Food Research International 131 (2020) 109027

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Food Research International

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Possibilities for using ohmic heating in Minas Frescal cheese production T

a,b a,b b b
Ramon S. Rocha , Ramon Silva , Jonas T. Guimarães , Celso F. Balthazar ,
Tatiana C. Pimentelc, Roberto P.C. Netod, Maria Inês B. Tavaresd, Erick A. Esmerinob,
Mônica Q. Freitasb, Leandro P. Cappatoe, Rodrigo N. Calvacantif, Flávio N. Rodriguesg,
⁎
Renata S.L. Raicesa, Marcia C. Silvaa, Adriano G. Cruza,
a
Instituto Federal de Educação, Ciência e Tecnologia do Rio de Janeiro (IFRJ), Departamento de Alimentos, 20270-021 Rio de Janeiro, Brazil
b
Universidade Federal Fluminense (UFF), Faculdade de Veterinária, 24230-340 Niterói, Rio de Janeiro, Brazil
c
Instituto Federal de Educação, Ciência e Tecnologia do Paraná (IFPR), 87703-536 Paranavaí, Paraná, Brazil
d
Universidade Federal do Rio de Janeiro (UFRJ), Instituto de Macromoléculas Professora Eloisa Mano (IMA), 21941-598 Rio de Janeiro, Brazil
e
Instituto Federal Goiano (IFGOiANO), Rio Verde, 75909-120 Goiás, Brazil
f
Universidade Estadual de Campinas (UNICAMP), Faculdade de Engenharia de Alimentos (FEA), 13083-000 Campinas, Brazil
g
Instituto Federal de Educação, Ciência e Tecnologia do Rio de Janeiro (IFRJ), Departamento de Física, 20270-021 Rio de Janeiro, Brazil

A R T I C LE I N FO A B S T R A C T

Keywords: The use of ohmic heating (OH, 4, 8, or 12 V/cm, 72–75 °C/15 s) for pasteurization of milk intended for the
Ohmic heating manufacture of Minas Frescal cheese was investigated. The cheeses were characterized for the gross composition,
Minas Frescal cheese bioactive compounds (antioxidant, antihypertensive and antidiabetic activities), fatty acid profile, rheological
Processing parameters, volatiles profile, and sensory acceptance. OH decreased the elasticity, hardness, and firmness while
improved the sensory acceptance of Minas Frescal cheese. In addition, higher antioxidant, antihypertensive and
antidiabetic activities values as well as higher concentrations of short, medium and long-chain fatty acids were
observed. Overall, OH can be considered a suitable technology to be used in milk for the Minas Frescal cheese
processing.

1. Introduction compounds (off-flavor) and reduction of nutritional losses and de-

Minas Frescal cheese is a soft, fresh white Brazilian cheese, slightly
salty, slightly acidic, and with a shelf life of 14–21 days (Cunha-Neto
et al., 2020). It presents high yield, simple manufacturing process, low
manufacturing cost and good consumers’ acceptance, being commonly
produced from pasteurized milk (Lollo et al., 2015).
Conventional heat processing stands out as the most widely used
technique to ensure the microbiological safety of milk intended for
cheese making. However, it has disadvantages such as heating at high
temperatures, loss of nutritional compounds, and sensory changes
(Ahmad et al., 2019; Coutinho et al., 2018). Heating has a negative
effect on bioactive compounds because many of these substances are
thermosensitive and degrade under pasteurization conditions (Ferreira
et al., 2019). Ohmic heating (OH) is defined as a process in which an
electric current is passed through materials with the primary purpose of
heating them by converting electrical energy into thermal energy, re-
sulting in a rapid and uniform increase in temperature (Cappato et al.,
2017). Thus, there is little formation of undesirable aromatic
gradation of bioactive compounds and fatty acids (Cappato et al.,
2018). Widespread commercial adoption of OH is dependent on reg-
ulatory approval by the Food and Drug Administration (FDA) in the
United States and other regulatory agencies around the world. FDA has
suggested its usage for pumpable foods, such as fruit juices, egg, milk,
and aseptic packaging (Jambrak, Donsì, Paniwnyk, & Djekic, 2019).
Therefore, more studies are needed to prove its efficiency (Moreno-
Vilet, Hernández-Hernández, & Villanueva-Rodríguez, 2018).
The variables employed in OH can directly influence the char-
acteristics of the processed products (Cappato et al., 2018), with em-
phasis on the intensity of electric field and electrical conductivity
(Cappato et al., 2017). Studies on the application of OH in dairy pro-
ducts are still scarce and generally related to the processing of goat's
milk (Pereira, Martins, & Vicente, 2008), lactose-hydrolyzed milk and
ice cream (Suebsiri, Kokilakanistha, Laojaruwat, Tumpanuvatr, &
Jittanit, 2019) and whey dairy beverages (Cappato et al., 2018; Ferreira
et al., 2019). To the best of our knowledge, there are no studies on the
effect of milk processing using OH technology on Minas frescal cheese

⁎
Corresponding author.
E-mail addresses: food@globo.com, adriano.cruz@ifrj.edu.br (A.G. Cruz).

