteolysis (Fathollahi, Hesari, Azadmardb, & Oustan, 2010). Some authors
M

386 suggest that the calcium bridges and the hydrophobic interactions are the bonds responsible

387 for stabilising the structure of the cheese’s protein network, whereas other authors indicate an
D

388 additional effect of the electrostatic interactions and the hydrogen bridges (Keim et al., 2006).
TE

389 The predominant bonds among the proteins in the curd at the stretching pH (pH 5.2)

390 were the hydrophobic interactions (Fig. 5A). The number of CaB was minimal, as was the
EP

391 number of disulphide bridges, which allowed the curd to undergo the stretching process,

considering that, at this pH, the number and/or the sum of the forces of all types of bonds was
C

392
AC

393 weaker. When the curd reached a pH of approximately 5.25, the number of calcium phosphate

394 bonds in the micelles (insoluble calcium) was minimal, which indicated that the percentage of

395 stabilised protein was minimal (Fig. 5B). The bonds among the micelles became weaker with

396 the pH decrease associated with the increase in the soluble calcium content, as a combined

397 effect, which caused the rigidity curd diminish, favouring fluidity when the tension of the

398 stretching process was applied in the curd.

17
ACCEPTED MANUSCRIPT
399 According to Yun, Kiely, Kindstedt, and Barbano (1993), the stretching stage of

400 mozzarella cheese is easier to achieve in the pH range between 5.0 and 5.3. In this range,

401 calcium phosphate becomes more dissociated and a decrease in the net charge on the proteins

402 helps increase the degree of hydrophobic interaction among the casein molecules, as also

403 shown in this study (Fig. 5). The stretching stage is probably responsible for the calcium

PT
404 bridges, the electrostatic interactions and the hydrogen bridges (Hinrichs & Keim, 2007) that

RI
405 are also predominant in mozzarella cheese (Fig. 5B). The additional effect of the electrostatic

406 interactions and/or the hydrogen bridges may have occurred because of the partial calcium

SC
407 removal during the stretching stage of the curd.

408 There was an increase in electrostatic interactions, hydrogen bonds and calcium bonds

409
U
between the curd at the stretching pH (CpH5.2) and the cheese after the stretching stage (Fig.
AN
410 5A,B). Therefore, the predominant bonds in the cheese were the calcium bonds, the
M

411 electrostatic interactions and the hydrogen bridges. These results agree with the casein micelle

412 model described by Horne (1998) and developed by Lucey et al. (2003) to explain the
D

413 cheeses’ texture. The same authors affirm that those bonds are the primary interactions that
TE

414 control the cheeses’ melting and behaviour at high temperatures. The force or the contribution

415 of each type of interaction is regulated by the residual charge in the casein molecule, the
EP

416 casein fractions and the storage temperature of the cheese. This residual charge is directly

influenced by the pH, the ionic force and the calcium bond (Lucey et al., 2003).
C

417
AC

418

419 4. Conclusions

420

421 There were changes in the structure and composition of the curd grains with

422 acidification. The protein matrix became more compact, and the size of the fat globules

423 decreased. The predominant bonds in the curd at the stretching pH were hydrophobic

18
ACCEPTED MANUSCRIPT
424 interactions, whereas the number of calcium bridges was minimal, which allowed the

425 stretching stage of the curd. The predominant bonds after the stretching stage were calcium

426 bridges, electrostatic interactions and hydrogen bridges. The additional effect of the

427 electrostatic interactions and hydrogen bridges were a consequence of the partial calcium

428 removal during the stretching stage of the curd. These results clarify important aspects of the

PT
429 bonds involved in the production of this type of cheese.

