Journal of Functional Foods 45 (2018) 435–443

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Journal of Functional Foods

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

Probiotic Minas Frescal cheese added with L. casei 01: Physicochemical and T
bioactivity characterization and effects on hematological/biochemical
parameters of hypertensive overweighted women – A randomized double-
blind pilot trial
Marcella F. Sperrya, Hugo L.A. Silvaa, Celso F. Balthazara, Erick A. Esmerinoa, Silvia Verruckb,
Elane S. Prudenciob, Roberto P.C. Netoc, Maria Inês B. Tavaresc, Jaqueline C. Peixotod,
Filomena Nazzaroe, Ramon S. Rochaf, Jeremias Moraesf, Aline S.G. Gomesf, Renata S.L. Raicesf,
⁎
Márcia C. Silvaf, Daniel Granatog, Tatiana C. Pimentelh, Mônica Q. Freitasa, Adriano G. Cruzf,
a
Universidade Federal Fluminense (UFF), Faculdade de Veterinária, 24230-340 Niterói, Brazil
b
Universidade Federal de Santa Catarina (UFSC), Departamento de Ciência e Tecnologia de Alimentos, 88034-001 Santa Catarina, Brazil
c
Universidade Federal do Rio de Janeiro (UFRJ), Instituto de Macromoléculas Professora Eloisa Mano (IMA), 21941-598 Rio de Janeiro, Brazil
d
Universidade Castelo Branco (UCB), Pós-Graduação em Nutrição clínica, 21710250, Rio de Janeiro, Brazil e Hospital Municipal Miguel Couto, Rio de Janeiro, Brazil
e
Istituto di Scieze dell’Alimentazione, CNR-ISA, Via Roma, 64, 83100 Avellino, Italy
f
Instituto Federal de Educação, Ciência e Tecnologia do Rio de Janeiro (IFRJ), Departamento de Alimentos, 20270-021 Rio de Janeiro, Brazil
g
Universidade Estadual de Ponta Grossa (UEPG), Departamento de Engenharia de Alimentos, 84030-900 Ponta Grossa, PR, Brazil
h
Instituto Federal do Paraná (IFPR), Campus Paranavaí, 87703-536 Paranavaí, PR, Brazil

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

Keywords: This study aimed to evaluate the effects of Lactobacillus casei 01 on quality parameters of Minas Frescal cheese
Minas Frescal cheese and to investigate the hematological and clinical effects of its regular consumption on hypertensive overweighed
Lactobacillus casei 01 women (n = 30, 50 g, 28 days). Microbiological (lactic acid bacteria and probiotic counts), physicochemical
Dairy foods parameters (proximate composition, pH, proteolysis, organic acid levels, and fatty acid profile), water mobility
Functional foods
(TD-NMR), bioactivity [antioxidant and angiotensin I-converting enzyme (ACE) inhibitory activity], and mi-
Clinical trial
crostructure were evaluated. The addition of L. casei 01 (108 CFU/g) did not change the proximate composition
Hypertension
and the microstructure of the cheeses, while a more suitable fatty acid profile, lower pH, and higher antioxidant
and ACE inhibitory activities, proteolysis and organic acid levels were observed. L. casei 01 improved the total
cholesterol, low-density lipoprotein-cholesterol, high-density lipoprotein-cholesterol, triacylglycerides, diastolic
and systolic pressure, hemoglobin, and hematocrit count of the hypertensive overweighed women.

1. Introduction
The growing demand for functional foods, in particular with respect
to probiotic dairy products, has driven the dairy industry to the de-
velopment of new foods (Reid, 2015). The wide variety and consump-
tion of cheese all over the world constitute a growing market for pro-
biotic cheese (Karimi, Sohrabvandi, & Mortazavian, 2012). It provides a
valuable alternative to fermented milks and yogurts as a food vehicle
for probiotic delivery because of potential advantages, such as making a
buffer against the high acidic environment in the gastrointestinal tract.
Therefore, this condition provides a more favorable environment for
probiotic survival throughout the gastric transit because of the higher
pH of the medium (Cruz, Buriti, de Souza, Faria, & Saad, 2009).
Cardiovascular diseases are the main causes of death worldwide,
and some diseases can increase their risk, including obesity and hy-
pertension, which is associated with high levels of total and low-density
lipoprotein cholesterol (LDL). These parameters are of paramount im-
portance to human’s health since they are directly associated with the
risk of hypertension, which can triple the risk of heart attacks
(Sharafedtinov et al., 2013).
Minas Frescal cheese is considered as one of the most popular
cheeses in Brazil, with an increased manufacturing yield, besides pro-
viding excellent conditions for survival and growth of probiotic strains
(Gomes et al., 2011; Dantas et al., 2016). Probiotic bacteria may be

⁎
Corresponding author.
E-mail address: food@globo.com (A.G. Cruz).