https://doi.org/10.1016/j.foodres.2020.109027
Received 1 December 2019; Received in revised form 20 January 2020; Accepted 21 January 2020
Available online 24 January 2020
0963-9969/ © 2020 Elsevier Ltd. All rights reserved.
R.S. Rocha, et al.
processing. Thus, the objective of this study was to evaluate the effect of
the intensity of ohmic heating parameters (OH, 4, 8, or 12 V/cm,
72–75 °C/15 s) of milk, on the quality parameters of Minas Frescal
cheese. Gross composition (moisture, protein and lipid), bioactive
compounds (antioxidant, antihypertensive, and antidiabetic activities),
physical characteristics (color and rheology), fatty acid profile, volatile
compounds, and sensory acceptance of the products were investigated.
2. Material and methods
2.1. Milk pasteurization by ohmic heating and conventional processing
Chilled raw milk (3.4% w/w) was purchased from the Barra Mansa
Milk Producers Cooperative, Barra Mansa - RJ and subjected to the
following treatments: conventional pasteurization (72–75 °C/15 s, Qc)
and ohmic heating (4 V/cm, 8 V/cm, and 12 V/cm, until reaching
72–75 °C/15 s, corresponding to the cheeses QI, QII, and QIII, respec-
tively) using the ohmic heating system located at the Food Processing
Laboratory of the Federal Institute of Rio de Janeiro. More detailis of
the ohmic system are available elsewhere (Costa et al., 2018). For
conventional pasteurization, the milks were heated using a water bath.
The ohmic heating equipment consisted of two 9 cm long “type 316”
stainless steel electrodes, a voltage source (DDP), a recipient for the
milk, a thermocouple, an agitator, and 3 multimeters for measuring the
variables (temperature, voltage and alternating current). The milk
(400 mL) was subjected to the ohmic treatments until it reached a
temperature of 72–75 °C/15 s. For all processes, after reaching the
temperature for the required time, the samples were immediately im-
mersed in an ice bath to promote rapid cooling to 7 °C, remaining at this
temperature for a maximum period of 4 h. A similar time–temperature
profile was adopted for all samples (Fig. 1, supplement material).
2.2. Energetic aspects
Energy expenditure was calculated as proposed by Martins et al.
(2019). The thermal load was calculated using Coxiella burnetii as the
Fig. 1. Temperature profiles of milk submitted to conventional processing and ohmic Heating for Minas Frescal cheese.
2
Food Research International 131 (2020) 109027
reference microorganism, with a z value (temperature range required
for the thermal destruction curve to cross a logarithmic cycle) of
4.34 °C. The heating curves were constructed using the experimental
data. The temperature profile was measured at 5 s intervals to the target
temperature of 72 °C, remaining at this temperature for 15 s. The
thermal load (F) was calculated as proposed by Costa et al. (2018),
based on the integrated lethal rate as a function of time.
2.3. Minas Frescal cheese processing
Minas Frescal cheese was produced according to the methodology
described by Dantas et al. (2016). For that, calcium chloride (0.2 g/
100 g, Labsynth, Sao Paulo, Brazil) and coagulant (3 g/L, Halamix,
Christian Hansen) were added to the pasteurized milk or OH-treated
milk, stirred, and kept at rest for 35 min at 37 °C for coagulation. Then,
the curd was cut, the whey was removed, and the remaining curd was
placed in 250 g plastic molds. The cheeses were dry-salted (0.8 g/100 g
NaCl, Labsynth), packaged, and stored in refrigerated chamber at 5 °C.
The products were evaluated on days 1, 7, and 14 of refrigerated sto-
rage. However, this study presented only the results from day 1 (ex-
cepted for TD-NMR), as the other characteristics remained stable during
storage and the differences were related to the type of processing used
(conventional or ohmic heating treatment).
2.4. Chemical composition, proteolytic activity and microbiological analysis
The chemical composition of cheeses (moisture, protein and fat) was
determined using traditional methods proposed by AOAC International
(2002). Proteolytic activity was determined after reaction with a re-
active solution (OPA). Amino acids and peptides were quantified by
absorbance readings of OPA derivatives at 340 nm (Sperry et al., 2018).
Mesophilic aerobes, total and thermotolerant coliforms and Salmonella
sp. were assessed according to the official methods (Brasil, 2003).
R.S. Rocha, et al.
2.5. Physical characteristics
2.5.1. Color
The color was determined using a portable colorimeter (CR-410,
MinoltaSensingKonica, Inc., Tokyo, Japan), obtaining the coordinates
L* (brightness), a* (green–red) and b* (blue-yellow). Chroma (C*,
Belsito et al., 2017), h index (Leclercq-Perlat, Corrieu, & Spinnler,
2004) and whiteness index (WI, Balthazar et al., 2017) were also cal-
culated.
2.5.2. Rheological properties
The rheological analyses of the cheese samples were carried out
according to the methodology described by Silva et al. (2017) using the
TA-XT21 Texture Analyzer (Stable Micro Systems Ltd., Surrey, UK)
previously calibrated with a 50 kg load cell, fixed platform, and a
36 mm diameter aluminum probe. Cylindrical samples (20 mm in
diameter, 24 mm height) were sampled, individually wrapped in PVC
film, and kept refrigerated at 10 ± 1 °C for at least 15 min before
analysis.
During the uniaxial compression test, the cheeses were compressed
to 80% of their height at a crosshead speed of 1 mm/s, and the force-
displacement data converted to true stress (σt) (Eq. (1)) and true strain
(εt) (Eq. (2)).
F (t ) F (t ) H (t )
σt = = added. The fatty acid profile was analyzed by gas chromatography
A (t ) A0 H0 (1)
H (t ) ⎞ H − ΔH ⎞
εt = ln ⎛ ⎜ | = | ln ⎛ 0
⎟ ⎜ ⎟
⎝ H0 ⎠ ⎝ H0 ⎠ (2)
where F(t) and H(t) correspond to the force applied and displacement
over time, respectively; A0 is the initial cross-sectional area and H0 is
the initial height of the sample; ΔH is the absolute deformation. The
parameters elasticity modulus (E), fracture stress (σf), fracture strain
(εf), and work of fracture (Wf) were calculated from the σt-εt curves. The
fracture point corresponds to the local maximum point (dσ/dε = 0) of
the curve data σt-εt, thus obtaining the σf and εf values. The modulus of
elasticity was calculated as the angular coefficient from the linear re-
gression of the data σt-εt to the point of fracture. The work of fracture
corresponds to the total fracture energy represented by the area below
the curve to the point of fracture.
The creep test was conducted by measuring the deformation before
the application of a constant force of 4.9 N for 180 s. After this period,
the force was removed, and the sample recovery was measured over
180 s. The results were expressed as compliance versus time, where
compliance corresponds to the ratio between the strain measured and
the initial tension applied (J(t) = γ(t)/σ0). The compliance data during
the creep phase were adjusted to Burgers model with 6 components, as
shown in Eq. (3).
t methodology described by Ferreira et al. (2019) and Cappato et al.
( ) (
J (t ) = J0 + J1 1 − e−t / τ1 + J2 1 − e−t / τ2 ) +
ηN (3)
where J(t) corresponds to compliance as function of time; J0 is the in-
stantaneous compliance of Maxwell spring portion; J1 and J2 are the
viscoelastic compliances representing compliance related to Kelvin-
Voigt element; τ1 and τ2 are the average times of delay associated with
the Kelvin- Voigt element; and ηN is the Newtonian viscosity associated
with Maxwell portion.
2.6. Bioactive compounds
The antioxidant activity of the samples was determined according to
the methodology described by Martins et al. (2018), through the re-
ducing ability of 1,1-diphenyl-2-picrylhydrazyl (DPPH). The inhibition
of Angiotensin-converting enzyme (ACE) was performed using the
methodology described by Ayyash, Abu-Jdayil, Hamed, and Shaker
(2018) and Cappato et al. (2018). The α-amylase and α-glucosidase
3
Food Research International 131 (2020) 109027
enzymes inhibition were determined according to the methodology of
Ayyash et al. (2018) and Ferreira et al. (2019).
2.7. Time domain nuclear magnetic resonance (TD-NMR)
TD-NMR measurements were performed in a low field spectrometer
operating at 23.4 MHz for proton (MARAN Ultra 0.54 T) with 7.5 μs
and 90° pulse length (Cappato et al., 2018). All cheese samples were
subjected to transversal relaxation time (T2) through the Carr-Purcell-
Meiboom-Gill (CPMG) pulse sequence with 5120 echoes, 600 μs be-
tween each echo and 8 scans with a waiting time of 2 s. Laplace Inverse
Transformation was used to determine the distribution curve of re-
laxation domains using the exponential decay curve obtained by echo
recording by WinDXP and OriginLab Origin Pro 8.5 softwares. The
cheese samples were grated for 3 s before analysis using a multi-
processor at medium speed (Walita, São Paulo, Brazil).
2.8. Fatty acid profile
Fat extraction was performed according to the methodology de-
scribed by Florence et al. (2012). For that, 1 g of the sample from each
treatment was weighed into Falcon tubes, and 50 μL of a standard so-
lution containing heptadecanoic acid and sorbic acid (6 mg/mL), 4 mL
methanol, 2 mL dichloromethane, and a small amount of BHT were
(Agilent Technologies® 5975C), according to methodology adapted
from Silveira et al. (2019). The gas flow rates used were 1.4 mL/min for
the carrier gas (H2); 30 mL/min for the auxiliary gas (N2) and 30 and
300 mL/min for H2 and flame synthetic air, respectively. The split ratio
was 1/80.
The detector and injector temperatures were 235 °C. The column
temperature was set at 65 °C for 4 min, followed by the first ramp of
16 °C/min to 185 °C, thus remaining for 12 min. A second ramp was
programmed from 20 °C/min to 235 °C, thus remaining for 14 min. The
peak areas detected by the equipment were determined by the Star
software (Varian). The injection volume was 2 uL. The identification of
fatty acids was based on the comparison of retention times of standard
methyl esters containing linoleic acid geometric isomers c9t11 and
t10c12 (189-19 and O-5626, Sigma, USA) and equivalent chain length
values (ECL) (Sperry et al., 2018).
The atherogenic index (AI, Batista et al., 2017), thrombogenic index
(TI, Batista et al., 2017), desired fatty acids (DFA, Barłowska et al.,
2018) and hypercholesterolemic saturated fatty acids (HSFA, Barłowska
et al., 2018) were calculated.
2.9. Volatile compounds
The profile of volatile compounds was determined according to the
(2018). For that, the cheeses were subjected to the separation of the
volatile constituents by gas chromatography (Agilent Technologies
5975C) from the injection of 1 μL of the extract. The extracts were
obtained using Sulpeco® divinylbenzene/carboxy/poly-
dimethylsiloxane (DVB/CAR/PDMS) fibers with 50/30 μm thickness
and 20 mL headspace vials in an automated CTC PAL Sampler (Agilent
Technologies SPME 120). The volatile compounds were identified using
the Agilent Mass Hunter Qualitative Analysis software (Agilent Tech-
nologies) and compared to the experimental spectra with the National
Institute of Standards and Technology Library (NIST/EPA/NIH Mass
Spectral Library, version 11, USA) using linear retention indices (LRI).
2.10. Sensory evaluation
Sensory evaluation was performed by an acceptance test using a 9-
point hedonic scale (1 = extremely disliked, 9 = extremely liked,
Lucatto et al., 2020) with 92 consumers (50 male, 42 female), with age
R.S. Rocha, et al. Food Research International 131 (2020) 109027