RI
430

431 Acknowledgements

SC
432

433 Author Mônica Correia Gonçalves thanks the National Council for Scientific and

434
U
Technologic Development (CNPq) for the financial support.
AN
435
M

436 References

437
D

438 AOAC. (2006a). Gerber method, method no. 2000.18. In W. Horwitz (Ed.), Official methods
TE

439 of analysis of AOAC International (18th edn., pp. 21–23). Gaithersburg, MD, USA:

440 AOAC International.


EP

441 AOAC. (2006b). Casein nitrogen content of milk, method no. 998.06. In W. Horwitz (Ed.),

Official methods of analysis of AOAC International (18th edn., p. 51). Gaithersburg,


C

442
AC

443 MD, USA: AOAC International.

444 AOAC. (2006c). Modified Mojonnier, method no. 989.05. In W. Horwitz (Ed.), Official

445 methods of analysis of AOAC International (18th edn., pp. 18–19). Gaithersburg, MD,

446 USA: AOAC International.

19
ACCEPTED MANUSCRIPT
447 AOAC. (2006d). Titrimetric method, method no. 920.124. In W. Horwitz (Ed.), Official

448 methods of analysis of AOAC International (18th edn., p. 73). Gaithersburg, MD,

449 USA: AOAC International.

450 AOAC. (2006e). Calcium, magnesium, and phosphorus in cheese, method no. 991.25. In W.

451 Horwitz (Ed.), Official methods of analysis of AOAC International (18th edn., p. 70).

PT
452 Gaithersburg, MD, USA: AOAC International.

RI
453 AOAC. (2006f). Direct forced air oven drying, method no. 990.19. In W. Horwitz (Ed.),

454 Official methods of analysis of AOAC International (18th ed., p. 35). Gaithersburg,

SC
455 MD, USA: AOAC International.

456 AOAC. (2006g). Kjeldahl method, method no. 2001.14. In W. Horwitz (Ed.), Official

457
U
methods of analysis of AOAC International (18th edn., pp. 72–73). Gaithersburg, MD,
AN
458 USA: AOAC International.
M

459 Bähler, B., & Hinrichs, J. (2013). Characterization of Mozzarella cheese curd by means of

460 capillary rheometry. International Journal of Dairy Technology, 66, 231–235.


D

461 Banville, V., Chabot, D., Power, N., Pouliot, Y., & Britten, M. (2016). Impact of thermo-
TE

462 mechanical treatments on composition, solids loss, microstructure, and rheological

463 properties of pasta filata type cheese. International Dairy Journal, 61, 155–165.
EP

464 Brasil. (2011). Ministério da Agricultura Pecuária e Abastecimento – MAPA – Instrução

Normativa n° 62, de 29 de setembro de 2011, que altera a IN 51 de 2002 e aprova os


C

465
AC

466 regulamentos técnicos de produção, identidade e qualidade do leite tipo a, do leite cru

467 refrigerado e seu transporte a granel. Brasília, Distrito Federal: Diário Oficial da

468 União em 29 de dezembro de 2011. Seção 1. [Ministry of Agriculture, Livestock and

469 Supply - MAPA - Normative Ruling No. 62, 29 September 2011 amending IN 51, 2002

470 and approving the technical regulations for production, identity and quality of cooled

471 raw milk, pasteurized milk and the technical regulation of cooled raw milk and

20
ACCEPTED MANUSCRIPT
472 transport in bulk tanks. Brasília, Federal District: Official Gazette, December 29,

473 2011. Section 1.].

474 Costa, R. G. B., Sobral, D., Paula, J. C. J., Silva, H. L. A., Guimarães, J. T., Cappato, L.

475 P., et al. (2017). Processamento de queijos. In A. G. Cruz, P. B. Zacarchenco, C.

476 A. F. Oliveira, & C. H. Corassin (Eds.), Processamento de Produtos Lácteos:

PT
477 queijos, leites fermentados, bebidas lácteas, sorvete, manteiga, creme de leite,

RI
478 doce de leite, soro em pó e lácteos funcionais (Vol 3, pp. 11–69). Rio de Janeiro,

479 Brazil: Elsevier Editora Ltda (In Portuguese).

SC
480 Fathollahi, I., Hesari, J., Azadmardb, S., & Oustan, S. (2010). Influence of proteolysis and

481 soluble calcium levels on textural changes in the interior and exterior of Iranian UF

482
U
white cheese during ripening. International Journal of Nutrition and Food
AN
483 Engineering, 66, 844–849.
M

484 Guinee, T. P. (2002). The functionality of cheese as an ingredient: A review. Australian

485 Journal of Dairy Technology, 57, 79–91.


D

486 Guinee, T. P., Feeney, E. P., Auty, M. A. E., & Fox, P. F. (2002). Effect of pH and calcium
TE

487 concentration on some textural and functional properties of Mozzarella cheese.