https://doi.org/10.1016/j.jff.2018.04.015
Received 24 January 2018; Received in revised form 10 April 2018; Accepted 11 April 2018
1756-4646/ © 2018 Elsevier Ltd. All rights reserved.
M.F. Sperry et al.
incorporated into Minas Frescal cheese because of its high water ac-
tivity, pH above 5.0, low salt content, and absence of synthetic pre-
servatives (Felicio et al., 2016). In addition, the ingestion of probiotic
Minas Frescal cheese has been shown to induce significant improve-
ments in hypertension (Lollo et al., 2015) and immune system para-
meters (Lollo et al., 2012) in vivo.
L. casei 01 is a probiotic culture associated to many health benefits,
such as antioxidant activity (Kim et al., 2005), immunostimulation
(Galdeano & Perdigón, 2006), decreases in cholesterol levels (Liong &
Shah, 2006), anticarcinogenic activity (Liu, Chu, Chou & Yu, 2011),
reduction in inflammation in the TNBS model of rat colitis (Ivanovska
et al., 2017), and decreases in the Clostridium difficile associated diar-
rhea (Auclair, Frappier & Milette, 2015). This strain can maintain the
viability above the minimum therapeutic level (> 6 log cfu/mL) in
diary matrices over the storage, such as yogurts (Pimentel, Garcia &
Prudencio, 2012), ice cream (Balthazar et al., 2018), and Prato cheese
(Silva et al., 2018). Furthermore, it has been shown to survive gastro-
intestinal conditions (Balthazar et al., 2018). However, up to now, there
is no report dedicated neither to the effects of L. casei 01 on techno-
logical parameters of Minas Frescal cheese nor to the beneficial effects
of the probiotic cheese on hypertensive overweight individuals.
In this scenario, the present study aimed to evaluate the effects of L.
casei 01 on the technological parameters of Minas Frescal cheese, and to
investigate the hematological and clinical effects of its regular con-
sumption on hypertensive overweighed women after 28 days.
2. Material and methods
2.1. Minas Frescal cheese processing
Cheese processing was performed in accordance with Gomes et al.
(2011), with slight modifications. Two different cheeses were manu-
factured: the conventional cheese (containing only the starter culture –
negative control) and the probiotic cheese (containing both the starter
and L. casei 01 – test sample). For each formulation, eighty liters of raw
milk containing 3.4% w/w fat (Instituto GPA, São Gonçalo, Brazil) were
pasteurized at 72 °C/15 s (Model pro110, Arpifrio, São Paulo, Brazil)
and then cooled to 37 °C. Calcium chloride (LABSYNTH, São Paulo,
Brazil) was added at a rate of 0.2 g/L milk. The starter culture (Lacto-
coccus lactis ssp. lactis and Lactococcus lactis ssp. cremoris - Sacco, Brazil)
and the probiotic strain Lactobacillus casei 01 (Chr. Hansen, Valinhos,
Brazil) were added (1% v/v, 7 - 8 log CFU/g) and the content was
homogenized manually. Powdered rennet (Halamix power, Chr Hansen,
Valinhos, São Paulo, Brasil) was added at a concentration of 3 g/L, and
the mixture was homogenized for 2 min. The resulting cheese-milk was
kept at 37 °C/40 min to coagulate. The curd was then cut, the whey run-
off and the remaining curd placed in 250 g plastic molds. The cheeses
were submitted to dry salting (0.8% w/v NaCl, LABSYNTH, São Paulo,
Brazil), and stored in cold chambers at 5 °C. The experimental protocol
Fig. 1. Experimental protocol for microbiological, physico-chemical and functional properties of conventional and probiotic cheeses.
436
Journal of Functional Foods 45 (2018) 435–443
for microbiological, physicochemical, and functional properties of
conventional and probiotic cheeses is presented in Fig. 1.
2.2. Microbiological counts
A portion of 25 g cheese and 225 mL sterile 0.1% peptone water (w/
v) (Oxoid, São Paulo, Brazil) were homogenized and serial dilutions
were made. The microbial counts were enumerated in triplicate using
the pour plate technique. For enumeration of L. lactis, the culture
medium M17 agar (Oxoid, São Paulo, Brazil) was used, which was
performed under aerobic conditions, incubated at 37 °C/72 h. The
enumeration of L. casei 01 was performed under anaerobic conditions
using MRS agar (Oxoid, São Paulo, Brazil) at 37 °C/72 h with
Vancomycin (Darukaradhya, Phillips, & Kailasapathy, 2006). Vanco-
mycin stock solution (2% w/v) was prepared with distilled water and
filter sterilized using a 0.45 µm membrane (Millipore Corporation) and
0.5 mL solution was added into 1 L base medium at the time of analysis.
2.3. Physicochemical analyses
The proximate composition (moisture, protein, and fat) of cheeses
was determined using traditional methods. Moisture was determined by
drying 5 g sample at 100–105 °C for 24 h. Protein was determined in
duplicate by the Kjeldahl method, by multiplying the nitrogen content
by the factor 6.38, and fat was quantified by the Gerber method (Felicio
et al., 2016). Results were expressed as g/100 g.
The pH measurements were carried out using a digital pH meter
(Micronal, B-375, Digimed, Piracicaba, São Paulo, Brasil), by inserting the
electrode directly into the triturated samples. The mineral content (cal-
cium and sodium) was determined by Inductively Coupled Plasma (ICP)
Optical Emission Spectrometry (Spectro Analytical Instruments, Kleve,
Germany) according to Felicio et al. (2016). The proteolytic activity was
determined after reaction with a reactive solution (OPA) containing so-
dium dodecyl sulfate, sodium tetraborate decahydrate, dithiothreitol, o-
phthalaldehyde, and ethanol. The amino acids and peptides released from
the lactic acid cultures were quantified by absorbance readings of the OPA
derivatives at 340 nm (Gomes et al., 2011).
The organic acids (lactic and acetic acids) were quantified by high-
performance liquid chromatography (HPLC) using an Aminex X-87H
column (300 mm, 7.8 mm, Bio-Rad Laboratories, Richmond, CA, USA)
and a guard column containing disposable H+ cartridges (Biorad-Rad
Laboratories) at 65 °C (Gomes et al., 2011). A sulfuric acid solution
(0.009 mol/L) was used as the mobile phase. The flow rate was 0.6 mL/
min, and UV–VIS detection was performed at 220 nm, with volume
injection of 25 μL (auto sampler injector). Standards of lactic and acetic
acids were prepared, and analytical curves were prepared using 6
evenly distributed concentrations (R2 > 0.99), and the chromato-
graphic peaks were integrated using the Millenium software. Standard
solutions of each acid were prepared tree times for plotting the
M.F. Sperry et al.
analytical curves. The concentrations used were 1, 2, 3, 4, 5 and 6 mg/
100 mL for lactic acid and 0, 1, 2, 3, 4 and 5 mg/100 mL for acetic acid.
2.4. Bioactivity
The angiotensin I-converting enzyme inhibitory (ACE) activity was
determined following the spectrophotometric assay (Torres-Llanez,
González-Córdova, Hernandez-Mendoza, Garcia, & Valllejo-Cordoba,