from 15 to 50 years, with majority (52%) aging 15–25 years. The
samples were served in cubes (15 g) at room temperature and mon-
adically coded with three random digits, using complete balanced
blocks. This project was approved by the IFRJ Ethics Committee (re-
gistration number: 72095317.0.0000.5268).
2.11. Statistical analysis
The experiment followed a completely randomized design and was
repeated three times, and the analyses were performed in triplicate.
Data were submitted to analysis of variance (ANOVA) and Tukey's test
(p < 0.05) using XLSTAT 2019.2 software (Adinsoft, Paris, France).
3. Results and discussion
3.1. Thermal profile and energy expenditure
The thermal profile of raw milk submitted to pasteurization or
ohmic heating is shown in Fig. 1. The application of 12 V/cm resulted
in a decrease in the time required to reach the process temperature
(3.17 min) when compared to the conventional heat treatment
(4.67 min). The application of 4 and 8 V/cm increased the time re-
quired to reach the process temperature (6.67 and 27 min, respec-
tively). The total energy used was 1290 kJ and 102.05, 141.64, and
118.38 kJ for the conventional treatment and the ohmic treatment at 4,
8 and 12 V/cm, respectively. The results indicate lower energy ex-
penditure with the ohmic treatment, while the time required to reach
the appropriate temperature is dependent on the intensity of the elec-
tric field used. The very significant increase in process time, as observed
for the low electric field (4 V/cm) may hinder the industrial application
of the technology (Costa et al., 2018).
The thermal load (F value) of pasteurization and ohmic heating
processes was 31.36 s, and 43.54 s (4 V/cm), 37.91 s (8 V/cm), and
25.60 s (12 V/cm), respectively. The thermal load must meet the cri-
teria established for dairy products, which report 15 s as minimum
value (Codex Alimentarius, 2003). The results indicate that all treat-
ments were efficient against C. burnetti, once the minimum values were
reached. However, F values increased in lower electric field treatments
(4 V/cm and 8 V/cm) due to longer time to reach the desired tem-
perature (72–75 °C) (Costa et al., 2018).
3.2. Chemical composition, proteolytic activity and microbiological analyses
Table 1 shows the results of chemical composition and proteolytic
activity of the cheeses. Minas Frescal cheeses showed chemical com-
position in the following range (g/100 g): moisture (56–61), protein
(14–18) and lipids (16.9–17.5), corroborating previous studies (Dantas
et al., 2016; Sperry et al., 2018). There was no effect of the type of milk
processing (pasteurization or ohmic heating) on the moisture and lipid
contents of the cheeses (p > 0.05, except QI for moisture). Cheeses
submitted to OH presented a lower protein content and a higher pro-
teolytic activity (p < 0.05). The application of the ohmic treatment
can increase the hydrolysis of proteins (Ferreira et al., 2019), resulting
in the formation of smaller peptides. The highest proteolysis can be
positive, since OH-treated cheeses would have greater digestibility and
a higher concentration of bioactive peptides (Sperry et al., 2018). The
cheeses made with milk submitted to 4 V/cm (QI) presented the lowest
protein content and the highest proteolytic activity (p < 0.05),
probably because of the highest time necessary to reach the processing
temperature, resulting in a cheese submitted to heating for a more
prolonged time.
The cheese made with pasteurized milk presented 3.34 log CFU/g
for mesophilic bacteria count and 2.80 log CFU/g for total coliforms,
and the cheeses made with OH-treated milk presented counts in the
range of 2.49–3.70 log CFU/g and 2.28–2.54 log CFU/g for mesophilic
bacteria count and total coliforms, respectively. The cheeses made with
milk treated by pasteurization or OH had < 3 MPN/g for thermo-
tolerant coliforms and no Salmonella sp. in 25 g of product. The mi-
crobial counts are in accordance with the Brazilian legislation for Minas
Frescal cheeses (Brasil, 1996), which establishes values < 5 log/g for
total and thermotolerant coliforms. Mesophilic bacteria counts are not
limited by legislation, but low counts indicate heat treatment efficiency
and good manufacturing practices. Therefore, the microbiological
safety of the cheeses prepared with milk treated by pasteurization or
OH could be observed.
3.3. Physical characteristics
3.3.1. Color parameters
The results of the color parameters are presented in Table 2. The
cheeses were light and slightly yellow in color (L* = 94.70–101.49,
b* = 9.02–11.74). Cheeses made with milk subjected to ohmic heating
at 4 and 8 V/cm (QI and QII) presented higher b* and C* values
(p < 0.05) when compared to the conventional cheese (Qc), indicating
that they became more yellowish and more intense in color. Chroma
(C*) is a quantitative color attribute directly related to the color in-
tensity of the samples (Belsito et al., 2017). The cheese submitted to
intermediate conditions (8 V/cm) also presented higher luminosity (L*,
p < 0.05). The cheese made from milk submitted to 12 V/cm pre-
sented all color parameters similar to the control (p > 0.05). The re-
sults indicate that milder ohmic processing conditions caused changes
in cheese color. The opaque white color of milk is related to the pre-
sence of proteins, especially casein. With the occurrence of proteolysis,
there is a change in protein composition, which may cause changes in
the color of products (Costa et al., 2019). Thus, the higher proteolysis of
OH-treated cheeses resulted in changes in the color of the samples QI
and QII (Table 1). Furthermore, the highest time elapsed until reaching
the pasteurization temperature in cheeses made with milk subjected to
ohmic heating at 4 and 8 V/cm could have contributed to the color
alterations (Fig. 1).
All cheeses presented similar h and WI parameters (p > 0.05),
indicating that the type of milk used in cheese making (pasteurized or
subjected to ohmic heating) has no impact on these color parameters.
The whiteness index (WI) correlates directly with consumer preference
for products characterized by the white color (Belsito et al., 2017), such
as Minas Frescal cheese. This result is interesting for commercialization