488 Journal of Dairy Science, 85, 1655–1669.


EP

489 Hinrichs, J., & Keim, S. (2007). Process-induced stabilizing bonds in fermented milk

products. Milchwissenschaft, 62, 422–425.


C

490
AC

491 Horne, D. S. (1998). Casein interactions: Casting light on the black boxes, the structure in

492 dairy products. International Dairy Journal, 8, 171–179.

493 Huppertz, T., & Kelly, A. L. (2006). Physical chemistry of milk fat globules. In P. F. Fox, &

494 P. L. H. McSweeney (Eds.), Advanced dairy chemistry. Vol. 3. Lipids (pp. 173–212.).

495 New York, NY, USA: Springer Science & Business Media, Inc.

21
ACCEPTED MANUSCRIPT
496 Jana, A. H., & Mandal, P. K. (2011). Manufacturing and quality of mozzarella cheese: A

497 review. International Journal of Dairy Science, 4, 199–226.

498 Johnston, K. A., Luckman, M. S., Lilley, H. G., & Smale, B. M. (1998). Effect of various

499 cutting and stirring conditions on curd particle size and losses of fat to the whey

500 during Cheddar cheese manufacture in Ost vats. International Dairy Journal, 8, 281–

PT
501 288.

RI
502 Joshi, N. S., Muthukumarappan, K., & Dave, R. I. (2003). Effect of calcium on

503 physicochemical properties of fat-free Mozzarella cheese. Journal of Food Science,

SC
504 68, 2289–2294.

505 Keim, S., Kulozik, U., & Hinrichs, J. (2006). Texture and stabilizing bonds in pressure-

506
U
induced, heat-induced and rennet-induced milk protein gels. Milchwissenschaft, 61,
AN
507 363–366.
M

508 Kindstedt, P. S. (2007). Low-moisture mozzarella cheese (LMMC). In P. L. H. McSweeney

509 (Ed.), Cheese problems solved (pp. 298–329). New York, NY, USA: CRC Press LLC,
D

510 Inc.
TE

511 Kindstedt, P., Carić, M., & Milanović, S. (2004). Pasta-filata cheeses. In P. F. Fox, P. L. H.

512 McSweeney, T. M. Cogan, & T. P. Guinee (Eds.), Cheese: Chemistry, physics and
EP

513 microbiology (Vol 2, pp. 251–277). London, UK: Elsevier Applied Press.

Lopez, C., Carmier, B., & Gassi, J. -Y., (2007). Development of the milk fat microstructure
C

514
AC

515 during the manufacture and ripening of Emmental cheese observed by confocal laser

516 scanning microscopy. International Dairy Journal, 17, 235–247.

517 Lopez-Sanchez, P., Svelander, C., Bialek, L., Schumm, S., & Langton, M. (2011). Rheology

518 and microstructure of carrot and tomato emulsions as a result of high-pressure

519 homogenization conditions. Journal of Food Science, 76, 130–140.

22
ACCEPTED MANUSCRIPT
520 Lourenço, J. P. M., & Wolfschoon-Pombo, A. F. (1982). Accuracy of volumetric

521 determination of casein in milk. Revista do Instituto de Laticínios Cândido Tostes, 37,

522 9–12 (In Portuguese).

523 Lucey, J. A., Johnson, M. E., & Horne, D. S. (2003). Perspectives on the basis of the rheology

524 and texture properties of cheese. Journal of Dairy Science, 86, 2725–2743.