2011). The inhibition was calculated using Eq. (1):
ACE inhibitory activity (%) = [(B−−A)/(B−−C)] × 100
where A is the absorbance in presence of ACE and ACE component, B is
the absorbance with ACE and without the ACE component, and C is the
absorbance without the ACE or ACE component (Torres-Llanez et al.,
2011).
The antioxidant activity was determined using the 2,2-diphenyl-1-
picrylhydrazyl (DPPH) radical-scavenging method following the ex-
perimental condition outlined by Lee, Jeewanthi, Park, and Paik
(2016). The DPPH radical-scavenging activity was calculated using Eq.
(2):
Antioxidant activity (%) = [1−(absorbancesample /absorbancecontrol)] × 100
2.5. 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. Transversal relaxation time (with a time constant
T2) was measured using Carr-Purcell-Meiboom-Gill (CPMG) pulse se-
quence with 8,192 echoes, 800 μs between each echo and 16 scans with
a waiting time of 10 s. Only even echoes were considered for ex-
ponential fitting to eliminate the errors associated with 90° pulses.
Inverse Laplace Transform of all acquired signal decay was performed
using WinDXP® software.
2.6. Fatty acid profile
The fatty acid profile (FA) was determined according to Felicio et al.
(2016). The fatty acid methyl esters were quantified by gas chromato-
graphy (GC) using an Agilent Technologies model 5975C gas chroma-
tograph (Santa Clara, California, USA) equipped with a flame-ioniza-
tion detector (FID), split injection (1:100), a fused silica capillary
column (Supelco SP-tm-2560, Supelco, Bellefonte, PA, USA) and a
programmed temperature gradient. The initial column temperature was
programmed at 65 °C/4 min, then rising to 185 °C at 16 °C/min, re-
maining at this temperature for 12 min followed by a second increase to
235 °C at 20 °C/min, remaining at this temperature for further 14 min,
giving a total time of 40 min. The gas (White Martins, São Paulo, Brazil)
flow rates were 1.4 mL/min for the stripping gas (H2), 30 mL/min for
the auxiliary gas (N2), and 30 and 300 mL/min respectively for the H2
and synthetic gas. The split was 1/80, the injection volume 2 µL, and
identification and quantification of the methylated samples were made
by comparison of their retention times with those of the respective
standards (SIGMA 05632 and SIGMA 189–19). Data were collected
using the MSD ChemStation software and the FA was classified as short-
chain FA (SCFA from C2 to C4), medium chain FA (MCFA from C6 to
C12) and long-chain FA (LCFA from C14 to C24). Atherogenic (AI) and
thrombogenic (TI) indices were also calculated, according to Eqs. (3)
and (4) (Batista et al., 2017).
AI = (C12: 0 + 4 × C14: 0 + C16: 0)
/[Σ MUFA + Σ PUFA (n−6) and (n−3)]
Journal of Functional Foods 45 (2018) 435–443
TI = (C14: 0: 0 + C18: 0)/[0.5 × ΣMUFA 0.5 Σ PUFA(n−6)
+ 3 × ΣPUFA(n−3) + (n−3)/(n−6)] (4)
2.7. Microstructural properties
Scanning electron microscopy (SEM) micrographs of the cheese
samples were obtained using a Jeol JSM 6390 LV scanning electron
microscope (Tokyo, Japan). The samples were fractured perpendicular
to their long axis, mounted on individual stubs with fracture face up-
(1) wards and coated with a fine gold layer using a Leica EM SCD 500
sputter coater (Wetzlar, Germany). All the samples were examined
using an accelerating voltage of 8 kV at a magnification of 100× (100
µm). For the visualization of the probiotic bacteria in the cheese sample
a Leica TCS-SP5 confocal laser scanning microscope (Wetzlar,
Germany) was used. The probiotic bacteria were stained with DAPI at a
concentration of 0.1 g/L in phosphate buffered saline (PBS buffer),
according to the methodology proposed by Russell, Newman, and
Williamson (1975). For this purpose, sections of the cheese, approxi-
mately 5 mm × 5 mm × 2 mm thick, were cut with a scalpel, placed on
a microscope slide and one drop of fluorescent probe added to the cut
surface. Probiotic bacteria were imaged at 405 nm laser excitation. The
image was acquired on CLSM using a 100× objective (HCX PL APO CS
(2) with a numerical aperture of 1.44 – oil immersion objective) with Leica
Type F immersion oil. Images were taken with a Leica microsystem
camera. The acquisition and processing of data were carried out using
the LAS AF Lite software.
2.8. Clinical protocol
The experimental clinical protocol for conventional and probiotic
cheeses is presented in Fig. 2. A 4-week double blind, randomized trial
was performed to assess the effects of probiotic Minas Frescal cheese on
hematological, inflammatory, and other metabolic indices (Valls et al.,
2015). For this purpose, a group of 30 participants (n = 15 individuals
consumed the probiotic cheese and n = 15 individuals consumed the
conventional cheese) were randomly selected from patients admitted to
the outpatient clinic of the Nutrition Service of Miguel Couto Municipal
Hospital, in Rio de Janeiro city, from September to October 2016. The
number of individuals is not large but suitable for a pilot test and in the
range used in previous studies (Songisepp et al., 2012; Sharafedtinov
et al., 2013; Beltrán-Barrientos et al., 2018).
The inclusion criteria for participation in the study were: to be female
aged between 35 and 72 years, to be overweighed and diagnosed with
arterial hypertension. Exclusion criteria were: a history of gastrointestinal
disease, food allergy or acute infection, use of any antimicrobial agent or
probiotic food and/or supplement within the preceding month, pregnancy,
or breast-feeding. The control group was characterized as being 53 ± 12
years old and 75.5 ± 7.5 kg while the probiotic group was characterized
as being 55.5 ± 12.5 years old and 75 ± 10 kg. Therefore, age and body
weight were matched between both groups.
All volunteers signed an informed consent in accordance with in-
stitutional policies and were informed that they could withdraw from
the study at any time. The trial was approved by the Ethics Committee
from the Miguel Couto Hospital. For the treatment group, the diet was
supplemented with 50 g/day of probiotic product (Minas Frescal
cheese) containing L. casei 01 while members of the control group
consumed the same amount of a probiotic-free Minas Frescal cheese
(negative control). Both cheeses were manufactured weekly for 28 days.
The effect of the diet supplemented with the probiotic cheese on
patients' health indices was evaluated. Hematological indices (he-
moglobin, erythrocytes, leukocytes, and lymphocytes), inflammatory
indices (white blood cell count), and metabolic indices (lipids: total
cholesterol, LDL- cholesterol, HDL- cholesterol, and triacylglycerides)
were determined using certified reference methods in the local clinical
(3)
laboratory at days 0 and 28.
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M.F. Sperry et al. Journal of Functional Foods 45 (2018) 435–443