Table 1
Color parameters of the Minas Frescal cheese using conventional processing and ohmic heating.
Tratamentos L* a* b* h C* WI

b b b a b
Qc 94.70 ± 0.20 5.60 ± 0.02 9.02 ± 0.02 35.51 ± 0.18 10.62 ± 0.01 88.13a ± 0.10
QI 97.31b ± 0.18 5.16c ± 0.02 11.39a ± 0.11 25.94a ± 0.15 12.50a ± 0.10 87.21a ± 0.07
QII 101.49a ± 0.60 5.90a ± 0.06 11.74a ± 2.74 24.45a ± 1.52 12.83a ± 1.89 87.07a ± 1.90
QIII 96.22b ± 3.02 5.48b ± 0.03 10.04ab ± 0.02 31.24a ± 0.14 11.43ab ± 0.03 87.71a ± 0.72

*Values are expressed as mean ± standard deviation. Different lowercase letters in the same line denote statistical difference (p < 0.05) between treatments
(cheeses) according the Tukey test. Qc, QI, QII, QIII = Qc, QI, QII, QIII = Minas frescal cheese submitted to conventional processing and Ohmic heating (4, 8, 12 V/
cm, 72–75 °C/15 s). L*, a*, and b* correspond to luminosity, yellow intensity, and red intensity, respectively. h∗ = hue angle, arctan (b∗/a∗), C∗ = Chroma,
(a∗2 + b∗2)1/2, WI = white index.