PT
525 Ma, X., James, B., Zhang, L., & Emanuelsson-Patterson, E. A. C. (2013). Correlating

RI
526 mozzarella cheese properties to its production processes and microstructure

527 quantification. Journal of Food Engineering, 115, 154–163.

SC
528 Metzger, L. E., Barbano, D. M., Rudan, M. A., & Kindstedt, P. S. (2000). Effect of milk

529 preacidication on low fat mozzarella cheese. I. Composition and yield. Journal of

530 Dairy Science, 83, 648–658.


U
AN
531 Michalski, M. -C., Michel, F., Sainmont, D., & Briard, V. (2001). Apparent ζ-potential as a
M

532 tool to assess mechanical damages to the milk fat globule membrane. Colloids and

533 Surfaces B. Biointerfaces, 23, 23–30.


D

534 Mizuno, R., Matsuda, T., Lucey, J. A., & Ichihashi, N. (2009). Effects of changes in the
TE

535 distribution of soluble and insoluble calcium on Mozzarella cheese.

536 Milchwissenschaft, 64, 169–172.


EP

537 Renda, A., Barbano, D. M., Yun, J., Kindstedt, P. S., & Mulvaney, S. J. (1997). Influence of

screw speeds of the mixer at low temperature on characteristics of Mozzarella cheese.


C

538
AC

539 Journal Dairy Science, 80, 1901–1907.

540 Richardson, G. H. (1985). Standard methods for examination of dairy products. Washington,

541 DC, USA: American Public Health Association.

542 Rowney, M., Roupas, P., Hickey, M., & Everett, D. (2003). The effect of compression,

543 stretching, and cooking temperature on free oil formation in Mozzarella curd. Journal

544 of Dairy Science, 86, 449–456.

23
ACCEPTED MANUSCRIPT
545 Taras, M. J. (1995). Standard methods for the examination of water and wastewater.

546 Washington, DC, USA: American Public Health Association.

547 Valle, J. L. E. D., Campos, S. D. D. S., Yotsunagi, K., & Souza, G. D. (2004). Influence of

548 the fat level in the functional properties of Mozzarella cheese. Food Science and

549 Technology, 24, 669–673.

PT
550 Walstra, P. (2006). Dairy Science and Technology (2nd edn.). New York, NY, USA: CRC

RI
551 Press LLC, Inc.

552 Yun, J. J., Kiely, L. J., Kindstedt, P. S., & Barbano, D. M. (1993). Mozzarella cheese: impact

SC
553 of milling pH on functional properties. Journal Dairy Science, 76, 3639–3647.

U
AN
M
D
TE
C EP
AC

24
ACCEPTED MANUSCRIPT
Figure legends

Fig. 1. Sampling stages of the standardised and pasteurised milk (PM)1, curd after

cutting (CC)2, curd after partial whey removal and whey from the partial removal (CR

and WR, respectively)3 and curd samples during acidification (CpH5.8, CpH5.6, CpH5.4,

PT
CpH5.2, CpH5.1)4, whey after total removal when the curd reached pH (WpH5.2)5, stretching

water (SW)6 and cheese (C)7.

RI
SC
Fig. 2. Evolution of pH (A) and soluble calcium (B) during the processing stages of

Mozzarella cheese. Curd after cutting (CC), curd after whey removal (CR), samples of

U
the curds during acidification (CpH5.8, CpH5.6, CpH5.4, CpH5.2, CpH5.1) and cheese (C).
AN
Average values with different superscript letters are significantly different by the
M

Tukey’s test (P ≤ 0.05). The cheese processing was repeated twice.


D

Fig. 3. Micrographs of the curds and cheese: A, curd after cutting (CC); B, curd after
TE

partial whey removal (CR); C, curd during acidification (CpH5.8); D, curd during

acidification (CpH5.2); E, cheese after the stretching stage (C).