Fig. 2. Experimental clinical protocol for conventional and probiotic cheeses.

2.9. Statistical analyses Table 1

Microbiological, physicochemical, and functional properties of conventional
All the cheese processing was performed three times, and the ana- and probiotic Minas Frescal cheeses.
lyses were performed in triplicate. The microbiological, physicochem- Conventional cheese Probiotic cheese
ical, and bioactive parameters were assessed by Student-t test for in-
dependent samples. For the responses obtained in the clinical trial, Lactococcus lactis 7.63a ± 0.2 7.71a ± 0.32
Lactobacillus casei 01 – 8.32 ± 0.67
mean values at days 0 and 28 were compared using the paired Student-t
pH 5.66a ± 0.1 5.15b ± 0.14
test (baseline vs data on day 28). Equality of variances was formally Proteolysis 0.38b ± 0.1 0.52a ± 0.05
checked by the F-test. All statistical analyses were performed using the Lactic acid 4.67b ± 0.2 5.32a ± 0.43
XLSTAT software for Windows version 2017.2 (Addinsoft, Paris, Acetic acid – 2.12 ± 0.54
France), considering p ≤ 0.05 (Nunes, Alvarenga, Sant'Ana, Santos, & Antioxidant activity 53.80b ± 6.1 70.89a ± 4.9
ACE-inhibitory activity 43.82b ± 3.3 61.20a ± 6.6
Granato, 2015).
Moisture 55.8a ± 0.2 54.7a ± 0.6
Proteins 17.2a ± 0.3 18.1a ± 0.3
3. Results and discussion Lipids 23.45a ± 0.15 24.04a ± 0.11
Na 285a ± 1.80 278a ± 1.34
Ca 605a ± 2.45 624a ± 1.67
3.1. Microbiological counts
*Values are expressed mean ± standard deviation (n=3).
a
The enumeration of lactic and probiotic cultures (L. lactis and L. Means with different lowercase superscripts in the same line indicate pre-
casei 01, respectively) is shown in Table 1. Similar L. lactis counts sence of statistical difference (p < 0.05) between cheeses. L. lactis and L. casei
(p > 0.05) were observed for both cheeses, therefore, the addition of L. 01 counts are expressed in log CFU/g. pH is dimensional. Proteolysis is ex-
casei 01 did not influence the growth of L. lactis (p > 0.05). The pro- pressed in absorbance340. Lactic acid and acetic acid concentrations are ex-
biotic cheese manufactured with L. casei 01 showed viable cells counts pressed in mg/100 g. Antioxidant activity and ACE-inhibitory activity are ex-
above 108 CFU/g, evidencing that the manufacturing process was ef- pressed in percentage of inhibition. Moisture, protein and fat are expressed as
fective for obtaining a probiotic Minas Frescal cheese. The counts were g/100 g. Na and Ca are expressed as mg/100 g.
b
within the range recommended for a product to be considered pro- Means with different lowercase superscripts in the same line indicate
biotic, that is, between 107 and 108 CFU/g (Felicio et al., 2016). Our presence of statistical difference (p < 0.05) between cheeses. L. lactis and L.
data showed the role of the cheese matrix as a suitable vehicle to casei 01 counts are expressed in log CFU/g. pH is dimensional. Proteolysis is
maintain the viability of probiotic bacteria throughout the processing, expressed in absorbance340. Lactic acid and acetic acid concentrations are ex-
corroborating the data reported by Yerlikaya & Ozer (2014) and Dantas pressed in mg/100 g. Antioxidant activity and ACE-inhibitory activity are ex-
pressed in percentage of inhibition. Moisture, protein and fat are expressed as
et al. (2016). However, there is no study relating the addition of L. casei
g/100 g. Na and Ca are expressed as mg/100 g.
01 in Minas Frescal cheese, which reveals the contribution of the pre-
sent study.
and probiotic Minas Frescal cheeses are shown in Table 1. Proteolysis is
related to the digestibility of the cheese and formation of bioactive
3.2. Proximate composition and physicochemical properties compounds and cheese flavor (Batista et al., 2015). Overall, the addi-
tion of L. casei 01 provided a lower pH (5.15 ± 0.14 vs 5.66 ± 0.1,
The proximate composition (moisture, protein, and fat values) and p ≤ 0.05), a higher proteolysis index (0.52 ± 0.05 vs 0.38 ± 0.1,
sodium and calcium contents are presented in Table 1. The addition of p ≤ 0.05), and increased lactic acid (5.32 ± 0.43 g vs 4.67 ± 0.2 mg/
L. casei 01 did not affect significantly (p > 0.05) the chemical com- 100 g, p ≤ 0.05), and acetic acid (2.12 ± 0.54 vs 0 mg/100 g) levels.
position (moisture, proteins, and lipids) and the mineral content (so- These findings suggest a careful selection of the probiotic strains for the
dium and calcium) of the samples when compared to the conventional manufacture of fresh cheeses as some changes can be observed.
cheese. Recent studies have also reported that the probiotic bacteria did Probiotic microorganisms probably used part of the lactose of the
not change the composition of Minas Frescal cheese (Felicio et al., milk to produce small amounts of organic acids during the processing of
2016; Dantas et al., 2016). the Minas Frescal cheese, resulting in lower pH and increased lactic and
The results of pH, proteolysis, and organic acids of the conventional