4
R.S. Rocha, et al. Food Research International 131 (2020) 109027

Table 2
Rheological parameters estimated by uniaxial compression and creep test of Minas Frescal cheese manufactured with conventional processing and ohmic heating.
Parameters Qc QI QII QIII

a c bcd
E (kPa) 15.66 ± 2.13 8.33 ± 0.04 7.84 ± 2.18 9.37b ± 2.01
εf (-) 0.59d ± 0.02 0.76a ± 0.09 0.63abc ± 0.10 0.65b ± 0.01
σf (kPa) 9.11a ± 1.14 5.75b ± 0.38 bc
4.55 ± 1.18 5.29c ± 0.01
Wf (kJ m−3) 2.68a ± 0.39 2.19a ± 0.42 1.42b ± 0.37 1.72b ± 0.01
J0 × 10−5 (Pa−1) 1.39a ± 0.28 1.52a ± 0.10 1.54a ± 0.06 1.49a ± 0.14
J1 × 10−5 (Pa−1) 8.71a ± 3.11 9.36a ± 0.73 8.64a ± 2.15 6.37a ± 2.67
τ 1 (–) 4.53b ± 1.13 6.44b ± 0.57 4.18b ± 1.02 11.83a ± 3.38
J2 × 10−5 (Pa−1) 5.69c ± 0.57 9.29a ± 0.79 6.10bc ± 2.40 9.03ab ± 3.63
τ2 (–) 28.10a ± 10.04 6.44c ± 0.57 9.79bc ± 3.25 4.72c ± 0.84
ηN × 106 (Pa·s) 3.03a ± 1.44 1.39b ± 0.11 2.09a ± 0.32 2.20a ± 0.24

*Values are expressed as mean ± standard deviation Different lowercase letters in the same line denote statistical difference (p < 0.05) between treatments
(cheeses) according the Tukey test. Qc, QI, QII, QIII = Qc, QI, QII, QIII = Minas frescal cheese submitted to conventional processing and Ohmic heating (4, 8, 12 V/
cm, 72–75 °C/15 s). E, εf, σf, Wf = Modulus of elasticity, Fracture strain, fracture stress and fracture work. J0, J1, τ, ηN = instantaneous compliance, viscoelastic
compliance, retarded time and Newtonian viscosity.

of products subjected to ohmic heating, once they exhibited similar WI
when compared to the conventional product.
3.3.2. Rheological parameters
Table 3 shows the uniaxial compression parameters for all samples,
calculated from the stress-strain curves. The conventional cheese (Qc)
showed significant (p < 0.05) higher modulus of elasticity (E), frac-
ture stress (σf) and fracture work (Wf) when compared to the other
samples. Thus, the ohmic heating processing led to a significant re-
duction of elasticity, hardness, and firmness of the cheeses, respec-
tively. This behavior may be due to a possible conformational change
on the protein molecules and their intermolecular interactions, which
can directly affect the water retention capacity and protein matrix
strength. Furthermore, it can be related to the higher proteolytic ca-
pacity of the cheeses made with milk submitted to OH (Table 1). The
control sample exhibited the lowest values of fracture strain (εf)
(p < 0.05) and, consequently, the highest crispness among the sam-
ples.
The compliance data of the samples during the creep test were fitted
by nonlinear regression to a six-component Burgers model
(0.982 < R2 < 0.999) and are presented in Table 3. These parameters
represent the rheological properties of viscoelastic materials, in which
cheese composition and process characteristics are the most likely in-
fluencing factors. The instantaneous compliance (J0) represents the
instantaneous voltage at the initial deformation time, probably due to
the structured portion of the non-disturbed protein network. Thus, a
higher J0 value reflected in a greater elastic deformation according to
the Hooke's law, indicating that the polypeptide interactions are com-
paratively free to rearrange in the network. In other words, the in-
stantaneous compliance is inversely related to the stiffness of the ma-
terial (Silva et al., 2017). No significant difference (p > 0.05) was
observed for J0 of all treatments.
The retardation time τ1 and τ2 were affected by the type of milk or
process parameters of OH (p < 0.05). Minas Frescal cheeses submitted
to OH presented lower τ2 than the conventional cheese (p < 0.05). In
general, the higher the retardation time of a system, the longer it takes
to reach full deformation on the application of shear stress (Olivares,
Zorrilla, & Rubiolo, 2009). Therefore, OH treatment decreased the time
to reach full deformation.
The Newtonian viscosity of the free dashpot (ηN) represents the
mechanical behavior of the fluid part of the system, that is, is associated
with the breakdown of the protein network structure and characterizes
the linear region of the viscous compliance. The (ηN) of the sample
treated at 4 V/cm (QI) was significantly lower in comparison to the
other samples (p < 0.05), which represents lower resistance to flow,
thus, a less solid-like behavior.
3.4. Bioactive compounds
The bioactive compounds values of Minas Frescal cheeses are shown
in Table 4. Cheeses made with milk submitted to ohmic heating at 4 and
8 V/cm (QI and QII) presented higher content of phenolic compounds
(86.2–92.6 µg/g), antioxidant activity (35.8–36.5%), α-amylase in-
hibitory activity (54.8–56.5%), α-glucosidase inhibitory activity
(68.7–69.5%) and ACE inhibitory activity (25.4–26.7%) when com-
pared to the cheese made with pasteurized milk (Qc, p < 0.05). The
results indicate that milder OH processing conditions increased the
functional properties of cheese. OH can increase the antioxidant capa-
city of dairy products by releasing certain bioactive compounds, such as
small peptides or phenolic compounds (Ferreira et al., 2019). In addi-
tion, the application of OH can influence unfolding, denaturation, and
aggregation kinetics of proteins, which may induce the formation of
bioactive peptides with ACE inhibitory activity (Costa et al., 2018). The
ACE inhibitory activity is important because it decreases the incidence
of hypertension and associated diseases (Rivera, Martin, & Landry,
2019). The presence of bioactive compounds in the serum fraction also
contributes as a binding component, showing affinity with the active
site of the α-amylase and α-glucosidase enzymes (Ferreira et al., 2019).
The α-amylase and α-glucosidase enzymes are located in the basolateral
sides of the intestine and are responsible for the hydrolysis of oligo-
saccharides and disaccharides, which are subsequently absorbed by the
intestine. The higher inhibition of these enzymes provided by OH may