EP

Fig. 4. Apparent zeta-potential of curd after cutting (1; CC), curd after partial whey
C
AC

removal (2; CR), curd during acidification (3; CpH5.8), curd during acidification (4;

CpH5.2) and cheese after the stretching stage (5; C). Average values with different

superscript letters are significantly different by the Tukey’s test (P ≤ 0.05).

Fig. 5. Predominant bond types in (A) the curd at stretching pH (CpH5.2) and (B) the

cheese after the stretching stage: P(SS), disulphide bonds; P(Hy), hydrophobic
ACCEPTED MANUSCRIPT
interactions; P(CaB), calcium bridges; P(EB + HB), electrostatic interaction + hydrogen

bonds; P(ug), unbound protein. The cheese processing was repeated twice.

PT
RI
U SC
AN
M
D
TE
C EP
AC
ACCEPTED MANUSCRIPT

Table 1

Composition of the standardised and pasteurised milk, whey after partial removal and whey at stretching pH (pH 5.2). a

PT
Chemical composition Samples

RI
PM CR WpH5.2 SW

SC
Acidity (%) 0.14 (0.00) 0.11 (0.00) 0.20 (0.00) 0.025 (0.00)
pH 6.50 (0.02) 6.32 (0.03) 5.22 (0.04) 5.26 (0.01)
Total solids (%) 10.9 (0.11) 5.99 (0.22) 6.38 (0.19) 0.75 (0.10)

U
Casein (%) 2.17 (0.16) 0.01 (0.00) 0.01 (0.00) 0.01 (0.00)

AN
Fat (%) 2.31 (0.17) 0.36 (0.03) 0.28 (0.07) 0.01 (0.10)
Total calcium (%) 0.150 (0.005) 0.071 (0.009) 0.072 (0.011) 0.024 (0.007)

M
Insoluble calcium (%) 0.090 (0.019) 0.040 (0.014) 0.020 (0.003) 0.020 (0.007)
Apparent zeta-potential, mV -8.48 (0.65) -8.78 (0.36) -8.14 (0.18) -6.45 (0.20)

D
TE
a
Abbreviations are: PM, standardised and pasteurised milk; CR, whey after partial removal; WpH5.2, whey at stretching pH; SW, stretching water.
EP
The batch was repeated twice. Values are means with standard deviations in parentheses.
C
AC
ACCEPTED MANUSCRIPT

Table 2

Composition of the curds and cheese during the production stages of Mozzarella cheese. a

PT
Composition Samples

RI
CC CR CpH5.8 CpH5.6 CpH5.4 CpH5.2 CpH5.1 Cheese

Moisture (%) 86.83 (0.68)a 76.50 (0.65)b 66.00 (1.44)c 63.13 (0.80)c 56.88 (0.87)d 54.50 (0.07)de 54.95 (0.56)de 52.63 (0.39)f

SC
Total calcium (%) 0.105 (0.008)c 0.425 (0.035)b 0.475 (0.003)ab 0.467 (0.008)ab 0.483 (0.003)ab 0.499 (0.009)a 0.502 (0.008)a 0.498 (0.00)a
Insoluble calcium (%) 0.089 (0.008)c 0.393 (0.035)b 0.436 (0.004)ab 0.422 (0.011)ab 0.429 (0.006)ab 0.449 (0.016)ab 0.433 (0.013)ab 0.456 (0.011)a

U
Casein (%) 2.77 (0.15) g 9.30 (0.52)f 15.48 (0.39)e 17.43 (0.41)ed 18.56 (0.38)cd 20.78 (0.19)bc 22.39 (0.01)ab 24.25 (1.54)a

AN
Total calcium/casein 0.038 (0.005)ab 0.045 (0.001)a 0.030 (0.00)bc 0.026 (0.00)cd 0.026 (0.00)cd 0.024 (0.00)cd 0.022 (0.00)d 0.020 (0.009)d

Insoluble calcium/casein 0.032 (0.004)b 0.042 (0.001)a 0.028 (0.00)bc 0.024 (0.00)cd 0.023 (0.00)cd 0.021 (0.00)cd 0.019 (0.00)d 0.018 (0.009)d