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M.F. Sperry et al. Journal of Functional Foods 45 (2018) 435–443

acetic acid contents (Batista et al., 2017). The higher acidity of the
probiotic cheese can be beneficial to the microbiological stability of the
product, with increases on the shelf life. This is particularly interesting
from the industry point of view, as Minas Frescal cheese is a product
with shortened shelf life, because of its high moisture content and low
salt content (Oliveira et al., 2017). Furthermore, the results indicate
that the probiotic culture has an enzymatic system with sufficient ac-
tivity for the growth in milk, and future studies could evaluate its use
with double function, as starter and probiotic cultures (Dantas et al.,
2016). However, the increased acidity and the presence of acetic acid
can impair the sensory acceptance of the products. The acetic acid can
contribute to the tart taste associated with vinegary, pungent, and
acidic taste (Batista et al., 2017).
The metabolic activity of the probiotic culture resulted in the in-
creased proteolytic activity on the probiotic Minas Frescal cheese. This
result could be positive, as the probiotic cheese would have higher
digestibility and bioactive peptides could be formed. However, it could
have a lower consumer acceptance, because of the increased total free
amino acid content, which contributes decisively to cheese flavor
(sweet, bitter, or malty) and can be precursors for the synthesis of other
flavors or volatile aroma, resulting in off-flavors (Batista et al., 2015).
It is noteworthy that, although the addition of probiotic strain re-
Fig. 3. Transversal relaxation domain distribution of conventional and pro-
sulted in changes in the physicochemical properties of Minas Frescal biotic cheese proportioned by time domain nuclear magnetic resonance.
cheese, the pH values (5.15), proteolysis (0.52), lactic acid content
(5.32 mg/100 g), and acetic acid content (2.12 mg/100 g) observed
were similar to previous studies with conventional and probiotic Minas dietary compounds, thus increasing the bioavailability of dietary anti-
Frescal cheeses, using other probiotic strains (Dantas et al., 2016; oxidants; (3) protective effects against oxidative stress by restoring gut
Felicio et al., 2016). microbiota (Amaretti et al., 2013), among others. Overall, the in-
corporation of probiotics in foods can be a suitable technological
3.3. Bioactivity strategy to supply dietary antioxidants, but more studies are needed to
demonstrate its antioxidant potential (Mishra et al., 2015).
The probiotic cheese presented higher ACE-I activity when com-
pared to the conventional cheese (61.20 ± 6.6 % and 43.82 ± 3.3 %, 3.4. Time domain nuclear magnetic resonance
respectively, p ≤ 0.05, Table 1) suggesting that the addition of L. casei
01 collaborated in a positive way to generate bioactive peptides. This Fig. 3 shows transversal relaxation domain distribution after Inverse
result is in agreement with Korhonen (2009), who found that the pro- Laplace Transform for each sample. Three domains can be observed for
biotic culture, together with the starter cultures, was capable of re- both samples related to confined water (T2,1), fat (T2,2) and bulk water
leasing different bioactive peptides during milk fermentation. However, (T2,3) and they were in accordance with previous study (Chen & Liu,
no study on the ACE-I activity in probiotic Minas Frescal cheese has 2012).
been reported so far. According to Gandhi and Shah (2016), ACE-in- The presence of the probiotic led to a greater alteration in the
hibitory peptides are biologically active peptides that possess anti-hy- proton relaxation of the fat, causing an increase of 106.3–130 ms, as
pertensive properties and have been detected in many fermented milk well as an increase in the percentage of protons in this domain. This
products. These compounds are released by bacterial proteinases and may indicate that L. casei 01 is located mainly in this relaxation en-
peptidases during fermentation. Studies on the ACE-inhibitory activity vironment, contributing to a better fat dispersion and, consequently,
and ACE-inhibiting peptides of different cheese varieties have already increasing its molecular mobility, increasing the T2,2 value. The re-
been conducted (Gandhi & Shah, 2016). Donkor, Henriksson, Vasiljevic duction observed in T2.3 also suggests some chemical interaction be-
and Shah (2005) reported that some probiotic strains possess proteo- tween the water molecules and L. casei 01, reducing its molecular
lytic activity in milk-based media and can produce potent ACE-in- mobility and its transverse relaxation time from 1364 to 975.1 ms. The
hibitory peptides during fermentation of milk. The proteolytic activity confined water (T2,1), present in the micropores of the cheese, did not
of L. casei has been studied and reviewed by other authors (Khalid & undergo significant changes because it was difficult to access (15.12 vs
Marth, 2010) and was observed in the present study. The main com- 12.75 ms).
ponents of this system are the cell envelope proteins; these enzymes are
responsible for extracellular hydrolysis of milk proteins during fer- 3.5. Fatty acid profile
mentation (Lozo et al., 2011) and production of peptides with ACE-
inhibitory activity (Rojas-Ronquillo et al., 2012). Table 2 shows the fatty acid profiles of the Minas Frescal cheeses.
The addition of L. casei 01 in Minas Frescal cheeses also led to a Overall, the Minas Frescal cheeses showed intermediate values for short
great improvement in the antioxidant activity when compared to the and medium chain fatty acids (SCFAs and MCFAs in the range of
conventional cheese (p ≤ 0.05), with mean values ranging from 62.12–76.54 and 116.85–127.36 mg/100 g of lipids respectively) and
53.80 ± 6.1% (conventional) to 70.89 ± 4.9% (probiotic) (Table 1). higher values for long chain fatty acids (2151.45 − 2298.38 mg/100 g
Despite Minas Frescal is a non-ripened cheese, a high antioxidant ac- fat).
tivity was observed in this study, probably because of the increased The addition of L. casei 01 generated different patterns, with sig-
proteolysis caused by probiotic bacteria. Some mechanisms associated nificant differences (p ≤ 0.05) in the probiotic and conventional Minas
to the antioxidant activity of probiotic cultures are: (1) promotion of Frescal cheese. Higher levels of medium and long-chain fatty acids
the production of antioxidant biomolecules such as exopolysaccharides (MCFAs and LCFAs) were observed in the probiotic cheese (p ≤ 0.05).
showing an in vitro antioxidant and free-radical scavenging activities; It has been reported that MCFAs exhibit antimicrobial activity for oral
(2) increased enzymatic activities involved in the transformation of microorganisms (Huang, Alimova, Myers, & Ebersole, 2011), and have