Table 3
Bioactive compounds of the Minas Frescal cheese manufactured with conventional processing and ohmic heating.
Cheese Total Phenolic Count DPPH α-glucosidase α-amylase ACE

c ab b b
Qc 62.2 ± 0.52 32.0 ± 0.24 64.6 ± 0.26 40.8 ± 0.27 12.5b ± 0.42
QI 92.6a ± 0.31 36.5a ± 0.66 69.5a ± 0.71 54.8a ± 0.39 26.7a ± 0.52
QII 86.2b ± 0.38 35.8a ± 0.18 68.7a ± 0.82 56.5a ± 0.81 25.4a ± 0.15
QIII 67.2c ± 0.2 36.8a ± 0.11 59.1bc ± 0.83 44.6b ± 0.20 18.1b ± 0.26

* Values are expressed as mean ± standard deviation. Different lowercase letters in the same line denote statistical difference (p < 0.05) between treatments
(cheeses) according the Tukey test. Qc, QI, QII, QIII = Minas frescal cheese submitted to conventional processing and Ohmic heating (4, 8, 12 V/cm, 72–75 °C/15 s).
Total Phenolic count are expressed in µg Acid Gallic/g while DDPH, the α-glucosidase, α-amylase and ACE are expressed in %.

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R.S. Rocha, et al. Food Research International 131 (2020) 109027

Table 4
Fatty acid profile of the Minas Frescal cheese manufactured with conventional processing and ohmic heating.
Fatty acid profile Qc QI QII QIII

c ab a
Butanoic (C4:0) 5.33 ± 0.2 6.24 ± 0.3 6.74 ± 0.5 5.56bc ± 0.4
Hexanoic (C6:0) 3.94b ± 0.5 4.72ab ± 0.7 4.92a ± 0.7 4.08b ± 0.9
Octanoic (C8:0) 1.34b ± 0.8 1.63ab ± 0.5 1.71a ± 0.4 1.47ªb ± 0.3
Docanic (C10:0) 3.08c ± 0.1 4.11a ± 0.3 4.03ab ± 0.4 3.37bc ± 0.6
Dodecanoic (C12:0) 2.08b ± 0.7 2.67a ± 0.9 2.45ab ± 0.7 2.15b ± 0.4
Tetradecanoic (C14:0) 8.96b ± 1.1 11.92a ± 1.2 11.49a ± 1.5 9.44b ± 1.8
Pentadecanoic (C15:0) 0.79ª ± 0.5 0.58b ± 0.6 0.44c ± 0.9 0.80a ± 0.7
Palmitic (C16:0) 22.82b ± 1.4 29.26a ± 1.4 29.13ª ± 1.3 23.65b ± 1.5
Palmitoleic (C16:1) 2.01ª ± 0.5 1.41b ± 0.4 1.17b ± 0.7 1.95a ± 0.7
Stearic (C18:0) 15.29b ± 1.3 16.76ab ± 1.6 18.37a ± 1.4 15.35b ± 1.4
Oleic (C18:1) 33.32a ± 1.1 20.02b ± 1.6 19.07a ± 1.4 31.36a ± 1.6
Conjugated linoleic (CLA) 0.75a ± 0.5 0.62ab ± 0.9 0.43b ± 0.8 0.75a ± 0.9
Short chain fatty acid (SCFA) 5.33c ± 0.4 6.24ab ± 0.6 6.74ª ± 0.7 5.56bc ± 0.7
Medium chain fatty acid (MFCA) 10.46b ± 0.4 13.14a ± 0.3 13.12ª ± 0.8 11.09ab ± 0.6
Long chain fatty acid (LCFA) 8.96b ± 1.1 11.92ª ± 1.5 11.49ª ± 1.3 9.44b ± 1.8
Monounsaturated fatty acid (MUFA) 35.34ª ± 1.2 21.44b ± 1.4 20.25b ± 1.4 33.31ª ± 1.7
Polyunsaturated fatty acid (PUFA) 0.75a ± 0.04 0.62ab ± 0.05 0.43b ± 0.04 0.75ª ± 0.6
AI 1.68b ± 0.04 3.61a ± 0.88 3.75a ± 0.04 1.87b ± 0.24
TI 2.61b ± 0.06 5.25a ± 1.22 5.71a ± 0.08 2.84b ± 0.06
DSA 51.38a ± 0.33 38.82b ± 4.04 39.05b ± 2.33 49.41a ± 0.43
HSFA 33.86b ± 0.41 43.85a ± 2.58 43.07a ± 1.37 35.24b ± 0.10

*Values are expressed as mean ± standard deviation (n = 6). Different lowercase letters in the same line denote statistical difference (p < 0.05) between
treatments (cheeses) according the Turkey test. The fatty acid composition was classified according to Felicio et al. (2016) who described SCFA as C2 to C4, MCFA as
C6 to C12 and LCFA as C14 to C24. Qc, QI, QII, QIII = Minas frescal cheese submitted to conventional processing and Ohmic heating (4, 8, 12 V/cm, 72–75 °C/15 s).
SFA: saturated fatty acid; MUFA: monounsaturated fatty acid; PUFA: polyunsaturated fatty acid. AI, TI = Atherogenic index, Thrombogenic index, respectively.
AI = (C12:0 + 4 C14:0 + C16:0)/[∑MUFA + ∑PUFA(n − 6) and (n − 3)]; TI = (C14:0 + C16:0 + C18:0)/[0.5 ∑MUFA + 0.5 ∑PUFA(n − 6) + 3
∑PUFA(n − 3) + (n − 3)/(n − 6)]. DFA = MUFA + PUFA + C18:0; HSFA = C12:0 + C14:0 + C16:0.

result in lower polysaccharide and disaccharide hydrolysis and glucose

absorption, contributing to the maintenance of blood glucose levels
(Zeng et al., 2016).
Minas Frescal Cheese processed from milk submitted to 12 V/cm
showed total phenolic content, antioxidant activity, α-amylase in-
hibitory activity, α-glucosidase inhibitory activity and ACE inhibitory
activity similar to the cheese made with pasteurized milk (p > 0.05).
In general, the results suggest that the use of larger electric fields in OH
resulted in degradation of the bioactive compounds of cheeses, similar
to that observed in the cheeses made from milk subjected to conven-
tional pasteurization. High OH intensity may increase the degradation
of bioactive compounds with antioxidant capacity (Costa et al., 2018).