M
Insoluble calcium/casein, dry 3.23 (0.47)b 4.22 (0.14)a 2.83 (0.04)bc 2.40 (0.00)cd 2.31 (0.01)cd 2.16 (0.04)cd 1.93 (0.05)d 1.88 (0.18)d
basis

D
d43 (µm) 7.58 (0.82)a 4.32 (0.27)bc 5.73 (0.35)ab - - 3.74 (0.01)bc - 2.79 (0.83)c

TE
EP
a
Abbreviations are: CC, curd after cutting; CR, curd after partial whey removal; CpH5.8, curd at pH 5.8; CpH5.6, curd at pH 5.6; CpH5.4, curd at pH

5.4; CpH5.2, curd at pH 5.2; CpH5.1, curd at pH 5.1; d43 (µm), average volume-weighted diameter. Values are means with standard deviation in
C
AC

parentheses; means with different superscript letters in the same row are significantly different by the Tukey’s test (P ≤ 0.05). The batch was

repeated twice.
ACCEPTED MANUSCRIPT

Standardised and Total whey


Curd salting
pasteurised milk1 removal5

Resting for
acidification Cooling in ice
Heating at 37 °C
until stretching bath
pH4

PT
Addition of
Partial whey
culture, CaCl2 Curd cutting Packaging7
removal3
and coagulant

RI
Stretching stage
Agitation Curd treatment Drying
of the curd6

SC
Coagulation Cooling in ice

U
Cutting2 Moulding
(35 min) bath
AN
Fig. 1.
M
D
TE
C EP
AC
ACCEPTED MANUSCRIPT

7.00
(A)
6.36a 6.33b

6.00 5.82c

PT
5.62d
5.40e
pH

RI
5.20f
5.09g
4.98h
5.00

U SC
4.00

AN
CC CD CpH5.8 CpH5.6 CpH5.4 CpH5.2 CpH5.1 C
Samples

M
Fig. 2. (A)

D
TE
C EP
AC
ACCEPTED MANUSCRIPT

0.080
(B) 0.068a
0.070
0.058ab
Soluble calcium (%)

0.060

PT
0.053abc
0.050 0.045bc 0.045bc

RI
0.039bc
0.040
0.031cd

SC
0.030

0.020 0.015d

U
0.010

AN
CC CD CpH5.8 CpH5.6 CpH5.4 CpH5.2 CpH5.1 C
Samples

M
Fig. 2B.

D
TE
C EP
AC
ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
M
D
TE
C EP
AC

Fig. 3. Micrographs of the curds and cheese: A, curd after cutting (CC); B, curd after partial

whey removal (CR); C, curd during acidification (CpH5.8); D, curd during acidification

(CpH5.2); E, cheese after the stretching stage (C).


ACCEPTED MANUSCRIPT

0.00
-1.00
-2.00

PT
Zeta-potencial (ζ)

-3.00
-4.00
-5.00

RI
-6.00
-7.00

SC
-6.48c
-8.00 -7.12bc
-9.00 -8.05ab
-8.13ab

U
-9.22a
-10.00

AN
1 2 3 4 5
Samples

M
Fig. 4.

D
TE
CEP
AC
ACCEPTED MANUSCRIPT

100 (A)
90
80
70
60

PT
50
Protein (%)

40
30

RI
20
10
0

SC
-10
-20
-30 P(SS) P(Hy) P(CaB) P(EB)+P(HB) P(ub)

U
-40

AN
-50
Curd at pH = 5.2 with 21.1% total protein

M
Fig. 5. (A)

D
TE
C EP
AC
ACCEPTED MANUSCRIPT

100
90 (B)
80
70
60

PT
50
Protein (%)

40
30

RI
20
10

SC
0
-10 P(SS) P(Hy) P(CaB) P(EB)+ P(HB) P(ub)
-20

U
-30
-40

AN
-50
Mozzarella cheese after the streching stage with 26.5% protein

M
Fig. 5. (B)

D
TE
C EP
AC