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Table 2
Fatty acids profile of conventional and probiotic Minas Frescal cheeses. G
Fi P
Fatty Acids Conventional cheese Probiotic cheese Fi
V
Butanoic acid (C4:0) 62.12a ± 6.2 76.54a ± 6.2
Docanic acid (C10:0) 56.59a ± 2.0 61.86a ± 0.4 G
Dodecanoic acid (C12:0) 60.26b ± 0.5 65.49a ± 0.7 G V
Tetradecanoic acid (C14:0) 210.69a ± 4.8 220.62a ± 1.6 P
Miristoleic acid (C14:1) 28.30a ± 3.6 35.60a ± 1.1
V V
Palmitic acid (C16:0)
Palmitoleic acid (C16:1)
746.65a ± 25.6
77.05a ± 3.0
800.53a ± 3.1
83.11a ± 0.1
Fi
Stearic acid (C18:0) 318.70b ± 4.4 350.43a ± 4.6
657.40b ± 0.8 677.28a ± 8.0 Fi
Oleic acid (C18:1c)
Conjugated linoleic acid (CLA) 62.10a ± 4.0 61.35a ± 3.8
P G
α-Linolenic acid (ALA) 50.56b ± 3.1 69.46a ± 1.5
Short-chain fatty acids (SCFA) 62.12a ± 6.2 76.54a ± 6.2 (a)
Medium-chain fatty acids (MFCA) 116.85b ± 2.5 127.36a ± 1.1
Long-Chain Fatty Acids (LCFA) 2151.45b ± 28.0 2298.38a ± 24.2
Monounsaturated fatty acid (MUFA) 762.75b ± 5.8 795.99a ± 9.3
Polyunsaturated fatty acid (PUFA) 112.66b ± 0.9 130.81a ± 5.3
Atherogenic index (IA) 1.88a 1.89a
Thrombogenic index (TI) 2.25a 2.15b

*
Values are expressed mean ± standard deviation.
a
Means with different lowercase superscripts in the same line indicate pre-
sence of statistical difference (p < 0.05) between cheeses. Fatty acids are ex-
pressed as mg/100 g fat. The fatty acid composition was classified SCFA as C4 to
C6, MCFA as C8 to C12 and LCFA as C14 to C24 in accordance with Felicio et al.
(2016). 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)].
b
Means with different lowercase superscripts in the same line indicate pre- (b)
sence of statistical difference (p < 0.05) between cheeses. Fatty acids are ex-
pressed as mg/100 g fat. The fatty acid composition was classified SCFA as C4 to
C6, MCFA as C8 to C12 and LCFA as C14 to C24 in accordance with Felicio et al.
(2016). 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)].

therapeutic potential in neurological disorders (Lei, Vacy, & Boon,

2016). The LCFA are associated with increased adhesion of the pro-
biotic cultures to the mucosal wall of the distal intestine portion
(Batista et al., 2017).
The probiotic cheeses were characterized by higher levels of
monounsaturated fatty acids (MUFAs), especially oleic acid (C18:1c).
MUFAs are protective against Metabolic Syndrome and Cardiovascular
Disease Risk Factors (Garaffo et al., 2011). The addition of L. casei 01 in
Minas Frescal cheese also resulted in higher PUFAs values when com-
pared to the conventional cheese (130.81 ± 5.3 vs 112.66 ± 0.9 mg/
100 g fat, respectively, p ≤ 0.05). Among the PUFAs, the conjugated
linoleic acid (CLA) and the α-Linolenic acid (ALA) stood out. CLA has
become the focus of many current studies, once its consumption inhibits
the carcinogenesis, atherosclerosis, reduces body fat content, improves
hyperinsulinemia, modulates the immune system, controls inflamma-
tion, and has antioxidant effects (Yuan, Chen, & Li, 2014). Probiotic
addition maintained CLA levels (p > 0.05) and increased the α-Lino-
lenic acid (ALA) content in the Minas Frescal cheeses (p ≤ 0.05). The
increase in the unsaturation degree of the fatty acids provided by
probiotic addition is a universally conserved adaptation response of
many microorganisms (Batista et al., 2017).
Concerning the saturated fatty acids (SFA, Table 2), the probiotic
cheese showed higher levels of stearic acid (C18:0) when compared to
the conventional cheese (350.43 ± 4.6 vs 318.70 ± 4.4 mg/100 g fat,
p ≤ 0.05). The same trend was observed for dodecanoic acid
(65.49 ± 0.7 vs 60.26 ± 0.5) (p ≤ 0.05).
Both Minas Frescal cheeses presented low AI (1.88-1.89) and TI
(2.25-2.15). The addition of probiotic culture resulted in the main-
tenance of the AI (p > 0.05), and decreased TI (p ≤ 0.05). The de-
crease in the TI is related to the improvement on the fatty acid profile of
the cheeses provided by the probiotic culture, with higher level of
(c)
Fig. 4. SEM micrographs of cheeses: (a) without probiotic bacteria and (b) with
probiotic bacteria. P: Protein matrix; G: Fat globule; V: Void space; Fi:
Filamentous structures. (c) The visualization of the probiotic bacteria in the
cheese by confocal laser scanning microscopy.
MUFA, PUFA, oleic acid and α-Linolenic acid (ALA). The lower TI
suggests that the probiotic Minas Frescal cheese would have lower
tendency to form clots in the blood vessels than the conventional
cheese.
Indeed, despite the higher content of saturated fatty acids (stearic
and docecanoic acids), probiotic cheeses do not seem to favor the lipids
adhesion to cells of the immune and circulatory systems, as the AI was
similar to that of the conventional product. Furthermore, the addition
of probiotic culture contributed to the improvement of the health as-
pects of the Minas Frescal cheeses, improving the fatty acid profile and
decreasing TI.
3.6. Microstructure
No significant differences on micrographs were observed between
the probiotic Minas Frescal cheese and the conventional cheese (Fig. 4a
and b). Overall, both cheeses presented a slightly compacted structure,
with void spaces. Fox, Guinee, Cogan, and McSweeney (2000) stated
that demineralization occurs by loss of calcium and phosphate in the