3.5. Time domain nuclear magnetic resonance (TD-MR)

Fig. 2 presents the TDN-NMR results. It could be observed two

different relaxation environments (domains) for all cheese samples. One
domain was located at the region of 0.1–100 ms (T2.1), and the other
(T2.2) in 100–700 ms. The OH-treatment of milk did not alter the basic
characteristics of the Minas Frescal cheese matrix. Cheeses made with
milk subjected to ohmic heating at 4 and 8 V/cm (QI and QII) presented
no significant variations in the matrix compared to the control (Qc),
both in relation to the number of relaxation domains or to the re-
spective relaxation times. Cheeses treated with 12 V/cm (QIII) showed Fig. 2. Distribution curves of transverse relaxation domains of TD-NMR of
more marked variations depending on the storage days for the popu- Minas Frescal cheese made with milk submitted conventional processing and
ohmic heating (D1, D7, D14 = 1, 7, 14 days of refrigerated storage).
lation of hydrogen in the confined water, with a reduction in the re-
laxation time from 15 to 10 ms, and a more pronounced reduction in
the percentage of free water as a function of aging. These reductions pasteurized milk (Qc, p < 0.05). The results are mainly related to the
indicate that the 12 V/cm treatment tends to provide a cheese more increase in butanoic, docanoic, tetradecanoic, and palmitic acid con-
susceptible to dryness during storage. tents, and the decrease of pentadecanoic and palmitoleic acids
(p < 0.05). Cheese made with milk submitted to 12 V/cm had a fatty
3.6. Fatty acid profile acid profile similar to cheese made with pasteurized milk (p > 0.05).
The results indicate that the fatty acid profile is altered in cheeses made
The results of the fatty acid profile are presented in Table 5. Cheeses with milk submitted to low or medium intensity electric fields (4 or
made with milk subjected to ohmic heating at 4 and 8 V/cm (QI and 8 V/cm), with an increase in saturated fatty acids content and a de-
QII) presented higher concentration of SCFA, MCFA, and LCFA and crease in unsaturated fatty acids. Saturated fatty acids may be more
lower MUFA concentration when compared to the cheeses made with stable under low or intermediate OH conditions when compared to the

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R.S. Rocha, et al. Food Research International 131 (2020) 109027

Table 5 Table 6
Volatile compounds profile of Minas Frescal cheese manufactured with con- Sensory acceptance of the Minas Frescal cheeses
ventional processing and ohmic heating. Minas Frescal cheese manufactured with con-
ventional processing and ohmic heating.
Cheeses
Cheese Overall liking
Groups Compounds Qc QI QII QIII
Qc 6.26b ± 1.24
Acids Acids QI 7.02a ± 1.32
Acetic acid – – – X QII 7.33a ± 1.01
Butanoic acid – – X X QIII 7.72a ± 1.12
3-Methylethyl ester butanoic acid – – – X
Oxalic acid, allyl isobutyl ester – X X X *Values are expressed as mean ± standard de-
Pentanoic acid, 2-methyl-, anhydride – X X X viation. Mean values from 92 consumers being
Butanoic acid, 3-methyl- X – X X
based on a 9-point hedonic scale (1 = dislike
Total 5 4 2 0
extremely; 5 = neither like nor dislike; 9 = like
Ketones Octane, 2,7-dimethyl- – X X X extremely. Means with different lowercase su-
Acetoin X X X – perscripts in the same column indicate presence
Methyl vinyl ketone – – X –
of statistical difference (p < 0.05, n = 92) ac-
4-Heptanone, 2,6-dimethyl- X X X –
cording the Tukey test. Qc, QI, QII, QIII = Qc, QI,
2-Butanone, 3-methoxy-3-methyl- – – – –
1-Pentene, 4,4-dimethyl- X – X – QII, QIII = Minas frescal cheese submitted to
1-Octene, 6-methyl- X X – X conventional processing and Ohmic heating (4, 8,
Ethanone, 1-cyclopropyl-2-(4-pyridinyl)- X – X 12 V/cm, 72–75 °C/15 s).
Propane, 2-methoxy- X X X –
Oxirane, (methoxymethyl)- – X X – (1.68–3.75), TI (2.61–5.71), and HSFA (33.86–43.85) levels, as well as
Heptane, 2,5,5-trimethyl- X X X –
Total 4 4 2 8
relatively high DFA levels (38.82–51.38).