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Table 3
Clinical indices after ingestion of conventional (probiotic free) and probiotic Minas Frescal cheeses for 28 days.
Clinical indexes Reference values1 Conventional Minas Frescal cheese Probiotic Minas Frescal cheese Extent of changes

2 2
Baseline (t = 0 days) After treatment P-value After treatment P-value Probiotic vs. Convencional
(t = 28 days) (t = 28 days) (P-value3)

Total cholesterol (mg/dL) < 200 201.3 ± 2.36 192.2 ± 1.55 0.65 188.8 ± 2.4 0.018 0.043
Total triglyceride (mg/dL) < 150 186.5 ± 1.12 179.3 ± 1.67 0.72 175.3 ± 1.12 0.03 0.034
LDL (mg/dL) < 100 160.5 ± 1.52 154.1 ± 1.11 0.55 149.5 ± 1.52 0.022 0.055
HDL (mg/dL) > 45 32.4 ± 0.89 34.2 ± 0.32 0.53 38.14 ± 1.89 0.015 0.034
Systolic BP (mmHg) 120 150.84 ± 1.83 136 ± 1.31 0.44 120.1 ± 1.83 0.001 0.0001
Diastolic BP (mmHg) 80–84 90.3 ± 0.56 92.4 ± 1.76 0.45 86.3 ± 1.56 0.001 0.0001
Hemoglobin (g/dL) 12–16 9.4 ± 0.78 10.7 ± 0.67 0.41 15.6 ± 0.78 0.012 0.032
Hematocrit (%) 36–48 28.5 ± 0.22 34.8 ± 0.68 0.41 38.2 ± 0.22 0.001 0.0001
Total leukocytes (cells/mm3) 4000–11,000 6345 ± 11 6585 ± 23 0.35 6556 ± 20 0.42 0.567
Total lymphocytes (cells/mm3) 880–4950 3456 ± 22 3567 ± 12 0.54 3735 ± 20 0.13 0.820
White Blood cells (millions cells/mL) 4.0–5.2 4.80 ± 2.45 4.70 ± 0.45 0.17 5.40 ± 1.15 0.12 0.430
Platelets (million cells/mm3) 150–450 238.20 ± 0.34 239.90 ± 1.62 0.24 234.30 ± 1.34 0.43 0.560

1
Values based on clinical laboratory references.
2
Probability values obtained by paired Student-t test to compare baseline vs conventional cheese group or baseline vs probiotic cheese group after 28 days.
3
Probability values obtained by unpaired Student-t test to compare conventional cheese group vs probiotic cheese group after 28 days.

casein micelle due to decrease in pH values. This compromises the
extension of the bound between micelles and, therefore, results in a less
compacted microstructure. Sousa e Silva et al. (2013) reported that
decreases in the void spaces may result in decreases in probiotic via-
bility due to damages on the bacterial cells.
The micrographs also showed that the fat globules were ‘trapped’
within the protein matrix. Filamentous structures were observed on the
surfaces of the fat globules. According to Lobato-Morales et al. (2007)
these structures may consist of casein chains and/or whey protein
chains and the deposition of these proteins onto a fat globule’s surface is
responsible for the integration of the fat globule onto the protein ma-
trix. Fig. 4C confirms the presence of the L. casei 01 added to the Minas
Frescal cheese.
3.7. Clinical trial
Significant differences were observed in the clinical indices between
the two groups (p ≤ 0.05) during 28 days of cheese intake (Table 3).
The consumption of probiotic cheeses led to a reduction in total cho-
lesterol (6.21%; 201.3 ± 2.36 vs 188.8 ± 2.4 mg/dL), LDL choles-
terol (6.85%; 160.5 ± 1.52 vs 149.5 ± 1.52 mg/dL), and triacylgly-
cerides (6%; 186.5 ± 1.12 vs 175.3 ± 1.12 mg/dL) and an increase of
HDL cholesterol (17.7%; 32.4 ± 0.89 vs 38.14 ± 1.89 mg/dL) after
28 days of consumption when compared to the baseline (day 0)
(p ≤ 0.05). On the other hand, the ingestion of the conventional Minas
Frescal cheese (control group) did not influence the cholesterol-related
parameters (p > 0.05). The hypocholesterolemic potential of some
probiotic strains (Ryan, Hanes, Schafer, Mikolai, & Zwickey, 2015;
Hjerpsted, Dragsted, & Tholstrup, 2016; Thushara, Gangadaran, Solati,
& Moghadasian, 2016) has been evaluated using human subjects. Sev-
eral cholesterol-lowering mechanisms of probiotics have been pro-
posed, including deconjugation catalyzed by bile salt hydrolase en-
zymes, binding of cholesterol onto probiotic cellular surface and
incorporation into their cell membrane, production of short-chain fatty
acids from oligosaccharides, co-precipitation of cholesterol with de-
conjugated bile, and cholesterol conversion to co-prostanol (Hjerpsted
& Tholstrup, 2015; Ishimwe, Daliri, Lee, Fang, & Du, 2015; Miremadi,
Sherkat, & Stojanovska, 2016; Thushara et al., 2016; Xavier-Santos,
Lima, Simão, Bedani, & Saad, 2018).
In addition, our findings showed that the ingestion of 50 g probiotic
Minas Frescal cheese per day for 28 days was able to attenuate the
systolic (SBP) and diastolic (DBP) blood pressure parameters
(p ≤ 0.05). The average SBP and DBP values ranged from
150.84 ± 1.83 and 90.3 ± 0.56, and 120.1 ± 1.83 and 86.3 ± 1.56
mm Hg for days 0 and 28 of consumption of probiotic Minas Frescal
cheese, respectively. The ingestion of the conventional Minas Frescal
cheese (control group) did not influence the SBP and DBP parameters
(p > 0.05). Hypertension or BP is defined as SBP above 140 mmHg
and DBP above 90 mmHg, being one of the key risk factors for an in-
dividual prone to many diseases including coronary heart disease,
cerebral hemorrhage, and renal and cardiac failure (Upadrasta &
Mademputi, 2016). Therefore, the result suggested that after 28 days of
consumption of the probiotic Minas Frescal cheese, the women had SBP
and DBP values lower than the levels considered as hypertension. Our
data are in-line with other clinical trials in which probiotic strains (L.
plantarum, L. animalis DPC6134) added into dairy foods and given to
obese hypertensive patients reduced the arterial BP (Hayes et al., 2007;
Sharafedtinov et al., 2013). Furthermore, they are consistent with
previous studies covering probiotic Minas Frescal cheese in an animal
model (Lollo et al., 2015) and fermented milk added with L casei strain
Shirota in older people (Aoyagi et al., 2017), who also observed a re-
duction in systolic and diastolic pressures due to the use of probiotic
strains.
The arterial BP-lowering effect may be related to vascular relaxa-
tion, which is in turn associated with the reduction of reactive species in
vascular biology. As the probiotic cheese containing L. casei had higher
in vitro antioxidant activity compared to the conventional (probiotic
free) cheese (Table 1), the in vivo BP-lowering effect seems reasonable.
The reduction of blood pressure is also associated with some peptides
derived from milk protein by probiotic fermentation, as these peptides
have hypotensive action and operate on renin–angiotensin system
having an ACE-inhibiting potential and a blood pressure lowering ef-
fect.
Upadrasta and Mademputi (2016) stated that regulation of hy-
pertension via administration of probiotics is cross-linked with several
different mechanisms, such as improving lipid levels, triglyceride le-
vels, bile acid deconjugation, and controlling body mass index. In ad-
dition, an increase in absorption of nutrients, phytoestrogens (act as
vasodilatory factors), and reduction in plasma glucose levels may also
influence the probiotic effect in BP regulation.
Gut dysbiosis has been linked to cardiovascular diseases including
hypertension (Adnan et al., 2017); thus, manipulation of the gut mi-
crobiota might constitute a novel treatment for hypertension, also
through the design of new functional food containing specific micro-
organisms. Different engineered probiotics were proposed to formulate
functional food with personalized effect giving rise to a lowering of
danger for human health, such as obesity and hypertension (Chua,
Kwok, Aggarwal, Sun, & Chang, 2017). In our case, a not engineered