Alcohols 1-Butanol 3-methyl acetate X – X –

3.7. Volatile compounds
Benzyl alcohol X – X X
Phenylethyl alcohol – – X –
1-Butanol, 3-methyl- X – X – The profile of volatile compounds of the Minas Frescal cheese
1,1-Ethanediol, diacetate X – X X samples is presented in Table 6. Twenty-nine volatile compounds were
Total 1 5 0 3 identified: 6 acids, 11 ketones, 5 alcohols, 1 terpene, and 6 ungrouped
Terpens D-Limonene X X X 10.90 compounds. All cheeses presented the compound 2-butanone, 3-
Total 0 0 0 1 methoxy-3-methyl, which is associated with sweet aroma (Bertuzzi,
Others Guanidine carbonate – X X – McSweeney, Rea, & Kilcawley, 2018).
Estragole – X X X Cheese made from milk subjected to 4 V/cm (QI) presented the
Cyclopropane, propyl- – X X X
highest number of volatile compounds (n = 16), possibly due to the
Cyclobutane, 1,2-bis(1-methylethenyl)-, trans- X – X X
4-Heptanone, 2,6-dimethyl- X – X X formation of new compounds and preservation of existing ones
1,3,5,7-Cyclooctatetraene X – X X (Ferreira et al., 2019). Indeed, 1-pentene, propane, 1-butanol 3-methyl
Total 3 3 0 1 acetate, benzylalcohol, 1-butanol 3-methyl, 1,1-ethanediol, diacetate,
and other compounds such as cyclobutane, 4-heptanone and 1,3,5,7-
LRI/=Linear Retention Index on CP-Wax 52 CB according to the Van der Dool
cyclooctatetraene were formed. These compounds are associated with
and Kratz equation. Organized by families. -=presence, X = absence. Qc, QI,
the aroma of fresh cheese (3-methyl 1-butanol, Ranadheera et al.,
QII, QIII = Minas frescal cheese submitted to conventional processing and
Ohmic heating (4, 8, 12 V/cm, 72–75 °C/15 s). 2019), and flowery and sweet aromas (benzyl alcohol, Celik, Cabaroglu,
& Krieger-Weber, 2019).
pasteurization process or more drastic OH conditions (Ferreira et al., Cheese made with milk subjected to 8 V/cm had the lowest number
2019). In addition, the low- or medium-intensity electric field may have (n = 4) of volatile compounds, due to the disappearance of a large
broken double bonds, altering the MUFA's molecular structure and number of compounds. Some compounds are important for cheese
decreasing its concentration (Ferreira et al., 2019). Saturated fatty acids aroma, such as phenyl ethyl alcohol (pleasant 'floral' aroma, Bozoudi
play an important role in cholesterol metabolism, insulin resistance, et al., 2018), while others are characteristic of off-flavor, such as bu-
and molecular mechanisms in the body, and the SFA levels are asso- tanoic acid (rancid cheese-like flavor).
ciated with obesity-related diseases, including osteoarthritis (Ferreira Cheese made with milk subjected to 12 V/cm had a similar number
et al., 2019). Short-chain fatty acids, in turn, have the ability to reduce of volatile compounds as the conventional product (n = 13), despite
the risk of cancer (Faraz et al., 2018). the different profiles. The cheese QIII showed the absence of acid group
Regarding health-related indices, the cheeses QI and QII presented compounds and increased amount of ketones and alcohols. Ketones are
higher AI, TI, and HSFA, and lower DFA levels (p < 0.05, Table 5). In generated from β oxidation of fatty acids and are usually present in
cheese made with milk submitted to 12 V/cm the health indexes were dairy products (Bozoudi et al., 2018). The presence of acetoin and 3-
maintained (AI, TI, HSFA, and DFA) (p > 0.05). These results are due methyl 1-butanol is related to the aroma of fresh, creamy, and buttery
to the thermal and non-thermal effects of OH and the interaction be- cheese (Ranadheera et al., 2019). The non-presence of some compounds
tween the food matrix components, which provided changes in the may contribute to the aroma of the products such as acetic acid (vi-
molecular structure, breaking double bonds and increasing saturated negar, pungent), 4-heptanone, 2,6 dimethyl (green) and 3-methyl bu-
fatty acid content (Ferreira et al., 2019). The thermal effect of OH is tanoic acid (rancid) (Ranadheera et al., 2019).
mainly related to the time necessary to reach the processing tempera-
ture, resulting in a prolonged time under heating. This parameter was 3.8. Sensory evaluation
higher for the cheese prepared with milk submitted to 4 V/cm (Fig. 1).
Therefore, the use of low and intermediate electric field can result in a The results of the sensory analysis are presented in Table 6. Cheeses
negative impact on both the fatty acid profile and health indices. scored 6.26–7.72 on a 9-point scale, indicating that the consumers liked
However, it is worth mentioning that all cheeses presented low AI slightly to moderately the products. Minas Frescal cheeses made with
milk subjected to ohmic heating (QI, QII, and QIII) presented greater

7
R.S. Rocha, et al.
overall acceptance (p < 0.05) when compared to the conventional
cheese (Qc). The results indicate that the color changes (Table 2), the
presence of a larger number of volatile compounds (Table 6, except for
QI) and the less elastic, firm and hard texture (Table 3) of the cheeses
QI, QII, and QIII can contribute for greater consumer acceptance. In-
deed, there are few reports regards the sensory tests of dairy products
manufactured using emerging technologies, such as ohmic heating, and
in this sense our data are very useful and bring relevant contribution
about the market potential of this product. Further studies should cover
projective methods (Judacewski et al., 2019) and innovative methods
based in consumer perception (Torres et al., 2017).
In addition, in vivo studies (animals or humans, Sarfraz et al., 2019)
aiming to evaluate the health effects of the consumption of OH-treated
cheeses in comparison to the products submitted to conventional pro-
cessing are welcome.
4. Conclusion
The OH treatment of milk to produce Minas Frescal cheese resulted
in a decrease in elasticity, hardness, and firmness and an improvement
in the sensory acceptance of the cheeses. Cheeses made with milk
submitted to OH at low and intermediate electric fields (4 and 8 V/cm)
had positive effects on the formation of bioactive compounds (total
phenolics, DPPH, ACE inhibitory activity, α-glucosidase, and α-amy-
lase), with a negative impact on the fatty acid profile. Cheese made
with OH-treated milk subjected to a high electric field (12 V/cm) pre-
sented similar characteristics to the cheese made with pasteurized milk,
with higher sensory acceptance, and shorter heating time (3.17 vs.
4.67 min), but did not contribute to a higher content of bioactive
compounds.
Therefore, OH has shown an interesting alternative for the manu-
facture of Minas Frescal cheese, with positive impacts on the functional
characteristics, sensory acceptance, and rheological properties.
Considering a multidimensional aspect, the use of the intermediate
electric field (8 V/cm, 72–75 °C) is recommended due to the shorter
processing time, which is compatible with the dairy industries.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
The authors are grateful to the finantial support of the Coordenação
de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) –
Finance Code 001, Fundação de Apoio a Pesquisa do Rio de Janeiro
(FAPERJ) and Instituto Federal de Educação, Ciência e Tecnologia do
Rio de Janeiro (IFRJ). A.G. Cruz, M.C.Silva, M.Q. Freitas and E.A.
Esmerino also thanks to Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPQ) for the grants.
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