441
M.F. Sperry et al.
strain of the probiotic L. casei was used to enhance the functional
properties of Minas Frescal cheese, in order to lead to a specific effect
on systolic and diastolic blood pressure.
The hemoglobin and hematocrit levels increased after the 4-week
consumption of probiotic cheese (15.6 ± 0.78 g/dL and
38.2 ± 0.22%, respectively, p ≤ 0.05) when compared to the baseline
values (9.4 ± 0.78 g/dL and 28.5%) and probiotic free cheese
(10.7 ± 0.67 g/dL and 34.8 ± 0.68%) (p ≤ 0.05). These values are
consistent with the findings obtained by Salahuddin, Akhter, Akter,
Miah, and Ahmad (2013) in Swiss Albino mice, who reported an in-
crease in hemoglobin after treatment with probiotic bacteria, and with
Santos, Guedes, Jesus, Santos, and Fiaconne (2017), who showed an in
vivo increase in the levels of hemoglobin, hematocrit, red blood cells,
total lymphocyte count, serum magnesium, albumin and total serum
cholesterol reduction by a synbiotic product. The increase in he-
moglobin and hematocrit levels could be attributed to the fact that
probiotics can increase the blood parameter values by hematopoietic
stimulation (Rajikkannu et al., 2015). Furthermore, the colonization of
the probiotic cultures in the intestine and the consequent decrease in
colonic pH is associated to the increase in iron absorption from the diet,
as the probiotic cultures can reduce ferric iron into highly absorbable
ferrous (Hoppe, Önning, Berggren & Hulthén, 2015). As iron is required
to produce red blood cells, and these are key components of he-
moglobin, the increased of iron absorption by probiotic consumption
could be associated to the increased levels of hemoglobin and hema-
tocrit.
Finally, for the other hematological indices (leukocytes, lympho-
cytes and platelets) and the inflammatory marker (white blood cells),
no significant differences were observed neither in the comparison to
the baseline, nor between the groups treated with conventional and
probiotic cheeses (p > 0.05). The results suggest that the probiotic
culture did not show any harmful changes on blood hematological and
inflammatory parameters and it could improve health condition by
enhancing concentration of hemoglobin and hematocrit count
(Abudabos, Murshed, Qaid, & Abdelrahman, 2016).
The results of this study state that a consumption of 50 g of a Minas
Frescal cheese added with L. casei 01 as probiotic culture was able to
reduce the cholesterol levels and the systolic and diastolic blood pres-
sure in hypertension overweight women. This is the first study that
evaluated this effect using the L. casei 01 in Minas Frescal cheese and its
application in a vivo study with hypertense individuals. Interventional
studies with a longer duration and with other targeted individuals
should be conducted to confirm these pilot results.
4. Conclusions
The addition of L. casei 01 in Minas Frescal cheese affected the pH,
proteolysis, organic acid levels, fatty acid profile, antioxidant, and ACE
inhibitory activities. However, the probiotification of Minas Frescal
cheese did not affect the proximate composition significantly. In an
interesting manner, proteolysis and ACE inhibitory activity correlated
positively with the presence of probiotic culture. In addition, the NMR
relaxation measurements show that the probiotic addition caused
changes in the molecular mobility due to the new intermolecular in-
teraction. This is a positive action and corroborates the changes ob-
served in the others quality parameters. The continuous ingestion of
Minas Frescal cheese containing L. casei 01 led to an improved lipid and
hematological profiles in hypertensive overweight women, which is an
interesting finding, as the product is widely consumed by Brazilians.
Future studies should focus on a clinical study with a larger number of
individuals aiming to evaluate the microbiota of the patients.
Acknowledgements
The authors gratefully thank to the Universidade Federal de Santa
Catarina (UFSC), especially to research supported by LCME-UFSC, such
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Journal of Functional Foods 45 (2018) 435–443
as scanning electron microscopy (SEM) and confocal laser scanning
microscopy. D. Granato thanks CNPq for a productivity grant (process
303188/2016-2).
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